Design of hybrid molecules as antimycobacterial compounds: Synthesis of isoniazid-naphthoquinone derivatives and their activity against susceptible and resistant strains of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2019.07.045

Isoniazid-naphthoquinone hybrids were synthesized and evaluated against a susceptible (H₃₇Rv) strain and two isoniazid-resistant strains (INH_R1 and INH_R2) of Mycobacterium tuberculosis. The antimycobacterial activity of the derivatives was determined based on the resazurin microtiter assay and their cytotoxicity in adhered mouse monocyte macrophage J774.A1 cells (ATCC TIB-67). Of the twenty-two compounds evaluated against the three strains of M. tuberculosis, twenty-one presented some activity against the H₃₇Rv and INH_R1 (katG S315T) or INH_R2 (inhA C(?5)T) strains. Compounds 1a, 2a, and 8a were effective against the INH_R1 strain, and compounds 1a, 1b, 2a, 3a, 5a, 5b and 8a were effective against the INH_R2 strain, with MICs in the range of 3.12–6.25 μg/mL. Compounds 1b and 5b were the most active against H₃₇Rv, with MIC of 0.78 μg/mL. Based on the selectivity index, 1b and 5b can be considered safe as a drug candidate compounds. These results demonstrate that quinoidal compounds can be used as promising scaffolds for the development of new anti-TB drugs and hybrids with activity against M. tuberculosis-susceptible and INH-resistant strains.

Full text of DOI:10.1016/j.bmc.2019.07.045

Journal Pre-Proof
Design of hybrid molecules as antimycobacterial compounds: Synthesis of iso-
niazid-naphthoquinone derivatives and their activity against susceptible and
resistant strains of Mycobacterium tuberculosis
Wallace J. Reis, Ícaro A.O. Bozzi, Matheus F. Ribeiro, Priscila C.B. Halicki,
Laís A. Ferreira, Pedro E. Almeida da Silva, Daniela F. Ramos, Carlos A. de
Simone, Eufrânio N. da Silva Júnior
PII:
S0968-0896(19)30641-8
https://doi.org/10.1016/j.bmc.2019.07.045
BMC 15029
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 May 2019
17 July 2019
26 July 2019
Please cite this article as: Reis, W.J., Bozzi, I.A.O., Ribeiro, M.F., Halicki, P.C.B., Ferreira, L.A., Almeida da Silva,
P.E., Ramos, D.F., de Simone, C.A., da Silva Júnior, E.N., Design of hybrid molecules as antimycobacterial
compounds: Synthesis of isoniazid-naphthoquinone derivatives and their activity against susceptible and resistant
strains of Mycobacterium tuberculosis, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.07.045
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JOURNAL PRE-PROOF
Design of hybrid molecules as antimycobacterial compounds: Synthesis
of isoniazid-naphthoquinone derivatives and their activity against
susceptible and resistant strains of Mycobacterium tuberculosis
a
a
a
b
Wallace J. Reis, Ícaro A. O. Bozzi, Matheus F. Ribeiro, Priscila C. B. Halicki, Laís
b
b
b
c
A. Ferreira, Pedro E. Almeida da Silva, Daniela F. Ramos, * Carlos A. de Simone,
and Eufrânio N. da Silva Júniora*
a.Institute of Exact Sciences, Department of Chemistry, Federal University of Minas
Gerais, Belo Horizonte, 31270-901, MG, Brazil. E-mail: eufranio@ufmg.br; Tel: +55
b.
3
1 34095720; Mycobacteria Laboratory, School of Medicine, Federal University of
Rio Grande, Rio Grande, 96200-190, RS, Brazil. E-mail: danielaramos@furg.br; Tel:
c.
+
55 53 32374634; Institute of Physics, University of São Paulo, São Carlos, 13560-
1
60, SP, Brazil.
Abstract: Isoniazid-naphthoquinone hybrids were synthesized and evaluated against a
susceptible (H37Rv) strain and two isoniazid-resistant strains (INHR1 and INHR2) of
Mycobacterium tuberculosis. The antimycobacterial activity of the derivatives was
determined based on the resazurin microtiter assay and their cytotoxicity in adhered
mouse monocyte macrophage J774.A1 cells (ATCC TIB-67). Of the twenty-two
compounds evaluated against the three strains of M. tuberculosis, twenty-one presented
some activity against the H37Rv and INHR1 (katG S315T) or INHR2 (inhA C(-15)T)
strains. Compounds 1a, 2a, and 8a were effective against the INHR1 strain, and
compounds 1a, 1b, 2a, 3a, 5a, 5b and 8a were effective against the INHR2 strain, with
MICs in the range of 3.12 to 6.25 µg/mL. Compounds 1b and 5b were the most active
against H37Rv, with MIC of 0.78 µg/mL. Based on the selectivity index, 1b can be
considered safe as a drug candidate compound. These results demonstrate that quinoidal
compounds can be used as promising scaffolds for the development of new anti-TB
drugs and hybrids with activity against M. tuberculosis-susceptible and INH-resistant
strains.
Keywords: quinone;
isoniazid-naphthoquinone derivatives; Mycobacterium
tuberculosis; tuberculosis; hybrid molecules; antimycobacterial compounds.
