2-((3,5-Dinitrobenzyl)thio)quinazolinones: Potent Antimycobacterial Agents Activated by Deazaflavin (F420)-Dependent Nitroreductase (Ddn)

Article abstract of DOI:10.1021/acs.jmedchem.0c01374

Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-thio-substituted quinazolinone 26. Structure-activity relationship (SAR) studies indicated a crucial role for both meta-nitro substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5, the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in cofactor F420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity (rv3547), indicating that reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.

Full text of DOI:10.1021/acs.jmedchem.0c01374

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Article
2‑((3,5-Dinitrobenzyl)thio)quinazolinones: Potent Antimycobacterial
Agents Activated by Deazaflavin (F420)‑Dependent Nitroreductase
(Ddn)
Yanlin Jian, He Eun Forbes, Fabian Hulpia, Martijn D. P. Risseeuw, Guy Caljon,
́
̀
Helene Munier-Lehmann, Helena I. M. Boshoff, and Serge Van Calenbergh*
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ABSTRACT: Swapping the substituents in positions 2 and 4 of the previously synthesized but yet undisclosed 5-cyano-4-
(methylthio)-2-arylpyrimidin-6-ones 4, ring closure, and further optimization led to the identification of the potent antitubercular 2-
thio-substituted quinazolinone 26. Structure−activity relationship (SAR) studies indicated a crucial role for both meta-nitro
substituents for antitubercular activity, while the introduction of polar substituents on the quinazolinone core allowed reduction of
bovine serum albumin (BSA) binding (63c, 63d). While most of the tested quinazolinones exhibited no cytotoxicity against MRC-5,
the most potent compound 26 was found to be mutagenic via the Ames test. This analogue exhibited moderate inhibitory potency
against Mycobacterium tuberculosis thymidylate kinase, the target of the 3-cyanopyridones that lies at the basis of the current
analogues, indicating that the whole-cell antimycobacterial activity of the present S-substituted thioquinazolinones is likely due to
modulation of alternative or additional targets. Diminished antimycobacterial activity was observed against mutants affected in
cofactor F420 biosynthesis (fbiC), cofactor reduction (fgd), or deazaflavin-dependent nitroreductase activity (rv3547), indicating that
reductive activation of the 3,5-dinitrobenzyl analogues is key to antimycobacterial activity.
line or linezolid.8 Thus, novel anti-TB agents are still needed to
tackle the spread and drug resistance burden of M. tuberculosis.
M. tuberculosis thymidylate kinase (MtbTMPK) is an enzyme
INTRODUCTION
■
Infection with Mycobacterium tuberculosis is currently one of the
top 10 leading causes of death by a single pathogen worldwide
involved in the synthesis of the DNA building block thymidine-
and leads to an estimated 10 million new tuberculosis (TB)
5′-triphosphate and is thus indispensable for mycobacterial
growth.9−12 The reported co-crystal structures of MtbTMPK
indicate it as an attractive target for identifying new anti-TB
agents.13−15
cases annually.1 The airborne transmission of M. tuberculosis
from an individual with active tuberculosis to a healthy
individual typically occurs via droplet nuclei containing a few
viable bacteria expelled through coughing. Although combina-
tion therapy with four first-line agents for the treatment of drug-
sensitive TB has decreased TB mortality, control of the disease is
hindered by the emergence of antibiotic-resistant strains as well
as by the pathogen’s ability to enter a persistent state, in which it
tolerates conventional antibiotics, even in the absence of genetic
drug resistance, necessitating long and complicated treatment
regimens.2,3 The newly approved agents bedaquiline,4 delam-
anid,5 and pretomanid6 have provided new hope for afflicted
patients. However, the first M. tuberculosis isolate resistant to
bedaquiline and delamanid was reported shortly after their
clinical introduction,7 and pretomanid only has a narrow use as it
is contraindicated in patients with hypersensitivity to bedaqui-
Through high-throughput screening, 3-cyanopyridones (Fig-
ure 1) were discovered as a new class of MtbTMPK inhibitors by
AstraZeneca in 2015.14 They reported that replacement of the
methylthio group of compound 1 with aromatic substituents
improved the inhibitory potency (Figure 1, 1 and 2).14
Received: August 5, 2020
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A

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Figure 1. Structures of reported MtbTMPK inhibitors (1−4), Pks13 inhibitors (5−8), and thioquinazolinone compounds (9, 10), as well as
compounds designed in this study.
a
Scheme 1. Synthesis of 13a−c, 16a, 16b, and 18
a
Reagents and conditions: (a) NaBH4, BF3·Et2O, tetrahydrofuran (THF); (b) CBr4, PPh3, ethyl acetate (EA); (c) NaBH4, THF; (d)
azobisisobutyronitrile (AIBN), DBDMH, CCl4, reflux.
However, none of these derivatives possessed whole-cell
activity.14 Starting from the reported compound 3, we
introduced an additional N atom, which resulted in moderately
active 2-aryl cyanopyrimidone analogues 4 (manuscript in
preparation).
Inspired by these observations, we interchanged the 4-
methylthio and 2-aromatic substituents of 4, leading to
cyanopyrimidone 21 (Figure 1). Moreover, the methylthio
group was expanded with a phenyl and a 3,5-dinitrophenyl ring,
a commonly used antimycobacterial pharmacophore,16,17 since
B
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a
Scheme 2. Synthesis of 21−23
a
Reagents and conditions: (a) thiourea, K2CO3, 3-bromobenzaldehyde, EtOH; (b) R = H, CH3I, NaOH, EtOH/H2O (2/1, v/v); R = Ph/3,5-
(NO2)2Ph, BnBr/13a, K2CO3, MeCN.
a
Scheme 3. Synthesis of 26−47, 50a−h, and 56a−d
a
Reagents and conditions: (a) (i) SOCl2, 90 °C, (ii) NH4SCN, acetone; (b) appropriate arylmethyl bromide, DMSO/MeCN (1/10, v/v), K2CO3;
(c) mCPBA, THF; (d) CH3I, K2CO3, MeCN; (e) thiourea, 200 °C; (f) (i)THF/dioxane (1/1, v/v), SOCl2, catalytic dimethylformamide (DMF);
(ii) NH4SCN, acetone/dioxane (15/10, v/v).
sterically demanding groups in the same position of cyanopyr-
idones were shown to be tolerated by AstraZeneca.14
Encouraged by the finding that a cyclization strategy to form
bicyclic (Figure 1, 5 → 6)18 or polycyclic motifs19 (Figure 1, 7
→ 8) can successfully improve the antimycobacterial activity
while maintaining inhibitory activity against the polyketide
synthase 13 (Pks13) target, and that thioquinazolinone
compounds such as 9 and 10 (Figure 1) were previously
shown to possess potent antitubercular activity,20,21 the phenyl
ring was fused to the pyrimidone ring to generate novel (S)-
quinazolinone derivatives.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of desired analogues 21−23, 26−
47, 50a−h, 56a−d, and 63a−d relied on the preparation of the
desired heterocycle core, followed by S-alkylation to install the
side chain (scheme 1−4). In cases where the required arylmethyl
bromide for S-alkylation was not commercially available, it was
prepared from either the corresponding alcohol (Scheme 1).22
Arylmethyl alcohols 12a−c, 15a, and 15b were prepared by
NaBH4 reduction of the corresponding commercially available
acids or aldehydes.23 Alternatively, 18 was prepared by radical
C
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a
Scheme 4. Synthesis of 63a−d
a
Reagents and conditions: (a) sulfuric acid, fuming nitric acid; (b) (i) SOCl2, reflux; (ii) NH3, CH2Cl2; (c) K2CO3, dimethyl sulfoxide (DMSO),
amine, 60 °C; (d) Pd/C, H2, EtOH; (e) KOH, CS2, EtOH/H2O, reflux; (f) 13a, DMSO/MeCN (1/10, v/v), K2CO3.
c
Table 1. MtbTMPK Inhibitory and Antimycobacterial Activity of Cyanopyrimidone and Thioquinazolinone Compounds
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis H37Rv in 7H9/
b
glucose liquid culture. Isoniazid was included as an internal control reference compound (MIC = 0.15 μM). Approximate value reaches upper
limit of solubility. Values are given in micromolar (μM), MtbTMPK IC50 values for which standard deviations (SD) are given are the calculated
c
mean values of at least two measurement results.
bromination with 1,3-dibromo-5,5-dimethylhydantoin
(DBDMH).24
(mCPBA, 1 equiv) yielded sulfoxide 46. Alternatively, 47 was
obtained via methylation27 of the same intermediate with CH3I.
Due to the poor solubility of aminonicotinic/aminoisonico-
tinic/aminopiconilic acids in thionyl chloride, a different
synthetic strategy was applied to gain access to the (S)-
pyridopyrimidinones. The aminonicotinic/aminoisonicotinic/
aminopiconilic acid was condensed directly with thiourea20,28 at
high temperatures to give 55a, 55c, and 55d. Since this method
only gave low conversion of 4-aminonicotinic acid to 55b
(monitored by liquid chromatography−mass spectrometry
(LC−MS)), for this analogue, we returned to the SOCl2-
mediated acid chloride formation, in a THF/dioxane mixture.
Alkylation of the resulting crude (S)-pyridopyrimidinone
intermediates 55a−d with 13a afforded the desired compounds
56a−56d.
The synthesis of 21−23 (Scheme 2) commenced with a
Knoevenagel condensation of 3-bromobenzaldehyde with ethyl
cyanoacetate, followed by a cyclization with thiourea under basic
conditions25 to afford the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyr-
imidine intermediate 20, which was immediately alkylated25
with iodomethane or (substituted) benzyl bromide to furnish
21−23.
Envisioned relatively large series of thioquinazolinones with
differently substituted arylmethyl moieties or various quinazo-
linone core modifications were synthesized through S-alkylation
of thioquinazolinone with the appropriate arylmethyl bromides
under basic conditions (Scheme 3).25 Condensation of
NH4SCN with anthranilic acid chloride derivatives, obtained
in situ from reaction of the (substituted) anthranilic acid with
thionyl chloride, afforded the thioquinazolinone intermediates
25, 49a−h, which were selectively S-alkylated with arylmethyl
bromides to furnish the desired compounds 26−45 and 50a−h.
Further oxidation26 of 26 with m-chloroperoxybenzoic acid
The synthesis of 6-substituted analogues 63a−d is shown in
Scheme 4. The introduction of a nitro group in position 6 of 3-
fluorobenzoic acid using fuming nitric acid led to intermediate
58,29 which was converted into an amide. After nucleophilic
aromatic substitution of amide 59 with a secondary amine, Pd/
D
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b
Table 2. Modifications of (S)-Dinitrobenzyl Side Chain
a
Ar
comp.
MIC 7H9
MRC-5 CC50
Ar
comp.
MIC 7H9
MRC-5 CC50
>64
3,5-(NO2)2Ph
Ph
2-thienyl
biphenyl
4-ClPh
4-MePh
4-MeOPh
3,4-Cl2Ph
3-ClPh
26
27
28
29
30
31
32
33
34
35
0.6
12.5
>50
>50
>50
>50
>50
>50
3.13
0.78
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2-ClPh
36
37
38
39
40
41
42
43
44
45
2.3
>50
12.5
>50
>50
≥50
≥50
>50
>50
>50
2-MeOPh
2-NO2Ph
3-NO2Ph
4-NO2Ph
5-nitrofuran-2-yl
5-nitrothiophen-2-yl
2, 4-(NO2)2Ph
2, 6-(NO2)2Ph
3-NO2-5-CF3Ph
>64
>64
>64
35
>64
>64
>64
>64
3-MeOPh
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis H37Rv in 7H9/
b
glucose liquid culture. Values are given in μM.
b
Table 3. Modifications of Quinazolinone Core
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis H37Rv in 7H9/
b
glucose liquid culture. Values are given in μM.
C-mediated hydrogenation afforded the desired substituted
anthranilic amide, which condensed30 with CS2 under basic
conditions to yield 6-substituted thioquinazolinone intermedi-
ates (62a−d). Subsequently, the desired compounds (63a−d)
were achieved through S-alkylation of 62a−d with 13a.
Biological Activity. Enzymatic Activity and Antimyco-
bacterial Activity against Wild-Type Strain. The synthesized
compounds were evaluated for their MtbTMPK inhibitory
potency (Table 1). Except for 23, the cyanopyrimidones (21,
22) showed moderate inhibitory activity. Quinazolinone 26 and
the corresponding sulfoxide 46 only poorly inhibited
MtbTMPK.
(Table 1). Cyanopyrimidone analogues 21 and 22 showed
moderate whole-cell activity, while introduction of two nitro
groups in the meta position (23) led to activity loss. Surprisingly,
the cyclized analogue 26 exhibited submicromolar antimyco-
bacterial activity in 7H9 medium, without showing cytotoxicity
against MRC-5 fibroblasts. Oxidation of 26 to the corresponding
sulfoxide (46) resulted in a drastic decrease of antimycobacterial
activity.
Encouraged by these results, we further focused on the
structure−antimycobacterial activity relationship and optimiza-
tion of 26 (Table 2; MtbTMPK inhibitory activity in Table S2).
Elimination of the nitro groups (27) led to a 20-fold decrease in
activity, while the 2-thienyl isostere 28 and the para-biphenyl
analogue 29 were inactive.
Despite their poor enzyme inhibitory potency, these
analogues mostly showed in vitro antimycobacterial activity
E
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b
Table 4. Modification of Quinazolinone Core by Introduction of Polar Moieties
a
Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M. tuberculosis H37Rv in 7H9/
b
glucose liquid culture. Values are given in μM.
Substitution in para position of the phenyl ring failed to
improve the antimycobacterial activity (30−32). In contrast,
selected substituents in ortho or meta position improved activity
(34−36). However, unlike analogue 26 which displayed
antimycobacterial activity in all growth media tested, com-
pounds 34−36 only showed activity when assayed in 7H9/
glucose medium, potentially indicating that their antimycobac-
terial activity might be associated with carbon source
metabolism31 (Table 5, SI7H9/glucose vs GAST/Fe = 6, 64, and 16
for 34, 35, and 36, respectively). Moreover, compounds 34−36
were inactive in media containing bovine serum albumin (BSA)
(albumin, dextrose, and catalase (ADC) supplement and 7H9/
glucose/BSA).32
Since the nitro groups of 26 proved important for
antimycobacterial activity, we investigated the effects of mono-
nitro-functionalized analogues. Remarkably, only ortho-sub-
stituted nitro analogue 38 displayed moderate activity. Also,
analogues with moieties with higher reduction potentials that
are more readily reduced such as nitrofuran (41) and
nitrothiophene (42) failed to show antimycobacterial activity.
Finally, we investigated the optimal positional arrangement of
the two nitro groups. We observed that the 3,5-dinitrophenyl
pattern was superior to two other isomers (2,4-analogue 43 and
2,6-analogue 44), which failed to inhibit antimycobacterial
growth at the highest concentration (50 μM). Replacement of
the 5-nitro with a trifluoromethyl group (45) also had a negative
impact on the activity.
