1H-Benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones: Design, synthesis and antitubercular activity

Article abstract of DOI:10.1016/j.ejmech.2018.06.005

Using a classical hybridization approach, a series of 1H-benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones were synthesized (39 examples) and evaluated as inhibitors of Mycobacterium tuberculosis growth. Chemical modification studies yielded potent antitubercular agents with minimum inhibitory concentration (MIC) values as low as 0.24 μM against M. tuberculosis H37Rv strain. Further, the synthesized compounds were active against four drug-resistant strains containing different levels of resistance for the first line drugs. These molecules were devoid of apparent toxicity to HepG2, HaCat, and Vero cells with IC_50s > 30 μM. Viability in mammalian cell cultures was evaluated using MTT and neutral red assays. In addition, some 3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or morphological alteration in zebrafish (Danio rerio) toxicity models. 3,4-Dihydroquinazolin-4-ones 9q and 9w were considered the lead compounds of these series of molecules with MIC values of 0.24 μM and 0.94 μM against M. tuberculosis H37Rv, respectively. Taken together, these data indicate that this class of compounds may furnish candidates for future development of novel anti-TB drugs.

Full text of DOI:10.1016/j.ejmech.2018.06.005

Accepted Manuscript
1H-Benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones: Design, synthesis and
antitubercular activity
Fernanda Souza Macchi, Kenia Pissinate, Anne Drumond Villela, Bruno Lopes
Abbadi, Valnês Rodrigues-Junior, Débora Dreher Nabinger, Stefani Altenhofen,
Nathalia Sperotto, Adílio da Silva Dadda, Fernanda Teixeira Subtil, Talita Freitas de
Freitas, Ana Paula Erhart Rauber, Ana Flávia Borsoi, Carla Denise Bonan, Cristiano
Valim Bizarro, Luiz Augusto Basso, Diógenes Santiago Santos, Pablo Machado
PII:
S0223-5234(18)30495-1
10.1016/j.ejmech.2018.06.005
EJMECH 10475
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2018
Revised Date: 18 April 2018
Accepted Date: 1 June 2018
Please cite this article as: F.S. Macchi, K. Pissinate, A.D. Villela, B.L. Abbadi, Valnê. Rodrigues-
Junior, Dé.Dreher. Nabinger, S. Altenhofen, N. Sperotto, Adí. da Silva Dadda, F.T. Subtil, T.F. de
Freitas, A.P. Erhart Rauber, Ana.Flá. Borsoi, C.D. Bonan, C.V. Bizarro, L.A. Basso, Dió.Santiago.
Santos, P. Machado, 1H-Benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones: Design, synthesis
and antitubercular activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.06.005.
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1H-Benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones: design, synthesis and
antitubercular activity
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Fernanda Souza Macchia,b, Kenia Pissinatea, Anne Drumond Villelaa, Bruno Lopes Abbadia,b,
Valnês Rodrigues-Juniora, Débora Dreher Nabingerb,d, Stefani Altenhofenb,d, Nathalia Sperottoa,c,
Adílio da Silva Daddaa,b, Fernanda Teixeira Subtila,b, Talita Freitas de Freitasa, Ana Paula Erhart
Raubera, Ana Flávia Borsoia, Carla Denise Bonanb,c,d, Cristiano Valim Bizarroa,b, Luiz Augusto
Bassoa,b,c, Diógenes Santiago Santosa,b, Pablo Machadoa,b,*
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aInstituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia
Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900,
Porto Alegre, Rio Grande do Sul, Brazil
bPrograma de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica
do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
cPrograma de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade
Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
dLaboratório de Neuroquímica e Psicofarmacologia, Pontifícia Universidade Católica do Rio
Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
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*Corresponding author.
E-mail address: pablo.machado@pucrs.br (Pablo Machado)

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Abstract
Using a classical hybridization approach, a series of 1H-benzo[d]imidazoles and 3,4-
dihydroquinazolin-4-ones were synthesized (39 examples) and evaluated as inhibitors of
Mycobacterium tuberculosis growth. Chemical modification studies yielded potent antitubercular
agents with minimum inhibitory concentration (MIC) values as low as 0.24 µM against M.
tuberculosis H37Rv strain. Further, the synthesized compounds were active against four drug-
resistant strains containing different levels of resistance for the first line drugs. These molecules
were devoid of apparent toxicity to HepG2, HaCat, and Vero cells with IC50s >30 µM. Viability
in mammalian cell cultures was evaluated using MTT and neutral red assays. In addition, some
3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or
morphological alteration in zebrafish (Danio rerio) toxicity models. 3,4-Dihydroquinazolin-4-
ones 9q and 9w were considered the lead compounds of these series of molecules with MIC
values of 0.24 µM and 0.94 µM against M. tuberculosis H37Rv, respectively. Taken together,
these data indicate that this class of compounds may furnish candidates for future development
of novel anti-TB drugs.
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Keywords: Mycobacterium tuberculosis, tuberculosis, molecular hybridization, drug-resistant
strains, SAR, cardiotoxicity.

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1. Introduction
Human tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium
tuberculosis (Mtb), and has been responsible for the deaths of thousands of people annually [1].
Only in 2015, 9.6 million new cases of the disease and 1.8 million deaths were reported by the
World Health Organization (WHO) worldwide [2]. The emergence of multidrug-resistant TB
(MDR-TB) and extensively drug-resistant TB (XDR-TB), HIV coinfection, and the elevated
number of individuals infected with latent or dormant bacilli have contributed to complicate this
scenario [1, 2]. The recommended treatment includes two months of isoniazid (INH), rifampicin
(RIF), ethambutol (ETH) and pyrazinamide (PZA), followed by four more months of INH and
RIF [3, 4]. Although it has a cure rate of up to 95%, the regime suffers with increasing number of
cases of individuals infected with drug-resistant strains [3]. In these cases, the treatment can to
be extended and requires the use of second-line drugs that are, in general, more expensive and
toxic [5]. Furthermore, the low levels of compliance with treatment, adverse effects, toxicity and
impossibility of co-administration with some antiretroviral drugs have limited the use of this
therapeutic strategy [6].
Within this context, there is an urgent need to obtain new therapeutic alternatives for
tuberculosis treatment; if possible, with innovative mechanisms of action capable of overcoming
the drug resistance concern. Although the approval of new drugs such as bedaquiline and
delamanid [7] for treatment of drug-resistant TB has brought some hope, the adaptive capacity of
Mtb has already led to the emergence of resistant strains for these drugs, evidencing the
continued need for new options [8].

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As part of our ongoing research we have studied the antimycobacterial activity of 2-quinolin-
4-yloxy)acetamides 1 (Figure 1) and their derivatives, with some encouraging in vitro results [9,
10]. The compounds have been active against resistant and non-resistant Mtb strains and have
exhibited selective inhibition of bacillus growth. In addition, lead molecule 1 (R = 4-propyl;
Figure 1) showed good membrane permeability, reasonable stability in intrinsic clearance
analysis, and synergistic in vitro effect with first line drug rifampin [10]. Recently, the
menaquinol cytochrome c oxidoreductase (bc1 complex) was proposed as a molecular target of
this chemical class by using whole-genome sequencing [11], corroborating other findings already
described in the literature [12]. It is important to note that although endowed with significant in
vitro results, initial data have indicated that 2-quinolin-4-yloxy)acetamides present low
bioavailability when orally administered to mice (unpublished results). This fact has prompt us to
evaluate new derivative structures with possible activity against Mtb. In this sense, SAR studies
have shown that acetamide moiety attached to the quinoline nuclei is part of the molecules’
pharmacophore, which is prone to be used in molecular hybridization-based approaches (Figure
1). In line with this purpose, 1H-benzo[d]imidazole and 3,4-dihydroquinazolin-4-one were used
as molecular scaffolds to evaluate the possibility of obtaining new anti-TB drug candidates by
hybridization between the titled heterocycles and acetamide group (Figure 1).
1H-
Benzo[d]imidazole and 3,4-dihydroquinazolin-4-one have been obtained as part of the structure
of compounds endowed with selective anti-TB activity. Our hypothesis was that the presence of
acetamide group attached to these heterocycles could provide novel compounds of optimized
activity. Lansoprazole sulfate (2) (Figure 1), the active metabolite from the drug lanzoprazole,
have exhibited significant activity against intracellular and in-broth cultures of Mtb with ICs50 of
0.59 µM and 0.46 µM, respectively [13]. Interestingly, whole-genome sequencing of resistant

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strains to LPZS revealed a unique nucleotide mutation in the b subunit of bc1 cytochrome [13].
3,4-Dihydroquinazolin-4-ones 3 have also been described as in vitro inhibitors of Mtb growth
with Minimal Inhibitory Concentration (MIC) as low as 4.76 µM (Figure 1). These compounds
were described to inhibit the Mycobacterium tuberculosis enoyl acyl carrier protein reductase, a
validated molecular target for TB drug development [14].
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Therefore, in an attempt to obtain new compounds with activity against drug-susceptible and
drug-resistant Mtb strains, new series of hybridized 1H-benzo[d]imidazoles and 3,4-
dihydroquinazolin-4-ones were synthesized and assayed against M. tuberculosis H37Rv. First,
the basic structural requirements for potency of compounds (SAR) were evaluated. Thereafter,
the most active structures against M. tuberculosis H37Rv were tested against a panel of clinically
isolated drug-resistant strains, and the viability of HepG2, HaCat, and Vero cells after exposure
to the compounds was determined. Finally, cardiotoxicity, neurotoxicity and possible
morphological alterations by exposure to the compounds using zebrafish (Danio rerio) models
were also evaluated.
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Figure 1. Molecular hybridization using acetamide moieties from 2-(quinolin-4-
yloxy)acetamides with 1H-benzo[d]imidazole and 3,4-dihydroquinazolin-4-one scaffolds.
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2. Results and Discussion
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The synthesis of the designed compounds was performed in two synthetic steps. First,
bromoacetamides 5 and 8 were obtained in an acylation reaction between primary or secondary
amines and bromoacetyl chloride according to already-reported protocols [9, 10]. It is important
to mention that the substituents were chosen based on the best antimycobacterial results
presented by the 2-(quinolin-4-yloxy)acetamides, including published [9, 10] and unpublished
data. The second step was accomplished through a second-order nucleophilic substitution
reaction (SN2). The 1H-benzo[d]imidazoles 6a–m were obtained from reaction of 2-
mercaptobenzoimidazole (4) and bromoacetamides 5a–m using potassium carbonate (K2CO3) as
base, according to a previously described method [15]. The reactants were stirred for 4 h at 40 °C
leading to products with 62–98% yields (Scheme 1). On the other hand, the synthesis of 3,4-
dihydroquinazolin-4-ones 9a–z was accomplished by reaction of 2-mercaptoquinazolin-4(3H)-
one (7) and bromoacetamides 5 and 8 in the presence of diisopropylethylamine (DIPEA) using
dimethylformamide (DMF) as solvent. The reaction mixtures were stirred for 16 h at 0–25 °C to
afford the desired products 9a–z with 35−89% yields (Scheme 2). Spectroscopic and
spectrometric data were obtained in agreement with the proposed structures (Supporting
Information).
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Comp. 5,6
R1
R2 Yield (%)
2-MeO-C6H4
4-pentyl-C6H4
4-heptyl-C6H4
2-naphthyl
H
H
H
H
83
96
88
88
a
b
c
d
H
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e
cyclohexyl
2-methylcyclohexyl
3-methylcyclohexyl
4-methylcyclohexyl
trans-4-methylcyclohexyl
Bn
H
H
H
H
H
H
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98
89
78
76
73
f
g
h
i
j
k
H
65
l
H
62
m
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Scheme 1. Reagents and conditions: i = K2CO3, CH3CN, 40 °C, 4 h.