3

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1
. Introduction
Tuberculosis (TB) is a contagious infectious disease caused mainly by the
pathogenic aerobic bacteria Mycobacterium tuberculosis (M. tuberculosis), which
1
usually establishes an infection in the lung of the host. According to the World Health
Organization, in 2017, there were an estimated 10.0 million new cases of TB and
4
60,000 cases of multidrug-resistant TB. Recently, this disease was classified along
with HIV as one of the main causes of death worldwide.2
The recommended therapeutic regimen for cases of drug-susceptible TB has a
2
cure rate of approximately 85%. Among the four first-line drugs used to treat these
cases (isoniazid (INH), rifampicin, ethambutol and pyrazinamide), INH is considered
the pillar of the therapeutic regimen due to its specificity, efficacy and low cost. In
addition, this drug is also used alone or in combination with other drugs in the
prophylactic treatment of latent TB and of TB in people living with HIV.3-5 However,
some limitations, such as the therapy length, the combination of several drugs, the
undesirable drug interactions and the possibility of side effects with variable levels of
severity, may cause treatment interruption, leading to treatment inefficacy, an increase
in the number of cases and acquired resistance; thus, the disease continues to be an
important social issue, mainly in populations at a low socioeconomic level.6,7
Considering the barriers to TB treatment, there are several strategies that are used in the
search for new anti-TB drugs, such as the discovery of new compounds, the use of
2
established drugs for new purposes, and the development of analogues/hybrids from
already known drugs that mainly aim to potentiate and reduce toxicity.6
Quinoidal compounds have been highlighted as potential scaffolds for the
development of anti-TB drugs.8 Among the naphthoquinones, lapachol and its
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derivatives present
a
broad spectrum of biological activities,9 including
antimycobacterial activity.10 Therefore, our group has recently developed useful
methods for the synthesis of functionalized bioactive naphthoquinones and
1
1
12
anthraquinones. Due to the important biological features of β-lapachone (β-lap), we
have used this molecule as a prototype for preparing compounds with remarkable
1
3
activity. Based on a strategy that utilizes modifications of the redox center and the A-
and C-rings, we and others have identified new entities with diverse antitumor and
trypanocidal activities.16 For instance, β-lap exhibited an MIC against M. tuberculosis
of 1.56 µg/mL. After modification of the redox center and C-ring of β-lap, its
1
4
phenazinic derivative was two times more active with MIC value of 0.78 µg/mL. In
this context, our group prepared phenazines,15,16 imidazoles,17 and β-lap and α-lap18
derivatives with pronounced anti-TB activity. The Carneiro group recently reported
insights from experimental and implicit/explicit solvation results related to α- and β-lap
isomerization in acidic media.19 The isomerization of β-lap to α-lap is an important
feature and allows the design of new para-quinoidal derivatives with high bioactivity20
17
(
Scheme 1A).
We are aware of the important features of compounds containing a hydrazone
moiety, such as potent bioactivity and photophysical characteristics. The Ishibashi,21
Morgan,22 Carvalho,23 Prinz,24 and Kakinuma groups have described the importance
of this class of compounds (Scheme 1B). Given our recent success in terms of the
synthesis of anti-TB compounds (Scheme 1A), herein, we describe an efficient strategy
for the synthesis of new hybrid lapachone-based isoniazid derivatives prepared via
redox center and C-ring modifications of lapachone derivatives (Scheme 1C), and we
evaluate their efficacy against 3 types of M. tuberculosis.
25
5

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Scheme 1. Overview.
2
. Results and discussion
2
.1. Chemistry
β-Lapachone-based compounds prepared from lapachol were the first class of
substances planned for in this work (Scheme 1). Initially, β-lap (1a), 3-iodo-β-
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lapachone (2a) and 3-bromo-β-lapachone (3a) were synthesized based on classic
methods that are well established in the literature.26 The naphthoquinone-based 1,2,3-
2
7
triazole 4a was obtained in four steps from lapachol. Initially, lapachol was cyclized to
β-lap, which was used for preparing 3,4-dibromo-β-lapachone. From the dibromo
compound, the azide derivative was then prepared and used in a click reaction with
phenylacetylene to produce 4a. Note that compound 4a was obtained in the racemic
form. The trans-stereochemistry was confirmed by X-ray crystallography analysis, as
previously reported in the literature.16,27 The choice of these quinones was based on
previous studies by our group9,13,16 and others28 that demonstrated the biological
potential of these compounds.29 As previously shown,30 the presence of heterocycles,
such as the 1,2,3-triazolic moiety attached to the quinoidal system, could be important
for the biological profile of these compounds.
Subsequently, isoniazid-naphthoquinone hybrid derivatives were easily prepared
by the reaction of the respective quinone and INH by reflux in acetic acid. Compounds
1
b-4b were obtained in moderate yields and isolated as crystalline solids (Scheme 2).
To the best of our knowledge, one protocol for synthesizing β-lap hydrazo
derivatives was already reported by Carvalho and later by our group.23,26 Carvalho and
coworkers described the reaction of phenylhydrazine hydrochloride and β-lap in dry
methanol. The formation of β-lapachone-6-phenyl-hydrazone at a 75% yield and β-
lapachone-5-phenyl-hydrazone at only a 3% yield were observed. Aware of the
possibility of the formation of a product with a hydrazo moiety at the C-5 or C-6
position, we prepared crystals suitable for X-ray crystallography studies of compounds
1
b and 2b via the formation of single crystals with slow evaporation methods. To our
delight, the structures of compounds 1b and 2b were solved by crystallographic
methods, which allowed the unambiguous confirmation of the formation of a product
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with the isoniazid portion at the C-6 position of the respective quinones (Figure 1). In
both molecules, the atoms of the naphthoquinonic ring are coplanar, and the largest
deviation [0.0217(2) Å] from the least-square plane is exhibited by atom C1 in 1a and
[
0.0384(3) Å] by atom C10 in 2a. In molecule 1a, the dihedral angle between the least-
square plane calculated for the atoms of the naphthoquinonic and heterocyclic
nitrogenated rings is 28.8(1)°, while for 2a, the dihedral angle is 3.73°. The pyran ring
3
1
exhibits an envelope configuration in both molecules with puckering parameters of q2
=
0.3471(2), q3 = -0.2836(9), Qt = 0. 4482(4), θ = 129.25(8), φ = -34.07(5) for 1a and
q2 = 0.3685, q3 = 0.2809(4), Qt = 0. 4633(4), θ = 52.68(5), φ = 148.94(5) for 2a. The
numbering of the structures solved by X-ray crystallography (Figure 1) is related to the
crystallographic information file (CIF).