In an attempt to increase the polarity of 26, we decided to
investigate all different aza-isomers (i.e., pyridopyrimidinones
56a−d, Table 4). Remarkably, only the 5-aza isomer (56a)
showed some antimycobacterial activity, while all other isomers
were inactive. Alternatively, several polar groups were
introduced at the 6-position (63a−d). The morpholine and
N-methylpiperazine derivatives (63c and 63d, respectively)
showed interesting whole-cell activity.
To further investigate whether the antimycobacterial activities
of compounds were metabolic-dependent, active compounds
were investigated under different growth conditions (Table 5).
Most dinitro-containing analogues (26, 47, 50b, 50f, 50g, 63c,
63d) performed well under different test conditions, potentially
reflecting less metabolic dependence, in contrast to non- or
mono-nitro compounds (27−38), which only displayed activity
in certain media. Comparison between BSA-free 7H9/glucose
medium and BSA-containing medium suggests that the
introduction of polar groups may effectively circumvent BSA
binding (63c, 63d). This observation was further supported by
the correlation analysis between RBSA/BSA‑free and clog P (Figure
2), where RBSA/BSA‑free value is lower with improved clog P.
Mutagenicity of Compound 26. Due to the mutagenic
potential caused by nitro groups, a 384-well microplate
“fluctuation” version of the classical reverse mutation Salmonella
typhimurium Ames test was performed for 26 in the absence and
presence of S9 metabolic activation (Table 6). Compound 26
exhibited genotoxic effect against TA98 strain in the absence of
S9, while no genotoxic effect was observed in the presence of S9.
Additionally, the genotoxic effect of 26 in the absence of S9
decreased with increased concentration, which may be due to
the bactericidal activity of 26 against S. typhimurium TA 98
strain. With the TA100 strain, no genotoxicity was observed,
regardless of metabolic activation.
Next, we turned our attention to modifications in the
quinazolinone core (Table 3). Methylation of the N3-amino
group of 26 (47) led to a small drop in activity (MIC = 1.6 μM).
Additional Cl substitution of the phenyl ring of the
quinazolinone core revealed that substitution on positions 7
and especially 6 is tolerated, while 5- or 8-substitution is
detrimental to activity (compare 50b/50c v. 50a/50d).
Changing the 6 Cl substituent of 50b (compounds 50e−h)
did not result in improved activity, although still affording
analogues with MIC values in the low micromolar range.
Antimycobacterial Activity against Mutant Strains.34−36
Based on the fact that 26 exhibited weak inhibitory potency
against MtbTMPK, we predicted that the whole-cell activity of
this (and structurally related) thioquinazolinone was likely due
F
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Table 5. Antimycobacterial Activity of Hits in Different
ab
Media
Figure 2. RBSA/BSA‑free vs calculated clog P (created by MATLAB): X-
axis, RBSA/BSA‑free. When RBSA/BSA‑free is in a range, the minimum value
was used; Y-axis, calculated clog P using SwissADME.33 The numbers
for each spot correspond to compound numbers in the structure−
activity relationship (SAR) tables.
Table 6. Evaluation of Mutagenicity via the Ames Fluctuation
Assay Performed with Compound 26 against TA100 and
TA98 Strains (Highest Concentration, 349 μM)
aa
Ames fluctuation assay
comp.
S9 activation
TA100
TA98
2-nitrofluorene
4-nitroquinoline N-oxide
aminoanthracene
−
−
+
n.d.
+
+
+
n.d.
+
26
26
−
+
−
−
+
−
a
−, negative mutagenicity; +, positive mutagenicity. n.d., not
determined. For all compounds, the statistical significance related to
mutagenicity was set at 0.05.
F
420-dependent nitroreductase Rv3547 and the Rv3547:A76E
mutant, all of which are resistant to pretomanid, were performed
to assess whether an activation mechanism is required for
antimycobacterial activity (Table 7). All aforementioned mutant
strains were resistant to all nitro-containing compounds studied,
providing evidence that these compounds undergo a similar
bioactivation mechanism as pretomanid37 (reductive activation
of the nitro group) (Table 7).
To support the notion that compound 26 and structurally
related compounds are prodrugs activated by F420-dependent
nitroreductase, resistant mutants were generated by plating of
M. tuberculosis on 7H11/OADC agar plates containing 3.75, 7.5,
and 15 μM concentrations of compound 26. As expected, the
frequency of spontaneous resistance to compound 26 was very
high, with a mutation frequency of 2.60 × 10−05 at 3.75 μM, 2.50
× 10−05 at 7.5 μM, and 1.50 × 10−06 at 15 μM (Supporting
Information, Table S4). Thirty-four 26-resistant mutants were
tested for their level of resistance by MIC determination against
compound 26 (Supporting information, Table S5), and all were
found to be more than 40-fold resistant compared with the
parental strain (H37Rv). Additionally, 33 out of 34 resistant
mutants generated to compound 26 were cross-resistant to
pretomanid and delamanid, while the susceptibility of these
mutants remained unchanged relative to isoniazid and
rifampicin, strongly further supporting that compound 26 is
activated by a mechanism similar to that of pretomanid and
delamanid.
a
Minimum inhibitory concentration (MIC) is the minimum
concentration required to inhibit >99% growth of M. tuberculosis
H37Rv in liquid culture and is given in μM. RBSA/BSA‑free = MIC
(7H9/glucose/BSA)/MIC (7H9/glucose).
b
to modulation of alternative or additional targets. Since the most
promising compounds possess a nitro group as in the anti-TB
drug pretomanid, MIC determinations in mutants deficient in
cofactor F420 biosynthesis (FbiC) or an enzyme involved in the
regeneration of reduced F420 (Fgd), as well as a mutant in the
G
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a
Table 7. Antimycobacterial Activities of 3,5-Dinitrobenzyl Compounds against Mutant Strains
a
fbiC mutant deficient in cofactor F420 biosynthesis; fgd mutant deficient in F420 reduction; rv3547 and rv3547:A76E mutants deficient in F420
-
dependent nitroreductase. Minimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99% growth of M.
tuberculosis H37Rv in 7H9/glucose liquid culture, and is given in μM.
Evaluations against selected mutants known to impede
reduction of nitro groups were less sensitive to 26 and related
compounds featuring identical S-(3,5-dinitrobenzyl) substitu-
ents, indicating that the antimycobacterial activities of these
compounds relied on the reductive activation of 3,5-
dinitrobenzyl moiety. The prodrug notion was further supported
by the observation that resistant mutations to compound 26
were cross-resistant to known prodrugs pretomanid and
delamanid. Taken together, thioquinazolinone analogues
prove to be potent antimycobacterial agents. Further
optimization efforts could be directed toward reducing the
protein binding of 35 or reducing the mutagenicity of 26.
CONCLUSIONS
■
We synthesized a large series of 38 thioquinazolinone analogues
structurally derived from an earlier cyanopyrimidone-type
MtbTMPK inhibitor. Several of these analogues displayed
antimycobacterial activity, with the 3,5-dinitrobenzyl analogue
26 possessing submicromolar activity. SAR exploration of 26
indicated that ortho/meta-substitution of the (S)-benzyl side
chain was tolerated, leading to the identification of the potent
non-nitro analogue 35, which, however, failed to display
antimycobacterial activity when assayed in different growth
media. Additionally, addition of BSA significantly lowered the
antimycobacterial activity of 35, suggesting high protein
binding. Next, modifications of the quinazoline core of 26
were investigated. It was observed that substitution in position 3,
6, or 7 is tolerated, leading to the discovery of analogues 63c and
63d with lower clog P. Most of the active compounds were
devoid of cytotoxicity against MRC-5, while the Ames
fluctuation assay demonstrated that dinitrobenzyl analogue 26
induced statistically significant reverse mutations without
metabolic activation in the TA98 strain.
EXPERIMENTAL SECTION
■
Enzymatic Assay. After expression and purification of MtbTMPK
as described by Munier-Lehmann et al.,38 the enzymatic assay was
conducted as previously reported.15 Briefly, at fixed concentrations of
ATP (0.5 mM) and dTMP (0.05 mM), compounds were evaluated at
different concentrations using the spectrophotometric assay described
by Blondin et al.39 The reaction medium consists of 50 mM Tris−HCl,
pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM NADH, 1 mM phosphoenol
pyruvate, and 2 units each of coupling enzymes (lactate dehydrogenase,
H
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pyruvate kinase, and nucleoside diphosphate kinase). The IC50 value
was calculated using KaleidaGraph to plot and fit the experimental data
points.
quantum coherence (HSQC), and heteronuclear multiple bond
correlation (HMBC) NMR spectrometry. Additionally, high-resolution
mass spectrometry (HRMS) was performed on a Waters LCT Premier
XE time-of-flight (TOF) mass spectrometer equipped with a standard
electrospray ionization (ESI) and modular LockSpray interface. The
purity (95%) of the tested compounds was determined by LC-MS
analysis (Waters AutoPurification system: a Waters Cortecs C18
column (2.7 μm, 100 × 4.6 mm2); a gradient system of formic acid in
H2O (0.2%, v/v)/MeCN; a flow rate of 1.44 mL/min; and a gradient of
95:5−0:100 in 10 min).
General Procedure A: Synthesis of (Substituted) Quinazolinone
Intermediates. According to a literature report,44 (substituted)
aminobenzoic acid (6.0 mmol) was refluxed in thionyl chloride (10
mL) for 2 h. The excess thionyl chloride was removed in vacuo, and the
crude acyl chloride was dissolved in acetone (10 mL) and added
dropwise to a solution of NH4SCN (6.9 mmol) in acetone (5.0 mL).
The reaction mixture was stirred at room temperature for 1 h, and the
obtained solid was collected through filtration. Then, the brown solid
was suspended in 2 M aq NaOH (15 mL) and filtered. Water (15 mL)
was added to the collected filtrate, and the mixture was acidified with 2
N aq HCl to pH 2. The obtained solid was collected by filtration, and
additionally washed with H2O/MeOH (1/1 v/v, 50 mL). The resulting
solid was used in the next step without further purification.
In Vitro Antitubercular Activity. The MIC values of all
compounds were determined as previously decribed.40−42 In brief, M.
tuberculosis H37Rv (ATCC 27294) and the mutant strains were grown
to OD650nm 0.2 in the respective medium prior to further 1000-fold
dilution in fresh medium. Drugs were twofold serially diluted in
duplicate in the medium of choice (50 μL/well) in a concentration
range spanning 100−0.049 μM in sterile 96-well U-bottom clear
polysteyrene microtiter plates. Isoniazid and DMSO were used as
positive and negative controls, respectively. An equal volume (50 μL) of
diluted cells was added to the plates with serial drug dilution. Plates
were sealed in ziplock bags and incubated at 37 °C. After 7−14 days, the
plates were read with enlarging inverted mirror plate reader. The MIC
was recorded as the concentration that fully inhibited all visible growth.
Generation of Compound 26-Resistant Mutants. Two
independent cultures of M. tuberculosis H37Rv (ATCC 27294) were
grown to mid-logarithmic phase. At that stage, the cells were harvested
and resuspended in fresh 7H9/ADC/Tween 80 medium to final cell
densities approximating 1 × 106, 1 × 107, 1 × 108, and 1 × 109 colony-
forming units (CFU)/mL. Dilutions of the suspensions were made, and
the resulting dilutions (100 μL) were plated on drug-free plates to
determine the actual cell density. To generate mutants against
compound 26, 100 μL of the four cell suspensions of the two
independent cultures was plated on 7H11/OADC agar plates
containing compound 26 at 3.75, 7.5, and 15 μM concentrations.
The plates were incubated at 37 °C for 4 weeks, and the colonies were
counted. Rifampicin was used as a control. A total of 34 colonies were
picked and inoculated in 7H9/ADC/Tween, and MICs were
determined for compound 26, pretomanid, delamanid, isoniazid, and
rifampicin to confirm their resistance to compound 26 and cross-
resistant to pretomanid and delamanid.
General Procedure B: Synthesis of Final Compounds. The
(substituted) thioquinazolinone/S-pyridopyrimidinone intermediate
(0.50 mmol) and K2CO3 (1.5 mmol) were dissolved in DMSO/MeCN
(1/10 v/v, 11 mL), and the appropriate arylmethyl bromide (0.45
mmol) was added in one portion. The resulting mixture was stirred at
room temperature for 1 h. After the consumption of starting material
was found by monitoring with TLC, water (100 mL) was added to the
reaction mixture. The resulting mixture was acidified with 1 N aq HCl
to pH 6. The generated solid was collected by filtration, dried in a high-
vacuum oil pump, and purified to give the desired compound.
1-(Bromomethyl)-3,5-dinitrobenzene (13a). According to a
literature report,23 to a suspension of NaBH4 (36 mg, 0.94 mmol) in
dry THF (10 mL) at 0 °C was added 3,5-dinitrobenzoic acid (0.10 g,
0.47 mmol), followed by BF3·Et2O (0.16 mL, 1.2 mmol). After
addition, the reaction mixture was warmed to room temperature and
stirred for 1 h, after which the reaction was quenched with 1 N aq HCl
(5.0 mL). The aqueous phase was extracted with CH2Cl2 (25 mL × 2),
and the organic layer was dried, concentrated, and purified to give
intermediate alcohol 12a (eluent system: 25% EA in petroleum ether,
In Vitro Genotoxicity Assay.43 Approximately 10 million bacteria
were exposed in triplicate to the test agent compound 26 (six
concentrations), a negative control (vehicle), and a positive control (2-
nitrofluorene against TA 98 in the absence of S9, 4-nitroquinoline N-
oxide against TA100 in the absence of S9, aminoanthracene against TA
98 and TA100 in the presence of S9) for 90 min in a medium containing
a low concentration of histidine (sufficient for about two doublings).
The cultures were then diluted into an indicator medium lacking
histidine, and dispensed into 48 wells of 384-well plates (micro-plate
format, MPF). The plates were incubated for 48 h at 37 °C, and cells
that underwent a reversion grew, resulting in a color change in the wells
with growth. The numbers of wells showing growth were counted and
compared to the vehicle control. An increase in the number of colonies
of at least twofold over baseline (mean + SD of the vehicle control) and
a dose response indicate a positive response. An unpaired, one-sided
Student’s t-test was used to identify the conditions that were
significantly different from the vehicle control. S9 fraction from the
livers of Aroclor 1254-treated rats was included in the incubation at a
final concentration of 4.5%. A reduced nicotinamide adenine
dinucleotide phosphate (NADPH)-regenerating system was also
included to ensure a steady supply of reducing equivalents. Strains
used in this study: S. typhimurium TA98: hisD3052, rfa, uvrB/pKM101,
detects frame-shift mutations; S. typhimurium TA100: hisG45, rfa,
uvrB/pKM101, detects base-pair substitutions.
