Design, synthesis and antimycobacterial activity of benzoxazinone derivatives and open-ring analogues: Preliminary data and computational analysis

Article abstract of DOI:10.1016/j.bmcl.2019.07.025

This study examines in depth benzoxazine nucleus for antimycobacterial property. We synthesized some benzoxazin-2-one and benzoxazin-3-one derivatives, which were tested for activity against a panel of Mycobacterium tuberculosis (Mtb) strains, including H37Ra, H37Rv and some resistant strains. Several compounds displayed a high antimycobacterial activity and the three isoniazid analogue derivatives 8a-c exhibited a MIC range of 0.125–0.250 μg/mL (0.37–0.75 μM) against strain H37Ra, therefore lower than the isoniazid reference drug. Two benzoxazin-2-one derivatives, 1c and 5j, together with isoniazid-analogue compound 8a, also revealed low MIC values against resistant strains and proved highly selective for mycobacterial cells, compared to mammalian Vero cells. To predict whether molecule 8a is able to interact with the active site of InhA, we docked it into the crystal structure; indeed, during the molecular dynamic simulation the compound never left the protein pocket. The more active compounds were predicted for ADME properties and all proved to be potentially orally active in humans.

Full text of DOI:10.1016/j.bmcl.2019.07.025

Accepted Manuscript
Design, synthesis and antimycobacterial activity of benzoxazinone derivatives
and open-ring analogues: preliminary data and computational analysis
Daniele Zampieri, Maria Grazia Mamolo, Julia Filingeri, Sara Fortuna,
Alessandro De Logu, Adriana Sanna, Davide Zanon
PII:
S0960-894X(19)30470-6
https://doi.org/10.1016/j.bmcl.2019.07.025
BMCL 26566
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 June 2019
12 July 2019
16 July 2019
Please cite this article as: Zampieri, D., Mamolo, M.G., Filingeri, J., Fortuna, S., De Logu, A., Sanna, A., Zanon,
D., Design, synthesis and antimycobacterial activity of benzoxazinone derivatives and open-ring analogues:
preliminary data and computational analysis, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.07.025
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and antimycobacterial activity of benzoxazinone derivatives and
open-ring analogues: preliminary data and computational analysis.
Daniele Zampieria*, Maria Grazia Mamoloa, Julia Filingeria, Sara Fortunaa, Alessandro De Logub, Adriana
Sannac and Davide Zanond.
aDepartment of Chemistry and Pharmaceutical Sciences, University of Trieste, P.le Europa 1-Via Giorgeri 1, 34127 Trieste, Italy.
bDepartment of Life and Environmental Sciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy.
cDepartment of Public Health, Clinical and Molecular Medicine, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy.
dPharmacy and Clinical Pharmacology Department, Institute for Maternal and Child Health IRCCS Burlo Garofolo, Via dell’Istria 65/1 34137, Trieste, Italy.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
This study examines in depth benzoxazine nucleus for antimycobacterial property. We
synthesized some benzoxazin-2-one and benzoxazin-3-one derivatives, which were tested for
activity against a panel of Mycobacterium tuberculosis (Mtb) strains, including H37Ra, H37Rv
and some resistant strains. Several compounds displayed a high antimycobacterial activity and the
three isoniazid analogue derivatives 8a-c exhibited a MIC range of 0.125-0.250 g/mL (0.37-0.75
M) against strain H37Ra, therefore lower than the isoniazid reference drug. Two benzoxazin-2-
one derivatives, 1c and 5j, together with isoniazid-analogue compound 8a, also revealed low MIC
values against resistant strains and proved highly selective for mycobacterial cells, compared to
mammalian Vero cells. To predict whether molecule 8a is able to interact with the active site of
InhA, we docked it into the crystal structure; indeed, during the molecular dynamic simulation the
compound never left the protein pocket. The more active compounds were predicted for ADME
properties and all proved to be potentially orally active in humans.
Keywords:
benzoxazinone
oxoacetamide
antimycobacterial activity
molecular dynamics
ADME
2009 Elsevier Ltd. All rights reserved.
More than seventy years have passed since the discovery of
streptomycin and sixty-six since isoniazid was synthesized and,
despite immense efforts made by the scientific community,
tuberculosis (TB) still remains one of the most widespread diseases
in the world.
Currently there are several mycobacterial targets such as 2-trans-
enoyl-ACP (CoA) reductase (InhA),2 alanine-racemase,3
metioninaminopeptidase (MetAP),4 isocitrate lyase (ICL),5
menaquinone-B (MenB),6 decaprenyl-phosphoryl--D-ribose
oxidase (DprE1)7 and others, most of which act on the
mycobacterial cell-wall.
