Synthesis, in vitro antibacterial activity against Mycobacterium tuberculosis, and reverse docking-based target fishing of 1,4-benzoxazin-2-one derivatives

Article abstract of DOI:10.1002/ardp.202000199

Seventeen 1,4-benzoxazin-2-ones bearing the enaminone moiety and three of their analogs were tested for the antibacterial activity against Mycobacterium tuberculosis (H37Rv). Minimal bactericidal concentrations (MBCs) were determined after 41 days of incu

Full text of DOI:10.1002/ardp.202000199

Received: 13 June 2020
Revised: 7 August 2020
Accepted: 11 September 2020
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DOI: 10.1002/ardp.202000199
F U L L P A P E R
Synthesis, in vitro antibacterial activity against
Mycobacterium tuberculosis, and reverse docking‐based target
fishing of 1,4‐benzoxazin‐2‐one derivatives
Ekaterina E. Stepanova
| Svetlana Yu. Balandina | Vera A. Drobkova |
Maksim V. Dmitriev | Irina V. Mashevskaya | Andrey N. Maslivets
Department of Chemistry, Perm State
University, Perm, Russia
Abstract
Seventeen 1,4‐benzoxazin‐2‐ones bearing the enaminone moiety and three of their
Correspondence
Ekaterina E. Stepanova and Andrey N.
Maslivets, Department of Chemistry, Perm
State University, ul. Bukireva 15, Perm,
Russian Federation, 614990.
analogs were tested for the antibacterial activity against Mycobacterium tuberculosis
(H37Rv). Minimal bactericidal concentrations (MBCs) were determined after
41 days of incubation by BACTEC. 1,4‐Benzoxazin‐2‐ones bearing the unsubstituted
benzo moiety showed the most promising activities (MBC = 5.00 µg/ml). For most
active compounds, antibacterial activities were determined daily during the 41 days.
The most promising compound showed a bacteriostatic effect at a concentration of
0.31 µg/ml on Day 4 of incubation, 0.62 µg/ml on Day 6, 2.50 µg/ml on Day 9, and
5.00 µg/ml on Day 41. All studied compounds, along with some of their reported
analogs, were docked to 35 proteins of M. tuberculosis to find their potent targets in
these organisms. As a result of reverse docking, aspartate 1‐decarboxylase, panD,
was selected as the most appropriate target. Docking of the most active compounds
to mutant panD from pyrazinamide‐resistant strains of M. tuberculosis implies that
they would not be active against these strains. Considering that most of pyr-
azinamide clinical resistance cases are due to loss‐of‐function mutations in pyr-
azinamidase, pncA, compounds from this study could be useful drugs for the
treatment of some cases of pyrazinamide‐resistant tuberculosis.
Email: caterina.stepanova@psu.ru (E. E. S.) and
koh2@psu.ru (A. N. M.)
Funding information
Government of Perm Krai; Ministry of Science
and Higher Education of the Russian
Federation, Grant/Award Number:
FSNF‐2020‐0008
K E Y W O R D S
1,4‐benzoxazin‐2‐one, antibacterial activity, molecular docking, Mycobacterium
tuberculosis, panD
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1
INTRODUCTION
is about 6 months, which imposes a serious economic burden both
for the patient and the state.
Tuberculosis (TB) is a communicable disease that is one of the top
10 causes of death worldwide and the leading cause of death from a
single infectious agent. Overcoming the global public health crisis of
multidrug‐resistant TB requires incessant search for new anti‐TB agents.
Anti‐TB drug development is complicated by the nature of the
TB pathogen, Mycobacterium tuberculosis (Mtb), which is char-
acterized by slow growth and high pathogenicity.[1] In addition, it is
important to consider economic factors, as TB treatment duration
1,4‐Benzoxazin‐2‐ones bearing the enaminone moiety (BOs;
Figure 1) have been thoroughly investigated for various biological ac-
tivities (antioxidant,[2] antimicrobial,[3,4] antimycotic,[3,5] anti‐Alzheimer's
disease,[6] platelet aggregation inhibitory,[7] and antimycobacterial[8,9]), as
they are attractive platforms for the development of inexpensive drugs
due to the simplicity of their synthesis and purification, the availability of
starting materials, and the possibility of their synthesis under mild green
conditions in gram‐scale quantities.[2,10–12]
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Arch Pharm. 2020;e2000199.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000199

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FIGURE 1 1,4‐Benzoxazin‐2‐ones bearing the enaminone moiety
Recently, BOs were found to exhibit significant antibacterial
activity against Mtb.[8,9] Detailed studies of structure–activity re-
lationships (SAR) of these compounds and their analogs (isosteres
and open‐ring analogs) were performed.[8,9]
dimethyl acetylenedicarboxylate (DMAD) with o‐aminophenol 2a
(Scheme 1). BO 1a was necessary as a starting point and a reference
compound for our study of evaluation of anti‐TB properties of novel
BOs and their analogs.
BOs examined for anti‐TB activity[8,9] belong to two chemically
different groups of enaminones, aroyl (R = Ar, Figure 1) and
alkoxycarbonyl‐bearing ones (R = OAlk, Figure 1). One more group of
BOs, alkyloyl‐bearing ones (R = Alk, Figure 1), was not investigated in
whole‐cell assays for the antibacterial activity against Mtb, despite
the fact that it was the first to be identified in high‐throughput
screen as MenB, 1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase, inhibitors
for the development of new anti‐TB agents.[8]
Ethoxycarbonyl‐bearing BOs 1b,c were synthesized by the con-
densation of diethyl oxaloacetate 3a with o‐aminophenols 2a,b
(Scheme 2).
Alkyloyl‐bearing BOs 1c–p were synthesized by the condensa-
tion of methyl acylpyruvates 3b,c with o‐aminophenols 2a–h
(Scheme 3). According to a similar procedure (Scheme 3), their
4‐fluorophenyl analog, BO 1q, was synthesized, starting from methyl
4‐fluorobenzoylpyruvate 3d.
Herein, we report a study on the anti‐TB activity of under-
valued BOs bearing the alkyloyl (R = tert‐Bu, iso‐Bu, Figure 1)
substituents and reverse docking evaluation of their potent
targets in cells of Mtb.
In addition, to expand the chemical space around target BO 3, we
designed its novel open‐ring analog, enamine 4; it was synthesized by
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
Methoxycarbonyl‐bearing BO 1a, which was reported as an anti‐TB
hit compound by Li et al.,[8] was synthesized by the reaction of
SCHEME 1 The synthesis of methoxycarbonyl‐bearing BO 1a.
Reagents and conditions: (a) MeOH, rt, 24 hr