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Comp. 5,8,9
R1
R2 Yield (%)
Ph
H
H
H
H
H
H
H
H
H
H
H
86
81
69
68
81
89
86
50
75
88
81
a
b
c
d
e
f
g
h
i
4-MeO-C6H4
4-Me-C6H4
4-F-C6H4
4-Cl-C6H4
4-Br-C6H4
4-I-C6H4
4-O2N-C6H4
4-propyl-C6H4
4-pentyl-C6H4
2-naphthyl
j
k
H
H
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80
l
m
cyclohexyl
cyclohexyl
H
Me
H
H
H
H
H
H
H
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41
n
o
p
q
r
s
t
u
v
2-methylcyclohexyl
3-methylcyclohexyl
4-methylcyclohexyl
trans-4-methylcyclohexyl
cyclopentyl
cycloheptyl
Bn
H
H
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w
59
x
H
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72
y
z
CH2-(CH2)3-CH2
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Scheme 2. Reagents and conditions: i = DIPEA, DMF, 0–25 °C, 16 h.

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The synthesized compounds 6a–m and 9a–z were tested in whole-cell assay against M.
tuberculosis H37Rv, using the first-line drug isoniazid as reference [16, 17]. The 1H-
benzo[d]imidazoles 6a–m presented only moderate activity against the bacillus under the tested
conditions (Table 1). Considering the data shown, one can conclude that cycloalkyl substituents
exhibited better activity than did aromatic groups. In addition, the extent of the chain using a
carbon with cycloalkyl or aromatic substituents did not lead to higher activity. The most active
compound 6j inhibited the Mtb growth with an MIC of 16.5 µM (5 µg/mL). This compound
shows the 4-methyl group in a trans position relative to the amidic nitrogen attached to the
cyclohexane ring. Interestingly, when a mixture of cis and trans isomers was used the activity of
the compound 6i was nearly 2.5-fold less than 1H-benzo[d]imidazole 6j. This finding
demonstrates a possible stereochemical preference for increasing antimycobacterial activity of
this class of compounds. Changing the methyl group to the 2- or 3-position of the cyclohexane
ring in the compounds 6g and 6h did not maintain the activity, with MIC values >33 µM (>10
µg/mL). It is noteworthy that the lipophilicities of 1H-benzo[d]imidazoles 6g–j are the same,
with CLogP of 3.81, denoting that structural factors, rather than the physicochemical properties,
appear to be linked to the activity of the molecules. The importance of the 4-methyl group
attached to the cyclohexane ring can be inferred by the result obtained for compounds 6f–h,
which were ineffective at the highest tested concentration (MIC >31.3 µM; >10 µg/mL). Finally,
chain extension using a methylene group in the compounds 6k–m and the presence of aromatic
substituents on the molecules 6a–e did not result in better activities when compared to
cyclohexyl derivatives (Table 1).
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Table 1. ClogP values and in vitro activities of the 1H-benzo[d]imidazoles 6a–m against M.
tuberculosis H37Rv.
MIC H37Rv
Entry ClogPa
µM
µg/mL
>10
>25
>25
>25
>10
>10
>10
>10
12.5
5
2.73 >31.9
5.86 >70.7
3.85 >65.5
4.42 >75.0
4.81 >29.6
3.29 >31.3
3.81 >33.0
3.81 >33.0
6a
6b
6c
6d
6e
6f
6g
6h
6i
3.81
3.81
41.2
16.5
6j
6k
6l
3.14 >33.6
3.91 82.4
>10
25
4.22 >31.5
-0.67
2.9
>10
0.3
6m
INH
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aClogP calculated by ChemBioDraw Ultra, version 13.0.0.3015. INH, isoniazid.
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In the second round of obtaining new drug candidates to treat tuberculosis, the antimycobacterial
activity of 3,4-dihydroquinazolin-4-ones 9a–z against M. tuberculosis H37Rv strain was
determined (Table 2). Once more, the cycloalkyl substituents presented the best inhibition
activities on bacillus growth, with MICs in the submicromolar range. Substituents containing
aromatic groups, which had produced highly potent compounds when present in the 2-(quinolin-
4-yloxy)acetamides, led to products with moderate or no activity at the highest concentrations
assayed. As expected, ClogP values of the 3,4-dihydroquinazolin-4-ones 9 were reduced when
compared to the analogs containing the quinoline scaffold. ClogP values were obtained ranging
from 1.38 to 4.63 for the synthesized compounds (Table 2). Variation using electron-donating,
electron-withdrawing or alkyl groups attached at the 4-position of the phenyl group in molecules
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9a–j yielded structures with moderate or no activity against Mtb. Increasing the molecular
volume by using a 2-naphthyl group in the 3,4-dihydroquinazolin-4-one 9k also failed to produce
satisfactory results (MIC >27.7 µM; >10 µg/mL). Afterwards, the importance of the planarity of
the naphthyl group was evaluated by the insertion of the tetrahydronaphthyl groups in the 9l and
9m structures. Whereas the stereoisomer of S configuration (9l) exhibited an MIC >27.4 µM
(>10 µg/mL), the R isomer (9m) was able to inhibit the bacillus growth with an MIC of 17.1 µM
(6.3 µg/mL). The apparent stereospecificity of the molecules will be rationalized when
subsequent studies reveal the molecular target responsible for the antimycobacterial activity.
This moderate MIC value with tetrahydronaphthyl group in 9m prompted us to investigate its
molecular simplification by the removal of the phenyl group and evaluation of the cyclohexyl
group. Indeed, 3,4-dihydroquinazolin-4-one 9n exhibited an MIC of 0.97 µM (0.31 µg/mL),
which was approximately 18-fold more potent than tetrahydronaphthyl-containing compound
9m. Moreover, this compound was almost 3-fold more potent than isoniazid drug (MIC = 2.9
µM; 0.3 µg/mL). These findings corroborated data already described in the literature [18].
Interestingly, the secondary amide seems to be crucial for the activity of the 3,4-
dihydroquinazolin-4-ones, as substitution of hydrogen for a methyl abolished completely the
antimycobacterial activity of compound 9o (MIC >75.4 µM; >25 µg/mL). Amidic hydrogen may
be involved in hydrogen bond(s) with a putative molecular target, stabilizing the protein-ligand
complex, or may be responsible for the correct conformation of the structure through
intramolecular stabilization. Following SAR evaluation, the presence of methyl at the 2-position
of the cyclohexyl ring did not significantly alter the activity of compounds, as 3,4-
dihydroquinazolin-4-one 9p showed an MIC of 0.94 µM (0.31 µg/mL). By contrast, 3-methyl-,
4-methyl-, and trans-4-methylcyclohexyl substituents yielded molecules with an MIC of 0.24