TB is one of the top 10 causes of death worldwide and, in the last
few years, it has been the leading cause of death from a single
infectious agent, ranking above HIV/AIDS. Diagnosis and
successful treatment of people with TB averts millions of deaths
each year (an estimated 54 million over the period 2000–2017), but
there are still large and persistent gaps in detection and treatment1.
Mycobacterium tuberculosis (Mtb) is the causative agent of the
disease, for which treatment regimens are both lengthy and toxic.
Furthermore, the spread of multidrug-resistant strains (MDR)
worsen the course of disease. In spite of some new drugs, such as
Bedaquiline and Delamanid, there is a pressing need for new
chemical entities with a clear mechanism of action and a selective
profile against Mtb, in particular against resistant strains.
Benzoxazine moiety is present in several structures endowed
with various activities, such as antiallergics,8 antimicrobials,9
antimycobacterials6 and antifungals.10 The antimycobacterial
activity of some benzoxazin-2-one compounds is due to the
inhibition of MenB, a synthetic pathway involving menaquinone,
one of the essential components of the electron transport chain in
many pathogens including mycobacteria (in humans this role is
played by ubiquinone). This is the starting point of the current work,
which focuses on exploring the chemical significance of the
benzoxazine nucleus for antimycobacterial activity.
 Corresponding author. Tel.: +39-040-5587858; fax: +39-040-52572; e-mail: dzampieri@units.it

(Z)-methyl-2-(2-oxo-2H-benzo[b][1,4]oxazin-3(4H)-
ylidene)acetate 1a was the lead compound of a series of derivatives
selected through HTPS by Li and coworkers.6 The authors studied
the effect of the substitution on the aromatic portion of the
benzoxazin-2-one core with different groups such as Cl-, F-, CH3-
NO2-, both on antimycobacterial activity and on enzymatic
inhibition (Fig. 1). In addition to the unsubstituted compound, which
exhibited the best value of enzymatic inhibition (IC50 = 10 M) and
MIC value 0.64 g/mL (2.9 M), the work highlighted the
importance of the substitution with a halogenated atom (F and Cl)
on the aromatic ring of the benzoxazin-2-one core, in particular they
found the same antimycobacterial activity for the 7-Cl substituted
derivative (1c) (MIC = 0.63 g/mL) and a decreased activity when
the substituent was in position C-6 (1b) (MIC = 5 g/mL) and a
slight increase of the enzymatic inhibition values (IC50 = 46.3 and
35.7 M respectively).
Figure 2. (a) Structure of benzoxazin-2-one compounds 2a-c; (b) Structure
of reduced compounds 3 and 4; (c) Structure of title compounds 5a-l.
Subsequently, we decided to synthesize the isoster (E)-methyl-2-
(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-ylidene)acetate
6
and some analogues substituted both on nitrogen atom and on
vinylic function, 7a-f and three INH-analogues 8a-c, fusing the
(substituted)benzoxazine-2,3-dione core with INH. Unexpectedly,
the reaction to obtain the benzoxazin-3-one-INH hybrids led to an
open-ring form, corresponding to the N-(2-hydroxyphenyl)-2-(2-
isonicotinoylhydrazinyl)-2-oxoacetamide 8a and its chloro-
substituted analogues 8b,c (Fig. 3, Fig. SI 1 and Scheme 1f).
Figure 1. Structure of benzoxazin-2-one reference compound 1a.
In order to assess the relevance of the chemical space around the
oxazine core and its importance for antimycobacterial activity, we
planned to synthesize reference compounds 1a-c and a second
generation of benzoxazin-2-one derivatives, and test them against
several mycobacterial strains. The aim was to evaluate
modifications such as the introduction of bulky substituents (2a,b),
or a heterocycle ring (2c) (Fig. 2a), as well as reductions of the
exocyclic double bond, 3 and the oxazinone carbonyl group, 4 of the
original lead compound 1a, as depicted in Fig. 2b. Furthermore, we
replaced the original ester functional group with several aroyl
groups, 5a-l (Fig. 2c).
Figure 3. Structure of the isosteric benzoxazin-3-one derivatives 6 and 7a-f
and oxoacetamide-INH compounds 8a-c.
The synthetic route to prepare all the benzoxazinone derivatives
is depicted in Scheme 1.