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2.2
Pharmacology/Biology
A series of 20 synthesized compounds was tested for activity against
pathogenic Mtb (H37Rv) using the standard BACTEC radiometric
growth assay. The effect of test compounds on the growth of Mtb
was monitored on Day 41. The lowest dilutions of the examined
compounds, in which no growth of Mtb was detected by BACTEC on
Day 41 of the assay, were considered as minimal bactericidal con-
centrations (MBCs; Table 1).
SCHEME 2 The synthesis of ethoxycarbonyl‐bearing BOs 1b,c.
Reagents and conditions: (a) EtOH, reflux, 5 min
On Day 41, 8 of 20 examined compounds were found to exhibit a
significant bactericidal activity against Mtb. Control tubes, containing
growth media, Mtb, and no test compounds, showed abundant
growth of mycobacteria.
The highest mycobactericidal activity was shown by compounds
having no substituents in 1,4‐benzoxazine moiety, BOs 1b,d,l,q, at a
concentration of 5.0 μg/ml, which is in agreement with previous SAR
studies of BOs.[8,9] Unexpectedly, BO 1a, reported earlier as an anti‐
TB hit compound,[8] did not fall into the group of the most active
compounds and showed a bactericidal effect at a concentration of
10 μg/ml. In our assay, replacement of methyl in compound 1a for
ethyl (compound 1b) resulted in an increase of bactericidal activity,
that is, MBC of methyl‐bearing compound 1a was 10 μg/ml and MBC
of ethyl‐bearing compound 1b was 5 μg/ml.
SCHEME 3 The synthesis of BOs 1d–q. Reaction and reagents:
(a) EtOH, reflux, 5 min
condensation of diethyl oxaloacetate 3a with N‐(2‐aminophenyl)
acetamide (Scheme 4).
Finally, we planned to study the anti‐TB activity of annelated
analogs of BO 1, pyrrolobenzoxazines 5a,b. They were prepared by
acylation of BOs 1b,q with oxalyl chloride (Scheme 5).[13,14] It should
be considered that pyrrolobenzoxazines 5a,b readily undergo
hydrolysis (Scheme 5),[15] which is easy to notice by the color change
(dark violet to yellow) when dissolved in media containing water. In
fact, the anti‐TB activity of pyrrolobenzoxazines 5a,b is not
investigated; however, the anti‐TB activity of their hydrolysis
products, 6a,b, is investigated.
Compound 1k, bearing 6‐Cl substituent in 1,4‐benzoxazine
moiety, showed a bactericidal effect at a concentration of 10 μg/ml.
Compounds 1m,p, bearing 6‐halogen (Br and Cl) substituents in
1,4‐benzoxazine moiety, showed a mycobactericidal activity at a
concentration of 20 μg/ml, and compound 1c, a 6‐Br analog of BO 1b,
did not show a bactericidal effect. The introduction of methyl‐ and
nitro‐substituents in 1,4‐benzoxazine moiety deteriorated the anti-
bacterial activity of BOs 1f,g,i,j,n,o. The same effect had a benzo‐
substituent in 1,4‐benzoxazine moiety of BO 1e. This confirms that
unsubstituted 1,4‐benzoxazine moiety is crucial for anti‐TB activity.
Annelated analogs of BOs, compounds 5a,b, and the open‐ring
analog of BOs, enamine 4, did not show bactericidal activity.
Incorporation of an alkyloyl substituent in BO 1 did not result in
the degradation of anti‐TB activity. A similar effect of incorporation
of 4‐fluorobenzoyl substituent was observed.
The examined tert‐Bu and iso‐Bu substituents in BO 1 showed a
similar effect on the anti‐TB activity. But simultaneous changes both
in alkyloyl substituents and 6‐halogen ones resulted in different ef-
fects. So, for 6‐Cl‐substituted BOs, tert‐Bu‐bearing BO 1k showed a
bactericidal effect at a concentration of 10 μg/ml, and its iso‐Bu
analog, BO 1m, showed a bactericidal effect at a concentration of
20 μg/ml.
SCHEME 4 The synthesis of the open‐ring analog of BO 1,
enamine 4. Reaction and reagents: (a) EtOH, reflux, 5 min
SCHEME 5 Synthesis and hydrolysis of
annelated analogs of BO 1,
pyrrolobenzoxazinones 5a,b. Reaction and
reagents: (a) Benzene, reflux, 90 min

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TABLE 1 Results of the anti‐TB assay by BACTEC
TABLE 1 (Continued)
Entry
Compound
1a
Structure
MBC (μg/ml)a
Entry
Compound
1n
Structure
MBC (μg/ml)a
1
10
14
>20
2
3
1b
1c
5
15
1o
>20
>20
16
17
1p
1q
20
5
4
5
6
7
1d (CCDC
5
2005170)
1e
1f
>20
>20
>20
18
19
4
>20
>20
1g
5a
8
1h
1i
>20
>20
>20
10
20
21
5b
>20
Isoniazid
1.25
9
10
11
1j
Abbreviations: MBC, minimal bactericidal concentration;
TB, tuberculosis.
aThe lowest dilution of the examined compound in which no bacterial
growth was detected on Day 41 of the assay.
1k
Compounds with the highest anti‐TB activity were additionally
tested by BACTEC, which detected the presence or absence of
growth of mycobacteria daily for 41 days (Figure 2). During the assay,
the growth rate of Mtb was compared in the control tube and in
tubes with a certain concentration of test substances.
12
13
1l
5
According to Figure 2, the studied compounds 1b,d,l,q had a
pronounced bactericidal effect against mycobacteria at a quite low
concentration of 5 μg/ml. At this concentration, the compounds
completely inhibited the growth of the TB pathogens, and the culture
growth was not observed throughout the incubation time. In control
tubes (medium containing Mtb), abundant growth of mycobacteria
was observed on the fourth day of the assay.
1m
20