Naturally, steric and electronic effects favor the formation of product at the C-6
position, but another important aspect worth mentioning is the observance of the
geometric Z-isomer rather than the E-isomer. The Z-isomer is likely favored because of
the stability of the six-membered structure formed by the intramolecular interaction
(
hydrogen bond) of the hydrogen of the hydrazo moiety with the neighboring carbonyl
group. This pattern of isomerism was commonly observed for the hydrazones obtained
from ortho-naphthoquinones.
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Scheme 2. Synthesis of β-lap analogs 1a-4a and their hybrid derivatives 1b-4b.
Figure 1. ORTEP-3 projection of compounds 1b and 2b showing atom labeling and
displacement ellipsoids drawn at the 50% probability level.
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Due to our knowledge of quinoidal chemistry and biology, we are aware of the
biological potential of nor-β-lap analogs.32,37 Therefore, we selected nor-β-lap (5a) and
its derivatives that were substituted with triazole (6a), arylamino (7a) and hydroxyl (8a)
groups to prepare the INH-hybrid compounds 5b-8b for antimycobacterial evaluation
(
Scheme 3). This second class of compounds was prepared from nor-lapachol. Our
group demonstrated the cytotoxicity and genetic toxicity of nor-β-lap (5a) in human
lymphocytes, HL-60 leukemia cells and murine V79 fibroblasts and its mechanism of
action to corroborate the importance of this compound as a prototype for a program of
3
3
drug development. On the other hand, the nor-β-lapachone-based 1,2,3-triazole (6a) is
3
4
a promising compound that exhibits efficient killing of cancer cell lines and parasites
3
0
such as Trypanosoma cruzi. Finally, quinones 7a and 8a were active against several
cancer cell lines and could be good choices for further derivatization.35 From the
selected nor-β-lapachones (5a-8a), the hybrid compounds 5b-8b were prepared at
moderate yields (Scheme 3). The synthesis was readily accomplished, but some
difficulty was encountered during the process of the purification of substance 6b due to
its low solubility in common organic solvents such as hexane and ethyl acetate. This
difficulty in the purification of compound 6b explains the low yield observed.
The last series of lapachones (9a-12a) was synthesized and thoroughly studied by the
Pinto and Castro groups (Scheme 4).36 Elingold and coworkers have described the
mechanism of action of compounds 9a and 10a in rat liver microsomal peroxidation.37
These results corroborate the importance of these compounds in biological terms.
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Scheme 3. Synthesis of nor-β-lap analogs 5a-8a and their hybrid derivatives 5b-8b.
Scheme 4. Synthesis of compounds 9a and 10a and their hybrid derivatives 9b and 10b
and α-lap derivatives 11a-11b and 12a-12b.
11

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2
.2. Biological activity
To identify new substances capable of eliminating Koch's bacillus, we prepared
new structural frameworks and evaluated their activity against M. tuberculosis.14-18 Our
goal was to focus on the synthesis of potential bioactive compounds via simple and
economically-viable synthetic routes. Since pathogenic bacillus species tend to be
sensitive to redox compounds, we therefore investigated the bioactivity of
naphthoquinones and their derivatives.16,18
Our strategy for the synthesis of new compounds was based on slight
modifications of the quinoidal system and the A- and C-rings of the naphthoquinonoid
compounds. These strategies allowed us to prepare, for instance, naphthoimidazoles and
naphthoxazoles with activity against rifampicin- and isoniazid-resistant strains of M.
tuberculosis. We demonstrated that fourteen heterocycles had high activity and
minimum inhibitory concentration (MIC) values in the range of 0.78 to 6.25 µg/mL
against strains of the bacillus. 17
It is important to emphasize that INH is one of the four frontline drugs used to
treat TB and is considered the pillar of the therapeutic regimen due mostly to its
specificity, efficacy and low cost.3,4,5 In addition, INH is a prodrug that requires
activation through oxidation by the enzyme catalase-peroxidase, which is encoded by
the katG gene, to generate the isonicotinoyl radical. This radical reacts covalently with
the NAD cofactor to form the active metabolite INH-NADH that inhibits InhA, which is
an essential enzyme for mycolic acid synthesis, to cause cell death.38
Resistance to INH is related to mutations in several genes, most of which occur
3
9
in katG and inhA. Approximately 50 to 80% of INH-resistant strains have mutations in
the katG gene that are responsible for the loss or reduction of catalase peroxidase
12

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activity during pro-drug activation.40 These mutations occur in approximately 10-40%
of INH-resistant strains, and in approximately 20% of cases they are found in the
promoter region of the gene.39,41
In this context, we evaluated the antimycobacterial activity of hybrid molecules
prepared via the redox center modification of INH and selected naphthoquinones
against susceptible and resistant M. tuberculosis strains. Hybrids of quinones and INH
and their precursors were evaluated by the broth microdilution technique. Of the 22
molecules evaluated against the three strains of M. tuberculosis, 21 derivatives were
active against the H37Rv (0.8-200 μg/mL) and INHR1 (3.12-200 μg/mL) strains, while
2
0 compounds were active against the INHR2 (3.12-200 μg/mL) strain (Table 1).