O
O
b)
X
O
O
COOCH3
a) (Yield: 45-61%)
From 1a
N
H
R1
(R1 = H, X = CH)
(Yield: 99%)
3
N
H
H3COOC
C
C
COOCH3
O
OH
COOCH3
c)
R1 = H, 6-Cl, 7-Cl
X =CH
R1 = H, 6Ph; Napht
X = N, CH
1a-c
2a-c
(Yield: 67%)
COOCH3
N
H
4
Ar
CH3
d)
O
OH
O
O
O
OCH2CH3
Ar
e)
(Yield: 12-99%)
(9a-l)
O
N
H
R1 = H; X = CH
O
Ar
Ar = Ph, 2-Cl, 3-Cl, 4-Cl, 2-CH3,
2-CH3, 3-CH3, 4-CH3, Ph-4-Ph,
4-Py, 3-Ind., 2-Tioph., 1-morph.
5a-l
O
O
O
R1 = H; X = CH
O
OH
Br
X
OH
g)
COOH
COOCH3
O
Br
f)
R1
R1 = H; X = CH
(Yield: 41%)
N
H
O
N
H
NH2
(10)
6
R1 = H, 4-Cl, 5-Cl,
6-Ph; Napht
X = CH, N
Ar
O
1)
Ar-CHO
O
O
h)
i)
j)
(Yield: 25-95%)
N
Z
R1 = H; X = CH
N
O
H
CH3-I or PhCH2-Cl
2)
(11)
Ar = Ph, 2-ClPh
Z = H, CH3, CH2Ph
7a-f
H
N
O
H
N
N
CONHNH2
R1
O
N
O
N
(INH)
O
N
O
O
H
N
H
N
k)
l)
R1
N
R1
H
R1 = H, 4-Cl, 5-Cl
X = CH
N
O
O
O
(Yield: 58-82%)
H
OH
(12a-l)
R1 = H, 4-Cl, 5-Cl
8a-c
Reagents and conditions: a) MeOH, rt, N2; b) EtOH, H2-Pd/C, rt; c) MeOH, NaBH4,rt; d) EtOH, NaOEt, rt; e) AcOH, reflux; f) 1,4-
Dioxane, rt; g) MeOH, Et3N, reflux; h) CH2Cl2, K2CO3, Bromoacetyl bromide, 0°C then rt; i) DMF, NaOMe, reflux; j) Acetone, K2CO3/KI
cat, reflux; k) Toluene, Oxalyl chloride, reflux; l) EtOH, reflux.
Scheme 1. Synthetic route of title compounds 1-8
The
lead
compound
(Z)-methyl
2-(2-oxo-2H-
aminonaphtalen-2-ol and 2,4-diaminophenol, the same synthetic
pathway led to analogue compounds 2a-c. All these derivatives are
in enamine form (Z configuration) due to the stabilization of the
intramolecular H-bond between oxazine NH group and exocyclic
carbonyl ester (Fig. 4); the treatment of compound 1a with H2/Pd-
benzo[b][1,4]oxazin-3(4H)-ylidene)acetate 1a and its halogenated
analogues 1b,c, were obtained from 2-aminophenol, 4-
chloroaminophenol or 5-chloroaminophenol and dimethyl but-2-
ynedioate;
starting
from
3-amino-[1,1'-biphenyl]-4-ol,3-

C10% or NaBH4 yield the corresponding reduced compounds 3 and
4, respectively. Series 5a-l was synthesized by Claisen condensation
between several arylethanones and diethyl oxalate in basic media
(NaOEt), and then the substituted butenoate intermediates 9a-l were
cyclized with 2-aminophenol in acetic acid. The synthesis of (E)-
atom such as nitrogen (2c) into the aromatic portion of benzoxazine
core, worsens the antimycobacterial activity.
Investigating more thoroughly the original benzoxazin-2-one
scaffold (1a), the corresponding reduced compounds 3 and 4
provided interesting information. In bgh<fact, the title compound 3,
carrying a reduced exocyclic double bond, showed a two-fold higher
MIC value 8 g/mL compared to the lead compound 1a, while
derivative 4, having a hydroxyl group in place of the carbonyl group
in position 2, proved to be a weak Mtb inhibitor (MIC = 64 g/mL).