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Performing the reverse docking procedure, we expected to find a
target with docking scores that would be maximal in the case of BOs
with unsubstituted 1,4‐benzoxazine moiety. To improve the sensi-
tivity of the reverse docking procedure, we docked compounds from
this paper together with the earlier reported anti‐TB BOs.[8] For
convenience, BOs from the study reported in Reference [8] are in-
dicated with the same numbers as in the source paper, but marked
with quotes (e.g., “2”). Compounds bearing a chiral atom were docked
as both enantiomers (6a and 6a+, 6b and 6b+).
Docking of BOs and their analogs to isocitrate lyase (icl1; Entry
23, Table 3) resulted in negative scores, which meant that ligands
were much larger than the cavity of the binding site. Thus, this
protein was not processed further.
FIGURE 2 Results of the antituberculosis assay by BACTEC (the
lowest dilution of the examined compound in which no bacterial
growth was detected on the monitoring day; the growth was
detected on Day 4 in control tubes without test compounds).
MBC, minimal bactericidal concentration
Obtained docking scores were normalized and distributed in five
categories to visualize ligand ranks to each protein (Figure 3).
According to Figure 3, compounds most active in biological assays,
BOs 1a,b,d,l,q, had highest normalized docking scores (top 25%) only
to aspartate 1‐decarboxylase (panD; Entry 22, Table 3). Considering
this, we assumed that a putative target of BO 1 in Mtb could be panD.
PanD catalyzes decarboxylation of L‐aspartate to result in
β‐alanine and CO2. This is a part of the pantothenate biosynthetic
pathway.[20]
Alkyloyl‐bearing compounds 1d,l exhibited a bacteriostatic effect
at the lowest concentration of 0.62 μg/ml on the Mtb during the first
6 days of the assay. Considering the slightly better effect of com-
pound 1d on the fourth day, it can be concluded that BO 1d is a
better candidate for drug development.
Selected BOs 1d,k,q were tested by resazurin microtiter assay
(REMA)[16,17] for anti‐TB activity (Table 2). The results were mon-
itored on Day 10, which were generally in agreement with the results
of BACTEC assay; the same SARs were observed. BOs 1d,q, bearing
no substituents in 1,4‐benzoxazine moiety, showed the lowest mini-
mum inhibition concentrations (MICs) of 0.97 μg/ml. Compound 1k,
bearing 6‐Cl substituent in 1,4‐benzoxazine moiety, showed MIC at a
higher concentration of 7.8 μg/ml.
Pyrazinamide (PZA) is a clinically effective anti‐TB drug. Re-
cently, PZA's mechanism of action was investigated by Sun et al.[21]
;
panD was found to be concurrently inhibited by pyrazinoic acid
(POA)[21] formed from PZA by the enzyme pyrazinamidase, pncA
(Rv2043c).[22] So, the substrate of panD is L‐aspartate and POA is its
concurrent inhibitor. BO 1 contains an aspartate‐like motif (Figure 4),
which, apparently, determined good docking scores of BO 1 to panD.
Docking characteristics of BOs 1a,b,d,l,q to cavity of panD are
summarized in Table 4. For comparison, similar characteristics of
POA (natural ligand of 6oyy, Figure 4) and 6‐chloro‐POA (natural
ligand of 6p02, Figure 4) are provided.
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2.3
Molecular docking
Four hydrogen bonds (HBs) contribute significantly to the bind-
ing between POA and panD: with Ala74, Ala75, and two with
Arg54.[21] In the best‐docked poses of BOs 1a,b,d,l,q, some of these
significant HBs were observed along with some other HBs and other
types of interactions (Figure 5). The best‐docked pose of BO 1l
showed an unfavorable bump of an alkyloyl substituent with Tyr22.
We suppose that this unfavorable interaction could be corrected by
flexibility of this amino acid residue or flexibility of alkyloyl sub-
stituent in BO 1l. The best‐docked poses of other BOs 1a,b,d,q had
no unfavorable interactions.
Identification of a target from a variety of candidate macromolecules
is a challenging task. Out of 890 known metabolic genes of Mtb,
structural data for only 140 of them are deposited in the structural
database PDB.[18]
To conjecture a potent target for BO 1, we used a reverse
docking strategy. For this purpose, we prepared a library of targets of
Mtb (H37Rv), based on a review by H. Tomioka et al.,[] summarizing
promising targets for anti‐TB drug discovery (Table 3).
Notably, docked BOs having a bulky substituent in 1,4‐benzoxazine
moiety suffered additional unfavorable interactions with the protein,
which could explain the deterioration of anti‐TB activity of such BOs in
comparison with unsubstituted ones (protein–ligand interactions in the
example of BO 1i are shown in Figure 6).
TABLE 2 Results of the anti‐TB assay by REMA
Entry
Compound
MIC (μg/ml)
0.97
1
2
3
4
1d
1k
7.8
Moreover, aza analog of BOs, quinoxalinone “12,” reported to be
inactive in anti‐TB assays,[8] was found not to form significant HBs
with Ala75 and Arg54 and to suffer an unfavorable interaction with
Tyr22 (Figure 7), in comparison with its analog, BO 1a (Figure 5b),
active in anti‐TB assay.[8] Similar SAR is known for PZA and POA.
1q
0.97
Isoniazid
0.032
Abbreviations: MIC, minimum inhibition concentration; REMA, resazurin
microtiter assay; TB, tuberculosis.

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TABLE 3 Targets of Mtb selected for
reverse docking
Entry
1
PDB ID
4ohu, 4tzk, 5g0t
1uzn
Protein (gene)
Enoyl‐ACP reductase (inhA)
2
β‐Ketoacyl‐ACP reductase (mabA)
β‐Ketoacyl‐ACP synthase (kasA)
β‐Ketoacyl‐ACP synthase (kasB)
β‐Ketoacyl‐ACP synthase (fabH)
Cyclopropane synthase (pcaA)
Cyclopropane synthase (cmaA1)
Cyclopropane synthase (cmaA2)
Cyclopropane synthase (mmaA2)
Polyketide synthase (pks13)
3
5ld8, 6p9l, 6p9m
2gp6
4
5
2qo1
6
1 l1e
7
1kpg, 1kph
1kpi
8
9
1tpy
10
11
12
13
6c4q
3ha5
S‐Adenosylmethionine‐dependent methyltransferase (mmaA4)
Acyl‐AMP ligase (fadD32)
5hm3
4ewl
N‐Acetyl‐1‐D‐myo‐inosityl‐2‐deoxy‐α‐D‐glucopyranoside
deacetylase (mshB)
14
15
16
17
18
19
1ozp, 1p0h
4n9w, 4nc9
1q9j
Mycothiol synthase (mshD)
Phosphatidyl mannosyltransferase (pimA)
Phthiocerol dimycocerosyl transferase (papA5)
Filamentation temperature‐sensitive protein (ftsZ)
1,4‐Dihydroxy‐2‐naphthoate‐coenzyme A synthase (menB)
5zue
4qij
6o0j
2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐
carboxylate synthase (menD)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
6ddp, 6nnh, 6nni
Dihydrofolate reductase (dfrA)
Pantothenate synthetase (panC)
Aspartate decarboxylase (panD)
Isocitrate lyase (icl1)
1mop, 1n2h, 4fzj
6p02, 6oyy, 6oz8
5dql
6ee1
Isocitrate lyase (icl2)
5ecv, 5h8u, 5t8g
5xnx
Malate synthase (glcB)
RelA protein (relA)
6c67
Adenosine kinase (Rv2202c)
Thymidylate kinase (tmk)
Dihydrolipoyl dehydrogenase (lpdC)
Iron‐dependent regulator (IdeR)
DosR regulator protein (DosR)
Citrate lyase (citE)
4unr
2a8x, 4m52
2isy
1zlj
1z6k
4tvm
Citrate synthase (gltA2)
2byo
Lipoprotein LppX (Rv2945c)
Transcription initiation complex (Rv3457c)
5uhb
So, PZA itself does not inhibit panD, and its oxygen analog, POA,
inhibits it (Figure 8).[21]
available in PDB (6p1y). This mutant protein is characterized by a smaller
volume of the active site.[21] Docking characteristics of BOs 1a,b,d,l,q and
POAs to cavity of mutant panD are summarized in Table 5. The best‐
docked poses of BOs 1a,b,d,l,q to mutant panD had lower docking scores
To evaluate the possibility of binding of BOs 1a,b,d,l,q to mutant
panD from PZA‐resistant strains, we performed their docking to a panD

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FIGURE 3 Categorized normalized
docking scores
in comparison with docking to non‐mutant panD and showed unfavorable
interactions with the protein (Figure 9). So, we can conclude that BOs
1a,b,d,l,q would be less active in nature to the mutant panD and would
not affect such PZA‐resistant strains. Nonetheless, most of the PZA
clinical resistance cases are due to loss‐of‐function mutations in pyr-
azinamidase (pncA).[22] Considering this, if our hypothesis about the me-
chanism of action of BO 1 is true, it could be a useful drug for treatment
of some cases of PZA‐resistant TB.
TABLE 4 Docking scores and binding energies of BOs 1a,b,d,l,q to
cavity of panD
Ligand
Scores
24.37a
23.08a
24.89a
25.79a
22.62a
26.75a
24.82b
ΔGbind (kJ/mol)
−29.21
1a
1b
−30.64
1d
−37.01
1l
−37.06
1q
−33.48
Natural ligand (6p02)
Natural ligand (6oyy)
−26.99
−25.17
Abbreviations: BO, 1,4‐benzoxazin‐2‐ones bearing the enaminone moiety;
panD, aspartate decarboxylase.
aDocked to 6p02.
bDocked to 6oyy.
FIGURE 4 Aspartate‐like motif in 1,4‐benzoxazin‐2‐ones bearing
the enaminone moiety 1 and pyrazinoic acid