4
2
11
We have previously reported that the insertion of halogens and hydroxyl or
alkyl groups43 into quinone rings leads to compounds with effective biological
activities. In this context, the Ferreira group has evaluated the activity of a series of α-
and β-pyran naphthoquinones and described their activity against M. tuberculosis.10
Their findings highlight the influence of small structural changes when preparing new
bioactive compounds. Based on these previous results, we selected β-lap (1a) and its
iodine, bromine and triazole derivatives 2a-4a as starting points for our biological
assays. Compound 1a exhibited MIC values of 6.25, 3.12 and 6.25 µg/mL against
H
37Rv, INHR1 and INHR2, respectively, and was the only compound active against
INHR1 (Table 1). The quinonoid derivatives 2a-4a exhibited MIC values in the range of
.56 to 12.5 µg/mL, demonstrating their biological activity. As expected, the C-ring
1
modification of β-lap produced the derivative 4a, which had an appended triazole
group. This compound, with an MIC value against H37Rv of 1.56 µg/mL, was
considered the most active of this first series of compounds when evaluated against the
bacillus.
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Molecules containing a triazole group have gained prominence due to their
antimycobacterial potential.44 Triazole acts to inhibit cell wall synthesis – a similar
mechanism of action to that of INH – and INH-azole hybrids can be considered potent
anti-TB agents.45
Based on our strategy, we evaluated the hybrid molecules 1b-4b that were
prepared from β-lap derivatives 1a-4a. Compounds 2b-4b were less potent than their
respective quinoidal precursors, but, to our delight, the hybrid derivative 1b exhibited a
MIC value against H37Rv of 0.78 µg/mL. Moreover, this compound had a 4-fold
reduced MIC against H37Rv compared to its precursor, whereas for the INH-resistant
strains, the MIC remained the same or increased 16-fold.
The second series of substances (5b-8b) was based on nor-β-lap 5a and its
derivatives 6a-8a. All evaluated compounds were active against the three strains of TB
studied herein, especially compounds 5a and 5b, which exhibited MIC values in the
range of 50 to 0.78 µg/mL. The hybrid derivative 5b was also almost four times more
active than its precursor 5a against the H37Rv strain. This result demonstrates the
efficiency of our strategy in the preparation of hybrid compounds.
Finally, we evaluated derivatives prepared from the C-allyl lawsones 9b, 10a
and 10b, the para-naphthoquinones 11a and 12a and the hybrid derivative 12b. The
1
8
non-active α-lap (12a) was not active, as was previously described by our group. This
latter series of compounds showed moderate activity, and none of the hybrid
compounds was more active than its respective precursor.
Regardless, the hybrid compounds have remained active, which demonstrates
that the modification of the redox center improves the activity exponentially in some
cases and does not produce inactive compounds in most of the examples studied,
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highlighting the potential of these compounds as important starting points for further
studies to discover new anti-TB strategies.
We also evaluated the cytotoxicity of the compounds against J774.A1 cells and
calculated the selectivity index (Table 1). In general, these compounds exhibited
moderate cytotoxicity. To our delight, the most active hybrid compounds, 1b and 5b,
exhibited low cytotoxicity and a good selectivity index (>250) against the H37Rv strain.
These compounds are currently being further investigated in our laboratories, and
detailed biological studies will be reported in due course.
Table 1 Activity of naphthoquinones (precursors) and isoniazid-naphthoquinone hybrid
compounds against three M. tuberculosis strains and cytotoxicity evaluation on J774A.1
cells lineage. SI: IC50/MIC.
IC50
MIC (µg/mL)
(
µg/mL)
SI (IC50/MIC)
Strain
Lineage
J774A.1
>200
Compound H37Rv INHR1 INHR2
H
37Rv INHR1 INHR2
1
1
2
2
3
3
4
a
b
a
b
a
b
a
6.25
0.78
3.12
100
3.12
50
6.25
6.25
6.25
100
>32
>64
>4
>32
>32
0.5
>200
>250
1.0
3.12
100
6.25
12.5
6.25
3.20
1.0
0.3
0.3
>16
0.2
30.76
1.93
0.3
0.3
6.25
12.5
1.56
3.12
12.5
12.5
0.3
0.6
>200
>16
0.9
>16
0.1
1.47
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4
5
5
6
6
7
7
8
8
9
9
b
a
b
a
b
a
b
a
b
a
b
200
3.12
0.78
6.25
12.5
12.5
6.25
3.12
25
200
12.5
50
>200
6.25
6.25
25
175
3.69
>200
2.11
101.47
3.67
>200
1.82
74.18
NE
0.9
1.2
>250
0.3
8.1
0.3
>32
0.6
3.0
NE
>16
0.2
1.5
1.5
0.7
NE
NE
NE
NE
0.9
0.3
>4
<0.9
0.6
>32
0.1
8.1
0.1
>2
25
0.1
8.1
0.1
>1
12.5
25
12.5
25
200
3.12
100
NE
12.5
25
100
6.25
25
0.6
0.7
NE
>16
0.1
0.4
0.7
0.2
NE
NE
NE
NE
0.3
3.0
NE
>8
NE
NE
25
12.5
12.5
12.5
12.5
25
>200
2.94
18.40
18.63
16.98
285*
NE
1
0a
0b
1a
1b
2a
2b
25
0.1
0.4
0.7
0.1
NE
NE
NE
NE
1
50
50
1
25
25
1
100
NE
> 200
≤0.03
2
200
NE
> 200
> 8
0.25
1
103.3*
> 200
1
Rifampicin ≤0.03
Isoniazid
0.03
NE
NE
MIC: Minimum inhibitory concentration; Values shown as IC50: Half maximal
inhibitory concentration; SI: Selectivity index; NE: Not evaluated. *Values presented
µM previously reported in the literature.18
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3
. Conclusions
The present work shows how a strategy utilizing redox center and C-ring
modifications in lapachone derivatives is a useful approach for the synthesis of novel
lead compounds for the treatment of tuberculosis. The synthesis of the compounds is
simple and can be performed with low cost and accessible reagents, which allows the
preparation of the compounds economically viable and accessible. Overall, the present
study contributes to the discovery of new strategies for TB treatment.