The latter data indicate the necessary presence of an H-bond
acceptor group at position C2 (Fig. 2b) of the benzoxazin-2-one
core, instead of an H-donor group (OH), while the increased
mobility of the ester side chain due to the saturation of the double
bond seems to have only a slight effect on the antimycobacterial
activity. Series 5a-l gave the best results of the benzoxazin-2-one
series, in terms of inhibition of mycobacteria growth, in fact all the
compounds proved to be more active, with MIC range of 2-4 g/mL,
than lead compound 1a. In particular, heterocyclic substituted
derivatives 5j-l were found to be the most active molecules with
MIC of 2 g/mL. The slight difference in terms of MIC values
within the series highlights the importance of the aromatic, bulky
substitution in the side chain of the benzoxazin-2-one scaffold in
place of an exclusively ester functional group and, particularly, the
significance of the heterocyclic substitution. This information
differs slightly from previous results in terms of antimycobacterial
activity of a small series of substituted (Z)-3-[2-(2-chlorophenyl)-2-
oxoetylidene]-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one
methyl
2-(3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-
ylidene)acetate 6 was carried out via 2-step reaction involving the
formation of intermediate (Z)-4-((2-hydroxyphenyl)amino)-2-
(methoxycarbonyl)-4-oxobut-2-enoic acid 10 obtained by the
reaction of 2-aminophenol and 3-bromofuran-2,5-dione in 1,4-
dioxane at room temperature; the subsequent cyclization with
triethylamine at reflux temperature provided the desired product 6
in E configuration (94%). Derivatives 7a-f were synthesized by
treating 2H-benzo[b][1,4]oxazin-3(4H)-one 11 with benzaldehyde
(7a), or 2-chlorobenzaldehyde (7b) in the presence of sodium
methoxide in DMF at reflux temperature and subsequently N-
alkylated with methyl iodide (7c,d) or benzyl chloride (7e,f). All the
derivatives synthesized were in Z configuration (Fig. 4) due to the
steric hindrance of the substituted phenyl moiety on the cyclic
carbonyl group. The INH-analogue molecules 8a-c were obtained
by treating isoniazid (INH) with 2H-benzo[b][1,4]oxazine-2,3(4H)-
dione intermediate 12a and the substituted 6-chloro 12b and 7-
chloro 12c intermediates, respectively, with a typical Schiff reaction
to yield the oxoacetamide compounds 8a-c, instead of the expected
benzoxazin-3-one-INH hybrids.
compounds;6 in fact the authors found that all the series proved to
be less active than lead ester compound 1a, against Mtb.
Curiously, isoster benzoxazin-3-one derivatives 6 and 7a-f are
weak inhibitors of Mtb, revealing MIC value of 64 g/mL and
contradicting the bioisosterism principle. INH analogue derivatives
8a-c were also tested against Mtb H37Ra and the data obtained
showed a MIC range of 0.125-0.250 g/mL, with the best inhibition
value for unsubstituted and 5-chloro-substituted derivatives 8a,b
(MIC = 0.125 g/mL or 0.37 M), more than twice as powerful of
INH in terms of micro molar MIC (0.125 g/mL or 0.91 M), thus
proving to be the best candidates of the entire series.
Figure 4. H-bond involved in benzoxazin-2-one compounds.
Lead compound (Z)-methyl-2-(2-oxo-2H-benzo[b][1,4]oxazin-
3(4H)-ylidene)acetate 1a, and its halogenated analogues 1b,c,
revealed MIC values of 4 g/mL for 1a, whereas values of 16
g/mL and 2 g/mL were found for 1b and 1c, respectively. The
bulky benzoxazin-2-one derivatives 2a,b, as well as the heterocycle
derivative pyridoxazin-2-one 2c, were devoid of antimycobacterial
activity (MIC > 64 g/mL). These findings confirm that the
increased steric hindrance (2a,b), or the presence of a protonable
Conversely to what happens with series 1a,c, where the position
of the electron-withdrawing substituent on the benzoxazine
aromatic core markedly influences antimycobacterial activity, with
oxoacetamide-INH derivatives 8a,c, this pattern is less significant.
Table 1. Antimycobacterial activity, MIC g/mL (M), of title compounds 1-8 against Mtb strain H37Ra and cytotoxicity of
compounds 1a-c, 3, 5a-l and 8a-c, in comparison with reference compounds INH against Vero cell line.