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µM (0.08 µg/ml), a potency increase of more than 4-fold compared to the non-substituted 9n.
Reduction in molecular volume by using a cyclopentyl group (9t) reduced the antitubercular
potency. Cyclopentyl-substituted 9t exhibited an MIC of 2.07 µM (0.63 µg/mL), which was 2.1-
fold less active than cyclohexyl-based compound 9n. The use of a cycloheptyl substituent again
yielded a structure with an MIC in the submicromolar range. 3,4-Dihydroquinazolin-4-one 9u
presented an MIC of 0.48 µM (0.16 µg/mL). In the same manner as for the 1H-
benzo[d]imidazoles series, the 3,4-dihydroquinazolin-4-one side chain was extended with an
additional methylene. First, using a benzyl group (9v) the MIC obtained was 4.8 µM (1.6
µg/mL), which was more promising than phenyl-substituted 9a. By contrast, the presence of
methylene separating the amidic nitrogen from the cyclohexyl ring did not alter the potency of
compound 9w (MIC = 0.94 µM; 0.31 µg/mL) compared to 9n (MIC = 0.97 µM; 0.31 µg/mL).
Another interesting observation was that the presence of an additional methyl group creating
stereogenic centers abolished the activity of the 3,4-dihydroquinazolin-4-ones 9x and 9y (MICs
>28.9 µM; >10 µg/mL). Finally, piperidinyl-containing compound 9z was devoid of activity at
the evaluated concentration (MIC >33.0 µM; >10 µg/mL), evidencing, once more, the necessity
of the secondary amide for the potent activity of the 3,4-dihydroquinazolin-4-ones.
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Table 2. ClogP values and in vitro activities of the 3,4-dihydroquinazolin-4-ones 9a-z against M.
tuberculosis H37Rv and clinical resistant isolates.
MIC H37Rv
MIC CDCT10
MIC CDCT16
MIC CDCT27
MIC CDCT28
Entry ClogPa
µM
µg/mL
>10
>10
>10
>10
10
µM
µg/mL
µM
µg/mL
µM
µg/mL
µM
µg/mL
2.02 >32.1
2.09 >29.3
2.52 >30.7
2.42 >30.4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9a
9b
9c
9d
9e
2.99
28.9
3.14 >25.6
3.40 >22.9
2.31 >28.1
3.57 >28.3
>10
>10
>10
>10
25
9f
9g
9h
9i
4.63
26.2
9j
3.19 >27.7
2.58 >27.4
17.1
0.97
2.27 >75.4
>10
>10
6.3
0.31 0.50
>25
9k
9l
2.58
2.07
-
0.31
-
9m
9n
9o
9p
9q
9r
0.16
-
0.97
-
0.25
-
0.08
-
0.5
-
0.16
-
-
2.58
2.58
2.58
2.58
1.51
2.62
1.92
2.68
0.94
0.24
0.24
0.24
2.07
0.48
4.8
0.31 0.95
0.08 0.94
0.08 0.24
0.08 0.12
0.63
0.16 0.48
1.6
0.31 0.93
0.31
0.31
0.08
0.04
-
3.9
0.9
0.48
0.24
-
1.3
0.31
0.16
0.08
-
0.96
0.24
0.12
0.12
-
0.31
0.08
0.04
0.04
-
1.9
0.93
0.24
0.12
-
0.63
0.31
0.08
0.04
-
9s
9t
-
0.16
-
0.93
-
0.31
-
0.24
-
0.08
-
0.48
-
0.16
-
9u
9v
9w
9x
9y
9z
-
0.94
0.31
-
-
0.93
0.31
-
-
0.48
-
0.16
-
0.93
-
0.31
-
2.99 >28.9
2.99 >28.9
1.38 >33.0
>10
>10
>10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-0.67
2.9
0.3 45.6
6.3
>729.2 >100
182.3
25
2.84
0.39
INH
229
aClogP calculated by ChemBioDraw Ultra, version 13.0.0.3015. INH, isoniazid.
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3,4-Dihydroquinazolin-4-ones with MIC values lower than 1 µM (9n, 9p–s, 9u and 9w) were
selected for further evaluation of their inhibitory activity against a panel of clinical isolate strains
(Table 2). The CDCT10 and CDCT16 strains are described as multidrug-resistant clinical

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isolates. CDCT10 presents resistance to drugs such as isoniazid, rifampin and ethambutol,
whereas CDCT16 strain exhibits resistance to isoniazid, rifampin, ethambutol and streptomycin.
Targeted sequencing from CDCT10 strain has revealed mutations in the rpoB and katG genes.
Also using targeted sequencing, mutations in the rpoB, katG and inhA regulatory region C(-15)T
were observed for CDCT16 strain. Additionally, CDCT27 was also evaluated; this drug-resistant
strain shows resistance to drugs such as isoniazid and ethambutol. CDCT27 has displayed
mutations in the katG gene. Finally, clinical isolate CDCT28 does not present resistance to the
first-line drugs, and targeted sequencing has also presented mutation in the rpoB gene. Notably,
the selected compounds exhibited identical activity or were even more potent against CDCT10,
CDCT27, and CDCT28 strains than against M. tuberculosis H37Rv strain (Table 2). By contrast,
3,4-dihydroquinazolin-4-ones 9p–r and 9u increased MIC values when assayed against CDCT16
strain. Only compounds 9n and 9w did not alter MIC values against this strain compared to those
presented for M. tuberculosis H37Rv. Although these results may suggest the participation of the
inhA gene product in the activity elicited by the compounds, the mutations in the clinical isolate
strains were obtained by target sequencing, and alterations elsewhere in the genome cannot be
excluded. Thus, inferences about the mechanism of action of the compounds based on
modifications in MIC values for these strains should be made with caution, and further studies
are needed to clarify this point. It is noteworthy that this class of compounds has been described
to target the membrane-bound type-II NADH dehydrogenase NdhA based on whole-genome
sequencing of resistant strains [19].
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Cellular viability was carried out after incubation with the test compounds using the neutral
red uptake assay [20] and MTT method [10] (Table 3). Exposing the HepG2, HaCat, and Vero
cell lineages to 3,4-dihydroquinazolin-4-ones 9n, 9p–s, 9u and 9w for 72 h did not significantly

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affect the cell viability [21]. The assayed concentration was at least 32 times higher than the
MICs of the synthesized compounds against M. tuberculosis H37Rv. These results suggest a
possible low toxicity of the compounds to mammalian cells and a likely high degree of
selectivity for Mtb.
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Table 3. Percentage of cell viability of HepG2, HaCat, and Vero cell lineages after exposition to
3,4-dihydroquinazolin-4-ones 9n, 9p–s, 9u and 9w.
% of cell viability ± SEMa
HepG2
HaCat
Vero
Neutral
Entry
Neutral
Neutral
MTT
MTT
MTT
red
red
red
86 ± 6
95 ± 4
100 ± 8
100 ± 8
94 ± 9
95 ± 8
97 ± 2
97 ± 3
90 ± 12
92 ± 4
9n
9p
9q
9r
94 ± 5 101 ± 3
99 ± 12 102 ± 7 95 ± 12
89 ± 6
89 ± 3
83 ± 7
95 ± 3
96 ± 3
97 ± 2
88 ± 7
94 ± 3
97 ± 4
88 ± 6
88 ± 3
89 ± 7
84 ± 4
88 ± 12
91 ± 4
89 ± 5
95 ± 5
95 ± 2
93 ± 6
94 ± 5
97 ± 3
9s
99 ± 3 105 ± 5 100 ± 10
79 ± 3 97 ± 3 98 ± 8
9u
9w
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aData are expressed as the mean cell viability ± SEM for each compound, tested at 10 µg/mL.
Results were obtained from mean values of triplicates of three independent experiments.
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The promising and selective activity showed by the compounds prompted us to investigate other
in vivo toxicological parameters such as cardiotoxicity, neurotoxicity and morphological
alterations, using zebrafish (Danio rerio) models [22–24]. In particular, there are possible
cardiac side effects under study attributed to the new antitubercular drug bedaquiline [25].
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Zebrafish embryos at 2 dpf (days post-fertilization) exposed to single dose of bedaquiline (72
µM) have presented significant alterations of the cardiac functions such as heart rate, stroke
volume, cardiac output and fractional shortening [26]. Therefore, cardiac risk assessment for
novel compounds should ideally be evaluated in the early drug discovery stages and the method
using zebrafish has proved to be a suitable protocol [27]. The 3,4-dihydroquinazolin-4-ones 9n,
9p–s, 9u and 9w were evaluated for the heartbeat rate in viable embryos at 2 and 5 dpf using
concentrations of 3, 15 and 20 µM (Table 4). Except for compounds 9p and 9r, the molecules
did not change the heartbeat rates in tested animals at 2 dpf at 3 µM concentration. By contrast,
at the highest dose assayed (20 µM) six of the seven structures tested induced changes in the
heartbeat rate of animals at 2 dpf. Notably, 3,4-dihydroquinazolin-4-one 9q did not alter
heartbeat rates in any of the concentrations tested. Considering animals at 5 dpf, only compound
9u was able significantly to alter the rate of heartbeats. This finding indicates an apparent cardiac
safety of compounds 9n, 9p–s, and 9w in animals at 5 dpf.
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Table 4. Cardiac evaluation of 3,4-dihydroquinazolin-4-ones 9n, 9p–s, 9u and 9w in viable
embryos at 2 and 5 dpf (days post-fertilization).
Zebrafish heart rate (Mean ± SD/min) – Embryos 2 dpf
Control
1% DMSO
3 µM
15 µM
20 µM
Entry
141.6 ± 15.8 147.3 ± 16.1 149.5 ± 16.3 155.0 ± 14.8**
141.6 ± 15.8 147.3 ± 16.1 156.3 ± 14.8** 151.7 ± 14.2
155.7 ± 14.1**
153.9 ± 16.1*
152.2 ± 8.5
9n
9p
9q
9r
9s
146.3 ± 9.3 148.8 ± 9.6 151.2 ± 7.1
151.7 ± 9.4
128.7 ± 21.7 134.4 ± 11.7 122.3 ± 9.6## 127.2 ± 6.6 146.4 ± 10.9**** / ##
128.7 ± 21.7 134.4 ± 11.7 135.5 ± 7.3
134.0 ± 6.1 150.2 ± 8.2**** / ###

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138.3 ± 10.7 139.9 ± 12.4 139.6 ± 15.0 136.5 ± 16.4
148.4 ± 10.8*
9u
138.3 ± 10.7 139.9 ± 12.4 140.7 ± 12.2 143.2 ± 14.1 152.6 ± 8.8**** / ###
9w
Zebrafish heart rate (Mean ± SD/min) – Embryos 5 dpf
Control
1% DMSO
3 µM
15 µM
20 µM
Entry
158.2 ± 12.7 157.4 ± 9.7 162.8 ± 10.5
156.7 ± 16.3 157.4 ± 9.7 162.6 ± 8.0
155.9 ± 12.5 159.1 ± 9.7 157.2 ±15.3
163.0 ± 8.4
163.4 ± 7.2
157.4 ± 9.3
158.4 ± 9.9
160.6 ± 12.3
160.9 ± 11.4
152.2 ± 10.6
153.8 ± 13.2
153. 8 ± 11.1
152.5 ± 11.0
9n
9p
9q
9r
148.1 ± 7.4 148.1 ± 11.3 147.7 ± 14.1 150.3 ± 13.5
148.1 ± 7.4 148.1 ± 11.3 149.2 ± 13.5 157.4 ± 18.6
153.2 ± 8.2 154.5 ± 6.8 160.1 ± 8.4* 160.4 ± 7.7*
9s
9u
9w
153.2 ± 8.2 154.5 ± 6.8 157.8 ± 9.0
158.9 ± 11.9
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*P < 0.5 compared with control group (Tukey post-test). **P < 0.01 compared with control group
(Tukey post-test). ****P < 0.0001 compared with control group (Tukey post-test). ##P < 0.01
compared with the 1% DMSO group (Tukey post-test). ###P < 0.001 compared with the 1%
DMSO group.
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Furthermore, the distance traveled by the animals was used as a parameter to evaluate
neurological impairment after exposure to the compounds. None of the evaluated compounds 9n,
9p–s, 9u, and 9w altered the locomotor activity of the animals (data not shown). Morphological
evaluation considered parameters such as body length, ocular distance, and surface area of the
eyes. Except for compound 9s which altered the body length of the larvae at 20 µM
concentration, none of the compounds in any of the tested concentrations (3, 15 and 20 µM)
shown modifications on the morphological parameters (data not shown). Finally, the larvae