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FIGURE 5 Protein–ligand interactions of 1,4‐benzoxazin‐2‐ones bearing the enaminone moiety 1a (a), 1b (b), 1d (c), 1l (d), 1q (e) with panD
(6p02). Red, unfavorable bumps; green, conventional hydrogen bonds; pale green, carbon hydrogen bonds; pale pink, alkyl and π–alkyl
interactions; violet: π–σ interactions
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3
CONCLUSION
promising activities (MBC = 5.00 µg/ml after 41 days of incubation). For
the four most active compounds, the bacteriostatic activity was de-
termined daily during the 41 days. The most promising compound 1d
showed a bacteriostatic effect at a concentration of 0.31 µg/ml on Day 4,
0.62 µg/ml on Day 6, 2.50 µg/ml on Day 9, and 5.00 µg/ml on Day 41.
We tested 17 BOs, their novel open‐ring analogs, and two annelated
analogs of BOs for in vitro antibacterial activity against Mtb (H37Rv). BOs
bearing the unsubstituted 1,4‐benzoxazin‐2‐one moiety showed the most

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FIGURE 8 Comparison of pyrazinamide (PZA) and quinoxalinone
“12” with their oxa analogs
FIGURE 6 Protein–ligand interactions of 1,4‐benzoxazin‐2‐ones
bearing the enaminone moiety 1i with panD (6p02). Red, unfavorable
bumps; green, conventional hydrogen bonds (HBs); pale green,
carbon HBs; pale pink, alkyl and π–alkyl interactions; violet, π–σ
interactions
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4
EXPERIMENTAL
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4.1
Chemistry
All studied compounds along with their reported analogs[8] were docked
to 35 proteins of Mtb (H37Rv) to find their potent targets. In the result of
reverse docking, aspartate 1‐decarboxylase, panD, was selected as the
most appropriate target. Docking of the most active compounds to mu-
tant panD from PZA‐resistant strains of Mtb implied that they would not
be active against these strains. Considering that most of the PZA clinical
resistance cases are due to loss‐of‐function mutations in pyrazinamidase,
pncA, these compounds could be useful drugs for the treatment of some
cases of pyrazinamide‐resistant TB.
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4.1.1
General
1H and 13C NMR (nuclear magnetic resonance) spectra (see the
Supporting Information) were acquired on a Bruker Avance‐III
spectrometer (400 and 100 MHz, respectively) in CDCl3 or di-
methyl sulfoxide (DMSO)‐d6 using hexamethyldisilazane or tet-
ramethylsilane signals (in 1H NMR) or solvent residual signals (in
1H NMR, 7.27 for CDCl3, 2.50 for DMSO‐d6; in 13C NMR, 77.00
for CDCl3, 39.51 for DMSO‐d6) as internal standards. Infrared
(IR) spectra were recorded on a PerkinElmer Spectrum Two
spectrometer from mulls in mineral oil. Melting points were
measured on a Mettler Toledo MP70 apparatus. The single‐
crystal X‐ray analysis was performed on an Xcalibur Ruby dif-
fractometer. Elemental analyses were carried out on a Vario
MICRO Cube analyzer. The purity of all compounds was ex-
amined using ultra‐high pressure liquid chromatography (UPLC)‐
ultraviolet (UV)‐mass spectrometry (MS); Waters ACQUITY
UPLC I‐Class system; Acquity UPLC BEH C18 column, grain size
TABLE 5 Docking scores and binding energies of 1,4‐benzoxazin‐
2‐ones bearing the enaminone moiety (BOs) 1a,b,d,l,q to cavity of
mutant panD (6p1y)
BO
Scores
21.87
22.80
22.86
26.06
24.21
26.15
24.63
ΔGbind (kJ/mol)
−27.91
1a
1b
−27.14
1d
−34.50
1l
−34.50
1q
−40.29
FIGURE 7 Protein–ligand interactions of quinoxalinone “12” with
panD (6p02). Red, unfavorable bumps; green, conventional hydrogen
bonds; pale green, carbon hydrogen bonds; pale pink, alkyl and
π–alkyl interactions; violet, π–σ interactions
Natural ligand (6p02)
Natural ligand (6oyy)
−26.66
−24.80

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FIGURE 9 Protein–ligand interactions of 1,4‐benzoxazin‐2‐ones bearing the enaminone moiety 1a (a), 1b (b), 1d (c), 1l (d), 1q (e) with mutant
panD (6p1y). Red, unfavorable interactions; green, conventional hydrogen bonds; pale green, carbon hydrogen bonds; pale pink, alkyl and π–alkyl
interactions; violet, π–σ interactions; blue, halogen bonds (fluorine); orange, π–cation interactions
of 1.7 μm; acetonitrile–water as eluents; flow rate of 0.6 ml/min;
ACQUITY UPLC PDA eλ detector (wavelength range of
230–780 nm; Xevo TQD mass detector; electrospray ionization
(ESI); positive and negative ion detection; ion source temperature
of 150°C; capillary voltage of 3.5–4.0 kV; cone voltage of
20–70 V; vaporizer temperature of 200°C.
Benzene and hexane were distilled over Na before the use. All
other solvents and reagents were purchased from commercial ven-
dors and were used as received.
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.