1
73 mg, 0.37 mmol, 78% yield). H NMR (300 MHz, CDCl3) δ ppm
4.98 (s, 2H, CH2), 8.52−8.73 (m, 2H, Ph), 8.87−9.08 (m, 1H, Ph). 13
C
NMR (75 MHz, CDCl3) δ ppm 63.0 (1C, CH2), 117.7 (1C, Ph), 122.5
(1C, Ph), 126.4 (2C, Ph), 145.4 (2C, Ph). To a solution of the
intermediate alcohol 12a, CBr4 (0.25 g, 0.74 mmol) in EA (10 mL) was
added PPh3 (0.19 g, 0.74 mmol), and the resulting mixture was stirred
at room temperature for 1 h according to a literature report.22 After
completion of the reaction, the mixture was evaporated and the residue
was purified to give 13a (eluent system: 10% EA in petroleum ether, 70
mg, 0.27 mmol, 73% yield). 1H NMR (300 MHz, CDCl3) δ ppm 4.63
(s, 2H, CH2), 8.60 (d, J = 2.1 Hz, 2H, Ph), 8.99 (t, J = 2.1 Hz, 1H, Ph).
13C NMR (75 MHz, CDCl3) δ ppm 29.3 (1C, CH2), 110.0 (1C, Ph),
118.6 (1C, Ph), 129.1 (2C, Ph), 142.0 (2C, Ph).
1-(Bromomethyl)-3-nitro-5-(trifluoromethyl)benzene (13b). Fol-
lowing the procedure described for 13a, NaBH4 (32 mg, 0.85 mmol), 3-
nitro-5-(trifluoromethyl)benzoic acid (0.10 g, 0.43 mmol), and BF3·
Et2O (0.14 mL, 1.1 mmol) in THF (10 mL) afforded 12b (eluent
system: 25% EA in petroleum ether, 85 mg, 0.38 mmol, 90% yield). 1H
NMR (300 MHz, CDCl3) δ ppm 3.32 (s, 1H, OH), 4.88 (s, 2H, CH2),
7.94 (s, 1H, Ph), 8.31 (s, 1H, Ph), 8.37 (s, 1H, Ph). 19F NMR (282
MHz, CDCl3) δ ppm −63.10 (s, 3 F). 13C NMR (75 MHz, CDCl3) δ
ppm 63.0 (1C, CH2), 119.4 (q, J = 3.4 Hz, 1C, Ph), 122.7 (q, J = 271.0
Hz, 1C, CF3), 124.2 (1C, Ph), 128.9 (q, J = 3.4 Hz, 1C, Ph), 132.1 (q, J
= 34.8 Hz, 1C, Ph), 144.7 (1C, Ph), 148.2 (1C, Ph). Then, 12b, CBr4
(0.26 g, 0.77 mmol) and PPh3 (0.20 g, 0.77 mmol) in EA (10 mL) gave
13b (eluent system: 10% EA in petroleum ether, 93 mg, 0.33 mmol,
In Vitro Cytotoxicity Assay. The cytotoxicity of compounds on
MRC-5 fibroblasts was performed exactly as previously reported using
neutral red uptake assay.15
Chemistry. All reagents and solvents were purchased from standard
commercial sources and were of analytical grade. All synthetic
compounds described in this study were checked with analytical thin-
layer chromatography (TLC) (Machery−Nagel-precoated F254 alumi-
num plates), visualized under UV light at 254 nm, and purified by
column chromatography (CC) on a Reveleris X2 (Grace) automated
flash unit. NMR data were recorded on a Varian Mercury 300/75 MHz
spectrometer or a Bruker Avance Neo 400/100 MHz spectrometer at
298.15 K using residual solvent signal as a reference. Confirmation of
compound structure was conducted with 1H, 13C, heteronuclear single
1
85% yield). H NMR (300 MHz, CDCl3) δ ppm 4.60 (s, 2H, CH2),
8.00 (s, 1H, Ph), 8.42 (s, 1H, Ph), 8.46 (t, J = 1.8 Hz, 1H, Ph). 13C NMR
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(75 MHz, CDCl3) δ ppm 29.8 (1C, CH2), 120.5 (q, J = 3.6 Hz, 1C, Ph),
122.5 (q, J = 272.9 Hz, 1C, CF3), 127.0 (1C, Ph), 131.4 (q, J = 3.4 Hz,
1C, Ph), 132.8 (q, J = 33.4 Hz, 1C, Ph), 141.3 (1C, Ph), 148.5 (1C, Ph).
1-(Bromomethyl)-2,4-dinitrobenzene (13c). Following the proce-
dure described for 13a, NaBH4 (36 mg, 0.94 mmol), 2,4-dinitrobenzoic
acid (0.10 g, 0.43 mmol), and BF3·Et2O (0.16 mL, 1.2 mmol) in THF
(10 mL) afforded 12c (eluent system: 25% EA in petroleum ether, 56
mg, 0.28 mmol, 60% yield). 1H NMR (400 MHz, methanol-d4) δ ppm
3.50 (s, 2H, CH2), 6.63 (d, J = 8.6 Hz, 1H, Ph), 7.00 (dd, J = 8.6, 2.4 Hz,
1H, Ph), 7.31 (d, J = 2.4 Hz, 1H, Ph). 13C NMR (101 MHz, CDCl3) δ
ppm 61.8 (1C, CH2), 120.4 (1C, Ph), 127.9 (1C, Ph), 130.3 (1C, Ph),
143.8 (1C, Ph), 146.9 (1C, Ph), 147.1 (1C, Ph). Then, 12c, CBr4 (0.19
g, 0.56 mmol) and PPh3 (0.15 g, 0.56 mmol) in EA (10 mL) gave 13c
(eluent system: 10% EA in petroleum ether, 66 mg, 0.25 mmol, 90%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 4.89 (s, 2H, CH2), 7.85 (d,
J = 8.5 Hz, 1H, Ph), 8.47 (dd, J = 8.5, 2.3 Hz, 1H, Ph), 8.90 (d, J = 2.3
Hz, 1H, Ph). 13C NMR (101 MHz, CDCl3) δ ppm 26.9 (1C, CH2),
121.0 (1C, Ph), 127.7 (1C, Ph), 133.9 (1C, Ph), 139.2 (1C, Ph), 147.7
(1C, Ph), 148.0 (1C, Ph).
solid was collected, taken up with boiling water, and filtered. The
aqueous phase was acidified to pH 5 with 1 N aq HCl, and the
precipitate was collected by filtration and dried in vacuo to afford the
desired compound, which was used in the next step without further
purification.
4-(3-Bromophenyl)-2-(methylthio)-6-oxo-1,6-dihydropyrimi-
dine-5-carbonitrile (21). According to a literature report,45 a solution
of 20 (1.0 g, 3.2 mmol), CH3I (0.20 mL, 3.2 mmol), and NaOH (0.13 g,
3.2 mmol) in EtOH/H2O (10/5 mL) was stirred at 60 °C for 1 h. After
cooling to room temperature, the reaction mixture was acidified to pH 5
with 1 N aq HCl. The obtained solid collected through filtration was
washed with water, dried, and purified to give 21 (eluent system: 5%
MeOH in CH2Cl2, 0.91 g, 2.8 mmol, 88% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.61 (s, 3H, CH3), 7.54 (t, J = 7.9 Hz, 1H, Ph), 7.82
(ddd, J = 8.0, 2.0, 0.9 Hz, 1H, Ph), 7.95 (dt, J = 7.9, 1.3 Hz, 1H, Ph),
8.09 (t, J = 1.8 Hz, 1H, Ph), 13.86 (s, 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 13.3 (1C, CH3), 93.5 (1C, C-5), 115.7 (1C, CN),
121.7 (1C, Ph), 127.7 (1C, Ph), 130.8 (1C, Ph), 131.1 (1C, Ph), 134.3
(1C, Ph), 137.6 (1C, Ph), 160.9 (1C, CO), 165.6 (1C, C-4), 167.1 (1C,
C-2). HRMS (ESI): m/z [M + H]+ calcd for [C12H8BrN3OS + H]+
323.9624, found 323.9643.
2-(Bromomethyl)-5-nitrofuran (16a). To a solution of 5-nitrofuran-
2-carbaldehyde (0.10 g, 0.71 mmol) in THF (10 mL) was added
NaBH4 (54 mg, 1.4 mmol), and the resulting mixture was stirred at
room temperature for 2 h. After consumption of the aldehyde starting
material, the reaction was quenched with saturated aq solution of Na+/
K+ tartaric acid (25 mL), and the aqueous layer was extracted with
CH2Cl2 (25 mL × 2). The organic layer was dried, concentrated, and
purified to afford 15a (eluent system: 25% EA in petroleum ether, 50
mg, 0.35 mmol, 50% yield). 1H NMR (400 MHz, CDCl3) δ ppm 2.27
(br. s., 1H, OH), 4.73 (s, 2H, CH2), 6.56 (d, J = 3.8 Hz, 1H, furyl), 7.29
(d, J = 3.6 Hz, 1H, furyl). 13C NMR (101 MHz, CDCl3) δ ppm 57.4
(1C, CH2), 110.6 (1C, furyl), 112.4 (1C, furyl), 151.9 (1C, furyl),
157.4 (1C, furyl). Following the procedure described for 13a, 15a, CBr4
(0.23 g, 0.70 mmol), and PPh3 (0.18 g, 0.70 mmol) in EA (10 mL) gave
16a (eluent system: 10% EA in petroleum ether, 59 mg, 0.28 mmol,
2-(Benzylthio)-4-(3-bromophenyl)-6-oxo-1,6-dihydropyrimidine-
5-carbonitrile (22). According to a literature report,25 to a stirred
suspension of 20 (0.5 g, 1.6 mmol) and K2CO3 (0.67 g, 4.9 mmol) in
MeCN (25 mL) was added BnBr (0.19 mL, 1.6 mmol). Stirring was
continued overnight. The volatiles were removed in vacuum. The crude
was taken up with water after it cooled to room temperature and
acidified to pH 5 with 1 N aq HCl. The obtained solid was purified to
give 22 (eluent system: 5% MeOH in CH2Cl2, 0.15 g, 0.37 mmol, 23%
1
yield). H NMR (300 MHz, DMSO-d6) δ ppm 4.50 (s, 2H, CH2),
7.20−7.36 (m, 3H, Ph), 7.36−7.43 (m, 2H, Ph), 7.52 (t, J = 15.8 Hz,
1H, Ph), 7.80 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H, Ph), 7.90 (ddd, J = 7.8, 1.2,
1.2 Hz, 1H, Ph), 8.00 (t, J = 1.8 Hz, 1H, Ph). 13C NMR (75 MHz,
DMSO-d6) δ ppm 34.3 (1C, CH2), 93.8 (1C, C-5), 115.5 (1C, CN),
121.7 (1C, Ph), 127.5 (1C, Ph), 127.7 (1C, Ph), 128.5 (2C, Ph), 129.0
(2C, Ph), 130.7 (1C, Ph), 131.2 (1C, Ph), 134.3 (1C, Ph), 136.4 (1C,
Ph), 137.4 (1C, Ph), 160.9 (1C, CO), 165.7 (1C, C-4), 166.1 (1C, C-
2). HRMS (ESI): m/z [M − H]− calcd for [C18H12BrN3OS − H]−
395.98112, found 395.9755.
4-(3-Bromophenyl)-2-((3,5-dinitrobenzyl)thio)-6-oxo-1,6-dihy-
dropyrimidine-5-carbonitrile (23). Following the same procedure
described for 22, 20 (0.50 g, 1.6 mmol), K2CO3 (0.67 g, 4.9 mmol), and
13a (0.42 g, 1.6 mmol) in MeCN (25 mL) afforded 23 (eluent system:
5% MeOH in CH2Cl2, 0.18 g, 0.37 mmol, 23% yield). 1H NMR (300
MHz, DMSO-d6) δ ppm 4.69 (s, 2H, CH2), 7.48 (t, J = 8.0 Hz, 1H, Ph),
7.75−7.85 (m, 2H, Ph), 7.90 (d, J = 1.8 Hz, 1H, Ph), 8.67 (s, 3H, Ph).
13C NMR (75 MHz, DMSO-d6) δ ppm 32.8 (1C, CH2), 115.9 (1C,
CN), 117.3 (1C, Ph), 121.7 (1C, Ph), 127.5 (1C, Ph), 129.4 (2C, Ph),
130.7 (1C, Ph), 130.8 (1C, Ph), 134.0 (1C, Ph), 137.6 (1C, Ph), 142.6
(1C, Ph), 147.8 (2C, Ph), 166.0 (1C, C-4), 166.1 (1C, C-2). C (C-5)
and C (CO) could not be observed. HRMS (ESI): m/z [M − H]− calcd
for [C18H10BrN5O5S − H]− 485.9513, found 485.9518.
2-Thioxo-2,3-dihydroquinazolin-4(1H)-one (25). Following Gen-
eral Procedure A, anthranilic acid (0.82 g, 6.0 mmol) in thionyl chloride
(10 mL) afforded acyl chloride, which was reacted with NH4SCN (0.53
g, 6.9 mmol) in acetone (15 mL) to give 25.
2-((3,5-Dinitrobenzyl)thio)quinazolin-4(3H)-one (26). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol) and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/10 v/v, 11
mL) afforded 26 (eluent system: 15% EA in toluene, 95 mg, 0.27 mmol,
59% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm 4.67 (s, 2H, CH2),
7.41 (t, J = 7.3 Hz, 1H, quinazolinone), 7.62 (d, J = 7.9 Hz, 1H,
quinazolinone), 7.80 (t, J = 7.3 Hz, 1H, quinazolinone), 7.99 (dd, J =
7.9, 1.2 Hz, 1H, quinazolinone), 8.66 (t, J = 2.2 Hz, 1H, Ph), 8.88 (d, J =
2.1 Hz, 2H, Ph), 12.63 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ
ppm 32.1 (1C, CH2), 117.3 (1C, Ph), 120.0 (1C, quinazolinone), 125.8
(1C, quinazolinone), 125.9 (1C, quinazolinone), 126.0 (1C,
quinazolinone), 130.1 (2C, Ph), 134.7 (1C, quinazolinone), 143.1
(1C, Ph), 147.6 (2C, Ph), 148.1 (1C, quinazolinone), 154.5 (1C,
1
82% yield). H NMR (400 MHz, CDCl3) δ ppm 4.47 (s, 2H, CH2),
6.63 (d, J = 3.6 Hz, 1H, furyl), 7.28 (d, J = 4.0 Hz, 1H, furyl). 13C NMR
(101 MHz, CDCl3) δ ppm 20.7 (1C, CH2), 112.4 (1C, furyl), 112.5
(1C, furyl), 151.9 (1C, furyl), 153.4 (1C, furyl).