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survival rate was not altered by exposure to 3,4-dihydroquinazolin-4-ones 9n, 9p–s, 9u, and 9w
in the experimental conditions used (data not shown).
Aiming at further in vivo effectiveness trials in rodents and pharmaceutical formulation studies
for oral administration, the stability of 3,4-dihydroquinazolin-4-ones 9n, 9p–s, 9u, and 9w in
aqueous medium was determined (Supporting information). The experiments were carried out
using 10% DMSO as co-solvent in PBS at 25 °C and 37 °C for up to 48 h. At room temperature
(25 °C), only compound 9s showed chemical instability. After 24 h, only 26% of the 3,4-
dihydroquinazolin-4-one 9s could be recovered. The other products remained stable over 48 h.
Elevation of temperature to 37 °C appeared to be crucial for chemical instability of compound
9r. After 6 h of incubation less than 70% of 9r was recovered. Once more, the 3,4-
dihydroquinazolin-4-one 9s presented instability under the experimental conditions tested. In
contrast, 3,4-dihydroquinazolin-4-ones 9n, 9p–q, 9u, and 9w were stable in aqueous medium for
48 h at 37 ° C in the evaluated conditions.
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3. Conclusion
In summary, herein was shown the synthesis of new series of hybridized 1H-
benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones and their in vitro antitubercular activity.
The simplicity of the route, easily accessible reactants and reagents, reasonable yields and high
purity make the synthetic protocols attractive. In addition, the synthesized compounds showed
potent and selective activity against drug-sensitive and drug-resistant Mtb strains, with no
apparent cytotoxicity to mammalian cells. The submicromolar antitubercular activity elicited by
3,4-dihydroquinazolin-4-ones, coupled with a preliminary outcome of low risk of cardiotoxicity
and neurotoxicity, suggests that this class of compounds may furnish candidates for future
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development of novel anti-TB drugs. Considering the data described so far, 3,4-
dihydroquinazolin-4-ones 9q and 9w are considered the lead compounds of this series of
synthesized molecules. New structural modifications of the 3,4-dihydroquinazolin-4-ones and
pharmaceutical formulation studies are in progress and these data will be reported to the
scientific community soon.
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4. Experimental Section
4.1 Synthesis and structure: apparatus and analysis
The commercially available reactants and solvents were obtained from commercial suppliers
and were used without additional purification. The reactions were monitored by thin-layer
chromatography (TLC) with Merck TLC Silica gel 60 F254. The melting points were measured
using a Microquímica MQAPF-302 apparatus. H and 13C NMR spectra were acquired on a
1
Avance III HD Bruker spectrometer (Pontifical Catholic University of Rio Grande do Sul).
Chemical shifts (δ) were expressed in parts per million (ppm) relative to DMSO-d6, which was
used as the solvent, and to TMS, which was used as internal standard. High-resolution mass
spectra (HRMS) were obtained for all the compounds on an LTQ Orbitrap Discovery mass
spectrometer (Thermo Fisher Scientific, Bremer, Germany). This system combines an LTQ XL
linear ion-trap mass spectrometer and an Orbitrap mass analyzer. The analyses were performed
through the direct infusion of the sample in MeOH/H2O (1:1) with 0.1% formic acid (flow rate
10 µL/min) in a positive-ion mode using electrospray ionization (ESI). For elemental
composition, calculations used the specific tool included in the Qual Browser module of
Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. Compound purity was determined

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using an Äkta HPLC system (GE Healthcare® Life Sciences) equipped with a binary pump,
manual injector and UV detector. Unicorn 5.31 software (Build 743) was used for data
acquisition and processing. The HPLC conditions were as follows: RP column 5 µm Nucleodur
C-18 (250 × 4.6 mm); flow rate 1.5ꢀmL/min; UV detection 254 nm; 100% water (0.1% acetic
acid) was maintained from 0 to 7 min and was followed by a linear gradient from 100% water
(0.1% acetic acid) to 90% acetonitrile/methanol (1:1, v/v) from 7 to 15 min (15–30 min) and
subsequently returned to 100% water (0.1% acetic acid) in 5 min (30–35 min) and maintained for
an additional 10 min (35–45 min). All the evaluated compounds were ≥ 90% pure.
4.2 General procedure for the synthesis of 1H-benzo-[d]-imidazoles 6a-m
The synthesis of these compounds was adapted from previously described methodology
[15]. The appropriately substituted bromoacetamide (1 mmol) was added to a mixture of 1H-
benzimidazole-2-thiol (1 mmol), potassium carbonate (K2CO3, 2 mmol) in acetonitrile (10 mL).
The reaction mixture was stirred at 40 °C (oil bath) for 4 h. The precipitated solid was filtered
off, washed with chloroform (3 × 20 mL) and dried under reduced pressure to afford the products
in good purity.
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4.2.1. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(2-methoxyphenyl)acetamide (6a): Yield 83%;
m.p.: 127.5 – 129.3 °C; HPLC 93% (tR = 15.90); 1H NMR (400 MHz, DMSO-d6)
δ ppm 3.67 (s,
3 H), 4.22 (s, 2 H), 6.88 (td, J = 8.2, 1.3 Hz, 1 H), 6.97 (td, J=8.2, 1.3 Hz, 1 H), 7.02 (dd, J = 8.2,
13
1.3 Hz), 7.13-7.17 (m, 2 H), 7.48-7.50 (m, 2 H), 8.10 (d, J = 8.1, 1 H), 9.94 (s, 1 H). C NMR
(101 MHz, DMSO-d6)
δ ppm 35.4, 55.6, 111.0, 120.3, 121.5, 124.1, 127.3, 148.7, 150.0, 166.8;
FTMS (ESI) m/z 314.0954 [M+H]+; calcd for C16H15N3O2S: 314.0958.
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4.2.2. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(4-pentylphenyl)acetamide (6b): Yield 96%; m.p.:
1
103.5 – 104.7 °C; HPLC 94% (tR = 19.10); H NMR (400 MHz, DMSO-d6)
δ ppm 0.83 (t, J
=7.0 Hz, 3 H), 1.25 (m, 4 H), 1.51 (m, 2 H), 2.49 (m, 2 H), 4.21 (s, 2 H), 7.09-7.11 (m, 4 H),
7.43-7.48 (m, 4 H), 10.58 (s, 1H). 13C NMR (101 MHz, DMSO-d6)
δ
ppm 13.8, 21.9, 30.6, 30.7,

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34.4, 36.1, 109.4, 113.8, 118.9, 121.0, 128.4, 136.6, 137.3, 140.0, 150.5, 166.1; FTMS (ESI) m/z
354.1630 [M+H]+; calcd for C20H23N3OS: 354.1635.
4.2.3. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(4-heptylphenyl)acetamide (6c): Yield 88%; m.p.:
1
142.6 – 143.7 °C; HPLC 93% (tR = 19.68); H NMR (400 MHz, DMSO-d6)
δ ppm 0.84 (t, J =
6.8 Hz, 4 H), 1.23 (d, J = 6.1 Hz, 8 H), 1.51 (m, 2 H), 2.50 (m, 2 H), 4.13 (s, 2 H), 7.03-7.06 (m,
2 H), 7.10 (d, J = 8.6 Hz, 2 H), 7.42-7.44 (m, 2 H), 7.48 (d, J = 8.6 Hz, 2 H). 13C NMR (101
MHz, DMSO-d6)
δ ppm 13.8, 22.0, 28.4, 30.9, 31.2, 34.5, 36.1, 109.4, 113.8, 118.8, 120.2,
122.0, 128.4, 132.6, 136.8, 137.2, 141.3, 151.9, 166.7; FTMS (ESI) m/z 382.1977 [M+H]+; calcd
for C22H27N3OS: 382.1948.
4.2.4. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(naphthalen-2-yl)acetamide (6d): Yield 88%;
m.p.: 101.7 – 102.2 °C; HPLC 92% (tR = 16.61); 1H NMR (400 MHz, DMSO-d6)
δ ppm 4.33 (s,
2 H), 7.10-7.14 (m, 2 H), 7.37-7.43 (td, J = 7.5, 1.5 Hz, 1 H), 7.44-7.47 (m, 3 H), 7.56-7.59 (dd,
J = 8.8, 2.2 Hz, 1 H), 7.81 (t, J = 8.8, 2 H), 7.86 (d, J = 9.0 Hz, 1 H), 8.29 (d, J = 1.7 Hz, 1 H),
13
10.73 (s, 1 H). C NMR (101 MHz, DMSO-d6)
δ ppm 36.2, 115.1, 119.6, 121.4, 124.6, 126.4,
127.2, 127.4, 128.4, 129.7, 133.3, 136.4, 149.8, 166.4; FTMS (ESI) m/z 334.1005 [M+H]+; calcd
for C19H15N3OS: 334.1009.
4.2.5. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(2,3-dihydro-1H-inden-5-yl)acetamide (6e): Yield
92%; m.p.: 89.7 – 90.6 °C; HPLC 90% (tR = 16.71); 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.97
(q, J = 7.4, Hz, 2 H), 2.79 (dt, J = 10.7, 7.4 Hz, 4 H), 4.23 (s, 2 H), 7.09-7.13 (m, 3 H), 7.26 (dd,
J = 8.1, 1.5 Hz, 1 H), 7.42-7.46 (m, 2 H), 7.49 (s, 1 H) 10.40 (s, 1 H). 13C NMR (101 MHz,
DMSO-d6)
δ ppm 25.0, 31.7, 32.4, 36.1, 115.2, 117.1, 121.3, 124.1, 137.0, 138.6, 144.1, 149.9,
165.8; FTMS (ESI) m/z 324.1161 [M+H]+; calcd for C19H19N3OS: 324.1165.
4.2.6. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-cyclohexylacetamide (6f): Yield 96%; m.p.: 132.9
– 134.0 °C; HPLC 90% (tR = 16.59); 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.08-1.25 (m, 5 H,
3’-H, 4’-H and 5’H), 1.45-1.48 (m, 1 H, 5’-H), 1.59-1.69 (m, 4 H, 2’-H and 6’-H), 3.54 (m, 1 H,
1’-H), 3.87 (s, 2 H, 8-H), 6.98-7.02 (m, 2 H, 5-H and 6-H), 7.35-7.39 (m, 2 H, 4-H and 7-H),
8.87 (s, 1 H, H-N). 13C NMR (101 MHz, DMSO-d6)
δ ppm 23.9, 25.2, 31.9, 35.1 (9-C), 47.5 (1’-
C), 113.8 (4-C and 7-C), 120.1 (5-C and 6-C), 141.5 (3a-C and 7a-C), 152.1 (2-C), 167.3 (10-C);
FTMS (ESI) m/z 290.1314 [M+H]+; calcd for C15H19N3OS: 290.1322.
4.2.7. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(2-methylcyclohexyl)acetamide (6g): Yield 98%;
1
m.p.: 114.8 – 115.5 °C; HPLC 91% (tR = 16.97 and 17.44); H NMR (400 MHz, DMSO-d6)
δ
ppm 0.74-0.75 (d, J = 6.8 Hz, 3 H), 0.79-0.80 (d, J = 6.6 Hz, 3 H), 0.84-0.86 (m, 1H), 0.96-1.02
(m, 1 H), 1.12-1.40 (m, 7 H), 1.58 (d, J = 11.7 Hz, 2 H), 1.64-1.75 (m, 4 H), 3.18-3.26 (m, 2 H),
3.88-4.03 (m, 4 H), 7.03-7.05 (m, 2 H), 7.06-7.09 (m, 2 H), 7.38-7.42 (m, 4 H), 8.41 (d, J = 4.4
Hz, 1 H), 8.88 (d, J = 4.4 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 18.8, 24.9, 25.2,
28.9, 29.7, 32.6, 33.7, 33.7, 34.9, 35.1, 37.1, 48.8, 53.7, 113.7, 120.3, 120.8, 140.2, 150.7, 166.8,
168.1; FTMS (ESI) m/z 304.1467 [M+H]+; calcd for C16H21N3OS: 304.1478.
4.2.8. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(3-methylcyclohexyl)acetamide (6h): Yield 89%;
1
m.p.: 128.7 – 129.4 °C; HPLC 90% (tR = 17.16 and 17.48); H NMR (400 MHz, DMSO-d6)
δ
ppm 0.70-0.98 (m, 5 H), 1.01-1.27 (m, 3 H), 1.37-1.58 (m, 4 H), 1.65 (d, J = 9.5 Hz, 2 H), 1.68-