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4.1.2
Procedure for the synthesis of BO 1a
C14H15NO3: C 68.56; H 6.16; N 5.71. Found: C 68.41; H 6.16; N 5.76. The
complete set of X‐ray diffraction data was deposited to the Cambridge
Crystallographic Data Centre (CCDC; Entry no. 2005170); it can be
requested at www.ccdc.cam.ac.uk.
DMAD (4.1 mmol, 0.5 ml) was added to
a stirring suspension
of o‐aminophenol 2a (4.1 mmol, 0.45 g) in methanol (5 ml).
The reaction mixture was stirred at room temperature overnight. The
formed yellow precipitate was filtered off, washed with methanol
(15 ml), and recrystallized from ethanol (50 ml) to give BO 1a.
(2Z)‐2‐(3,3‐Dimethyl‐2‐oxobutylidene)‐1,2‐dihydro‐3H‐naphtho‐
[2,1‐b][1,4]oxazin‐3‐one (1e)
1‐Aminonaphthalen‐2‐ol 2h hydrochloride was utilized in general
procedure instead of 1‐aminonaphthalen‐2‐ol 2h. Yield: 0.74 g (63%);
yellow solid; mp 168–169°C (ethanol). 1H NMR (DMSO‐d6): δ = 13.75
(s, 1H), 7.98 (m, 1H), 7.87 (m, 1H), 7.73 (m, 2H), 7.59 (m, 1H), 7.41 (m,
1H), 6.44 (s, 1H), and 1.24 (s, 9H) ppm. 13C NMR (DMSO‐d6):
δ = 206.7, 155.7, 139.7, 138.0, 130.3, 128.7, 127.6, 126.1, 123.7,
120.9, 118.8, 117.5, 116.3, 91.9, 42.3, and 26.9 (3C) ppm. IR:
Methyl (2Z)‐(2‐oxo‐2H‐1,4‐benzoxazin‐3(4H)‐ylidene)acetate (1a)[9]
Yield: 0.68 g (76%); yellow solid; mp 163–165°C (ethanol; mp[9]
164–168°C). 1H NMR (DMSO‐d6): δ = 10.66 (s, 1H), 7.50 (m, 1H),
7.18 (m, 2H), 7.04 (m, 1H), 5.64 (s, 1H), and 3.72 (s, 3H) ppm. IR:
ν = 3,337, 1,768, 1,723, and 1,666 cm−1. MS (ESI+): m/z calcd for
C
11H9NO4+H+: 220.06 [M+H]+; found: 220.02. Anal. calcd (%) for
C11H9NO4: C 60.28; H 4.14; N 6.39. Found: C 60.12; H 4.16; N 6.41.
ν = 3,177, 1,758, and 1,631 cm−1
. MS (ESI+): m/z calcd for
C18H17NO3+H+: 296.13 [M+H]+; found: 296.13. Anal. calcd (%) for
C18H17NO3: C 73.20; H 5.80; N 4.74. Found: C 73.41; H 5.68; N 4.75.
|
4.1.3
General procedure for the synthesis of
BOs 1b–q
(3Z)‐3‐(3,3‐Dimethyl‐2‐oxobutylidene)‐7‐nitro‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1f)
Alkyl acylpyruvate 3a–d (4.0 mmol) was added to a suspension of o‐
aminophenol 2a–h (4.0 mmol) in ethanol (10 ml). The reaction mix-
ture was refluxed for 5 min and cooled to ambient temperature. The
formed yellow precipitate was filtered off, washed with ethanol
(5 ml), and recrystallized to give BO 1b–q.
Yield: 0.82 g (71%); yellow solid; mp 201–202°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.22 (s, 1H), 8.04 (m, 2H), 7.75 (m, 1H), 6.50 (s, 1H), and
1.19 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 206.6, 155.4, 141.4, 140.1,
137.3, 130.8, 120.8, 116.4, 111.9, 95.2, 42.8, and 26.4 (3C) ppm. IR:
ν = 3,182, 1,768, and 1,646 cm−1
C14H14N2O5+H+: 291.10 [M+H]+; found: 291.12. Anal. calcd (%) for
14H14N2O5: C 57.93; H 4.86; N 9.65. Found: C 58.12; H 4.80; N 9.63.
.
MS (ESI+): m/z calcd for
Ethyl (2Z)‐(2‐oxo‐2H‐1,4‐benzoxazin‐3(4H)‐ylidene)acetate (1b)[10]
Yield: 0.63 g (71%); yellow solid; mp 110–112°C (ethanol; mp[10]
116–117°C). 1H NMR (CDCl3): δ = 10.70 (s, 1H), 7.14 (m, 2H), 7.00 (m,
2H), 5.94 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), and 1.33 (t, J = 7.2 Hz, 3H) ppm.
C
(3Z)‐3‐(3,3‐Dimethyl‐2‐oxobutylidene)‐6‐nitro‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1g)
IR: ν = 3,214, 1,760, and 1,661 cm−1
12H11NO4+H+: 234.08 [M+H]+; found: 234.12. Anal. calcd (%) for
.
MS (ESI+): m/z calcd for
Yield: 0.84 g (73%); yellow solid; mp 194–195°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.19 (s, 1H), 8.63 (m, 1H), 7.89 (m, 1H), 7.40 (m, 1H),
6.43 (s, 1H), and 1.19 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 206.0, 155.5,
145.0, 144.1, 137.6, 125.4, 117.7, 117.0, 112.0, 93.9, 42.5, and 26.5
(3C) ppm. IR: ν = 3,194, 1,768, and 1,633 cm−1. MS (ESI+): m/z calcd for
C14H14N2O5+H+: 291.10 [M+H]+; found: 291.10. Anal. calcd (%) for
C
C12H11NO4: C 61.80; H 4.75; N 6.01. Found: C 61.98; H 4.76; N 6.01.
Ethyl (2Z)‐(6‐bromo‐2‐oxo‐2H‐1,4‐benzoxazin‐3(4H)‐
ylidene)acetate (1c)
Yield: 0.97 g (78%); yellow solid; mp 137–139°C (ethanol). 1H NMR
(DMSO‐d6): δ = 10.67 (s, 1H), 7.83 (m, 1H), 7.15 (m, 2H), 5.64 (s, 1H),
4.19 (q, J = 7.1 Hz, 2H), and 1.26 (t, J = 7.1 Hz, 3H) ppm. 13C NMR
(DMSO‐d6): δ = 167.7, 155.5, 139.1, 137.9, 126.2, 124.5, 118.3, 118.0,
C14H14N2O5: C 57.93; H 4.86; N 9.65. Found: C 58.19; H 4.89; N 9.64.
(3Z)‐6‐Bromo‐3‐(3,3‐dimethyl‐2‐oxobutylidene)‐3,4‐dihydro‐2H‐
1,4‐benzoxazin‐2‐one (1h)
116.5, 89.9, 59.6, and 14.1 ppm. IR: ν = 3,179, 1,771, and 1,668 cm−1
.
Yield: 0.95 g (73%); yellow solid; mp 153–154°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.14 (s, 1H), 7.85 (m, 1H), 7.18 (m, 2H), 6.38 (s, 1H),
and 1.18 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 206.1, 155.8, 139.9,
138.0, 125.8, 125.2, 118.8, 118.0, 116.5, 93.2, 42.4, and 26.6 (3C)
ppm. IR: ν = 3,177, 1,765, and 1,633 cm−1. MS (ESI+): m/z calcd for
C14H14BrNO3+H+: 324.02, 326.02 [M+H]+; found: 324.04, 326.04.
Anal. calcd (%) for C14H14BrNO3: C 51.87; H 4.35; N 4.32. Found: C
52.03; H 4.33; N 4.12.
MS (ESI+): m/z calcd for C12H10BrNO4+H+: 311.99, 313.99 [M+H]+;
found: 311.96, 313.96. Anal. calcd (%) for C12H10BrNO4: C 46.18; H
3.23; N 4.49. Found: C 46.00; H 3.30; N 4.43.
(3Z)‐3‐(3,3‐Dimethyl‐2‐oxobutylidene)‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1d)[14]
Yield: 0.81 g (83%); yellow solid; mp 86–88°C (ethanol; mp[14] 80–81°C).
1H NMR (DMSO‐d6): δ = 12.33 (s, 1H), 7.48 (m, 1H), 7.19 (m, 2H), 7.08 (m,
1H), 6.36 (s, 1H), and 1.18 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 206.1,
156.0, 140.6, 138.6, 125.1, 124.0, 123.0, 116.3, 116.2, 92.1, 42.3, and
26.7 (3C) ppm. IR: ν = 3,180, 1,772, and 1,632 cm−1. MS (ESI+): m/z calcd
for C14H15NO3+H+: 246.11 [M+H]+; found: 246.12. Anal. calcd (%) for
(3Z)‐3‐(3,3‐Dimethyl‐2‐oxobutylidene)‐6‐methyl‐3,4‐dihydro‐2H‐
1,4‐benzoxazin‐2‐one (1i)
Yield: 0.84 g (81%); yellow solid; mp 142–143°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.31 (s, 1H), 7.26 (m, 1H), 7.08 (m, 1H), 6.89 (m, 1H),