2-(Bromomethyl)-5-nitrothiophene (16b). Following the proce-
dure described for 16a, 5-nitrothiophene-2-carbaldehyde (0.10 g, 0.64
mmol) and NaBH4 (48 mg, 1.3 mmol) in THF (10 mL) afforded 15b
(eluent system: 25% EA in petroleum ether, 76 mg, 0.48 mmol, 75%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 2.47 (s, 1H, OH), 4.88 (d, J
= 0.9 Hz, 2H, CH2), 6.93 (dt, J = 4.2, 1.0 Hz, 1H, thienyl), 7.81 (d, J =
4.1 Hz, 1H, thienyl). 13C NMR (101 MHz, CDCl3) δ ppm 60.2 (1C,
CH2), 123.4 (1C, thienyl), 128.8 (1C, thienyl), 150.8 (1C, thienyl),
153.4 (1C, thienyl). Then, 15b, CBr4 (0.32 g, 0.96 mmol) and PPh3
(0.25 g, 0.96 mmol) in EA (10 mL) gave 16b (eluent system: 10% EA in
petroleum ether, 70 mg, 0.31 mmol, 66% yield). 1H NMR (400 MHz,
CDCl3) δ ppm 4.63 (s, 2H, CH2), 7.07 (d, J = 4.3 Hz, 1H, thienyl), 7.79
(d, J = 4.3 Hz, 1H, thienyl). 13C NMR (101 MHz, CDCl3) δ ppm 24.5
(1C, CH2), 127.0 (1C, thienyl), 128.4 (1C, thienyl), 148.2 (1C,
thienyl), 151.8 (1C, thienyl).
2-(Bromomethyl)-1,3-dinitrobenzene (18). According to a liter-
ature report,24 2,6-dinitrotoluene (0.97 g, 5.4 mmol), AIBN (30 mg,
0.18 mmol), and DBDMH (2.9 g, 10 mmol) were suspended in CCl4
(50 mL), and the resulting mixture was refluxed overnight. After
cooling to room temperature, the reaction mixture was diluted with
CH2Cl2 (0.15 L), concentrated in vacuo and the resulting residue was
purified to give 18 (eluent system: 10% EA in petroleum ether, 0.38 g,
1.5 mmol, 27% yield). 1H NMR (400 MHz, CDCl3) δ ppm 4.95 (s, 2H,
CH2), 7.69 (t, J = 8.2 Hz, 1H, Ph), 8.11 (d, J = 8.1 Hz, 2H, Ph). 13
C
NMR (101 MHz, CDCl3) δ ppm 19.9 (1C, CH2), 127.5 (1C, Ph),
128.7 (2C, Ph), 130.1 (1C, Ph), 150.2 (2C, Ph).
6-(3-Bromophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimi-
dine-5-carbonitrile (20). According to a literature report,25 to a stirred
solution of 3-bromobenzaldehyde (0.80 mL, 6.9 mmol), ethyl
cyanoacetate (0.78 g, 6.9 mmol), and thiourea (0.52 g, 6.9 mmol) in
EtOH was added K2CO3 (1.1 g, 8.3 mmol). Stirring was continued at
reflux overnight. After cooling to room temperature, the pale yellow
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quinazolinone), 161.0 (1C, CO). HRMS (ESI): m/z [M − H]− calcd
Ph), 7.36 (d, J = 8.0 Hz, 2H, Ph), 7.43 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H,
quinazolinone), 7.60 (d, J = 8.1 Hz, 1H, quinazolinone), 7.78 (ddd, J =
8.3, 7.1, 1.6 Hz, 1H, quinazolinone), 8.04 (dd, J = 7.9, 1.13 Hz, 1H,
quinazolinone), 12.56 (br. s., 1H, NH). 13C NMR (101 MHz, DMSO-
d6) δ ppm 20.7 (1C, CH3), 33.4 (1C, CH2), 120.0 (1C, quinazolinone),
125.7 (1C, quinazolinone), 126.0 (2C, quinazolinone), 129.0 (2C, Ph),
129.1 (2C, Ph), 134.1 (1C, Ph), 134.6 (1C, quinazolinone), 136.5 (1C,
Ph), 148.3 (1C, quinazolinone), 155.3 (1C, quinazolinone), 161.1 (1C,
CO). HRMS (ESI): m/z [M + H]+ calcd for [C16H14N2OS + H]+
283.0900, found 283.0898.
for [C15H10N4O5S − H]− 357.0299, found 357.0300.
2-(Benzylthio)quinazolin-4(3H)-one (27). Following General Pro-
cedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5 mmol), and
benzyl bromide (53 μL, 0.45 mmol) in DMSO/MeCN (1/10 v/v, 11
mL) afforded 27 (eluent system: 15% EA in toluene, 78 mg, 0.29 mmol,
65% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm 4.49 (s, 2H, CH2),
7.15−7.34 (m, 3H, Ph), 7.39 (t, J = 7.5 Hz, 1H, quinazolinone), 7.47 (d,
J = 7.3 Hz, 2H, Ph), 7.58 (d, J = 7.9 Hz, 1H, quinazolinone), 7.73 (t, J =
8.0 Hz, 1H, quinazolinone), 8.04 (d, J = 7.9 Hz, 1H, quinazolinone),
12.59 (br. s., 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 33.6
(1C, CH2), 120.0 (1C, quinazolinone), 125.6 (1C, quinazolinone),
126.0 (2C, quinazolinone), 127.3 (1C, Ph), 128.4 (2C, Ph), 129.2 (2C,
Ph), 134.5 (1C, quinazolinone), 137.3 (1C, Ph), 148.3 (1C,
quinazolinone), 155.2 (1C, quinazolinone), 161.2 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C15H12N2OS + H]+ 269.0743, found
269.0753.
2-((4-Methoxybenzyl)thio)quinazolin-4(3H)-one (32). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 4-methoxybenzyl bromide (90 mg, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 32 (eluent system: 15% EA in
toluene, 55 mg, 0.18 mmol, 41% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 3.71 (s, 3H, OCH3), 4.44 (s, 2H, CH2), 6.85−6.90 (m, 2H,
Ph), 7.37−7.45 (m, 3H, quinazolinone, Ph), 7.60 (d, J = 8.1 Hz, 1H,
quinazolinone), 7.78 (ddd, J = 8.3, 7.04, 1.6 Hz, 1H, quinazolinone),
8.03 (dd, J = 7.9, 1.3 Hz, 1H, quinazolinone), 12.54 (br. s., 1H, NH).
13C NMR (101 MHz, DMSO-d6) δ ppm 33.1 (1C, CH2), 55.0 (1C,
OCH3), 113.8 (2C, Ph), 120.0 (1C, quinazolinone), 125.7 (1C,
quinazolinone), 126.0 (2C, quinazolinone), 128.9 (1C, Ph), 130.4 (2C,
Ph), 134.6 (1C, quinazolinone), 148.3 (1C, quinazolinone), 155.4 (1C,
quinazolinone), 158.5 (1C, Ph), 161.2 (1C, CO). HRMS (ESI): m/z
[M + H]+ calcd for [C16H14N2O2S + H]+ 299.0849, found 299.0858.
2-((3,4-Dichlorobenzyl)thio)quinazolin-4(3H)-one (33). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 3,4-dichlorobenzyl bromide (65 μL, 0.45 mmol) in
DMSO/MeCN (1/10 v/v, 11 mL) afforded 33 (eluent system: 15% EA
in toluene, 0.11 g, 0.32 mmol, 72% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.49 (s, 2H, CH2), 7.40−7.47 (m, 1H, quinazolinone), 7.49−
7.54 (m, 1H, Ph), 7.55−7.63 (m, 2H, quinazolinone, Ph), 7.75−7.85
(m, 2H, quinazolinone, Ph), 8.04 (dd, J = 7.9, 1.3 Hz, 1H,
quinazolinone), 12.63 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.2 (1C, CH2), 120.0 (1C, quinazolinone), 125.7
(1C, quinazolinone), 125.8 (1C, quinazolinone), 126.1 (1C,
quinazolinone), 129.6 (1C, Ph), 129.7 (1C, Ph), 130.5 (1C, Ph),
130.7 (1C, Ph), 131.3 (1C, Ph), 134.6 (1C, quinazolinone), 139.3 (1C,
Ph), 148.1 (1C, quinazolinone), 155.0 (1C, quinazolinone), 161.3 (1C,
CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H10Cl2N2OS + H]+
336.9964, found 336.9981.
2-((3-Chlorobenzyl)thio)quinazolin-4(3H)-one (34). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 3-chlorobenzyl bromide (59 μL, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 34 (eluent system: 15% EA in
toluene, 96 mg, 0.32 mmol, 70% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.51 (s, 2H, CH2), 7.29−7.39 (m, 2H, Ph), 7.41−7.51 (m,
2H, quinazolinone, Ph), 7.58−7.65 (m, 2H, quinazolinone, Ph), 7.74−
7.84 (m, 1H, quinazolinone), 8.05 (dd, J = 7.9, 1.3 Hz, 1H,
quinazolinone), 12.62 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.8 (1C, CH2), 120.0 (1C, quinazolinone), 125.7
(1C, quinazolinone), 125.9 (1C, quinazolinone), 126.1 (1C,
quinazolinone), 127.2 (1C, Ph), 127.9 (1C, Ph), 129.1 (1C, Ph),
130.2 (1C, Ph), 132.8 (1C, Ph), 134.7 (1C, quinazolinone), 140.3 (1C,
Ph), 148.2 (1C, quinazolinone), 154.9 (1C, quinazolinone), 161.1 (1C,
CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H11ClN2OS + H]+
303.0354, found 303.0356.
2-((Thiophen-2-ylmethyl)thio)quinazolin-4(3H)-one (28). Follow-
ing General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 2-(bromomethyl)thiophene (49 μL, 0.45 mmol) in
DMSO/MeCN (1/10 v/v, 11 mL) afforded 28 (eluent system: 15% EA
1
in toluene, 87 mg, 0.32 mmol, 70% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 4.74 (s, 2H, CH2), 6.94 (dd, J = 5.1, 3.5 Hz, 1H,
thienyl), 7.15 (d, J = 2.6 Hz, 1H, thienyl), 7.38 (dd, J = 5.1, 1.1 Hz, 1H,
thienyl), 7.45 (t, J = 7.5 Hz, 1H, quinazolinone), 7.63 (d, J = 8.0 Hz, 1H,
quinazolinone), 7.80 (t, J = 7.5 Hz, 1H, quinazolinone), 8.05 (dd, J =
7.9, 1.3 Hz, 1H, quinazolinone), 12.60 (br. s., 1H, NH). 13C NMR (101
MHz, DMSO-d6) δ ppm 28.3 (1C, CH2), 120.0 (1C, quinazolinone),
125.8 (1C, quinazolinone), 126.0 (1C, thienyl), 126.1 (2C,
quinazolinone), 126.6 (1C, thienyl), 127.4 (1C, thienyl), 134.7 (1C,
quinazolinone), 140.0 (1C, thienyl), 148.2 (1C, quinazolinone), 154.8
(1C, quinazolinone), 161.2 (1C, CO). HRMS (ESI): m/z [M + H]+
calcd for [C13H10N2OS2 + H]+ 275.0308, found 275.0303.
2-(([1,1′-Biphenyl]-4-ylmethyl)thio)quinazolin-4(3H)-one (29).
Following General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 4-(bromomethyl)biphenyl (0.11 g, 0.45 mmol)
in DMSO/MeCN (1/10 v/v, 11 mL) afforded 29 (eluent system: 15%
1
EA in toluene, 44 mg, 0.13 mmol, 28% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 4.55 (s, 2H, CH2), 7.32−7.38 (m, 1H, Ph), 7.40−
7.48 (m, 3H, quinazolinone, Ph), 7.56−7.67 (m, 7H, quinazolinone,
Ph), 7.79 (ddd, J = 8.3, 7.1, 1.6 Hz, 1H, quinazolinone), 8.05 (dd, J =
7.9, 1.2 Hz, 1H, quinazolinone), 12.61 (br. s., 1H, NH). 13C NMR (101
MHz, DMSO-d6) δ ppm 33.2 (1C, CH2), 120.0 (1C, quinazolinone)
125.7 (1C, quinazolinone), 126.0 (2C, quinazolinone), 126.6 (2C, Ph),
126.7 (2C, Ph), 127.4 (1C, Ph), 128.9 (2C, Ph), 129.8 (2C, Ph), 134.7
(1C, quinazolinone), 136.7 (1C, Ph), 139.1 (1C, Ph), 139.7 (1C, Ph),
148.3 (1C, quinazolinone), 155.2 (1C, quinazolinone), 161.2 (1C,
CO). HRMS (ESI): m/z [M + H]+ calcd for [C21H16N2OS + H]+
345.1056, found 345.1060.
2-((4-Chlorobenzyl)thio)quinazolin-4(3H)-one (30). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 4-chlorobenzyl bromide (92 mg, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 30 (eluent system: 15% EA in
toluene, 0.10 g, 0.34 mmol, 75% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.49 (s, 2H, CH2), 7.31−7.40 (m, 2H, Ph), 7.43 (ddd, J = 8.0,
7.1, 1.1 Hz, 1H, quinazolinone), 7.49−7.56 (m, 2H, Ph), 7.61 (d, J = 8.1
Hz, 1H, quinazolinone), 7.78 (ddd, J = 8.3, 7.0, 1.6 Hz, 1H,
quinazolinone), 8.04 (dd, J = 7.9, 1.2 Hz, 1H, quinazolinone), 12.60
(br. s., 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.7 (1C,
CH2), 120.0 (1C, quinazolinone), 125.7 (1C, quinazolinone), 126.0
(2C, quinazolinone), 128.3 (2C, Ph), 131.0 (2C, Ph), 131.8 (1C, Ph),
134.7 (1C, quinazolinone), 136.8 (1C, Ph), 148.3 (1C, quinazolinone),
154.9 (1C, quinazolinone), 161.1 (1C, CO). HRMS (ESI): m/z [M +
H]+ calcd for [C15H11ClN2OS + H]+ 303.0354, found 303.0346.
2-((4-Methylbenzyl)thio)quinazolin-4(3H)-one (31). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and p-methylbenzyl bromide (83 mg, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 31 (eluent system: 15% EA in
toluene, 70 mg, 0.25 mmol, 55% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 2.26 (s, 3H, CH3), 4.46 (s, 2H, CH2), 7.12 (d, J = 7.9 Hz, 2H,
2-((3-Methoxybenzyl)thio)quinazolin-4(3H)-one (35). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 3-methoxybenzyl bromide (63 μL, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 35 (eluent system: 15% EA in
toluene, 80 mg, 0.27 mmol, 60% yield). 1H NMR (300 MHz, DMSO-
d6) δ ppm 3.70 (s, 3H, OCH3), 4.45 (s, 2H, CH2), 6.80 (dd, J = 8.2, 2.1
Hz, 1H, Ph), 6.99−7.09 (m, 2H, Ph), 7.21 (t, J = 7.5 Hz, 1H, Ph), 7.41
(t, J = 7.5 Hz, 1H, quinazolinone), 7.59 (d, J = 8.2 Hz, 1H,
quinazolinone), 7.76 (t, J = 8.2 Hz, 1H, quinazolinone), 8.01 (d, J = 7.6
Hz, 1H, quinazolinone), 12.55 (br. s., 1H, NH). 13C NMR (75 MHz,
DMSO-d6) δ ppm 33.5 (1C, CH2), 55.0 (1C, OCH3), 112.8 (1C, Ph),
114.8 (1C, Ph), 120.0 (1C, quinazolinone), 121.3 (1C, Ph), 125.7 (1C,
quinazolinone), 126.0 (1C, quinazolinone), 126.0 (1C, quinazoli-
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01374
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
none), 129.5 (1C, Ph), 134.7 (1C, quinazolinone), 138.8 (1C, Ph),
148.3 (1C, quinazolinone), 155.2 (1C, quinazolinone), 159.2 (1C, Ph),
161.1 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C16H14N2O2S
+ H]+ 299.0849, found 299.0848.