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1.77 (m, 2 H), 3.47-3.57 (m, 1 H), 3.98 (s, 2 H), 7.09-7.13 (m, 2 H) 7.41-7.46 (m, 2 H), 8.24 (d,
13
J = 4.4 Hz, 1 H), 8.44 (d, J = 4.4 Hz, 1 H). C NMR (101 MHz, DMSO-d6)
δ ppm 20.0, 21.6,
22.2, 24.3, 26.4, 29.8, 31.2, 31.8, 33.4, 33.8, 35.0, 35.3, 38.1, 41.0, 44.5, 48.2, 113.7, 121.2,
150.0, 166.3, 166.9; FTMS (ESI) m/z 304.1467 [M+H]+; calcd for C16H21N3OS: 304.1478.
4.2.9. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(4-methylcyclohexyl)acetamide (6i): Yield 78%;
m.p.: 98.2 – 99.0 °C; HPLC 96% (tR = 17.12 and 17.41); 1H NMR (400 MHz, DMSO-d6)
δ ppm
0.66 (d, J = 6.1 Hz, 3 Hcis), 0.84 (d, J = 6.4 Hz, 3 Htrans), 0.88-0.97 (m, 2 Htrans), 1.08-1.17 (m, 4
Hcis/trans), 1.26-1.44 (m, 7 Hcis/trans), 1.53-1.58 (m, 2 Hcis), 1.63 (dd, J = 12.5, 2.7 Hz, 2 Htrans), 1.77
(dd, J = 12.5, 2.7 Hz, 2 Htrans), 3.3-3.4 (m, 2 Hcis/trans), 3.8 (s, 2 Hcis), 3.9 (s, 2 Htrans), 6.96-7.05
(m, 2 Hcis), 7.02-7.05 (m, 2 Htrans), 7.35-7.41 (m, 4 Hcis/trans), 8.68 (s, 1 Htrans), 9.13 (d, J = 4.4 Hz,
13
1 Hcis). C NMR (101 MHz, DMSO-d6)
δ
ppm 21.4cis, 22.0trans, 29.0cis, 29.1cis, 30.8cis, 31.3trans
,
,
32.0trans, 33.3trans, 34.7cis, 35.1trans, 44.2cis, 48.1trans, 113.7trans, 113.9cis, 119.6cis, 120.3trans
141.0trans, 142.2cis, 151.6trans, 153.1cis, 167.0trans, 168.0cis; FTMS (ESI) m/z 304.1467 [M+H]+;
calcd for C16H21N3OS: 304.1478.
4.2.10. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(trans-4-methylcyclohexyl)acetamide (6j): Yield
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76%; m.p.: 144.5 – 145.2 °C; HPLC 91% (tR = 17.17); H NMR (400 MHz, DMSO-d6)
δ ppm
0.84 (d, J = 6.4 Hz, 3 H), 0.88-0.98 (m, 2 H), 1.10-1.18 (m, 2 H), 1.22-1.39 (m, 1 H), 1.63 (d, J =
12.7 Hz, 2 H), 1.76 (d, J = 9.5 Hz, 2 H), 3.43 (m, 1 H), 3.92 (s, 2 H), 7.05-7.07 (m, 2 H), 7.40-
7.41 (m, 2 H), 8.55 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 22.0, 31.3, 32.0, 33.3, 35.2,
48.1, 113.8, 120.5, 120.6, 140.7, 151.2, 166.9; FTMS (ESI) m/z 304.1496 [M+H]+; calcd for
C16H21N3OS: 304.1478.
4.2.11. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-benzylacetamide (6k): Yield 73%; m.p.: 175.5 –
177.0 °C; HPLC 91% (tR = 15.98); 1H NMR (400 MHz, DMSO-d6)
δ
ppm 4.10 (s, 2 H), 4.32 (d,
J = 6.1 Hz, 2 H), 7.12-7.19 (m, 2 H), 7.21-7.25 (m, 5 H), 7.44-7.46 (m, 2 H), 8.82 (t, J = 5.4 Hz,
1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 34.9, 42.4, 121.3, 126.6, 127.0, 128.1, 138.9,
149.7, 167.3; FTMS (ESI) m/z 298.1029 [M+H]+; calcd for C16H15N3OS: 298.1009.
4.2.12. 2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(cyclohexylmethyl)acetamide (6l): Yield 65%;
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m.p.: 166.7 – 167.1 °C; HPLC 90% (tR = 17.21); H NMR (400 MHz, DMSO-d6)
δ ppm 0.77-
0.88 (m, 2 H), 1.10-1.14 (m, 4 H), 1.34-1.35 (m, 1 H), 1.55-1.69 (m, 6 H), 2.94 (ddd, J = 17.1,
10.8, 6.6 Hz, 2 H), 4.00 (s, 2 H), 7.09-7,14 (m, 2 H), 7.41-7.45 (m, 2 H), 8.32 (s, 1 H). 13C NMR
(101 MHz, DMSO-d6)
δ ppm 25.3, 25.9, 30.1, 34.9, 37.3, 45.1, 113.7, 121.2, 139.6, 149.9,
167.3; FTMS (ESI) m/z 304.1474 [M+H]+; calcd for C16H21N3OS: 304.1478.
4.2.13. (S)-2-((1H-Benzo[d]imidazol-2-yl)thio)-N-(1-cyclohexylethyl)acetamide (6m): Yield
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62%; m.p.: 139.7 – 140.2 °C; HPLC 92% (tR = 17.59); H NMR (400 MHz, DMSO-d6)
δ ppm
0.81-1.10 (m, 7 H), 1.22-1.26 (m, 1 H), 1.52-1.60 (m, 5 H), 3.55-3.60 (m, 1 H), 3.82-3.99 (m, 2
H), 7.02 (dd, J = 5.9, 3.2 Hz, 2 H), 7.37 (dd, J = 6.1, 3.2 Hz, 2 H), 8.56 (s, 1 H). 13C NMR (101
MHz, DMSO-d6)
δ ppm 17.4, 25.7, 25.8, 28.2, 28.6, 35.0, 42.3, 48.9, 113.7, 120.3, 141.0, 151.5,
167.3; FTMS (ESI) m/z 318.1621 [M+H]+; calcd for C17H23N3OS: 318.1635.
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4.3 General procedure for the synthesis of 3,4-dihydroquinazolin-4-ones 9a-z