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6.34 (s, 1H), 2.28 (s, 3H), and 1.17 (s, 9H) ppm. 13C NMR (DMSO‐d6):
δ = 206.1, 156.1, 138.7, 134.6, 123.7, 123.5, 116.3, 115.9, 92.1, 42.3, 26.7
(3C), and 20.3 ppm. IR: ν = 3,161, 1,771, and 1,636 cm−1. MS (ESI+): m/z
calcd for C15H17NO3+H+: 260.13 [M+H]+; found: 260.18. Anal. calcd (%)
for C15H17NO3: C 69.48; H 6.61; N 5.50. Found: C 69.21; H 6.60; N 5.53.
6.13 (s, 1H), 2.39 (d, J = 7.1 Hz, 2H), 2.28 (s, 3H), 2.09 (m, 1H), and
0.92 (d, J = 6.6 Hz, 6H) ppm. 13C NMR (DMSO‐d6): δ = 200.7, 156.0,
138.7, 137.4, 134.7, 123.6, 123.5, 116.3, 115.9, 96.7, 51.4, 25.3, 22.3
(2C), and 20.3 ppm. IR: ν = 3,189, 1,768, 1,758, and 1,641 cm−1. MS
(ESI+): m/z calcd for C15H17NO3+H+: 260.13 [M+H]+; found: 260.15.
Anal. calcd (%) for C15H17NO3: C 69.48; H 6.61; N 5.50. Found: C
69.34; H 6.67; N 5.53.
(3Z)‐3‐(3,3‐Dimethyl‐2‐oxobutylidene)‐5‐methyl‐3,4‐dihydro‐2H‐
1,4‐benzoxazin‐2‐one (1j)
Yield: 0.86 g (83%); yellow solid; mp 121–122°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.77 (s, 1H), 7.06 (m, 3H), 6.39 (s, 1H), 2.34 (s, 3H), and
1.19 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 207.0, 155.7, 140.9, 139.2,
126.3, 123.5, 122.8, 122.1, 114.2, 92.0, 42.4, 26.8 (3C), and 15.6 ppm. IR:
ν = 3,172, 1,771, 1,756, and 1,631 cm−1. MS (ESI+): m/z calcd for
(3Z)‐5‐Methyl‐3‐(4‐methyl‐2‐oxopentylidene)‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1o)
Yield: 0.85 g (76%); yellow solid; mp 99°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.76 (s, 1H), 7.11 (m, 2H), 7.03 (m, 1H), 6.19 (s,
1H), 2.42 (d, J = 7.1 Hz, 2H), 2.34 (s, 3H), 2.11 (m, 1H), and 0.93 (d,
J = 6.8 Hz, 6H) ppm. 13C NMR (DMSO‐d6): δ = 201.6, 155.6, 141.0,
138.1, 126.3, 123.5, 122.8, 122.2, 114.2, 96.6, 51.3, 25.4, 22.3
(2C), and 15.6 ppm. IR: ν = 3,167, 1,768, and 1,633 cm−1. MS (ESI
+): m/z calcd for C15H17NO3+H+: 260.13 [M+H]+; found: 260.14.
Anal. calcd (%) for C15H17NO3: C 69.48; H 6.61; N 5.50. Found: C
69.69; H 6.63; N 5.47.
C
15H17NO3+H+: 260.13 [M+H]+; found: 260.17. Anal. calcd (%) for
C15H17NO3: C 69.48; H 6.61; N 5.50. Found: C 69.27; H 6.57; N 5.54.
(3Z)‐6‐Chloro‐3‐(3,3‐dimethyl‐2‐oxobutylidene)‐3,4‐dihydro‐2H‐
1,4‐benzoxazin‐2‐one (1k)
Yield: 0.85 g (76%); yellow solid; mp 149–150°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.15 (s, 1H), 7.72 (m, 1H), 7.21 (m, 1H), 7.07 (m, 1H),
6.39 (s, 1H), and 1.18 (s, 9H) ppm. 13C NMR (DMSO‐d6): δ = 206.1,
155.8, 139.5, 138.0, 128.7, 125.5, 122.3, 117.6, 115.9, 93.2, 42.4, and
26.6 (3C) ppm. IR: ν = 3,177, 1,763, and 1,636 cm−1. MS (ESI+): m/z
calcd for C14H14ClNO3+H+: 280.07 [M+H]+; found: 280.09. Anal.
calcd (%) for C14H14ClNO3: C 60.12; H 5.05; N 5.01. Found: C 60.32;
H 5.01; N 5.22.
(3Z)‐6‐Bromo‐3‐(4‐methyl‐2‐oxopentylidene)‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1p)
Yield: 0.92 g (71%); yellow solid; mp 130–131°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.06 (s, 1H), 7.84 (m, 1H), 7.17 (m, 2H), 6.17 (s, 1H), 2.41
(d, J = 7.1 Hz, 2H), 2.09 (m, 1H), and 0.93 (d, J = 6.6 Hz, 6H) ppm. 13C NMR
(DMSO‐d6): δ = 200.8, 155.7, 140.0, 136.6, 125.9, 125.2, 118.7, 118.0,
116.5, 97.8, 51.5, 25.2, and 22.3 (2C) ppm. IR: ν = 3,192, 1,766, and
1,646 cm−1. MS (ESI+): m/z calcd for C14H14BrNO3+H+: 324.02, 326.02
[M+H]+; found: 324.03, 326.03. Anal. calcd (%) for C14H14BrNO3:
C 51.87; H 4.35; N 4.32. Found: C 51.99; H 4.23; N 4.32.
(3Z)‐3‐(4‐Methyl‐2‐oxopentylidene)‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1l)
Yield: 0.77 g (79%); yellow solid; mp 108–110°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.25 (s, 1H), 7.48 (m, 1H), 7.19 (m, 2H), 7.08 (m, 1H),
6.14 (s, 1H), 2.40 (d, J = 7.1 Hz, 2H), 2.09 (m, 1H), and 0.93 (d, J = 6.8 Hz,
6H) ppm. 13C NMR (DMSO‐d6): δ = 200.7, 155.9, 140.7, 137.4, 125.1,
124.0, 123.0, 116.2, 116.2, 96.8, 51.4, 25.2, and 22.3 (2C) ppm. IR:
(3Z)‐3‐[2‐(4‐Fluorophenyl)‐2‐oxoethylidene]‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1q)[23]
Yield: 0.90 g (80%); yellow solid; mp 186–187°C (toluene; mp[23]
190–192°C). 1H NMR (DMSO‐d6): δ = 12.78 (s, 1H), 8.10 (m, 2H),
7.59 (m, 1H), 7.35 (m, 2H), 7.24 (m, 2H), 7.14 (m, 1H), and 6.87 (s,
1H) ppm. 13C NMR (DMSO‐d6): δ = 188.0, 165.8, 163.3, 155.8, 141.1,
139.9, 134.7 (2C), 130.0, 129.9, 125.2, 123.8, 123.6, 116.7, 116.3,
115.8, 115.6, and 92.4 ppm. IR: ν = 3,100, 1,752, and 1,622 cm−1. MS
(ESI+): m/z calcd for C16H10FNO3+H+: 284.07 [M+H]+; found: 284.05.
Anal. calcd (%) for C16H10FNO3: C 67.84; H 3.56; N 4.94. Found: C
68.02; H 3.55; N 4.89.
ν = 3,177, 1,758, and 1,641 cm−1
C14H15NO3+H+: 246.11 [M+H]+; found: 246.13. Anal. calcd (%) for
14H15NO3: C 68.56; H 6.16; N 5.71. Found: C 68.79; H 6.06; N 5.77.
.
MS (ESI+): m/z calcd for
C
(3Z)‐6‐Chloro‐3‐(4‐methyl‐2‐oxopentylidene)‐3,4‐dihydro‐2H‐
1,4‐benzoxazin‐2‐one (1m)
Yield: 0.84 g (75%); yellow solid; mp 135–136°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.07 (s, 1H), 7.71 (m, 1H), 7.21 (m, 1H), 7.07 (m, 1H),
6.17 (s, 1H), 2.41 (d, J = 6.8 Hz, 2H), 2.10 (m, 1H), and 0.93 (d, J = 6.6 Hz,
6H) ppm. 13C NMR (DMSO‐d6): δ = 200.8, 155.7, 139.5, 136.6, 128.7,
125.5, 122.3, 117.6, 115.9, 97.8, 51.5, 25.1, and 22.3 (2C) ppm. IR:
|
4.1.4
Procedure for the synthesis of the open‐ring
ν = 3,204, 1,763, and 1,648 cm−1
.
MS (ESI+): m/z calcd for
analog of BO (4)
C14H14ClNO3+H+: 280.07 [M+H]+; found: 280.08. Anal. calcd (%) for
C14H14ClNO3: C 60.12; H 5.05; N 5.01. Found: C 60.24; H 5.12; N 5.03.
Diethyl oxaloacetate 3d (4.0 mmol, 0.75 g) was added to a suspension
of N‐(2‐aminophenyl)acetamide (4.0 mmol, 0.6 g) in ethanol (10 ml).
The reaction mixture was refluxed for 5 min and then cooled to
ambient temperature. The formed yellow precipitate was filtered off,
washed with ethanol (5 ml), and recrystallized from ethanol (10 ml) to
give compound 4.
(3Z)‐6‐Methyl‐3‐(4‐methyl‐2‐oxopentylidene)‐3,4‐dihydro‐2H‐1,4‐
benzoxazin‐2‐one (1n)
Yield: 0.81 g (73%); yellow solid; mp 89–90°C (ethanol). 1H NMR
(DMSO‐d6): δ = 12.25 (s, 1 H), 7.26 (m, 1H), 7.09 (m, 1H), 6.89 (m, 1H),