2-((4-Nitrobenzyl)thio)quinazolin-4(3H)-one (40). Following Gen-
eral Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5 mmol),
and p-nitrobenzyl bromide (97 mg, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 40 (eluent system: 15% EA in toluene, 0.10 g,
0.32 mmol, 72% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.61 (s,
2H, CH2), 7.40−7.49 (m, 1H, quinazolinone), 7.62 (d, J = 8.1 Hz, 1H,
quinazolinone), 7.76−7.83 (m, 3H, quinazolinone, Ph), 8.03 (dd, J =
7.9, 1.3 Hz, 1H, quinazolinone), 8.15−8.22 (m, 2H, Ph), 12.65 (s, 1H,
NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.7 (1C, CH2), 120.0
(1C, quinazolinone), 123.4 (2C, Ph), 125.8 (1C, quinazolinone), 126.0
(1C, quinazolinone), 126.1 (1C, quinazolinone), 130.5 (2C, Ph), 134.7
(1C, quinazolinone), 146.2 (1C, Ph), 146.5 (1C, Ph), 148.2 (1C,
quinazolinone), 154.6 (1C, quinazolinone), 161.1 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C15H11N3O3S + H]+ 314.0594, found
314.0586.
2-(((5-Nitrofuran-2-yl)methyl)thio)quinazolin-4(3H)-one (41).
Following General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 16a (93 mg, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 41 (eluent system: 15% EA in toluene, 0.11 g,
0.35 mmol, 78% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.68 (s,
2H, CH2), 6.87 (d, J = 3.8 Hz, 1H, furyl), 7.45 (td, J = 7.5, 1.1 Hz, 1H,
quinazolinone), 7.59−7.67 (m, 2H, quinazolinone, furyl), 7.79 (ddd, J
= 8.3, 7.0, 1.6 Hz, 1H, quinazolinone), 8.04 (dd, J = 7.9, 1.2 Hz, 1H,
quinazolinone), 12.71 (br. s., 1H, NH). 13C NMR (101 MHz, DMSO-
d6) δ ppm 26.0 (1C, CH2), 112.9 (1C, furyl), 114.2 (1C, furyl), 120.0
(1C, quinazolinone), 126.0 (1C, quinazolinone), 126.1 (2C,
quinazolinone), 134.7 (1C, quinazolinone), 148.1 (1C, quinazoli-
none), 150.9 (1C, furyl), 155.8 (2C, quinazolinone, furyl), 161.1 (1C,
CO). HRMS (ESI): m/z [M + H]+ calcd for [C13H9N3O4S + H]+
304.0387, found 304.0377.
2-(((5-Nitrothiophen-2-yl)methyl)thio)quinazolin-4(3H)-one (42).
Following General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21
g, 1.5 mmol), and 16b (0.10 g, 0.45 mmol) in DMSO/MeCN (1/10 v/
v, 11 mL) afforded 42 (eluent system: 15% EA in toluene, 31 mg, 0.097
mmol, 22% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.76 (s, 2H,
CH2), 7.28 (d, J = 4.1 Hz, 1H, thienyl), 7.48 (t, J = 7.4 Hz, 1H,
quinazolinone), 7.67 (d, J = 8.0 Hz, 1H, quinazolinone), 7.84 (t, J = 7.4
Hz, 1H, quinazolinone), 7.97 (d, J = 4.1 Hz, 1H, thienyl), 8.07 (d, J =
7.6 Hz, 1H, quinazolinone), 12.73 (br. s., 1H, H). 13C NMR (101 MHz,
DMSO-d6) δ ppm 28.5 (1C, CH2), 120.1 (1C, quinazolinone), 125.9
(1C, quinazolinone), 126.1 (1C, quinazolinone), 126.2 (1C,
quinazolinone), 127.9 (1C, thienyl), 129.4 (1C, thienyl), 134.9 (1C,
quinazolinone), 147.9 (1C, quinazolinone), 150.0 (1C, thienyl), 150.7
(1C, thienyl), 154.5 (1C, quinazolinone), 161.1 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C13H9N3O3S2 + H]+ 320.0158, found
320.0173.
2-((2,4-Dinitrobenzyl)thio)quinazolin-4(3H)-one (43). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 13c (0.12 g, 0.45 mmol) in DMSO/MeCN (1/10 v/v, 11
mL) afforded 43 (eluent system: 15% EA in toluene, 85 mg, 0.24 mmol,
53% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.86 (s, 2H, CH2),
7.39−7.50 (m, 1H, quinazolinone), 7.59 (d, J = 7.9 Hz, 1H,
quinazolinone), 7.80 (ddd, J = 8.3, 7.1, 1.6 Hz, 1H, quinazolinone),
8.00 (dd, J = 7.9, 1.1 Hz, 1H, quinazolinone), 8.21 (d, J = 8.6 Hz, 1H,
Ph), 8.51 (dd, J = 8.6, 2.4 Hz, 1H, Ph), 8.73 (d, J = 2.5 Hz, 1H, Ph),
12.65 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 30.0 (1C,
CH2), 120.0 (1C, quinazolinone), 120.1 (1C, Ph), 125.9 (1C,
quinazolinone), 126.0 (2C, quinazolinone), 127.6 (1C, Ph), 134.3
(1C, Ph), 134.7 (1C, quinazolinone), 140.3 (1C, Ph), 146.5 (1C, Ph),
148.0 (1C, Ph), 148.4 (1C, quinazolinone), 154.3 (1C, quinazolinone),
161.0 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H10N4O5S
+ H]+ 359.0445, found 359.0461.
2-((2-Chlorobenzyl)thio)quinazolin-4(3H)-one (36). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 1-(bromomethyl)-2-chlorobenzene (58 μL, 0.45 mmol) in
DMSO/MeCN (1/10 v/v, 11 mL) afforded 36 (eluent system: 15% EA
1
in toluene, 99 mg, 0.33 mmol, 73% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 4.60 (s, 2H, CH2), 7.28−7.36 (m, 2H, Ph), 7.41−
7.47 (m, 1H, quinazolinone), 7.47−7.53 (m, 1H, Ph), 7.66 (d, J = 8.0
Hz, 1H, quinazolinone), 7.69−7.75 (m, 1H, Ph), 7.76−7.83 (m, 1H,
quinazolinone), 8.04 (dd, J = 7.9, 1.3 Hz, 1H, quinazolinone), 12.61 (s,
1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 31.6 (1C, CH2),
120.0 (1C, quinazolinone), 125.8 (1C, quinazolinone), 126.0 (2C,
quinazolinone), 127.3 (1C, Ph), 129.4 (2C, Ph), 131.7 (1C, Ph), 133.4
(1C, Ph), 134.7 (1C, Ph), 134.7 (1C, quinazolinone), 148.2 (1C,
quinazolinone), 154.7 (1C, quinazolinone), 161.1 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C15H11ClN2OS + H]+ 303.0354, found
303.0360.
2-((2-Methoxybenzyl)thio)quinazolin-4(3H)-one (37). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and o-methoxybenzyl bromide (90 mg, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 37 (eluent system: 15% EA in
toluene, 78 mg, 0.26 mmol, 58% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 3.84 (s, 3H, OCH3), 4.45 (s, 2H, CH2), 6.89 (td, J = 7.4, 0.9
Hz, 1H, Ph), 7.02 (d, J = 7.9 Hz, 1H, Ph), 7.28 (td, J = 7.9, 1.75 Hz, 1H,
Ph), 7.43 (t, J = 7.5 Hz, 1H, quinazolinone), 7.48 (dd, J = 7.5, 1.6 Hz,
1H, Ph), 7.63 (d, J = 8.1 Hz, 1H, quinazolinone), 7.79 (ddd, J = 8.3, 7.1,
1.5 Hz, 1H, quinazolinone), 8.04 (dd, J = 8.0, 1.3 Hz, 1H,
quinazolinone), 12.52 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 28.8 (1C, CH2), 55.5 (1C, OCH3), 110.9 (1C, Ph),
119.9 (1C, quinazolinone), 120.3 (1C, Ph), 124.6 (1C, Ph), 125.6 (1C,
quinazolinone), 126.0 (2C, quinazolinone), 129.1 (1C, Ph), 130.6 (1C,
Ph), 134.7 (1C, quinazolinone), 148.4 (1C, quinazolinone), 155.7 (1C,
quinazolinone), 157.2 (1C, Ph), 161.2 (1C, CO). HRMS (ESI): m/z
[M + H]+ calcd for [C16H14N2O2S + H]+ 299.0849, found 299.0848.
2-((2-Nitrobenzyl)thio)quinazolin-4(3H)-one (38). Following Gen-
eral Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5 mmol),
and 2-nitrobenzyl bromide (97 mg, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 38 (eluent system: 15% EA in toluene, 98 mg,
0.31 mmol, 70% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm 4.75 (s,
2H, CH2), 7.39 (t, J = 7.5 Hz, 1H, quinazolinone), 7.49 (t, J = 7.8, Hz,
1H, Ph), 7.57 (d, J = 7.9 Hz, 1H, quinazolinone), 7.67 (t, J = 7.5 Hz, 1H,
Ph), 7.75 (t, J = 7.9 Hz, 1H, quinazolinone), 7.87 (d, J = 7.6 Hz, 1H,
Ph), 7.94−8.03 (m, 2H, quinazolinone, Ph), 12.56 (br. s., 1 H). 13C
NMR (75 MHz, DMSO-d6) δ ppm 30.8 (1C, CH2), 120.4 (1C,
quinazolinone), 124.7 (1C, Ph), 125.8 (1C, quinazolinone), 125.9 (1C,
quinazolinone), 126.0 (1C, quinazolinone), 128.9 (1C, Ph), 132.7 (1C,
Ph), 133.2 (1C, Ph), 133.7 (1C, Ph), 134.6 (1C, quinazolinone), 148.1
(1C, quinazolinone), 148.4 (1C, Ph), 154.7 (1C, quinazolinone), 161.1
(1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H11N3O3S + H]+
314.0594, found 314.0607.
2-((3-Nitrobenzyl)thio)quinazolin-4(3H)-one (39). Following Gen-
eral Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5 mmol),
and 1-(bromomethyl)-3-nitrobenzene (97 mg, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 39 (eluent system: 15% EA in
toluene, 88 mg, 0.28 mmol, 62% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.62 (s, 2H, CH2), 7.40−7.48 (m, 1H, quinazolinone), 7.57−
7.68 (m, 2H, quinazolinone, Ph), 7.75−7.85 (m, 1H, quinazolinone),
7.99 (d, J = 7.8 Hz, 1H, Ph), 8.03 (dd, J = 7.9, 1.3 Hz, 1H,
quinazolinone), 8.10 (dd, J = 8.1, 1.5 Hz, 1H, Ph), 8.43−8.49 (m, 1H,
Ph), 12.64 (br. s., 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm
32.5 (1C, CH2), 120.0 (1C, quinazolinone), 122.1 (1C, Ph), 124.1 (1C,
Ph), 125.8 (1C, quinazolinone), 125.9 (1C, quinazolinone), 126.0 (1C,
quinazolinone), 129.8 (1C, Ph), 134.7 (1C, quinazolinone), 136.0 (1C,
Ph), 140.5 (1C, Ph), 147.5 (1C, Ph), 148.2 (1C, quinazolinone), 154.7
(1C, quinazolinone), 161.1 (1C, CO). HRMS (ESI): m/z [M + H]+
calcd for [C15H11N3O3S + H]+ 314.0594, found 314.0595.
2-((2,6-Dinitrobenzyl)thio)quinazolin-4(3H)-one (44). Following
General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3 (0.21 g, 1.5
mmol), and 18 (0.12 g, 0.45 mmol) in DMSO/MeCN (1/10 v/v, 11
mL) afforded 44 (eluent system: 15% EA in toluene, 0.13 g, 0.36 mmol,
81% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 5.00 (s, 2H, CH2),
7.43 (t, J = 7.6 Hz, 1H, quinazolinone), 7.52 (d, J = 8.0 Hz, 1H,
quinazolinone), 7.75−7.85 (m, 2H, quinazolinone, Ph), 8.00 (dd, J =
7.9, 1.2 Hz, 1H, quinazolinone), 8.33 (d, J = 8.3 Hz, 2H, Ph), 12.60 (s,
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01374
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1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 25.6 (1C, CH2),
120.0 (1C, quinazolinone), 125.5 (1C, quinazolinone), 126.0 (1C,
quinazolinone), 126.0 (1C, quinazolinone), 127.9 (1C, Ph), 129.4 (2C,
Ph), 130.1 (1C, Ph), 134.6 (1C, quinazolinone), 147.9 (1C,
quinazolinone), 150.4 (2C, Ph), 154.1 (1C, quinazolinone), 160.9
(1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H10N4O5S + H]+
359.0445, found 359.0432.
2-((3-Nitro-5-(trifluoromethyl)benzyl)thio)quinazolin-4(3H)-one
(45). Following General Procedure B, 25 (89 mg, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13b (0.13 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 45 (eluent system: 15% EA in toluene, 0.11 g,
0.28 mmol, 60% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.66 (s,
2H, CH2), 7.40−7.51 (m, 1H, quinazolinone), 7.61 (d, J = 8.1 Hz, 1H,
quinazolinone), 7.75−7.88 (m, 1H, quinazolinone), 8.02 (dd, J = 7.9,
1.3 Hz, 1H, quinazolinone), 8.34 (s, 1H, Ph), 8.46 (s, 1H, Ph), 8.77 (s,
1H, Ph), 12.65 (br. s., 1H, NH). 19F NMR (377 MHz, DMSO-d6) δ
ppm −61.35 (s, 3 F). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.3
(1C, CH2), 119.1 (q, J = 4.5 Hz, 1C, Ph), 120.0 (1C, quinazolinone),
122.9 (q, J = 272.1 Hz, 1C, CF3), 125.7 (1C, quinazolinone), 125.9
(1C, quinazolinone), 126.1 (1C, quinazolinone), 128.2 (1C, Ph), 129.9
(q, J = 33.8 Hz, 1C, Ph), 132.5 (q, J = 3.8 Hz, 1C, Ph), 134.7 (1C,
quinazolinone), 142.8 (1C, Ph), 147.9 (1C, Ph), 148.1 (1C,
quinazolinone), 154.6 (1C, quinazolinone), 161.1 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C16H10F3N3O3S + H]+ 382.0468,
found 382.0471.