4
. Experimental
General experimental procedures: All chemicals were obtained from commercial
sources and used without further purification. Solvents were distilled and when required
were dried by distillation according to standard procedure. Melting points were
obtained on a Thomas Hoover apparatus and are uncorrected. Column chromatography
was performed on silica gel (SilicaFlash G60 UltraPure 60-200 µm, 60 Å). Infrared
spectra were recorded on a Shimadzu FTIR Spectrometer IR Prestige-21. H and 13
1
C
NMR were recorded at room temperature using a Bruker AVANCE DRX200 and
DRX400 MHz, in the solvents indicated, with tetramethylsilane (TMS) as internal
reference. Chemical shifts (δ) are given in parts per million (ppm) and coupling
constants (J) in Hertz (Hz). The mass spectrometer was operated in the positive ion
mode. A standard atmospheric pressure photoionization (APPI) source was used to
generate the ions. The sample was injected using a constant flow (3 µL/min). The
solvent was an acetonitrile/methanol mixture. The APPI-Q-TOF MS instrument was
calibrated in the mass range of 50-3000 m/z using an internal calibration standard (low
17

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concentration tuning mix solution) supplied by Agilent Technologies. Data were
processed employing Bruker Data Analysis software version 4.0. Compounds were
named following IUPAC rules as applied by ChemBioDraw Ultra (version 12.0).
Chemistry: Lawsone was acquired from Sigma-Aldrich (St. Louis, MO, USA). C-allyl
lawsone was prepared from lawsone as previously reported.46 Lapachol (2-hydroxy-3-
(
3’-methyl-2’-butenyl)-1,4-naphthoquinone) was extracted from the heartwood of
Tabebuia sp. (Tecoma). Initially, a saturated aqueous sodium carbonate solution was
added to the sawdust of ipe. After the formation of the lapachol sodium salt,
hydrochloric acid was added, allowing the precipitation of lapachol. After filtration, a
yellow solid was obtained. This solid was purified by a series of recrystallizations with
4
7
48
appropriate solvents. Nor-lapachol was prepared by Hooker oxidation reaction. From
lapachol, β-lapachone (1a), 3-iodo-β-lapachone (2a), 3-bromo-β-lapachone (3a) and β-
lapachone-based 1,2,3-triazole (4a) were prepared as previously described in the
literature.27 From nor-lapachol, nor-β-lapachone (5a), nor-β-lapachone-based 1,2,3-
triazole (6a), 3-hydroxyl-nor-β-lapachone (7a) and arylamino nor-β-lapachone (8a)
30
were also prepared as previously described by our group. Compounds derived from C-
allyl lausone 9a and 10a and para-naphthoquinones 11a and 12a were prepared
following methods previously described by us and others.29
General procedure for the preparation of isoniazid-naphthoquinone hybrids:
Isonicotinylhydrazide (0.70 mmol) was added all at once to a solution of the respective
quinone (0.31 mmol) in acetic acid (5 mL). The mixture was stirred and heated to reflux
it, and it was monitored by TLC until complete consumption of the starting material
occurred. The residue was poured into water, and the precipitate was filtered and
washed with water. After drying, the crude hydrazones were purified by column
18

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chromatography in silica gel via elution with a gradient mixture of hexane and ethyl
acetate with increasing polarity.
(
Z)-N'-(2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-
ylidene)isonicotinohydrazide (1b): Compound 1b was obtained as orange solid
-
1
(
(
129.78 mg, 87% yield). m.p (°C) = 210-212. IR (solid, cm ) ν: (KBr) 3416 (w), 1702
1
s), 1598 (m), 1393(s). H NMR (200 MHz, CDCl
3
) δ: 16.3 (s, 1H), 8.39 (d, J = 8,0 Hz,
1
2
H), 7.95 (d, J = 8.0 Hz, 1H), 7.59-7.51 (m, 2H), 7.42-7.34 (m, 3H), 7.19-7.12 (m, 1H),
1
3
.69 (t, J = 6.6 Hz, 2H), 1.90 (t, J = 6.6 Hz, 2H), 1.47 (s, 6H). C NMR (100 MHz,
) δ: 205.7, 183,6, 179.4, 159.8, 142.7, 132.8, 129.4, 128.9, 127.7, 125.8, 124.5,
CDCl
3
+
+
1
24.4, 122.6, 121.5, 116.0, 110.9, 98.1, 31.8, 26.7, 16.1. MS (EI ): (m/z) [M+H] 362.1.
]+: 362.3.