Cpd
X
Y
R1
Z
M. tuberculosis ATCC25197 H37Ra
MIC CC50
S.I.a

g/mL (M)
4 (18)
g/mL (M)
500 (> 2281)
413 (1628)
405 (1596)
ntb
1a
1b
1c
2a
2b
C=O
C=O
C=O
C=O
C=O
C=CH-COOCH3
C=CH-COOCH3
C=CH-COOCH3
C=CH-COOCH3
C=CH-COOCH3
H
H
H
H
H
H
> 125
26
6-Cl
7-Cl
6-Ph
16 (63)
2 (7.9)
202
nt
64 (145)
> 64 (> 238)
nt
nt
2c
C=O
C=CH-COOCH3
H
> 64 (> 217)
nt
nt
3
C=O
CH-CH2-COOCH3
C=CH-COOCH3
C=CHCO-Ph
C=CHCO-2-ClPh
C=CHCO-3-ClPh
C=CHCO-4-ClPh
C=CH-CO-o-tolyl
C=CH-CO-m-tolyl
C=CH-CO-p-tolyl
C=CH-CO-4-PhPh
C=CH-CO-4-Pyr
C=CH-CO-3-Indol
C=CH-CO-2-Thiof
C=CH-CO-1-Morph
C=O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5-Cl
4-Cl
-
H
8 (36)
342 (1546)
nt
43
4
CH-OH
H
64 (289)
4 (15)
nt
5a
5b
5c
5d
5e
5f
C=O
H
> 250 (> 942)c
472 (1573)
482 (1606)
375 (1251)
> 500 (> 1790)
> 500 (> 1790)
461 (1650)
> 125 (> 366)c
> 500 (> 1878)
> 500 (> 1643)
> 500 (> 1845)
310 (1138)
nt
> 62
118
120
94
C=O
H
4 (13)
C=O
H
4 (13)
C=O
H
4 (13)
C=O
H
4 (14)
> 125
> 125
115
> 30
> 125
> 250
> 250
155
nt
C=O
H
4 (14)
5g
5h
5i
C=O
H
4 (14)
C=O
H
4 (12)
C=O
H
4 (15)
5j
C=O
H
2 (6.5)
5k
5l
C=O
H
2 (7.3)
C=O
H
2 (7.3)
6
C=CH-COOCH3
H
64 (291)
64 (270)
64 (238)
64 (127)
64 (224)
64 (196)
64 (177)
0.125 (0.42)
0.125 (0.37)
0.250 (0.75)
0.125 (0.91)
7a
7b
7c
7d
7e
7f
C=CH-Ph
C=O
H
nt
nt
C=CH-2ClPh
C=O
H
nt
nt
C=CH-Ph
C=O
CH3
nt
nt
C=CH-2ClPh
C=O
CH3
nt
nt
C=CH-Ph
C=O
CH2-Ph
nt
nt
C=CH-2ClPh
C=O
CH2-Ph
nt
nt
8a
8b
8c
INH
-
-
-
-
-
-
-
-
-
423 (1409)
402 (1201)
390 (1165)
>1000 (>7300)
3384
3316
1560
>8000
-
-
-
aCC50/MIC H37Ra; bnot tested; cMaximum solubility (DMSO) beyond which it precipitates in the medium.

The most interesting compounds were chosen to evaluate their in
vitro cytotoxicity in the mammalian Vero cell line using the MTT
colorimetric assay. The results are reported both as CC50 and
Selectivity Index, S.I. (Table 1). The S.I. is an evaluation of the
selectivity of the molecules between eukaryotic and mycobacterial
cells, thus a value of safety of our title compounds. According to a
study of Hartkoorn and coworkers11 on TB drug susceptibility,
antimycobacterial activity is considered to be specific when the
selectivity index is > 10 and our compounds turned out to be much
more selective than required. In fact, the collected data are in the
range of 125-500 g/mL in terms of CC50 and a selectivity index of
26-3384. Interestingly, the most active molecules against strain
H37Ra, 1a-c and 8a-c, also proved to be the safest with CC50
ranging from 390 to 500 g/mL.
the pyrazinamide-, rifampicin- and streptomycin-resistant strains.
Indeed, all the compounds exhibited a marked antimycobacterial
activity, with MIC range of 1-16 g/mL. The best results were
obtained by 7-chloro- and indolyl-derivatives of the benzoxazine-2-
one series, 1c and 5j, both with MIC values of 1 g/mL.
The X-ray structure of the enzyme inhibitor complex has been
known for two decades (1ZID)12 showing the InhA bound to an
INH-adduct. INH is in fact activated forming an acyl pyridine
adduct with NAD (or INH-NAD). Further structures for InhA bound
to an INH-adduct include 2PR213 with INH-NADP, 2IDZ14 with
NADH-INH and 2NV6, an isoniazid resistant S94A mutant with
INH-NAD adduct.15 By overlapping them the INH units closely
overlay in the protein pocket. It is clear from the crystal structures
that there are two possible positions for conjugated rings: one
occupied by the INH and the other one occupied by the adenine.