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Diethyl 2‐(2‐acetamidoanilino)but‐2‐enedioate (4)
compounds of 1,000 μg/ml. The aliquots of obtained solutions were
treated by twofold serial dilutions and added to mycobacteria growth
indicator tubes (MGITs) in quantities that provide the final concentra-
tions (µg/ml): 20.0, 10.0, 5.0, 2.5, 1.25, 0.6, and 0.31. Each MGIT tube
contained 7 ml of Middlebrook 7H9 sterile nutrient broth. Then, 0.8 ml
of ВАСТЕС MGIT OADC (oleic acid, albumin, dextrose, and catalase)
growth supplement was added to each tube. In addition to the liquid
medium, the tubes contained an oxygen‐free fluorochrome, tris‐4,7‐
diphenyl‐1,10‐phenanthroline ruthenium chloride pentahydrate (Ru
[dpp]), placed at the bottom of the tube and coated with silicone.
A 1.0‐McFarland suspension of Mtb (5 × 108 microbial cells per 1 ml)
was prepared using a densitometer. Next, a solution was prepared by
diluting the initial suspension 10 times with a sterile physiological saline
solution, thus resulting in 5 × 107 microbial cells per 1 ml. Then, a sus-
pension of Mtb (0.5 ml) was added to each of the above‐prepared MGITs.
In parallel, the inoculum of Mtb was loaded into control MGITs with
Middlebrook 7H9 broth (0.5 ml), each containing no test compounds. For
control, similar experiments were carried out with isoniazid (isonicotinic
acid hydrazide, 99%; Sigma‐Aldrich).
Yield: 0.85 g (66%); yellow solid; mp 99–101°C (ethanol). 1H NMR
(DMSO‐d6): δ = 9.88 (s, 1H), 9.40 (s, 1H), 7.26 (m, 1H), 7.13 (m, 2H), 6.81
(m, 1H), 5.22 (s, 1H), 4.12 (q, J = 7.1 Hz, 2H), 4.01 (q, J = 7.1 Hz, 2H), 2.08
(s, 3H), 1.23 (t, J = 7.1 Hz, 3H), and 0.96 (t, J = 7.1 Hz, 3H) ppm. 13C NMR
(DMSO‐d6): δ = 168.7, 167.4, 163.8, 148.0, 135.1, 130.6, 125.7, 125.1,
124.8, 122.6, 93.4, 61.5, 59.3. 22.8, 14.1, and 13.2 ppm. IR: ν = 3,184,
1,741, 1,686, and 1,663 cm−1. MS (ESI+): m/z calcd for C16H20N2O5+H+:
321.15 [M+H]+; found: 321.19. Anal. calcd (%) for C16H20N2O5: C 59.99;
H 6.29; N 8.74. Found: C 60.21; H 6.19; N 8.92.
|
4.1.5
Procedures for the syntheses of the
annelated analogs of BOs (5a,b)
Oxalyl chloride (6 mmol, 0.5 ml) was added to a suspension of BO 1q
(4 mmol, 1.1 g) in 20 ml of benzene. The reaction mixture refluxed for
110 min (gaseous HCl evolved) and then cooled to ambient tem-
perature. The formed dark violet solid of compound 5a was fil-
tered off.
All tubes were incubated at 37°C and analyzed by ВАСТЕС
MGIT 960. If the compound is active against Mtb, it inhibits its
growth and suppresses fluorescence of Ru(dpp), whereas, in the
control tube, growth is not inhibited and, accordingly, the level of
fluorescence in this tube is pronounced. During bacterial growth, free
oxygen is consumed inside the tubes and replaced with CO2. As free
oxygen is consumed, inhibition of the fluorochrome, Ru(dpp), is
stopped. Fluorescence becomes detectable when the test tube is ir-
radiated with UV light and is automatically registered by photo-
sensors of ВАСТЕС MGIT 960. The minimum dilution of the
examined compound, in which the growth was not registered by
ВАСТЕС, was taken as MBC. All assays were carried out in duplicate.
Oxalyl chloride (6 mmol, 0.5 ml) was added to a suspension of BO
1b (4 mmol, 1.1 g) in 10 ml of benzene. The reaction mixture refluxed
for 110 min (gaseous HCl evolved) and then cooled to ambient
temperature. The solvent was removed under vacuum. The resulting
dark violet solid of compound 5b was grinded with hexane.
3‐(4‐Fluorobenzoyl)‐1H‐pyrrolo[2,1‐c][1,4]benzoxazine‐1,2,4‐
trione (5a)[23]
Yield: 1.22 g (91%); dark violet solid; mp 212–214°C (decomp., benzene;
mp[23] 214–216°C). 1H NMR (DMSO‐d6): δ = 8.38 (m, 1H), 8.22 (m, 2H),
and 7.35 (m, 5H) ppm. MS (ESI+): m/z calcd for C18H8FNO5+H2O+H+:
356.06 [M+H2O+H]+; found: 356.10. Anal. calcd (%) for C18H8FNO5: C
64.10; H 2.39; N 4.15. Found: C 64.12; H 2.44; N 4.32.
|
4.2.2
Tuberculostatic activity by REMA
Ethyl 1,2,4‐trioxo‐2,4‐dihydro‐1H‐pyrrolo[2,1‐c][1,4]benzoxazine‐3‐
carboxylate (5b)[24]
A 1.0‐McFarland suspension of Mtb (H37Rv) was prepared and 0.1 ml
was added to each of the following bottles: A direct control (bottle
containing diluent, DMSO, but no test compound), a control con-
taining a 1:100 organism dilution (also without test compound), and
each concentration with the test compound.
Yield: 0.84 g (73%); dark violet solid; mp 156°C (decomp., hexane;
mp[24] 156–157°C). 1H NMR (CDCl3): δ = 7.21–7.34 (m, 4H), 4.42 (m,
2H), and 1.39 (m, 3H) ppm. MS (ESI+): m/z calcd for C14H9NO6+H2O
+H+: 306.06 [M+H2O+H]+; found: 306.02. Anal. calcd (%) for
C14H9NO6: C 58.54; H 3.16; N 4.88. Found: C 58.34; H 2.91; N 4.81.
From a culture of Mtb, which was in a log phase of growth in the
Löwenstein–Jensen nutrient medium, a 1.0‐McFarland suspension
was prepared with a sterile physiological saline solution and 0.2%
solution of Tween 80. The resulting suspension (50 μl) was trans-
ferred into a tube with 7H9 broth and OADC additive to give Mtb
concentration of 1.5 × 106 CFU/ml.
|
4.2
Pharmacological/biological assays
|
4.2.1
Bactericidal activity by BACTEC
Initial stock solutions (1,000 μg/ml) and subsequent dilutions of test
compounds were prepared in DMSO. Further dilutions were performed
by twofold dilutions of the stock solution with pure DMSO, that is, so-
lutions with concentrations of 500, 250, 125, 62.5, 31.25, 15.63, and
7.81 μg/ml were obtained. The resulting solutions of the studied com-
pounds (3.1 μl) were loaded into the wells of a 96‐well plate, with each
containing a culture medium (97 μl). Then, a suspension of Mtb (100 μl),
The bactericidal activity of chemical compounds against Mtb (H37Rv)
was studied by a standard ВАСТЕС MGIT 960 radiometric growth
system (Becton Dickinson).
Initial stock solutions of test compounds (10 mg/ml) were pre-
pared in DMSO. Then, they were diluted with Middlebrook 7H9
sterile nutrient broth (9 ml) to result in the concentration of test