Cald. for [C21
H
20
N
3
O
3
(
Z)-N'-(3-iodo-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-
ylidene)isonicotinohydrazide (2b): Compound 2b was obtained as orange solid
-
1
(
(
112.50 mg, 85% yield). m.p (°C) = 209-211. IR: (solid, cm ) ν: (KBr) 3420 (w), 1657
1
m), 1625 (m), 1390 (s). H NMR (200 MHz, CDCl
3
) δ: 16.15 (s, 1H), 9.20-8.72 (m,
2
H), 8.50-8.29 (s, 1H), 7.86-7.87 (m, 3H), 7.65-7.43 (m, 2H), 4.40 (dd, J = 5.5 and 8.5
Hz, 1H), 3.39 (dd, J = 5.5 and 17.9 Hz, 1H), 3.17 (dd, J = 8.5 and 17.9 Hz, 1H), 1.72 (s,
3
1
2
H), 1.65 (s, 3H). 13C NMR (100 MHz, CDCl
3
) δ: 213.3, 182.9, 181.2, 163.7, 162.2,
50.7, 139.4, 131.4, 130.8, 129.4, 126.2, 123.3, 121.5, 110.6, 81.0, 30.3, 28.1, 26.8,
+
+
O
]+: 488.2.
4.8. MS (EI ): (m/z) [M+H] 488.0 Cald. for [C21
H19IN
3
3
(
Z)-N'-(3-bromo-2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[h]chromen-6(5H)-
ylidene)isonicotinohydrazide (3b): Compound 3b was obtained as orange solid
-
1
(
(
116.62 mg, 85% yield). m.p. (C°) = 209-211. IR: (solid, cm ) ν: (KBr) 3442 (w), 1692
1
m), 1606 (m), 1390 (s). H NMR (200 MHz, CDCl
3
) δ: 16.16 (s, 1H), 8.95-8.80 (m,
19

JOURNAL PRE-PROOF
2
H), 8.49-8.33 (m, 1H), 7.96-7.80 (m, 3H), 7.59-7.46 (m, 2H), 4.43-4,21 (m, 1H), 3.28
(
dd, J = 5.0 and 17.9 Hz, 1H), 3.03 (dd, J = 7.5 and 17.9 Hz, 1H), 1.66 (s, 3H), 1.61 (s,
1
3
3
1
H). C NMR (100 MHz, CDCl
3
) δ: 196.1, 181.6, 168.3, 162.3, 162.2, 150.5, 150.4,
40.2, 131.1, 128.9, 124.7, 123.6, 122.1, 121.9, 109.9, 81.0, 50.2, 28.0, 26.4, 23.4. MS
+
+
O
]+: 441.3.
(
EI ): (m/z) [M+H] 441.0. Cald. for [C21
H
19BrN
3
3
(
Z)-N'-3-bromo-2,2-dimethyl-5-oxo-4-(4-phenyl-1H-1,2,3-triazol-1-yl)-3,4-dihydro-
H-benzo[h]chromen-6(5H)-ylidene)isonicotinohydrazide (4b): Compound 4b was
2
-1
obtained as yelllow solid (50.28 mg, 40% yield). m.p. (°C) = 275-276. IR (solid, cm )
1
ν: (KBr) 3420 (w), 1657 (m), 1625 (m). H NMR (400 MHz, CDCl
3
) δ: 15.55 (s, 1H),
8
7
5
.92-8.69 (m, 2H), 8.08-8.14 (m, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.91 (d, J = 7.7 Hz, 2H),
.70-7.76 (m, 3H), 7.66-7.52 (m, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 1H),
13
.85 (d, J = 8.5 Hz, 1H), 4.98 (d, J = 8.5 Hz, 1H), 1.76 (s, 3H), 1.57 (s, 3H). C NMR
) δ: 209.3, 204.1, 179.2, 167.7, 150.7, 132.5, 132.4, 130.9, 129.7,
28.9, 128.8, 124.6, 123.4, 123.0, 122.6, 117.7, 116.3, 96.7, 84.2, 75.4, 29.7, 26.8, 20.6.
(
100 MHz, CDCl
3
1
+
+
O
]+: 583.4.
MS (EI ): (m/z) [M] 583.0. Cald. for [C29
H23BrN
6
3
(
Z)-N'-(2,2-dimethyl-4-oxo-2,3-dihydronaphtho[1,2-b]furan-5(4H)-
ylidene)isonicotinohydrazide (5b): Compound 5b was obtained as orange solid
-
1
(
(
114.15 mg, 75% yield) m.p. (C°) = 248-250. IR (solid, cm ) ν: (KBr) 3420 (m), 3120
1
m), 1698 (m), 1405 (s). H NMR (400 MHz, CDCl
3
) δ: 16.36 (s, 1H), 9.04-8.78 (m,
2
7
H), 8.36 (d, J = 1.7 Hz, 1H), 7.93-7.77 (m, 2H), 7.65 (d, J = 7.0 Hz, 1H), 7.48 (dt, J =
1
3
.0 and 14.5 Hz, 2H), 5,07 (m, 1H), 2.94 (s, 1H), 1.70 (s, 3H), 1,53 (s, 3H). C NMR
(
100 MHz, CDCl
3
) δ: 200.6, 179.3, 169.5, 163.5, 152.8, 150.4, 139.6, 137.4, 133.5,
+
1
31.4, 129.2, 125.1, 123.9, 122.7, 121.6, 113.8, 93.7, 38.8, 28.3. MS (EI ): (m/z) [M+
+
O
]+: 348.3.
H] 348.1. Cald. for [C20
H
18
N
3
3
20

JOURNAL PRE-PROOF
(
Z)-N'-(2,2-dimethyl-4-oxo-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-
dihydronaphtho[1,2-b]furan-5(4H)-ylidene)isonicotinohydrazide (6b): Compound
1
6
b was obtained as orange solid (46.23 mg, 35% yield). m.p. (°C) = 239-240. H NMR
(
(
(
200 MHz, CDCl
3
) δ: 14.04 (s, 1H), 8.95-8.60 (m, 2H), 8.45-8.28 (m, 1H), 7.90-7.60
m, 7H), 7.45-7.30 (m, 3H), 6.20-6.05 (m, 1H), 1.72 (s, 3H), 1.27 (s, 3H). 13C NMR
100 MHz, CDCl
3
) δ: 211.8, 191.0, 179.8, 160.6, 156.9, 135.3, 131.7, 130.4, 130.3,
1
29.7, 129.4, 129.3, 128.9, 128.4, 128.3, 125.7, 124.0, 92.4, 68.3, 29.7, 27.6, 21.4.