2-((3,5-Dinitrobenzyl)sulfinyl)quinazolin-4(3H)-one (46). To a
solution of 26 (0.10 g, 0.28 mmol) in THF (10 mL) was added
mCPBA (75%, 63 mg, 0.28 mmol), and the resulting mixture was stirred
at room temperature for 2 h. After concentration in vacuo, the residue
was purified to give 46 (eluent system: 25% EA in toluene, 86 mg, 0.23
mmol, 83% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.76 (d, J =
12.9 Hz, 1H, CH2), 4.95 (d, J = 12.9 Hz, 1H, CH2), 7.53−7.74 (m, 2H,
quinazolinone), 7.88 (t, J = 7.1 Hz, 1H, quinazolinone), 8.12 (d, J = 7.0
Hz, 1H, quinazolinone), 8.47 (d, J = 1.9 Hz, 2H, Ph), 8.78 (t, J = 2.0 Hz,
1H, Ph), 12.43 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm
56.3 (1C, CH2), 118.2 (1C, Ph), 121.8 (1C, quinazolinone), 126.3 (1C,
quinazolinone), 126.9 (1C, quinazolinone), 128.1 (1C, quinazoli-
none), 131.0 (2C, Ph), 133.9 (1C, Ph), 135.0 (1C, quinazolinone),
146.8 (1C, quinazolinone), 147.5 (2C, Ph), 157.5 (1C, quinazolinone),
160.8 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C15H10N4O6S
− H]− 373.0248, found 373.0244.
reacted with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give
49c.
8-Chloro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49d). Fol-
lowing General Procedure A, 3-chloroanthranilic acid (1.0 g, 6.0 mmol)
in thionyl chloride (10 mL) afforded acyl chloride, which was reacted
with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give 49d.
6-Bromo-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49e). Fol-
lowing General Procedure A, 5-bromoanthranilic acid (1.3 g, 6.0
mmol) in thionyl chloride (10 mL) afforded acyl chloride, which was
reacted with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give
49e.
6-Methyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49f). Fol-
lowing General Procedure A, 5-methylanthranilic acid (0.91 g, 6.0
mmol) in thionyl chloride (10 mL) afforded acyl chloride, which was
reacted with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give
49f.
6-Fluoro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49g). Fol-
lowing General Procedure A, 5-fluoroanthranilic acid (0.93 g, 6.0
mmol) in thionyl chloride (10 mL) afforded acyl chloride, which was
reacted with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give
49g.
6-Nitro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49h). Follow-
ing General Procedure A, 5-nitroanthranilic acid (1.1 g, 6.0 mmol) in
thionyl chloride (10 mL) afforded acyl chloride, which was reacted with
NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give 49h.
5-Chloro-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-one (50a).
Following General Procedure B, 49a (0.11 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50a (eluent system: 15% EA in toluene, 0.11 g,
0.28 mmol, 59% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.67 (s,
2H, CH2), 7.42 (dd, J = 7.8, 0.9 Hz, 1H, quinazolinone), 7.58 (dd, J =
8.2, 0.9 Hz, 1H, quinazolinone), 7.73 (t, J = 8.0 Hz, 1H, quinazolinone),
8.68 (t, J = 2.1 Hz, 1H, Ph), 8.88 (d, J = 2.0 Hz, 2H, Ph), 12.69 (s, 1H,
NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.1 (1C, CH2), 116.9
(1C, quinazolinone), 117.3 (1C, Ph), 125.4 (1C, quinazolinone), 128.3
(1C, quinazolinone), 130.1 (2C, Ph), 132.7 (1C, quinazolinone), 134.5
(1C, quinazolinone), 143.0 (1C, Ph), 147.7 (2C, Ph), 150.5 (1C,
quinazolinone), 155.6 (1C, quinazolinone), 159.1 (1C, CO). HRMS
(ESI): m/z [M − H]− calcd for [C15H9ClN4O5S − H]− 390.9909,
found 390.9908.
6-Chloro-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-one (50b).
Following General Procedure B, 49b (0.11 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50b (eluent system: 15% EA in toluene, 91 mg,
0.23 mmol, 51% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.68 (s,
2H, CH2), 7.45 (dd, J = 8.5, 2.1 Hz, 1H, quinazolinone), 7.70 (d, J = 2.0
Hz, 1H, quinazolinone), 7.99 (d, J = 8.5 Hz, 1H, quinazolinone), 8.68
(t, J = 2.1 Hz, 1H, Ph), 8.91 (d, J = 2.1 Hz, 2H, Ph), 12.79 (s, 1H, NH).
13C NMR (101 MHz, DMSO-d6) δ ppm 32.3 (1C, CH2), 117.3 (1C,
Ph), 118.8 (1C, quinazolinone), 125.1 (1C, quinazolinone), 126.0 (1C,
quinazolinone), 128.1 (1C, quinazolinone), 130.2 (2C, Ph), 139.2 (1C,
quinazolinone), 142.9 (1C, Ph), 147.6 (2C, Ph), 149.1 (1C,
quinazolinone), 156.5 (1C, quinazolinone), 160.4 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C15H9ClN4O5S + H]+ 393.0055, found
393.0045.
7-Chloro-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-one (50c).
Following General Procedure B, 49c (0.11 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50c (eluent system: 15% EA in toluene, 70 mg,
0.18 mmol, 40% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.69
(br. s., 2H, CH2), 7.53−7.76 (m, 1H, quinazolinone), 7.77−7.90 (m,
1H, quinazolinone), 7.93 (br. s., 1H, quinazolinone), 8.68 (br. s., 1H,
Ph), 8.89 (br. s., 2H, Ph), 12.84 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.2 (1C, CH2), 117.3 (1C, Ph), 121.3 (1C,
quinazolinone), 125.0 (1C, quinazolinone), 128.0 (1C, quinazoli-
none), 130.0 (1C, quinazolinone), 130.1 (2C, Ph), 134.8 (1C,
quinazolinone), 142.9 (1C, Ph), 146.8 (1C, quinazolinone), 147.7
(2C, Ph), 155.4 (1C, quinazolinone), 160.1 (1C, CO). HRMS (ESI):
m/z [M + H]+ calcd for [C15H9ClN4O5S + H]+ 393.0055, found
393.0063.
2-((3,5-Dinitrobenzyl)thio)-3-methylquinazolin-4(3H)-one (47).
To a solution of 26 (0.10 g, 0.28 mmol) and K2CO3 (0.12 g, 0.83
mmol) in MeCN (10 mL) was added iodomethane (34 μL, 0.55
mmol), and the resulting mixture was stirred at room temperature for 1
h. The reaction mixture was adjusted to pH 5−6 with 1 N aq HCl, and
the concentrated mixture was purified to give 47 (eluent system: 15%
1
EA in toluene, 38 mg, 0.10 mmol, 37% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 3.48 (s, 3H, CH3), 4.77 (s, 2H, CH2), 7.45 (t, J = 7.7
Hz, 1H, quinazolinone), 7.68 (d, J = 7.9 Hz, 1H, quinazolinone), 7.84
(t, J = 7.5 Hz, 1H, quinazolinone), 8.05 (dd, J = 7.9, 1.3 Hz, 1H,
quinazolinone), 8.69 (t, J = 2.1 Hz, 1H, Ph), 8.95 (d, J = 2.1 Hz, 2H,
Ph). 13C NMR (101 MHz, DMSO-d6) δ ppm 30.1 (1C, CH3), 33.7
(1C, CH2), 117.4 (1C, Ph), 118.7 (1C, quinazolinone), 125.6 (1C,
quinazolinone), 126.0 (1C, quinazolinone), 126.4 (1C, quinazoli-
none), 130.3 (2C, Ph), 134.7 (1C, quinazolinone), 142.7 (1C, Ph),
146.6 (1C, quinazolinone), 147.6 (2C, Ph), 156.1 (1C, quinazolinone),
160.6 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for [C16H12N4O5S
+ H]+ 373.0601, found 373.0603.
5-Chloro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49a). Fol-
lowing General Procedure A, 6-chloroanthranilic acid (1.0 g, 6.0 mmol)
in thionyl chloride (10 mL) afforded acyl chloride, which was reacted
with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give 49a.
6-Chloro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49b). Fol-
lowing General Procedure A, 5-chloroanthranilic acid (1.0 g, 6.0 mmol)
in thionyl chloride (10 mL) afforded acyl chloride, which was reacted
with NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) to give 49b.
7-Chloro-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (49c). Fol-
lowing General Procedure A, 4-chloroanthranilic acid (1.0 g, 6.0
mmol) in thionyl chloride (10 mL) afforded acyl chloride, which was
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01374
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
8-Chloro-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-one (50d).
Following General Procedure B, 49d (0.11 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50d (eluent system: 15% EA in toluene, 0.12 g,
0.31 mmol, 68% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.72 (s,
2H, CH2), 7.39 (t, J = 7.9 Hz, 1H, quinazolinone), 7.95 (ddd, J = 9.2,
7.9, 1.4 Hz, 2H, quinazolinone), 8.69 (t, J = 2.1 Hz, 1H, Ph), 8.94 (d, J =
2.1 Hz, 2H, Ph), 12.91 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ
ppm 32.3 (1C, CH2), 117.4 (1C, Ph), 121.7 (1C, quinazolinone), 125.1
(1C, quinazolinone), 126.1 (1C, quinazolinone), 129.3 (1C,
quinazolinone), 129.7 (2C, Ph), 134.7 (1C, quinazolinone), 143.2
(1C, Ph), 144.5 (1C, quinazolinone), 147.8 (2C, Ph), 156.2 (1C,
quinazolinone), 160.6 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd
for [C15H9ClN4O5S + H]+ 393.0054, found 393.0063.
6-Bromo-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-one (50e).
Following General Procedure B, 49e (0.13 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50e (eluent system: 15% EA in toluene, 0.14 g,
0.32 mmol, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.69 (s,
2H, CH2), 7.58 (d, J = 8.8 Hz, 1H, quinazolinone), 7.97 (dd, J = 8.7, 2.4
Hz, 1H, quinazolinone), 8.06 (d, J = 2.4 Hz, 1H, quinazolinone), 8.68
(t, J = 2.1 Hz, 1H, Ph), 8.89 (d, J = 2.1 Hz, 2H, Ph), 12.84 (s, 1H, NH).
13C NMR (101 MHz, DMSO-d6) δ ppm 32.2 (1C, CH2), 117.3 (1C,
Ph), 118.1 (1C, quinazolinone), 121.7 (1C, quinazolinone), 128.1 (1C,
quinazolinone), 128.2 (1C, quinazolinone), 130.1 (2C, Ph), 137.5 (1C,
quinazolinone), 142.9 (1C, Ph), 147.1 (1C, quinazolinone), 147.7 (2C,
Ph), 155.5 (1C, quinazolinone), 159.9 (1C, CO). HRMS (ESI): m/z
[M − H]− calcd for [C15H9BrN4O5S − H]− 434.9404, found 434.9408.
2-((3,5-Dinitrobenzyl)thio)-6-methylquinazolin-4(3H)-one (50f).
Following General Procedure B, 49f (96 mg, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50f (eluent system: 15% EA in toluene, 86 mg,
0.23 mmol, 51% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.40 (s,
3H, CH3), 4.67 (s, 2H, CH2), 7.55 (d, J = 8.3 Hz, 1H, quinazolinone),
7.64 (dd, J = 8.3, 1.8 Hz, 1H, quinazolinone), 7.80 (s, 1H,
quinazolinone), 8.67 (t, J = 2.1 Hz, 1H, Ph), 8.89 (d, J = 2.0 Hz, 2H,
Ph), 12.56 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 20.7
(1C, CH3), 32.1 (1C, CH2), 117.3 (1C, Ph), 119.7 (1C,
quinazolinone), 125.4 (1C, quinazolinone), 125.7 (1C, quinazoli-
none), 130.1 (2C, Ph), 135.6 (1C, quinazolinone), 135.9 (1C,
quinazolinone), 143.1 (1C, Ph), 146.2 (1C, quinazolinone), 147.6
(2C, Ph), 153.4 (1C, quinazolinone), 161.0 (1C, CO). HRMS (ESI):
m/z [M − H]− calcd for [C16H12N4O5S − H]− 371.0455, found
371.0458.
none), 128.9 (1C, quinazolinone), 130.1 (2C, Ph), 142.7 (1C, Ph),
144.1 (1C, quinazolinone), 147.7 (2C, Ph), 152.0 (1C, quinazolinone),
159.6 (1C, quinazolinone), 160.2 (1C, CO). HRMS (ESI): m/z [M −
H]− calcd for [C15H9N5O7S − H]− 402.0150, found 402.0148.
2-Thioxo-2,3-dihydropyrido[3,2-d]pyrimidin-4(1H)-one (55a).
According to a literature report,20,28 a mixture of 3-aminopicolinic
acid (0.83 g, 6.0 mmol) and thiourea (2.3 g, 30 mmol) was stirred at 200
°C for 2 h. After cooling to 40 °C, water (50 mL) was added to the
reaction mixture and the resulting mixture was stirred at room
temperature overnight. After filtration, the filtrate was collected and
concentrated to give crude 55a, which was used in the next step without
purification.
2-Thioxo-2,3-dihydropyrido[4,3-d]pyrimidin-4(1H)-one (55b). To
a solution of 4-aminonicotinic acid (0.83 g, 6.0 mmol) in THF/dioxane
(1/1 v/v, 20 mL) were added SOCl2 (1.0 mL, 21 mmol) and catalytic
DMF (8.0 μL), and the resulting mixture was stirred at room
temperature for 3 h.46 After completion of acyl chloride formation, a
solution of NH4SCN (0.53 g, 6.9 mmol) in acetone (15 mL) and
dioxane (10 mL) was added to the reaction mixture. The resulting
mixture was stirred at 80 °C for 2 h and filtered when it was hot. The
filter cake was washed with CH2Cl2 (20 mL) and dried to give 55b,
which was used in the next step without purification.
2-Thioxo-2,3-dihydropyrido[3,4-d]pyrimidin-4(1H)-one (55c).
Following the procedure described for 55a, 3-aminoisonicotinic acid
(0.83 g, 6.0 mmol) and thiourea (2.3 g, 30 mmol) afforded 55c, which
was in the filter cake.
2-Thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (55d).
Following the procedure described for 55a, 2-aminonicotinic acid
(0.83 g, 6.0 mmol) and thiourea (2.3 g, 30 mmol) afforded 55d, which
was in the filter cake.
2-((3,5-Dinitrobenzyl)thio)pyrido[3,2-d]pyrimidin-4(3H)-one
(56a). Following General Procedure B, 55a (90 mg, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 56a (eluent system: 5% MeOH in
CH2Cl2, 79 mg, 0.22 mmol, 49% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.70 (s, 2H, CH2), 7.81 (dd, J = 8.3, 4.3 Hz, 1H, pyridyl), 8.02
(d, J = 7.5 Hz, 1H, pyridyl), 8.62−8.75 (m, 2H, pyridyl, Ph), 8.90 (d, J =
1.8 Hz, 2H, Ph), 12.93 (br. s., 1H, NH). 13C NMR (101 MHz, DMSO-
d6) δ ppm 32.4 (1C, CH2), 117.3 (1C, Ph), 129.0 (1C, pyridyl), 130.1
(2C, Ph), 133.9 (1C, pyridyl), 137.1 (1C, pyridyl), 142.9 (1C, Ph),
145.1 (1C, pyridyl), 147.7 (2C, Ph), 148.2 (1C, pyridyl), 155.6 (1C,
NCS), 159.6 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for
[C14H9N5O5S + H]+ 360.0397, found 360.0395.