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2-Mercaptoquinazolin-4(3H)-one (1 mmol) was diluted in N,N-dimethylformamide (5 mL)
with the addition of diisopropylethylamine (DIPEA, 1.1 mmol) under an argon atmosphere at
0ꢀ°C. The solution was stirred for 5 min and bromacetamine (1.1 mmol) was added. After 30
min, the temperature was elevated to 25 °C. The reaction mixture was stirred for additional 18 h
and the precipitated product filtered off, washed with water and dried under vacuum. The
products were obtained in satisfactory purity without the need for additional purification.
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4.3.1. 2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-phenylacetamide (9a): Yield 86%; m.p.:
233.5 – 234.3 °C; HPLC 99% (tR = 16.51 min); 1H NMR (400 MHz, DMSO-d6)
δ ppm 4.17 (s,
2 H, 9-H), 7.04 (t, J = 7.3 Hz, 1 H, 4’-H), 7.30 (t, J = 7.3 Hz, 2 H, 3’-H), 7.40 (td, J = 8.1, 1.5
Hz, 1 H, 6-H), 7.47 (d, J = 8.1 Hz, 1 H, 8-H), 7.59 (d, J = 7.6 Hz, 2 H, 2’-H), 7.73 (td, J = 7.6,
1.6 Hz, 1 H, 7-H), 8.02 (dd, J = 8.1, 1Hz, 1 H, 5-H), 10.34 (s, 1 H, NH), 12.68 (s, 1 H, NH, 3-H).
13C NMR (101 MHz, DMSO-d6)
δ ppm 35.1 (9-C), 119.1 (2’-C), 119.9 (4a-C), 123.4 (4’-C),
125.6 (5-C), 125.9(8-C), 128.7 (3’-C), 134.6 (7-C), 138.8 (1’-C), 148.2 (8a-C), 155.2 (2-C),
161.0 (4-C), 165.8 (10-C); FTMS (ESI) m/z 312.0797 [M+H]+; calcd for C16H13N3O2S:
312.0801.
4.3.2. N-(4-Methoxyphenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9b): Yield
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81%; m.p.: 214.1 – 216.9 °C; HPLC 95% (tR = 16.34 min); H NMR (400 MHz, DMSO-d6)
δ
ppm 3.70 (s, 3 H), 4.14 (s, 2 H), 6.87 (d, J = 9.0 Hz, 2 H), 7.38-7.42 (m, 1 H), 7.47-7.50 (m, 3
H), 7.74 (td, J = 8.3, 1.5 Hz, 1 H), 8.02 (dd, J = 7.8, 1.5 Hz, 1 H), 10.20 (s, 1 H), 12.66 (s, 1 H).
13C NMR (101 MHz, DMSO-d6)
δ ppm 35.0, 55.1, 113.9, 119.9, 120.7, 125.7, 125.8, 126.0,
132.0, 134.6, 148.2, 155.3, 155.3, 161.0, 165.3; FTMS (ESI) m/z 342.0873 [M+H]+; calcd for
C17H15N3O3S: 342.0907.
4.3.3. 2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(p-tolyl)acetamide (9c): Yield 69%; m.p.:
247.9 – 250.1 °C; HPLC 96% (tR = 17.00 min); 1H NMR (400 MHz, DMSO-d6)
δ ppm 2.23 (s, 3
H), 4.15 (s, 2 H), 7.10 (d, J = 8.1 Hz, 2 H), 7.40 (td, J = 8.0, 1.5 Hz, 1 H), 7.47 (d, J = 8.6 Hz, 3
H), 7.73 (td, J = 8.4, 1.5 Hz, 1 H), 8.02 (dd, J = 7.9, 1.5 Hz, 1 H), 10.35 (s, 1 H), 12.67 (s, 1 H).
13C NMR (101 MHz, DMSO-d6)
δ ppm 20.4, 35.1, 119.1, 119.9, 125.7, 125.8, 126.0, 129.1,
132.3, 134.6, 136.4, 148.2, 155.2, 161.0, 165.6; FTMS (ESI) m/z 326.0927 [M+H]+; calcd for
C17H15N3O2S: 326.0958.
4.3.4. N-(4-Fluorophenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9d): Yield
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68%; m.p.: 243.6 – 244.8 °C; HPLC 97% (tR = 16.62 min); H NMR (400 MHz, DMSO-d6)
δ
ppm 4.16 (s, 2 H), 7.11-7.17 (m, 2 H), 7.40 (td, J = 8.1, 1.5 Hz, 1 H), 7.46 (d, J = 8.1 Hz, 1 H),
7.58-7.63 (m, 2 H), 7.73 (td, J = 7.6, 1.6 Hz, 1 H), 8.02 (dd, J = 8.1, 1.5 Hz, 1 H), 10.40 (s, 1 H),

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12.67 (s, 1 H). C NMR (101 MHz, DMSO-d6)
δ
ppm 35.0, 115.3 (d, JCF = 22.0 Hz), 119.9,
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120.9 (d, JCF = 8.0 Hz), 125.7, 125.8, 126.0, 134.6, 135.2 (d, JCF = 2.9 Hz), 148.2, 155.2,
156.8 (d, JCF = 239.8 Hz), 161.0, 165.8; FTMS (ESI) m/z 330.0674 [M+H]+; calcd for
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C16H12FN3O2S: 330.0707.
4.3.5. N-(4-Chlorophenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9e): Yield
81%; m.p.: 262.7 – 264.1 °C; HPLC 93% (tR = 17.21 min); H NMR (400 MHz, DMSO-d6)
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δ
ppm 4.18 (s, 2 H), 7.37 (d, J = 8.0, 2 H), 7.41 (td, J = 8.1, 1.1 Hz, 1 H), 7.46 (d, J = 8 Hz,1 H),
7.63 (d, J = 8.0, 2 H), 7.75 (td, J = 8.0, 1.1 Hz, 1 H), 8.03 (dd, J = 7.8, 1.5 Hz, 1 H), 10.49 (s, 1
H), 12.68 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 35.1, 119.9, 120.7, 125.7, 126.0,
127.0, 128.7, 134.6, 137.9, 140.4, 148.1, 155.3, 161.1, 166.0; FTMS (ESI) m/z 346.0403
[M+H]+; calcd for C16H13ClN3O2S: 346.0412.
4.3.6. N-(4-bromophenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9f): Yield 89%;
m.p.: 236.2 – 238.1 °C; HPLC 92% (tR = 17.26 min); H NMR (400 MHz, DMSO-d6)
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δ ppm
4.17 (s, 2 H), 7.40 (td, J = 8.1, 1.5 Hz, 1 H), 7.44 (d, J = 8.1 Hz, 1 H), 7.48 (d, J = 8.8 Hz, 2 H),
7.57 (d, J = 8.8 Hz, 2 H), 7.73 (td, J = 7.6, 1.6 Hz, 1 H), 8.02 (dd, J = 7.8, 1.5 Hz, 1 H), 10.48 (s,
1 H), 12.67 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 35.1, 114.9, 119.8, 121.0, 125.6,
126.0, 131.5, 134.6, 138.2, 148.0, 155.2, 161.0, 166.0; FTMS (ESI) m/z 391.9843 [M+H]+; calcd
for C16H12BrN3O2S: 391.9886.
4.3.7. N-(4-Iodophenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9g): Yield 86%;
m.p.: 233.5 – 235.5 °C; HPLC 91% (tR = 17.44 min); H NMR (400 MHz, DMSO-d6)
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δ ppm
4.17 (s, 2 H), 7.39-7.47 (m, 4 H), 7.64 (d, J = 7.6 Hz, 2 H), 7.74 (td, J = 8.5, 1.5 Hz, 1 H), 8.03
(dd, J = 8.1, 1.5 Hz,1 H), 10.36 (s, 1 H), 12.58 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ
ppm
34.9, 86.6, 119.8, 121.3, 125.5, 125.8, 134.4, 137.2, 138.5, 147.9, 155.1, 160.9, 165.9.; FTMS
(ESI) m/z 437.9723 [M+H]+; calcd forC16H12IN3O2S: 437.9768.
4.3.8. N-(4-Nitrophenyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9h): Yield
50%; m.p.: 235.8 – 238.1°C; HPLC 95% (tR = 16.75 min); H NMR (400 MHz, DMSO-d6)
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δ
ppm 4.26 (s, 2 H), 7.40-7.44 (m, 2 H), 7.74 (td, J = 7.6, 1.6 Hz, 1 H), 7.88 (d, J = 9.7 Hz, 2 H),
8.04 (dd, J = 7.8, 1.5 Hz, 1 H), 8.25 (d, J = 9.7 Hz, 2 H), 11.00 (s, 1 H), 12.73 (s, 1 H). 13C NMR
(101 MHz, DMSO-d6)
δ ppm 35.3, 118.8, 119.9, 125.0, 125.7, 126.1, 134.6, 142.3, 145.0, 148.1,
155.1, 161.0, 167.0; FTMS (ESI) m/z 357.0617 [M+H]+; calcd for C16H12N4O4S: 357.0652.
4.3.9. 2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(4-propylphenyl)acetamide (9i): Yield 75%;
m.p.: 209.8 – 212.1 °C; HPLC 95% (tR = 17.83 min); H NMR (400 MHz, DMSO-d6)
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δ ppm
0.87 (t, J = 8.1 Hz, 3 H), 1.51-1.60 (m, 2 H), 2.47-2.52 (m, 2 H), 4.19 (s, 2 H), 7.13 (d, J = 7.6
Hz, 2 H), 7.42 (t, J = 7.6 Hz, 1 H), 7.51 (d, J = 6.8 Hz, 3 H), 7.76 (t, J = 7.6 Hz, 1 H), 8.05 (d, J
= 7.8 Hz, 1 H), 10.29 (s, 1 H), 12.70 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 13.5, 24.0,
35.1, 36.6, 119.2, 119.9, 125.7, 125.8, 126.0, 128.5, 134.6, 136.6, 137.2, 148.2, 155.3, 161.0,
165.6; FTMS (ESI) m/z 354.1236 [M+H]+; calcd for C19H19N3O2S: 354.1271.
4.3.10. 2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(4-pentylphenyl)acetamide (9j): Yield
88%; m.p.: 218.2 – 219.6 °C; HPLC 94% (tR = 18.52 min); H NMR (400 MHz, DMSO-d6)
ppm 0.85 (t, J = 8.0 Hz, 3 H), 1.20-1.28 (m, 4 H), 1.51-1.57 (m, 2 H), 2.49-2.54 (m, 2 H), 4.17
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δ