Reference compounds 1a-c and the most active molecules of the
two series, heterocyclic derivatives 5j-l and the INH analogue
derivatives 8a-c, were also tested against Mtb strain H37Rv and
several resistant strains as reported in Table 2.
To predict whether one of the most interesting compounds of our
series, the INH-analogue 8a, is able to interact with the active site
of the protein pocket, we docked it into the site, and starting from
the resulting configuration we ran 250 ns molecular dynamics (MD)
simulation in full water solvent.
Table 2. Antimycobacterial activity, MIC g/mL (M) of
compounds 1a-c, 5j-l and 8a-c in comparison with reference
compound isoniazid (INH) against strain H37Rv and four
resistant strains of M. tuberculosis.
We chose to work with the wild type 2Å resolution 2IDZ.14 We
first tested our method by removing the highly flexible ligand and
dock it back into the protein pocket. With the genetic algorithm and
simulated annealing of AutoDock it was not possible to obtain a
molecule with the expected orientation, a problem not encountered
with AutoDock Vina which led to a first ranked conformation with
RMSD 0.72 Å compared to the crystal structure and a predicted
binding affinity of -12.4 Kcal/mol (Fig. SI 2).
MIC g/mL (M)
Cpd
ATCC
27294
H37Rv
ATCC
35822
INH-Ra
ATCC
35828
Pyr-Rb
ATCC
35838
Rifa-Rc
ATCC
35820
Str-Rd
1a
1b
1c
1 (4.5)
8 (31)
1 (3.9)
> 32 (> 146)
> 32 (> 126)
> 32 (> 126)
1 (4.5)
8 (31)
1 (3.9)
2 (9.1)
16 (63)
1 (3.9)
1 (4.5)
16 (63)
1 (3.9)
We then docked compound 8a in the same pocket obtaining first
ranked conformation with a predicted binding affinity of -8.6
kcal/mol (Fig. SI 5a). Among the other top ranked complexes, we
identified 4 other similar conformations with RMSD < 1.30 Å in
comparison with the first one and with binding energies between -
8.5 kcal/mol and -8.4 kcal/mol (Fig. SI 3). The remaining poses,
though occupying the same site, are flipped compared to the others,
as the hydroxyphenyl group occupies the position of the
isonicotinoyl group and vice versa.
5j
1 (3.3)
4 (14.7)
2 (7.3)
1 (3.3)
1 (3.1)
1 (3.1)
0.25 (1.8)
> 32 (> 105)
32 (118)
1 (3.3)
1 (3.3)
4 (14.7)
2 (7.3)
1 (3.3)
4 (12.0)
4 (12.0)
1 (3.3)
5k
5l
4 (14.7)
1 (3.6)
4 (14.7)
2 (7.3)
In the top-ranking binding pose (Fig. SI 4a), the isonicotinoyl
group occupies the same adenine position of the original binder (Fig.
SI 4b). All the amino acids that held compound 8a in place but one
were also found to interact with the original ligand. Both molecules
were held in place thanks to a hydrogen bond with Ile21, and with
compound 8a forming an additional hydrogen bond with Gly96.
> 32 (> 117)
> 32 (> 106)
> 32 (> 96)
> 32 (> 96)
> 32 (> 233)
8a
8b
8c
INH
2 (6.7)
8 (26.7)
8 (24.0)
8 (24.0)
We then performed 250 ns MD simulation on the complex
formed by InhA and the top ranked binding pose of compound 8a.
The ligand topologies built with ATB16 led to a minimized
molecular geometry with a RMSD of 0.1721 Å compared to the
semi-empirical quantum chemistry minimization, thus validating
the generated force field parameters. Along the MD trajectory we
re-scored the complex with the same scoring function used for
generating the docked poses (Fig. SI 5b).
4 (12.0)
4 (12.0)
0.25 (1.8)
0.25 (1.8) 0.25 (1.8)
aIsoniazid-resistant strain; bPyrazinamide-resistant strain; cRifampicin-
resistant strain; dStreptomycin-resistant strain.
During the simulation, the VINA energy values reached -12.8
kcal/mol. The lowest energy conformation corresponded to a shift
in the location of compound 8a in the protein pocket associated with
a clear variation in protein conformation (Fig. SI 5c) and the
presence of an intermolecular hydrogen bond, and two hydrogen
bonds with the protein one with Ser 94 and one with Gly 96 (Fig. SI
4c).