(
Z)-N'-(3-hydroxy-2,2-dimethyl-4-oxo-2,3-dihydronaphtho[1,2-b]furan-5(4H)-
ylidene)isonicotinohydrazide (7b): Compound 7b was obtained as yellow solid (89.02
-
1
mg, 60% yield). m.p. (°C) = 248-250. IR: (solid, cm ) ν: (KBr) 3408 (w), 3173(w)
1
1
8
2
698 (m),1420 (s). H NMR (400 MHz, CDCl
3
) δ: 16.30 (s, 1H), 10.03 (s, 2H), 8.95-
.75 (m, 1H), 8.60-8.31 (m, 1H), 7.87 (d, J = 4.8 Hz, 2H), 7.65 (dd, J = 7.9 and 17.3 Hz,
1
3
H), 7.58-7.41 (m, 1H), 5.07 (s, 1H), 1.70 (s, 3H), 1.53 (s, 3H). C NMR (100 MHz,
CDCl
3
) δ: 209.3, 204.1, 183.2, 176.5, 167.7, 166.0, 132.5, 132.4, 130.9, 129.7, 124.6,
+
+
1
23.0, 122.6, 117.7, 116.3, 84.2, 75.4, 29.8, 26.8, 20.7. MS (EI ): (m/z) [M+H] 364.1.
]+: 364.3.
Cald. for [C20
H
18
N
3
O
4
(
Z)-N'-(2,2-dimethyl-4-oxo-3-(phenylamino)-2,3-dihydronaphtho[1,2-b]furan-
(4H)-ylidene)isonicotinohydrazide (8b): Compound 8b was obtained as brown solid
5
-
1
(
82.41 mg, 60% yield) m.p. (°C) = 245-247. IR (solid, cm ) ν: (KBr) 3428 (m),
1
3
0
7
3
1
419(m), 1619 (m), 1403 (s). H NMR (400 MHz, CDCl
3
) δ: 14.18 (s, 1H), 8.81 (d, J =
.5 Hz, 2H), 8.48-8.40 (m, 1H), 7.78 (d, J = 4.8 Hz, 2H), 7.68-7.52 (m, 3H), 7.44 (t, J =
.5 Hz, 1H), 7.26 (dd, J = 8.9 and 1.4 Hz, 2H), 6.66-6.84 (m, 3H), 4.89 (s, 1H), 1.67 (s,
H), 1.63 (s, 3H). 13C NMR (100 MHz; CDCl
47.4, 144.3, 134.9, 134.0, 132.3, 129.6, 128.8, 124.3, 123.0, 122.5, 118.0, 114.3,
) δ: 194.1, 193.8, 193.6, 182.4, 170.2,
3
21

JOURNAL PRE-PROOF
+
+
1
12.9, 96.8, 91.4, 64.0, 62.2, 60.9, 27.4, 21.8. MS (EI ): (m/z) [M+H] 439.1. Cald. for
[
C
26
H
23
N
4
O
]+: 439.4.
3
(
Z)-N'-(2-methyl-4-oxo-2,3-dihydronaphtho[1,2-b]furan-5(4H)-
ylidene)isonicotinohydrazide (9b): Compound 9b was obtained as brown solid
-
1
(
(
101.15 mg, 65% yield). m.p. (°C) = 248-250. IR (solid, cm ) ν: (KBr) 3438 (w), 3122
1
w), 1694 (s), 1414 (s). H NMR (400 MHz, CDCl
3
) δ: 16.25 (s, 1H), 8.86-8.97 (m,
2
H), 8.58-8.38 (m, 1H), 7.96-7.78 (m, 2H), 7.73 (d, J = 7.6 Hz, 1H), 7.60 (dd, J = 7.6
and 14.7 Hz, 1H), 7.51 (td, J = 0.9 and 7.6 Hz, 1H), 5.39-5.27 (m, 1H), 3.34 (dd, J = 9.6
13
and 15,1 Hz, 1H), 2.79 (dd, J = 7.1 and 15.1 Hz, 2H) 1.62 (d, J = 6.4 Hz, 3H). C NMR
(
100 MHz, CDCl ) δ: 179.6, 175.0, 170.0, 150.3, 150.1. 137.8, 133.7, 129.5, 129.1,
3
+
+
1
3
28.8, 125.5, 124.0, 122.8, 114.3, 110.5, 84.9, 33.3, 22.1. MS (EI ): (m/z) [M+H]
]+: 334.3.
34.1. Cald. for [C19
H
16
N
3
O
3
(
Z)-N'-(4-oxo-2-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-2,3-dihydronaphtho[1,2-
b]furan-5(4H)-ylidene)isonicotinohydrazide (10b): Compound 10b was obtained as
-1
purple solid (86.67 mg, 65% yield). m.p. (°C) = 208-210. IR (solid, cm ) ν: (KBr) 3438
1
(
m), 1690 (m), 1625 (w), 1406 (s). H NMR (400 MHz, CDCl
3
) δ: 15.3 (s, 1H), 8.75-
8
3
.98 (m, 2H), 8.50-8.18 (m, 2H), 7.72-7.95 (m, 5H), 7.51-7.66 (m, 2H), 7.48-7.31 (m,
H), 5.66-5.40 (m, 1H), 4.96-4.64 (m, 2H), 3.55-3.34 (m, 1H), 3.00-3.26 (m, 1H).