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(s, 2 H), 7.12 (d, J = 8.3 Hz, 2 H), 7.42 (t, J = 7.9, 1 H), 7.48-7.50 (m, 3 H), 7.75 (td, J = 7.6, 1.6
Hz, 1 H), 8.04 (dd, J = 7.9, 1.3 Hz, 1 H), 10.27 (s, 1 H), 12.69 (s, 1 H). 13C NMR (101 MHz,
DMSO-d6)
δ ppm 13.8, 21.9, 30.6, 30.8, 34.5, 35.1, 119.2, 119.9, 125.7, 125.8, 126.0, 128.4,
134.6, 136.5, 137.4, 148.2, 155.2, 161.0, 165.6; FTMS (ESI) m/z 382.1546 [M+H]+; calcd for
C21H23N3O2S: 382.1584.
4.3.11. N-(Naphthalen-2-yl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9k): Yield
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81%; m.p.: 219.3 – 221.5 °C; HPLC 95% (tR = 17,37 min); H NMR (400 MHz, DMSO-d6)
δ
ppm 4.25 (s, 2 H), 7.36-7.43 (m, 3 H), 7.47 (t, J = 7.3 Hz, 2 H), 7.62 (d, J = 8.8 Hz, 1 H), 7.72 (t,
J = 8.2 Hz, 1 H), 7.80 (dd, J = 11.1, 8.2 Hz, 1 H), 7.86 (d, J = 8.8 Hz, 1 H), 8.03 (d, J = 8.1 Hz, 1
13
H), 8.29 (s, 1 H), 10.57 (s, 1 H), 12.70 (s, 1 H). C NMR (101 MHz, DMSO-d6)
δ ppm 35.2,
115.3, 119.8, 119.9, 124.6, 125.7, 125.8, 126.0, 126.4, 127.3, 127.4, 128.4, 129.8, 133.4, 134.6,
136.5, 148.2, 155.3, 161.1, 166.1; FTMS (ESI) m/z 362.0942 [M+H]+; calcd forC20H15N3O2S:
362.0958.
4.3.12. (S)-2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(1,2,3,4-tetrahydronaphthalen-2-
1
yl)acetamide (9l): Yield 37%; m.p.: 244.1 – 245.0 °C; HPLC 93% (tR = 17.31 min); H NMR
(400 MHz, DMSO-d6)
δ ppm 1.67-1.73 (m, 2 H), 1.87-1.91 (m, 2 H), 2.67-2.78 (m, 2 H), 3.95-
4.04 (m, 2 H), 4.94-5.00 (m, 1 H), 7.01 (t, J = 7.3 Hz, 1 H), 7.08 (d, J = 7.1 Hz, 1 H), 7.11-7.17
(m, 2 H), 7.41-7.46 (m, 2 H), 7.77 (t, J = 7.7 Hz, 1 H), 8.04 (d, J = 7.8 Hz, 1 H), 8.60 (d, J = 8.8
Hz, 1 H), 12,66 (s, 1H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 19.9, 28.6, 29.6, 33.9, 46.9,
119.9, 125.6, 125.6, 125.8, 125.9, 126.6, 128.0, 128.6, 134.4, 136.9, 137.1, 148.2, 155.3, 161.0,
166.3; FTMS (ESI) m/z 366.1269 [M+H]+; calcd for C20H19N3O2S: 366.1271.
4.3.13. (R)-2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(1,2,3,4-tetrahydronaphthalen-2-
yl)acetamide (9m): Yield 80%; m.p.: 241.2 – 242.7 °C; HPLC 96% (tR = 17.28 min); 1H NMR
(400 MHz, DMSO-d6)
δ ppm 1.66-1.72 (m, 2 H), 1.85-1.92 (m, 2 H), 2.64-2.73 (m, 2 H), 3.94-
4.03 (m, 2 H), 4.96-5.00 (m, 1 H), 6.98-7.16 (m, 4 H), 7.40-7.44 (m, 2 H), 7.76 (t, J = 7.1 Hz, 1
13
H), 8.03 (d, J = 7.8 Hz, 1 H), 8.58 (d, J = 8.6 Hz, 1 H), 12.66 (s, 1 H). C NMR (101 MHz,
DMSO-d6)
δ ppm 19.9, 28.6, 29.6, 33.9, 46.9, 119.9, 125.6, 125.6, 125.8, 125.9, 126.6, 128.0,
128.6, 134.4, 136.9, 137.1, 148.2, 155.3, 161.0, 166.3.; FTMS (ESI) m/z 366.1268 [M+H]+;
calcd for C20H19N3O2S: 366.1271.
4.3.14. N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9n): Yield 65%;
1
m.p.: 219.0 – 221.3 °C; HPLC 91% (tR = 16.17 min); H NMR (400 MHz, DMSO-d6)
δ ppm
1.08-1.30 (m, 5 H), 1.52 (m, 1 H), 1.65-1.74 (m, 4 H), 3.50-3.60 (m, 1 H, 1’-H), 3.92 (s, 2 H, 9-
H), 7.42 (td, J = 8.1, 1.5 Hz, 1 H, 6-H), 7.50 (d, J = 8.1 Hz, 1 H, 8-H), 7.77 (td, J = 7.6, 1.6 Hz, 1
H, 7-H), 8.03 (dd, J = 7.8, 1.5 Hz, 1 H, 5-H), 8.13 (d, J = 7.6 Hz, 1 H, HN), 12.65 (s, 1 H, NH, 3-
H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 24.3, 25.1, 32.2, 34.1, 47.9, 119.9, 125.6, 125.7,
125.9, 134.5, 148.2, 155.3, 161.0, 165.8; FTMS (ESI) m/z 318.1277 [M+H]+; calcd for
C16H19N3O2S: 318.1271.
4.3.15. N-Cyclohexyl-N-methyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(9o):
Yield 52%; m.p.: 184.2 – 185.2 °C; HPLC 97% (tR = 17.43 min); H NMR (400 MHz, DMSO-
d6) ppm 1.07-1.11 (m, 2 H), 1.25-1.35 (m, 4 H), 1.43-1.47 (m, 5 H), 1.48-1.57 (m, 4 H), 1.72-
1.78 (m, 5 H), 2.74 and 3.01 (s, 2x NCH3), 3.78 and 4.21 (m, 2x CH), 4.23 and 4.34 (s, 2x CH2),
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7.40-7.48 (m, 4x ArH), 7.74-7.79 (m, 2x ArH), 8.04 (d, J = 8.1, 2x ArH), 12.61 (s, 1 H, 2x HN).
13C NMR (101 MHz, DMSO-d6)
ppm 24.7, 24.9, 25.1, 25.2, 27.2, 29.1, 29.6, 30.2, 33.1, 34.0,
δ
52.5, 56.1, 119.9, 125.5, 125.6, 125.7, 125.8, 125.9, 126.0, 134.5, 134.6, 148.2, 148.3, 155.2,
155.5, 161.0, 166.3; FTMS (ESI) m/z 332.1419 [M+H]+; calcd for C17H20N3O2S: 332.1427.
4.3.16. N-(2-Methylcyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
Yield 55%; m.p.: 230.2 – 232.8 °C; HPLC 91% (tR = 17.03 and 17.17 min); 1H NMR (400 MHz,
DMSO-d6) ppm 0.81 (J = 6.6 Hz, 3 H), 0,93-1.02 (m, 1 H), 1.10-1,39 (m, 4 H), 1.59 (d, J =
(9p):
δ
11.2 Hz, 1 H), 1.69 (q, J = 13 Hz, 2 H), 3.23-3.29 (m, 1 H), 3.89-4.01 (m, 2 H), 7.42 (t, J = 7.5
Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.77 (t, J = 7.6 Hz, 1 H), 8.04 (d, J = 8.1 Hz, 2 H), 12.66 (s, 1
H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 19.0, 25.0, 25.3, 32.7, 33.8, 34.1, 37.2, 49.2, 53.8,
119.9, 125.6, 125.8, 126.0, 134.5, 148.3, 155.4, 161.0, 166.1; FTMS (ESI) m/z 332.1414
[M+H]+; calcd for C17H21N3O2S: 332.1427.
4.3.17. N-(3-Methylcyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
Yield 35%; m.p.: 219.2 – 223.2 °C; HPLC 94% (tR = 17.24 and 17.47 min); 1H NMR (400 MHz,
DMSO-d6) ppm 0.71-0.90 (m, 7 H), 1.00-1.10 (m, 1 H), 1.20-1.29 (m, 2 H), 1.35-1.51 (m, 2
(9q):
δ
H), 1.58 (d, J = 12.5 Hz, 1 H), 1.75 (d, J = 11.7 Hz, 2 H), 3.52-3.54 (m, 1 H), 3.91 (s, 2 H), 7.42
(t, J = 8.1, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.77 (t, J = 7.7 Hz, 1 H), 8.04 (d, J = 8.1 Hz, 1 H),
8.12 (d, J = 7.1 Hz, 1 H), 12.64 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 22.2, 24.4,
31.3, 31.9, 33.8, 34.2, 41.2, 48.3, 120.0, 125.6, 125.8, 126.0, 134.4, 148.3, 155.3, 161.0, 165.9;
FTMS (ESI) m/z 332.1415 [M+H]+; calcd for C17H21N3O2S: 332.1427.
4.3.18. N-(4-Methylcyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(9r):
Yield 45%; m.p.: 199.2 – 200.8 °C; HPLC 90% (tR = 17.23 min); H NMR (400 MHz, DMSO-
d6) ppm 0.80 (d, J = 6.6 Hz, 3 H), 0.84 (d, J = 6.6 Hz, 1 H), 0.89-0.99 (m, 1H), 1.14-1.23 (m, 4
1
δ
H), 1.39-1.48 (m, 6 H), 1.55-1.65 (m, 3 H), 1.77 (d, J = 11.0 Hz, 1 H), 3.5 (m, 1 H)4.2 (s, 2 H),
7.3-7.4 (m, 3 H), 7.4 (t, J = 7.3 Hz, 2 H), 7.6 (d, J = 8.8 Hz, 1 H), 7.7 (t, J = 8.2 Hz, 1 H), 7.8
(dd, J = 11.1, 8.2 Hz, 1 H), 7.9 (d, J = 8.8 Hz, 1 H), 8.0 (d, J = 8.1 Hz, 1 H), 8.3 (s, 1 H), 10.6 (s,
1 H), 12.7 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 35.2, 115.3, 119.8, 119.9, 124.6,
125.7, 125.8, 126.0, 126.4, 127.3, 127.4, 128.4, 129.8, 133.4, 134.6, 136.5, 148.2, 155.3, 161.1,
166.1; FTMS (ESI) m/z 332.1414 [M+H]+; calcd for C17H21N3O2S: 332.1427.
4.3.19. N-(trans-4-Methylcyclohexyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9s):
1
Yield 59%; m.p.: 223.4 – 225.8 °C; HPLC 91% (tR = 17.29 min); H NMR (400 MHz, DMSO-
d6)
δ ppm 0.85 (d, J = 6.6 Hz, 3 H), 0.92-0.99 (m, 2 H), 1.13-1.23 (m, 2 H), 1.26-1.35 (m, 1 H),
1.65 (d, J = 11.7 Hz, 2 H), 1.76 (d, J = 11.0 Hz, 2 H), 3.47-3.50 (m, 1 H), 3.91 (s, 2 H), 7.42 (t, J
= 8.1 Hz, 1 H), 7.49 (d, J = 8.1 Hz, 1 H), 7.77 (td, J = 7.7, 1.5 Hz, 1 H), 8.03 (dd, J = 8.1, 1.5 Hz,
1 H), 8.11 (d, J = 7.1 Hz, 1 H), 12.65 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 22.0,
31.3, 32.1, 33.4, 34.1, 48.2, 119.9, 125.6, 125.8, 125.9, 134.5, 148.3, 155.3, 161.0, 165.9; FTMS
(ESI) m/z 332.1433 [M+H]+; calcd for C17H21N3O2S: 332.1427.
4.3.20. N-Cyclopentyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9t): Yield 60%;
1
m.p.: 214.5 – 242.9 °C; HPLC 98% (tR = 16.38 min); H NMR (400 MHz, DMSO-d6)
δ ppm
1.38-1.41 (m, 2 H), 1.47-1.51 (m, 2 H), 1.57-1.64 (m, 2 H), 1.73-1.81 (m, 2 H), 3.90 (s, 2 H),
3.95-4.03 (m, 1 H), 7.41 (td, J = 8.1, 1.5 Hz, 1 H), 7.48 (d, J = 8.1 Hz, 1 H), 7.76 (td, J = 8.4, 1.5