All the compounds displayed the same previous trend against
H37Rv with MIC range of 1-8 g/mL with the best values for the
three INH-analogue molecules 8a-c (1 g/mL), while all the
derivatives proved to be virtually ineffective against the INH-
resistant strain. The most interesting results were obtained against

The system equilibrates after 120 ns at which point compound 8a
assumes a new conformation. This can be appreciated by looking at
the root mean square deviation (RMSD) of compound 8a calculated,
compared to a fixed protein backbone (Fig. 5a): at the beginning of
the simulation the compound migrated to a new position, and further
rearranged. The change in compound configuration seems not
associated with major protein rearrangements, as indicated by the
little fluctuations observed in the protein backbone RMSD (Fig. 5b).
Also, the protein reaches equilibrium at 120 ns. Over the last 100 ns
of the MD simulation, the backbone root mean squared fluctuation
(RMSF, Fig. 5c) indicates that only few residues were found above
0.15 nm with the majority not reaching 0.10 nm. In the same time
interval, the average VINA score, calculated over the data of Fig.
5b, equals -10.3 kcal/mol.
To understand the details of the equilibrium configuration we
looked at the lowest energy pose found along the last 100 ns of the
MD trajectory. That is at t = 193.9 ns with a VINA score of -12.4
kcal/mol (Fig. SI 5b). In this configuration compound 8a reached a
steady state in the protein pocket (Fig. 5d). The compound was held
in place thanks to four hydrogen bonds (Fig. 5e), while one
intermolecular hydrogen bond kept the molecule in a fixed
configuration. Six protein side chains contribute to the binding, and
a flexible loop was capable of adapting to the ligand conformation
and instant position in the pocket (Fig. 5f), resulting in a concerted
movement that enabled binding interactions to maintain their
strength.
Figure 6. (a) Contributions to the total binding energy averaged over 150-
250 ns time interval. (b) close ups on compound 8a with highlighted amino
acids with positive (shades of red) and negative (shades of blue) contribution to
the binding energy larger than ± 0.5 kcal/mol, snapshot taken at t = 193.9 ns.
All the data in the left panels refer to 150-250 ns time interval.
We finally performed a MM-PBSA analysis to clarify the nature
of the energetic contributions to the protein/compound interaction.
Using the MM-PBSA method, binding free energy (∆G) was
calculated as follows:17
Δ퐺 = Δ퐸 − Δ푆 + Δ퐺solv with Δ퐸 = 퐸vdW + 퐸el and
Δ퐺solv = Δ퐺polar + Δ퐺apolar
Where 퐸vdW is the van der Waals energy contribution and 퐸el is
the electrostatic energy contribution to the ligand/protein binding
energy ∆E, T is the temperature, ∆S the variation in entropy between
unbound and bound state, and Δ퐺solv the desolvation free energy
with its polar Δ퐺polarand apolar Δ퐺apolar contributions (the latter here
calculated with the solvent accessible surface area, SASA, method).
As we neglect here the entropic contribution, we call the term Δ퐸 +
Δ퐺solv simply total binding energy.
Figure 5. Trajectories analysis: (a) compound 8a RMSD with in the frame
of the protein backbone (black) and its running average (green); (b) protein
backbone RMSD (black) and its running average (cyan); (c) protein backbone
RMSF calculated over the last 100 ns (black) with values above 0.15 nm
highlighted in blue; (d) simulation snapshot at t = 193.9 ns where protein
residues with backbone RMSF larger than 0.15 nm are highlighted in blue; (e)
schematic diagram of the interaction between InhA and compound 8a at t =
193.9 ns (f) close ups on compound 8a at t = 193.9 ns. In (e-f) protein residues
interacting with the target by van der Waals interactions are highlighted in
red/orange, while those forming hydrogen bonds are green. Hydrogen bonds
distances measured in nm are also indicated.
The MM-PBSA analysis (Fig. 6) revealed that both van der
Waals and electrostatic contributions kept compound 8a in place
(Fig. 6). Amino acid energetic contributions on average larger than
± 0.5 kcal/mol (Fig. 6b) were associated with Ile 202, Phe 149, Glu
219, Gly 96, Ile 194, Phe 97, Ile 95, Met 199, Pro 156, with only
Asp 148 clearly opposing the binding (details in SI, Fig. SI 6).