2-((3,5-Dinitrobenzyl)thio)pyrido[4,3-d]pyrimidin-4(3H)-one
(56b). Following General Procedure B, 55b (90 mg, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 56b (eluent system: 5% MeOH in
CH2Cl2, 44 mg, 0.12 mmol, 27% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.72 (s, 2H, CH2), 7.50 (d, J = 5.6 Hz, 1H, pyridyl), 8.69 (t, J
= 2.1 Hz, 1H, Ph), 8.81 (d, J = 5.6 Hz, 1H, pyridyl), 8.90 (d, J = 2.0 Hz,
2H, Ph), 9.14 (s, 1H, pyridyl), 13.02 (s, 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.4 (1C, CH2), 115.9 (1C, pyridyl), 117.4 (1C, Ph),
119.2 (1C, pyridyl), 130.1 (2C, Ph), 142.6 (1C, Ph), 147.7 (2C, Ph),
149.4 (1C, pyridyl), 152.8 (1C, pyridyl), 153.6 (1C, pyridyl), 160.3
(1C, NCS), C (CO) could not be observed. HRMS (ESI): m/z [M +
H]+ calcd for [C14H9N5O5S + H]+ 360.0397, found 360.0403.
2-((3,5-Dinitrobenzyl)thio)pyrido[3,4-d]pyrimidin-4(3H)-one
(56c). Following General Procedure B, 55c (90 mg, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded crude 56c. After filtration, the solid
was suspended in DMSO (2.0 mL) and the suspension was stirred at
room temperature for 0.5 h. 56c was obtained through filtration (91 mg,
0.25 mmol, 56% yield). 1H NMR (400 MHz, trifluoroacetic acid-d) δ
ppm 4.74 (s, 2H, CH2), 8.61−8.75 (m, 2H, pyridyl), 8.82 (s, 2H, Ph),
8.93 (br. s., 1H, Ph), 9.36 (s, 1H, pyridyl). 13C NMR (101 MHz,
trifluoroacetic acid-d) δ ppm 34.2 (1C, CH2), 119.0 (1C, Ph), 126.1
(1C, pyridyl), 130.8 (2C, Ph), 132.9 (1C, pyridyl), 136.5 (1C, pyridyl),
141.8 (1C, Ph), 143.8 (1C, pyridyl), 146.9 (1C, pyridyl), 149.2 (2C,
Ph), 160.9 (1C, NCS), 163.8 (1C, CO). HRMS (ESI): m/z [M + H]+
calcd for [C14H9N5O5S + H]+ 360.0397, found 360.0394.
2-((3,5-Dinitrobenzyl)thio)-6-fluoroquinazolin-4(3H)-one (50g).
Following General Procedure B, 49g (98 mg, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50g (eluent system: 15% EA in toluene, 0.13 g,
0.35 mmol, 78% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.68 (s,
2H, CH2), 7.65−7.76 (m, 3H, quinazolinone), 8.68 (t, J = 2.1 Hz, 1H,
Ph), 8.90 (d, J = 2.0 Hz, 2H, Ph), 12.78 (s, 1H, NH). 19F NMR (377
MHz, DMSO-d6) δ ppm -114.34 (s, 1 F). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.2 (1C, CH2), 110.8 (d, J = 23.3 Hz, 1C,
quinazolinone), 117.3 (1C, Ph), 121.2 (d, J = 9.0 Hz, 1C,
quinazolinone), 123.2 (d, J = 23.6 Hz, 1C, quinazolinone), 128.5 (d,
J = 7.9 Hz, 1C, quinazolinone), 130.1 (2C, Ph), 143.0 (1C, Ph), 145.04
(1C, quinazolinone), 147.7 (2C, Ph), 154.1 (1C, quinazolinone), 158.2
(1C, CO), 160.6 (d, J = 16.9 Hz, 1C, quinazolinone). HRMS (ESI): m/
z [M − H]− calcd for [C15H9FN4O5S − H]− 375.0205, found
375.0211.
2-((3,5-Dinitrobenzyl)thio)-6-nitroquinazolin-4(3H)-one (50h).
Following General Procedure B, 49h (0.11 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 50h (eluent system: 15% EA in toluene, 0.11 g,
0.28 mmol, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.74 (s,
2H, CH2), 7.80 (d, J = 9.0 Hz, 1H, quinazolinone), 8.58 (dd, J = 8.9, 2.7
Hz, 1H, quinazolinone), 8.66−8.74 (m, 2H, quinazolinone, Ph), 8.91
(d, J = 2.1 Hz, 2H, Ph), 13.15 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.4 (1C, CH2), 117.4 (1C, Ph), 120.1 (1C,
quinazolinone), 122.3 (1C, quinazolinone), 128.2 (1C, quinazoli-
N
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2-((3,5-Dinitrobenzyl)thio)pyrido[2,3-d]pyrimidin-4(3H)-one
(56d). Following General Procedure B, 55d (90 mg, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 56d (eluent system: 5% MeOH in
CH2Cl2, 0.12 g, 0.33 mmol, 72% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.77 (s, 2H, CH2), 7.46 (dd, J = 7.9, 4.6 Hz, 1H, pyridyl), 8.40
(dd, J = 7.9, 2.0 Hz, 1H, pyridyl), 8.70 (t, J = 2.1 Hz, 1H, Ph), 8.86−8.93
(m, 3H, pyridyl, Ph), 12.98 (br. s., 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.1 (1C, CH2), 115.3 (1C, pyridyl), 117.4 (1C, Ph),
121.7 (1C, pyridyl), 129.8 (2C, Ph), 135.7 (1C, pyridyl), 142.8 (1C,
Ph), 147.8 (2C, Ph), 155.8 (1C, pyridyl), 157.7 (1C, pyridyl), 158.8
(1C, NCS), 161.8 (1C, CO). HRMS (ESI): m/z [M + H]+ calcd for
[C14H9N5O5S + H]+ 360.0397, found 360.0402.
5-Fluoro-2-nitrobenzoic Acid (58). According to a literature
report,29 to a solution of 3-fluorobenzoic acid (0.79 g, 5.6 mmol) in
sulfuric acid (5.0 mL) at 0 °C was added fuming nitric acid (0.31 mL,
6.7 mmol). The resulting mixture was stirred at room temperature for 1
h. Then, the solution was poured to water (75 mL) and extracted with
EA (0.15 L × 2). The combined organic layers were dried and
concentrated to give 58 without purification (0.79 g, 4.3 mmol, 76%
yield). 1H NMR (400 MHz, methanol-d4) δ ppm 7.45 (ddd, J = 9.0, 7.7,
2.8 Hz, 1H, Ph), 7.55 (dd, J = 8.3, 2.8 Hz, 1H, Ph), 8.03 (dd, J = 9.0, 4.6
Hz, 1H, Ph). 19F NMR (377 MHz, methanol-d4) δ ppm -106.2 (s, 1 F).
13C NMR (101 MHz, methanol-d4) δ ppm 118.1 (d, J = 23.5 Hz, 1C,
J = 9.4, 2.9 Hz, 1H, Ph), 7.52 (br. s., 1H, NH2), 7.87 (br. s., 1H, NH2),
7.97 (d, J = 9.4 Hz, 1H, Ph). 13C NMR (101 MHz, DMSO-d6) δ ppm
46.5 (2C, morpholinyl), 65.7 (2C, morpholinyl), 111.9 (1C, Ph), 112.5
(1C, Ph), 126.5 (1C, Ph), 135.1 (1C, Ph), 136.4 (1C, Ph), 153.8 (1C,
Ph), 168.4 (1C, CO).
5-(4-Methylpiperazin-1-yl)-2-nitrobenzamide (60d). Following
the procedure described for 60a, 59 (0.50 g, 2.7 mmol), K2CO3
(0.56 g, 4.1 mmol), and N-methylpiperazine (0.36 mL, 3.3 mmol) in
DMSO (2.0 mL) afforded 60d (eluent system: 15% MeOH in CH2Cl2,
0.56 g, 2.1 mmol, 78% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm
2.21 (s, 3H, CH3), 2.34−2.46 (m, 4H, piperazinyl), 3.29−3.48 (m, 4H,
piperazinyl), 4.85 (s, 2H, NH2), 6.85 (d, J = 2.0 Hz, 1H, Ph), 6.93 (dd, J
= 9.3, 2.3 Hz, 1H, Ph), 7.84 (d, J = 9.3 Hz, 1H, Ph). 13C NMR (101
MHz, DMSO-d6) δ ppm 45.6 (1C, CH3), 46.4 (2C, piperazinyl), 54.2
(2C, piperazinyl), 112.2 (1C, Ph), 112.3 (1C, Ph), 126.2 (1C, Ph),
135.4 (1C, Ph), 138.5 (1C, Ph), 153.5 (1C, Ph), 169.3 (1C, CO).
6-(Dimethylamino)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one
(62a). To a stirred solution of 60a (0.29 g, 1.4 mmol) in EtOH (40 mL)
was added Pd−C (10%) under N2. H2 gas was introduced to the
reaction mixture using a double-folded balloon. Upon completion, the
reaction mixture was filtered through a Celite pad and the filter cake was
washed with MeOH (20 mL). The filtrate was concentrated to give 61a
1
without purification (0.23 g, 1.3 mmol, 93% yield). H NMR (400
MHz, DMSO-d6) δ ppm 2.73 (s, 6H, N(CH3)2), 5.88 (s, 2H, NH2),
6.61 (d, J = 8.8 Hz, 1H, Ph), 6.80 (dd, J = 8.8, 2.8 Hz, 1H, Ph), 6.91 (d, J
= 2.8 Hz, 1H, Ph), 7.01 (s, 1H, NH2), 7.74 (s, 1H, NH2). 13C NMR
(101 MHz, DMSO-d6) δ ppm 41.9 (2C, N(CH3)2), 113.7 (1C, Ph),
114.7 (1C, Ph), 117.6 (1C, Ph), 120.1 (1C, Ph), 141.6 (1C, Ph), 142.3
(1C, Ph), 171.5 (1C, CO). According to a literature report,30 KOH
(0.24 g, 4.3 mmol) was suspended in EtOH (10 mL) and the
suspension was stirred at room temperature for 0.5 h to dissolve the
KOH. Then, CS2 (0.26 mL, 4.3 mmol) was added to the stirred solution
and the resulting mixture was stirred for another 0.5 h to produce a
yellow suspension. Then, 61a (0.51 g, 2.9 mmol), EtOH (5.0 mL), and
water (0.40 mL) were added to the mixture in succession. The reaction
mixture was refluxed overnight. After cooling to room temperature, the
volatile material was removed in vacuo. The mixture was adjusted to pH
6−7 with 1 N aq HCl. The precipitate was collected through filtration
and dried over an oil pump to give 62a without purification.
Ph), 119.7 (d, J = 23.5 Hz, 1C, Ph), 128.2 (d, J = 9.3 Hz, 1C, Ph), 132.6
(d, J = 9.3 Hz, 1C, Ph), 146.1 (1C, Ph), 165.7 (d, J = 254.2 Hz, 1C, Ph),
167.2 (1C, CO).
5-Fluoro-2-nitrobenzamide (59). 58 (2.0 g, 11 mmol) was refluxed
in thionyl chloride (10 mL) for 2 h. The excess thionyl chloride was
removed in vacuo. The crude acyl chloride was dissolved in CH2Cl2 (25
mL), followed by addition of NH3 (excess) through a balloon. The
precipitate was filtered to afford 59 without purification (1.2 g, 6.5
mmol, 60% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.50−7.58
(m, 2H, Ph), 7.80 (br. s., 1H, NH2), 8.13 (dd, J = 9.8, 4.8 Hz, 1H, Ph),
8.19 (br. s., 1H, NH2). 19F NMR (377 MHz, DMSO-d6) δ ppm -104.3
(s, 1 F). 13C NMR (101 MHz, DMSO-d6) δ ppm 116.2 (d, J = 24.8 Hz,
1C, Ph), 117.3 (d, J = 24.8 Hz, 1C, Ph), 127.3 (d, J = 11.0 Hz, 1C, Ph),
135.7 (d, J = 8.8 Hz, 1C, Ph), 143.3 (1C, Ph), 163.7 (d, J = 254.8 Hz,
1C, Ph), 165.4 (1C, CO).
5-(Dimethylamino)-2-nitrobenzamide (60a). According to a
literature report,47 to a solution of 59 (0.50 g, 2.7 mmol) and K2CO3
(0.79 g, 5.7 mmol) in DMSO (2.0 mL) at 60 °C was added
dimethylamine hydrochloride (0.27 g, 3.3 mmol). After 1 h, the
reaction was quenched with water (0.10 L). The resulting mixture was
extracted with EA (0.10 L × 2), and the combined organic layers were
dried, concentrated, and purified to afford 60a (eluent system: 25% EA
in petroleum ether, 0.30 g, 1.4 mmol, 52% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 3.08 (s, 6H, N(CH3)2), 6.58 (d, J = 2.9 Hz, 1H, Ph),
6.75 (dd, J = 9.4, 2.9 Hz, 1H, Ph), 7.49 (br. s., 1H, NH2), 7.85 (br. s.,
1H, NH2), 7.96 (d, J = 9.4 Hz, 1H, Ph). 13C NMR (101 MHz, DMSO-
d6) δ ppm 39.8 (2C, N(CH3)2), 109.8 (1C, Ph), 110.5 (1C, Ph), 126.7
(1C, Ph), 133.0 (1C, Ph), 136.8 (1C, Ph), 153.3 (1C, Ph), 168.8 (1C,
CO).
2-Nitro-5-(piperidin-1-yl)benzamide (60b). Following the proce-
dure described for 60a, 59 (0.50 g, 2.7 mmol), K2CO3 (0.56 g, 4.1
mmol), and piperidine (0.32 mL, 3.3 mmol) in DMSO (2.0 mL)
afforded 60b (eluent system: 25% EA in petroleum ether, 0.58 g, 2.3
mmol, 86% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.53−1.67
(m, 6H, piperidyl), 3.44−3.52 (m, 4H, piperidyl), 6.80 (d, J = 2.9 Hz,
1H, Ph), 6.97 (dd, J = 9.5, 2.9 Hz, 1H, Ph), 7.48 (s, 1H, NH2), 7.84 (s,
1H, NH2), 7.93 (d, J = 9.4 Hz, 1H, Ph). 13C NMR (101 MHz, DMSO-
d6) δ ppm 23.8 (1C, piperidyl), 24.9 (2C, piperidyl), 47.6 (2C,
piperidyl), 111.5 (1C, Ph), 112.2 (1C, Ph), 126.9 (1C, Ph), 133.6 (1C,
Ph), 136.9 (1C, Ph), 153.4 (1C, Ph), 168.6 (1C, CO).