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Docking procedure
prepared as described above, was added to all wells of the plate. Final
concentrations of test compounds in wells of a 96‐well plate were 15.6,
7.8, 3.9, 1.95, 0.97, 0.49, 0.24, and 0.12 μg/ml; the final concentration of
DMSO in wells was 1.6%. An Mtb culture without addition of compounds
(100 μl of pure medium + 100 μl of a suspension of Mtb) and a culture of
Mtb with DMSO (97 μl of a pure medium + 3.1 μl of DMSO + 100 μl of a
suspension of Mtb) were used as a positive control. For control, similar
experiments were carried out with isoniazid (initial stock solution and
subsequent dilutions of isoniazid were prepared in water; final con-
centrations in wells of a 96‐well plate were 0.007, 0.015, 0.031, 0.063,
0.125, 0.25, and 0.5 μg/ml).
4.3.3
Ligand–protein docking was performed in GOLD suit (2020.0 CSD
Release).
For protein structures having a native ligand (substrate or inhibitor),
the binding site was determined as a space within a radius of 10–20 Å
from the cavity of the deleted native ligand. For protein structures
without a native ligand, the binding site was predicted by the comparison
of docking solutions of ligand 3a in the whole protein; the place where
the ligand was most often located was considered as a binding site, and
the final docking procedure for all examined ligands was performed in the
space within a radius of 14–17 Å from a selected solvent‐accessible atom
from the predicted cavity for ligand 3a.
The plates were incubated at 37°C for 7 days. Then, 50 μl of a
resazurin (7‐hydroxy‐3H‐phenoxazin‐3‐one 10‐oxide) solution (with the
addition of Tween 80) was added to the wells and the incubation was
continued at 37°C. The results were monitored after 24, 48, and 72 hr.
The minimum dilution of the examined compound in which there was no
change in the blue‐violet color of resazurin in all replicates was taken as
MIC. All assays were carried out in triplicate.
Ligands were set as flexible with rotatable bonds. For ligands
bearing the BO motif, rotatable bonds were overridden to make en-
aminone pattern flat and rigid (it was necessary to take into account the
fact that BOs exist as enamino tautomers with Z‐configuration of the
exocyclic C═C, which is stabilized by the intramolecular chelate‐type
HB). Proteins were treated as rigid. For superimposed proteins, en-
semble docking was applied to consider possible protein flexibility.
ChemScore was used as a fitness function. If the top three solutions
were within 1 Å, the number of genetic algorithm runs was terminated
early. All other parameters and options in GOLD were used as default.
All calculations were carried out in triplicate. Statistical treatment
was carried out using GraphPad Prism 8 (8.0.1 release) program. Best
docking poses for each ligand were saved as *.pdb files in Hermes. Then
protein–ligand interactions were determined by BIOVIA Discovery Stu-
dio Visualizer (Discovery Studio 2020 v20.1.0.19295) using default
parameters and options.
|
4.3
Molecular docking
|
4.3.1
Ligands preparation
Two‐dimensional structures of ligands were generated using the
ChemDraw panel in ChemBio3D Ultra 14.0. 3D structures of ligands
were generated by ChemBio3D Ultra 14.0. Energy minimization of ligand
structures was performed by «MM2 minimize» function implemented in
ChemBio3D Ultra 14.0. The SYBYL2 (*.mol2) format of structures was
saved and used for docking study. In each structure bearing BO motif,
serial numbers of atoms corresponding to the motif were set manually in
ChemBio3D Ultra 14.0 to match each other in each structure.
ACKNOWLEDGMENTS
This study was supported by the Ministry of Science and Higher
Education of the Russian Federation (FSNF‐2020‐0008) and the
Government of Perm Krai. The authors thank the Ural Research
Institute of Phthisiopulmonology (Yekaterinburg, Russia) for per-
forming biological studies in vitro by REMA and Clinical Phthisio-
pulmonological Medical Center (Krasnokamsk, Russia) for performing
biological studies in vitro by BACTEC.
|
4.3.2
Proteins preparation
The crystal structures of proteins were downloaded from the Protein
Data Bank (PDB; www.rcsb.org). Proteins preparation for docking
was performed using the GOLD Docking Wizard in GOLD suit
(2020.0 CSD Release). The proteins' preparation included the addi-
tion of hydrogen atoms, deletion of water molecules, and deletion of
ligands (except for cofactors). For proteins available at PDB as sev-
eral separate structures, two or three different structures were
downloaded and superimposed at A‐chains in GOLD Docking Wizard
to best match weighting of 10.0 to consider protein flexibility.
Before docking to 6p1y, a tetramer of 6p1y was prepared by su-
perimposition with chains of each subunit of a 6p02 tetramer in Hermes,
the 3D visualizer provided with the CSD‐Discovery Suite (2020.0 CSD
Release), to best match weighting of 1.0. All waters and ligands in 6p1y
were deleted and the resulting 6p1y structures were saved separately as
*.pdb files in Hermes. The resulting *.pdb files were combined in Notepad
for Windows 10 and saved as a single *.pdb file, which was re‐saved in
Hermes to afford the *.pdb file that was used for docking.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interest.
ORCID
Ekaterina E. Stepanova
http://orcid.org/0000-0002-5851-3082
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How to cite this article: Stepanova EE, Balandina SY,
Drobkova VA, Dmitriev MV, Mashevskaya IV, Maslivets AN.
Synthesis, in vitro antibacterial activity against Mycobacterium
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