We computationally verified whether the identified compound
8a was capable of fitting in the protein pocket. The docking result

led to an estimated score of -8.6 kcal/mol corresponding to Kd 0.50
M at room temperature, a value that after 150 ns MD simulation
reduced to an average of -10.3 kcal/mol (Kd = 28 nM). Analysis of
the MD trajectory revealed that compound 8a is held in its binding
site by four hydrogen bonds (Fig. 5e), while Asp 148 opposes
binding due to weak electrostatic and van der Waals not capable of
counteracting the unfavorable desolvation. This was also observed
in the MM-PBSA contributions to the total binding energy (Fig. 6b).
antimycobacterial activity and a potentially promising new class of
antitubercular agents.
Abbreviations
ADME, absorption metabolism distribution excretion; CC50,
cytotoxic concentration 50%; DprE1, decaprenyl-phosphoryl--D-
ribose oxidase; HBA, h-bond acceptor; HBD, h-bond donor; IC50,
inhibition concentration 50%; INH, isoniazid; InhA, 2-trans-enoyl-
ACP (CoA) reductase; MD, molecular dynamics; MDR, multidrug
All the derivatives endowed with antimycobacterial activity
(MIC < 64 g/mL) were evaluated in silico (Table SI1) for
prediction of drug likeness properties,18 using the most common
pharmacokinetic parameters, on the basis of the extended version of
Lipinski’s rule of five (RO5).19 The RO5 extended criterion means
that an orally active drug should not violate more than one of the
following requirements: MW ≤ 500; HBA and HBD (related to
membrane permeability) ≤ 10 and ≤ 5, respectively; logP and logS
(related to intestinal absorption) ≤ 5; PSA ≤ 140 Å. All the evaluated
compounds, in comparison with lead compound 1a and INH as
reference standard, exhibited good drug-likeness properties, as all
the values fell within the ranges of RO5.
resistant
strains;
Men-B,
menaquinone-B;
MetAP,
metioninaminopeptidase; MIC, minimum inhibitory concentration;
MM-PBSA, molecular mechanics Poisson-Boltzmann surface area;
Mtb, Mycobacterium tuberculosis; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; MW, molecular weight;
NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide
adenine dinucleotide reduced form; RMSD, root mean squared
deviation; RMSF, root mean squared fluctuation; SASA, solvent
accessible surface area; TB, tuberculosis.
Acknowledgments
In conclusion, this study builds on a previous exploratory study
investigating the oxazinone core for antimycobacterial activity
started by Li and coworkers.6 We synthesized different series of
compounds both with benzoxazin-2-one (2a-c, 3, 4, and 5a-l) and
benzoxazin-3-one (6, 7a-f) scaffolds. Also, we synthesized three
new open-ring analogues, such as oxoacetamide derivatives,
presenting isoniazid moiety (8a-c). All the title compounds were
evaluated against several mycobacterial strains, including strain
H37Ra, H37Rv and four strains resistant to first-line drugs used for
TB treatment. One of the most interesting compounds found was
derivative 8a, a combined molecule of N-(2-hydroxypheny)-2-
oxoacetamide fragment and INH. In order to predict whether
compound 8a was able to interact with the active site of the 2-trans-
enoyl-ACP (CoA) reductase (InhA), the isoniazid biological target,
we docked it into the crystal structure, and then we performed 250
ns MD simulation in full water solvent. During the simulation, the
compound never left the protein pocket, proving strong interactions
with the active site of the enzyme. However, further study will be
required to fully characterize the new system.
The financial support of FRA 2016 Research Fund University of
Trieste-Italy (owner: Dr. Daniele Zampieri), is gratefully
acknowledged. We also acknowledge the CINECA Awards N.
HP10C70TG1, 2018, for the availability of high performance
computing resources and support. The authors would like to thank
Dr. Fabio Hollan (Dep. Of Chemistry and Pharmaceutical Sciences-
Univ. of Trieste) for MS data.
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Supplementary Material
Supplementary material that may be helpful in the review process
should be prepared and provided as a separate electronic file. That
file can then be transformed into PDF format and submitted along
with the manuscript and graphic files to the appropriate editorial
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Highlights:
 Benzoxazin-2-one and -3-one derivatives were synthesized and biologically evaluated
 Antimycobacterial evaluation (MIC) and cytotoxicity (IC50) have been reported
 Oxoacetamide-Isoniazid analogues were obtained and biologically evaluated
 Compound 8a has been computationally studied

Graphical Abstract
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Design, synthesis and antimycobacterial
activity of benzoxazinone derivatives and
open-ring analogues: preliminary data and
computational analysis.
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Daniele Zampieria*, Maria Grazia Mamoloa, Julia Filingeria, Sara Fortunaa, Alessandro De Logub, Adriana
Sannac and Davide Zanond.