6-(Piperidin-1-yl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one
(62b). Following the procedure described for 62a, 60b (0.57 g, 2.3
mmol) and Pd−C (10%) in EtOH (40 mL) under H2 afforded 61b
(0.48 g, 2.2 mmol, 96% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm
1.39−1.54 (m, 2H, piperidyl), 1.61 (q, J = 5.6 Hz, 4H, piperidyl), 2.82−
3.02 (m, 4H, piperidyl), 6.05 (br. s., 2H, NH2), 6.59 (d, J = 8.8 Hz, 1H,
Ph), 6.79−6.94 (m, 1H, Ph), 6.98 (br. s., 1H, NH2), 7.06 (br. s., 1H,
Ph), 7.72 (br. s., 1H, NH2). 13C NMR (101 MHz, DMSO-d6) δ ppm
23.8 (1C, piperidyl), 25.7 (2C, piperidyl), 51.9 (2C, piperidyl), 114.2
(1C, Ph), 117.2 (2C, Ph), 123.3 (1C, Ph), 142.2 (1C, Ph), 144.1 (1C,
Ph), 171.4 (1C, CO). Then, KOH (0.18 g, 3.2 mmol), CS2 (0.19 mL,
3.2 mmol) and 61b (0.47 g, 2.1 mmol) in EtOH/water (15 mL/0.3
mL) gave 62b without purification.
6-Morpholino-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (62c).
Following the procedure described for 62a, 60c (0.51 g, 2.0 mmol)
and Pd−C (10%) in EtOH (40 mL) under H2 afforded 61c (0.30 g, 1.4
mmol, 67% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.89−3.00
(m, 4H, morpholinyl), 3.68−3.74 (m, 4H, morpholinyl), 6.08 (s, 2H,
NH2), 6.62 (d, J = 8.9 Hz, 1H, Ph), 6.91 (dd, J = 8.8, 2.7 Hz, 1H, Ph),
7.01 (br. s., 1H, NH2), 7.05 (d, J = 2.8 Hz, 1H, Ph), 7.74 (br. s., 1H,
NH2). 13C NMR (101 MHz, DMSO-d6) δ ppm 50.6 (2C,
morpholinyl), 66.3 (2C, morpholinyl), 114.1 (1C, Ph), 116.1 (1C,
Ph), 117.4 (1C, Ph), 122.3 (1C, Ph), 141.0 (1C, Ph), 144.3 (1C, Ph),
171.3 (1C, CO). Then, KOH (0.11 g, 2.0 mmol), CS2 (0.12 mL, 2.0
mmol) and 61c (0.29 g, 1.3 mmol) in EtOH/water (15 mL/0.20 mL)
gave 62c without purification.
5-Morpholino-2-nitrobenzamide (60c). Following the procedure
described for 60a, 59 (0.50 g, 2.7 mmol), K2CO3 (0.56 g, 4.1 mmol),
and morpholine (0.29 mL, 3.3 mmol) in DMSO (2.0 mL) afforded 60c
(eluent system: 5% MeOH in CH2Cl2, 0.51 g, 2.0 mmol, 75% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm 3.36−3.44 (m, 4H, morpholinyl),
3.66−3.76 (m, 4H, morpholinyl), 6.89 (d, J = 2.8 Hz, 1H, Ph), 7.02 (dd,
6-(4-Methylpiperazin-1-yl)-2-thioxo-2,3-dihydroquinazolin-
4(1H)-one (62d). Following the procedure described for 62a, 60d (0.55
g, 2.1 mmol) and Pd−C (10%) in EtOH (40 mL) under H2 afforded
61d (0.45 g, 1.9 mmol, 92% yield). 1H NMR (400 MHz, DMSO-d6) δ
ppm 2.20 (br. s., 3H, CH3), 2.43 (br. s., 4H, piperazinyl), 2.85−3.02 (m,
O
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4H, piperazinyl), 6.06 (br. s., 2H, NH2), 6.61 (d, J = 8.6 Hz, 1H, Ph),
6.89 (d, J = 8.1 Hz, 1H, Ph), 7.00 (br. s., 1H, NH2), 7.05 (br. s., 1H, Ph),
7.76 (br. s., 1H, NH2). 13C NMR (101 MHz, DMSO-d6) δ ppm 45.8
(1C, CH3), 50.1 (2C, piperazinyl), 54.9 (2C, piperazinyl), 114.2 (1C,
Ph), 116.3 (1C, Ph), 117.3 (1C, Ph), 122.5 (1C, Ph), 141.1 (1C, Ph),
144.1 (1C, Ph), 171.4 (1C, CO). Then, KOH (0.15 g, 2.8 mmol), CS2
(0.17 mL, 2.8 mmol) and 61d (0.43 g, 1.8 mmol) in EtOH/water (15
mL/0.30 mL) gave 62d without purification.
(1C, quinazolinone), 143.3 (1C, Ph), 147.6 (2C, Ph), 148.9 (1C,
quinazolinone), 150.6 (1C, quinazolinone), 161.2 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C20H20N6O5S + H]+ 457.1289, found
457.1283.
ASSOCIATED CONTENT
■
sı
* Supporting Information
6-(Dimethylamino)-2-((3,5-dinitrobenzyl)thio)quinazolin-4(3H)-
one (63a). Following General Procedure B, 62a (0.11 g, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 63a (eluent system: 20% EA in
toluene, 0.18 g, 0.44 mmol, 98% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 2.97 (s, 6H, N(CH3)2), 4.64 (s, 2H, CH2), 7.08 (d, J = 3.0
Hz, 1H, quinazolinone), 7.31 (dd, J = 9.0, 3.0 Hz, 1H, quinazolinone),
7.51 (d, J = 9.0 Hz, 1H, quinazolinone), 8.66 (t, J = 2.1 Hz, 1H, Ph),
8.88 (d, J = 2.1 Hz, 2H, Ph), 12.37 (s, 1H, NH). 13C NMR (101 MHz,
DMSO-d6) δ ppm 32.0 (1C, CH2), 40.1 (2C, N(CH3)2), 105.0 (1C,
quinazolinone), 117.2 (1C, Ph), 120.6 (1C, quinazolinone), 120.6 (1C,
quinazolinone), 126.7 (1C, quinazolinone), 130.0 (2C, Ph), 139.4 (1C,
quinazolinone), 143.4 (1C, Ph), 147.6 (2C, Ph), 148.4 (1C,
quinazolinone), 148.9 (1C, quinazolinone), 161.3 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C17H15N5O5S + H]+ 402.0867, found
402.0869.
2-((3,5-Dinitrobenzyl)thio)-6-(piperidin-1-yl)quinazolin-4(3H)-
one (63b). Following General Procedure B, 62b (0.13 g, 0.50 mmol),
K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/
MeCN (1/10 v/v, 11 mL) afforded 63b (eluent system: 15% EA in
toluene, 0.18 g, 0.41 mmol, 91% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 1.55 (d, J = 4.4 Hz, 2H, piperidyl), 1.59−1.70 (m, 4H,
piperidyl), 3.12−3.28 (m, 4H, piperidyl), 4.64 (s, 2H, CH2), 7.28 (d, J =
1.8 Hz, 1H, quinazolinone), 7.45−7.57 (m, 2H, quinazolinone), 8.66
(t, J = 2.0 Hz, 1H, Ph), 8.87 (d, J = 2.0 Hz, 2H, Ph), 12.42 (s, 1H, NH).
13C NMR (101 MHz, DMSO-d6) δ ppm 23.7 (1C, piperidyl), 25.0 (2C,
piperidyl), 32.1 (1C, CH2), 49.2 (2C, piperidyl), 108.3 (1C,
quinazolinone), 117.2 (1C, Ph), 120.5 (1C, quinazolinone), 124.3
(1C, quinazolinone), 126.6 (1C, quinazolinone), 130.0 (2C, Ph), 140.9
(1C, quinazolinone), 143.3 (1C, Ph), 147.6 (2C, Ph), 149.4 (1C,
quinazolinone), 150.2 (1C, quinazolinone), 161.2 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C20H19N5O5S + H]+ 442.1180, found
442.1184.
2-((3,5-Dinitrobenzyl)thio)-6-morpholinoquinazolin-4(3H)-one
(63c). Following General Procedure B, 62c (0.13 g, 0.50 mmol), K2CO3
(0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45 mmol) in DMSO/MeCN (1/
10 v/v, 11 mL) afforded 63c (eluent system: 5% MeOH in CH2Cl2,
0.18 g, 0.42 mmol, 92% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm
3.18 (br. s., 4H, morpholinyl), 3.75 (br. s., 4H, morpholinyl), 4.65 (br.
s., 2H, CH2), 7.31 (br. s., 1H, quinazolinone), 7.55 (br. s., 2H,
quinazolinone), 8.67 (br. s., 1H, Ph), 8.89 (br. s., 2H, Ph), 12.47 (br. s.,
1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.1 (1C, CH2),
48.2 (2C, morpholinyl), 66.0 (2C, morpholinyl), 108.1 (1C,
quinazolinone), 117.3 (1C, Ph), 120.5 (1C, quinazolinone), 123.6
(1C, quinazolinone), 126.7 (1C, quinazolinone), 130.1 (2C, Ph), 141.5
(1C, quinazolinone), 143.3 (1C, Ph), 147.6 (2C, Ph), 149.0 (1C,
quinazolinone), 150.7 (1C, quinazolinone), 161.1 (1C, CO). HRMS
(ESI): m/z [M + H]+ calcd for [C19H17N5O6S + H]+ 444.0973, found
444.0966.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01374.
Determination of IC50 values of compounds 21−23, 26,
27, 30−40, 45−47, 50b, and 50c (Table S1);
antimycobacterial activity of compounds 21−23, 28−
33, 37, 39−46, 50a, 50d, 56b−d, 63a, and 63b under
different media (Table S2); frequency of resistance for
compound 26 and rifampicin (Table S3); MIC values of
compound 26, pretomanid, delamanid, isoniazid, and
rifampicin against wild-type strain and mutants generated
1
to compound 26 (Table S4); H, 13C, and 19F NMR
spectra of compounds 21−23, 26−47, 50a−50h, 56a−
56d, and 63a−d (Table S5); HSQC and HMBC spectra
of compounds 46 and 47; purity of final compounds
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Serge Van Calenbergh − Laboratory for Medicinal Chemistry
(FFW), Ghent University, B-9000 Gent, Belgium;
orcid.org/0000-0002-4201-1264; Phone: +32 (0)9 264
81 24; Email: serge.vancalenbergh@ugent.be; Fax: +32 (0)
9 264 81 46
Authors
Yanlin Jian − Laboratory for Medicinal Chemistry (FFW),
Ghent University, B-9000 Gent, Belgium
He Eun Forbes − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Disease, National Institutes of Health,
Bethesda, Maryland 20892, United States
Fabian Hulpia − Laboratory for Medicinal Chemistry (FFW),
Ghent University, B-9000 Gent, Belgium; orcid.org/0000-
0002-7470-3484
Martijn D. P. Risseeuw − Laboratory for Medicinal Chemistry
(FFW), Ghent University, B-9000 Gent, Belgium
Guy Caljon − Laboratory of Microbiology, Parasitology and
Hygiene, University of Antwerp, B-2610 Antwerp, Belgium
́
̀
Helene Munier-Lehmann − Unit of Chemistry and
Biocatalysis, Department of Structural Biology and Chemistry,
Institut Pasteur, CNRS UMR3523, 75015 Paris, France
Helena I. M. Boshoff − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Disease, National
Institutes of Health, Bethesda, Maryland 20892, United
States; orcid.org/0000-0002-4333-206X
2-((3,5-Dinitrobenzyl)thio)-6-(4-methylpiperazin-1-yl)-
quinazolin-4(3H)-one (63d). Following General Procedure B, 62d
(0.14 g, 0.50 mmol), K2CO3 (0.21 g, 1.5 mmol), and 13a (0.12 g, 0.45
mmol) in DMSO/MeCN (1/10 v/v, 11 mL) afforded 63d (eluent
system: 5% MeOH in CH2Cl2, 93 mg, 0.20 mmol, 45% yield). 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.24 (s, 3H, CH3), 2.41−2.59 (m, 4H,
piperazinyl), 3.18−3.23 (m, 4H, piperazinyl), 4.64 (s, 2H, CH2), 7.29
(d, J = 2.3 Hz, 1H, quinazolinone), 7.47−7.58 (m, 2H, quinazolinone),
8.66 (t, J = 2.1 Hz, 1H, Ph), 8.88 (d, J = 2.1 Hz, 2H, Ph), 12.01−12.81
(m, 1H, NH). 13C NMR (101 MHz, DMSO-d6) δ ppm 32.1 (1C, CH2),
45.6 (1C, CH3), 47.8 (2C, piperazinyl), 54.4 (2C, piperazinyl), 108.2
(1C, quinazolinone), 117.2 (1C, Ph), 120.4 (1C, quinazolinone), 123.9
(1C, quinazolinone), 126.6 (1C, quinazolinone), 130.1 (2C, Ph), 141.2
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01374
Notes
The authors declare no competing financial interest.
P
https://dx.doi.org/10.1021/acs.jmedchem.0c01374
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Prasad, S.; Gill, A.; Ugarkar, B. G.; Venkatraman, J.; Read, J.; Panda, M.
Structure guided lead generation for M. tuberculosis thymidylate kinase
(Mtb TMK): discovery of 3-cyanopyridone and 1,6-naphthyridin-2-
one as potent inhibitors. J. Med. Chem. 2015, 58, 753−766.
(15) Song, L.; Merceron, R.; Gracia, B.; Quintana, A. L.; Risseeuw, M.
D. P.; Hulpia, F.; Cos, P.; Ainsa, J. A.; Munier-Lehmann, H.; Savvides, S.
N.; Van Calenbergh, S. Structure guided lead generation toward
nonchiral M. tuberculosis thymidylate kinase inhibitors. J. Med. Chem.
2018, 61, 2753−2775.
(16) Karabanovich, G.; Dusek, J.; Savkova, K.; Pavlis, O.; Pavkova, I.;
Korabecny, J.; Kucera, T.; Vlckova, H. K.; Huszar, S.; Konyarikova, Z.;
Konecna, K.; Jand’ourek, O.; Stolarikova, J.; Kordulakova, J.; Vavrova,
K.; Pavek, P.; Klimesova, V.; Hrabalek, A.; Mikusova, K.; Roh, J.
Development of 3,5-dinitrophenyl-containing 1,2,4-triazoles and their
trifluoromethyl analogues as highly efficient antitubercular agents
inhibiting decaprenylphosphoryl-beta-D-ribofuranose 2’-oxidase. J.
Med. Chem. 2019, 62, 8115−8139.
ACKNOWLEDGMENTS
■
This work was supported by the China Scholarship Council
(grant number 201607060021) and in part by the Intramural
Research Program of the NIAID, NIH. S.V.C. thanks the
Hercules Foundation (project AUGE/17/22 “Pharm-NMR”).
ABBREVIATIONS
■
EA, ethyl acetate; ADC, albumin, dextrose, and catalase
supplement; dTMP, thymidine monophosphate; Pks13, poly-
ketide synthase 13
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