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Hz, 1 H), 8.02 (dd, J = 8.1, 1.5 Hz, 1 H), 8.20 (d, J = 7.1 Hz, 1 H), 12.64 (s, 1 H). 13C NMR (101
MHz, DMSO-d6)
δ ppm 23.3, 32.1, 34.0, 50.6, 119.9, 125.6, 125.7, 125.9, 134.5, 148.2, 155.3,
161.0, 166.2; FTMS (ESI) m/z 304.1119 [M+H]+; calcd for C15H17N3O2S: 304.1114.
4.3.21. N-Cycloheptyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9u): Yield 76%;
1
m.p.: 223.0 – 223.3 °C; HPLC 97% (tR = 17.26 min); H NMR (400 MHz, DMSO-d6)
δ ppm
1.33-1.55 (m, 10 H), 1.75-1.16 (m, 2 H), 3.72-3.75 (m, 1 H), 3.91 (s, 2 H), 7.42 (td, J = 8.0, 1.2
Hz, 1 H), 7.49 (d, J = 8.1 Hz, 1 H), 7.77 (td, J = 8.4, 1.5 Hz, 1 H), 8.01 (dd, J = 7.8, 1.5 Hz, 1 H),
8.16 (d, J = 7.8 Hz, 1 H), 12.65 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 23.6, 27.7,
34.0, 50.0, 119.9, 125.6, 125.7, 125.9, 134.5, 148.2, 155.3, 161.0, 165.5; FTMS (ESI) m/z
332.1432 [M+H]+; calcd for C17H21N3O2S: 332.1427.
4.3.22. N-Benzyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9v): Yield 41%; m.p.:
188.8 – 191.6 °C; HPLC 96% (tR = 16.32 min); 1H NMR (400 MHz, DMSO-d6)
δ ppm 4.03 (s,
2 H), 4.31 (d, J = 6.1 Hz, 2 H), 7.18-7.26 (m, 5 H), 7.41-7.48 (m, 2 H), 7.77 (td, J = 7.7, 1.5 Hz,
1 H), 8.04 (dd, J = 7.8, 1.5 Hz, 1 H), 8.71 (t, J = 5.7 Hz, 1 H), 12.64 (s, 1 H). 13C NMR (101
MHz, DMSO-d6)
δ ppm 33.7, 42.4, 119.9, 125.6, 125.9, 126.6, 126.9, 128.1, 134.5, 139.0, 148.2,
155.1, 161.0, 166.9; FTMS (ESI) m/z 326.0924 [M+H]+; calcd for C17H15N3O2S: 326.0958.
4.3.23. N-(Cyclohexylmethyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9w): Yield
1
46%; m.p.: 231.0 – 231.9 °C; HPLC 97% (tR = 17.20 min); H NMR (400 MHz, DMSO-d6)
δ
ppm 0.78-0.87 (m, 2 H), 1.05-1.10 (m, 3 H), 1.36-1.40 (m, 1 H), 1.57-1.64 (m, 5 H), 2.93 (t, J =
6.4 Hz, 2 H), 3.93 (s, 2 H), 7.42 (td, J = 8.1, 1.5 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.76 (td, J =
13
7.7, 1.5 Hz, 1 H), 8.03 (dd, J = 7.8, 1.2 Hz, 1 H), 8.15 (t, J = 7.8 Hz, 1 H), 12.64 (s, 1 H). C
NMR (101 MHz, DMSO-d6)
δ ppm 25.2, 25.8, 30.2, 33.8, 37.4, 45.1, 119.9, 125.6, 125.8, 125.9,
134.4, 148.2, 155.2, 161.0, 166.7; FTMS (ESI) m/z 332.1432 [M+H]+; calcd for C17H21N3O2S:
332.1427.
4.3.24. (S)-N-(1-Cyclohexylethyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide
(9x):
Yield 59%; m.p.: 232.5 – 236.7 °C; HPLC 91% (tR = 17.42 min); H NMR (400 MHz, DMSO-
d6) ppm 0.84-1.13 (m, 7 H), 1.01 (d, J = 6.8 Hz, 3 H), 1.22-1.26 (m, 1 H), 1.54-1.69 (m, 6 H),
1
δ
3.58-3.67 (m, 1H), 3.89 (d, J = 14.9 Hz, 1 H), 3.98 (d, J = 14.9 Hz, 1 H), 7.42 (t, J = 7.5 Hz, 1
H), 7.51 (d, J = 8.1 Hz, 1 H), 7.77 (t, J = 7.1 Hz, 1 H), 7.97 (d, J = 7.8 Hz, 1 H), 8.04 (d, J = 8.6
Hz, 1 H), 12.65 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 17.5, 18.4, 25.6, 25.8, 28.5,
28.7, 34.0, 42.4, 49.0, 56.0, 119.5, 119.9, 125.6, 125.8, 125.9, 134.5, 148.2, 155.3, 161.0, 166.1;
FTMS (ESI) m/z 346.1577 [M+H]+; calcd for C18H23N3O2S: 346.1584.
4.3.25. (R)-N-(1-Cyclohexylethyl)-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (9y):
1
Yield 82%, m.p.:217.2 – 217.9 °C; HPLC 91% (tR= 17.81); H NMR (400 MHz, DMSO-d6)
δ
ppm 0.83-0.95 (m, 2 H), 1.00 (d, J = 6.8 Hz, 3 H), 1.03-1.16 (m, 2 H), 1.23-1.29 (m, 1 H), 1.53-
1.69 (m, 5 H), 3.57-3.64 (m, 1 H), 3.89 (d, J =14.9 Hz, 1 H), 3.98 (d, J = 14.9 Hz, 1 H), 7.42 (t, J
= 7.5 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 1 H), 7.77 (t, J = 7.6 Hz, 1 H), 7.97 (d, J = 7.8 Hz, 1 H), 8.04
(d, J = 8.6 Hz, 1 H), 12.66 (s, 1 H). 13C NMR (101 MHz, DMSO-d6)
δ ppm 17.5, 25.6, 25.8,
28.5, 28.7, 34.0, 42.4, 49.0, 119.9, 125.6, 125.8, 125.9, 134.5, 148.2, 155.3, 161.0, 166.0; FTMS
(ESI) m/z 346.1606 [M+H] +; calcd for C18H23N3O2S: 346.1584.

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4.3.26. 2-((2-Oxo-2-(piperidin-1-yl)ethyl)thio)quinazolin-4(3H)-one (9z): Yield 72%; m.p.:
118.5 – 121.0 °C; HPLC 98% (tR = 16.34 min); 1H NMR (400 MHz, DMSO-d6)
ppm 1.46 (m,
δ
2 H), 1.62 (m, 4 H), 3.55 (m, 2 H), 3.51-3.56 (m, 2 H), 4.29 (s, 2 H), 7.42 (t, J = 7.5 Hz, 1 H),
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NMR (101 MHz, DMSO-d6)
δ ppm 23.8, 25.2, 26.0, 33.3, 42.7, 46.6, 119.9, 125.6, 125.8, 126.0,
134.5, 148.2, 155.4, 161.0, 165.0; FTMS (ESI) m/z 304.1117 [M+H]+; calcd for C15H17N3O2S:
304.1140.
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4.4 Mycobacterium tuberculosis inhibition assay
The measurement of the minimum inhibitory concentration (MIC) for each tested
compound was performed in 96-well U-bottom polystyrene microplates. Isoniazid (positive
control) and the compound solutions were prepared at 1 mg/mL concentration in
dimethylsulfoxide (DMSO). They were diluted in Middlebrook 7H9 medium containing 10%
ADC (albumin, dextrose, catalase) to a concentration between 10 and 50 µg/mL of each
compound containing 5% DMSO. Serial two-fold dilutions of each drug in 100 µL of
Middlebrook 7H9 medium containing 10% ADC were prepared directly in 96-well plates at
concentration ranges of 25.0 to 0.05 µg/mL, 10.0 to 0.02 µg/mL, or 0.02 to 0.0004 µg/mL.
Growth controls containing no antibiotics, and sterile controls without inoculation, were
included. The MIC was determined for M. tuberculosis H37Rv ATCC 27294 reference strain
(American Type Culture Collection, Rockville, Md.) and for the clinical isolates CDCT10
(1009/09), CDCT16 (630/08), CDCT27 (0128/09) and CDCT28 (1051/10). Mycobacterium
strains were grown in Middlebrook 7H9 containing 10% OADC (oleic acid, albumin, dextrose
and catalase) and 0.05% Tween 80. The cells were vortexed with sterile glass beads (4 mm) for 5
min to disrupt any clumps and were allowed to settle for 20 min. The supernatant was measured
spectrophotometrically at an absorbance of 600 nm. The Mtb suspensions were divided into
aliquots and stored at –20 °C. Each suspension was appropriately diluted in Middlebrook 7H9
broth containing 10% ADC to achieve an optical density at 600 nm of 0.006, and 100 µL was