+
+
O
]+: 477.1597.
HRMS (EI ): (m/z) [M+H] 477.1560. Cald. for [C27
H
21
N
6
3
(
Z)-N'-(2,2-dimethyl-4-oxo-2,3-dihydronaphtho[2,3-b]furan-9(4H)-
ylidene)isonicotinohydrazide (11b): Compound 11b was obtained as yellow solid
-1
(
91.32 mg, 60% yield) m.p. (°C) = 220-222. IR (solid, cm ) ν: (KBr) 3437 (s), 3299 (s),
1
1
1
693 (m), 1636 (s), 1541 (s), 1410 (m). H NMR (200 MHz, CDCl
3
) δ: 12.22 (s, 1H),
1.46 (s, 1H), 8.88 (d, J = 1.3 Hz, 2H), 8.51 (d, J = 1.5 Hz, 1H), 8.13 (dd, J = 4.3 Hz,
22

JOURNAL PRE-PROOF
1
2
1
3
H), 7.40-7.90 (m, 4H), 3.00 (s, 2H), 1.70 (s, 6H). 13C NMR (100 MHz, CDCl
3
) δ:
22.0, 181.6, 179.1, 174.0, 156.1, 149.2, 149.1, 132.1, 131.0, 129.9, 125.7, 123.5,
+
+
+
19.7, 108.8, 94.5, 39.1, 28,6. MS (EI ): (m/z) [M+H] 348.1. Cald. for [C20
H
18
N
3
O ] :
3
48.3.
(
Z)-N'-(2,2-dimethyl-5-oxo-3,4-dihydro-2H-benzo[g]chromen-10(5H)-
ylidene)isonicotinohydrazide (12b): Compound 12b was obtained as yellow solid
-
1
(
(
67.13 mg, 45% yield) m.p. (°C) = 208-210. IR (solid, cm ) ν: (KBr) 3330 (m), 1693
1
m), 1610 (m), 1385 (s), 1267(s). H NMR (200 MHz, CDCl3, DMSO-d
6
) δ: 12.82 (s,
1
7
H), 12.17 (s, 1H), 9.04-8.78 (m, 2H), 8.49-8.64 (m, 1H), 8.15 (d, J = 8.8 Hz, 1H),
13
.45-7.91 (m, 4H), 2.72 (t, J = 6.5 Hz, 2H), 1.93 (t, J = 6.5 Hz, 2H), 1.57 (s, 6H). C
) δ: 197.1, 183.3, 154.5, 150.9, 150.8, 149.8, 140.3,
33.2, 132.2, 129.7, 125.6, 124.9, 124.8, 123.7, 123.6, 120.9, 117.3, 80.0, 31.1, 26.8,
NMR (100 MHz, CDCl3, DMSO-d
6
1
1
+
+
O
]+: 362.3.
6.8. MS (EI ): (m/z) [M+H] 362.1. Cald. for [C21
H
20
N
3
3
X-ray crystal structure analysis: X-ray diffraction data collection was performed on
an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on a κ-goniostat)
using graphite-monochromated MoKα radiation (0.71073 Å) at room temperature. Data
collection was carried out using COLLECT software49 at up to 50° in 2θ. The
integration and scaling of the reflections and the correction for Lorentz and polarization
5
0
effects were performed with the HKL DENZO-SCALEPACK software programs. The
structures of the compounds were solved by direct methods with SHELXS-97.51 The
models were refined by full-matrix least-squares on F using SHELXL-97.52 The
program ORTEP-353 was used for the graphic representation, and the program
2
5
4
WINGX was used to prepare the materials for publication. All H atoms were located
based on geometric considerations (C–H = 0.93-0.97 Å) and refined with Uiso(H) =
23

JOURNAL PRE-PROOF
1
.5Ueq (C-methyl) or 1.2Ueq (other). Ortep-3 diagrams of the molecules are shown in
Figure 1. The reference numbers for the compounds are CCDC 1891836 and 1891837.
Copies of the available material can be obtained, free of charge, by application to the
Director, CCDC, 12 Union Road, Cambridge CH21EZ, UK (fax: +44-1223-336-033, e-
mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
Strains and inoculum: Three M. tuberculosis strains were used to evaluate the
antimycobacterial activity of the INH derivatives in vitro: a pansusceptible control
strain, H37Rv (ATCC 27294); a strain that is monoresistant to INH because of a
mutation in the katG gene (S315T), INHR1 (ATCC 35822); a strain that is also resistant
to INH because of a C(-15)T mutation in the promoter region of the inhA gene, INHR2
.
The strains were cultured with the Ogawa-Kudoh method for up to 14 days at 37 °C.
The inoculum for each strain was prepared according to the 1.0 McFarland standard (3 x
1
08 UFC/mL). Then, the mixture was diluted at a ratio of 1:20 in Middlebrook 7H9
Broth.
Antimycobacterial activity: The antimycobacterial activity of the INH derivatives was
determined by the resazurin microtiter assay.55 To perform the assay, 100 μL of
MiddleBrook 7H9 broth enriched with 10% OADC was added to each well of a 96-well
plate, and serial two-fold dilutions of each compound were performed directly in the
plate at concentrations that ranged from 200 to 0.4 μg/mL. In sequence, 100 μL of the
inoculum was added to each well. Growth controls containing no compounds and
sterility controls without inoculum were also included. After seven days of incubation at
3
7 °C, 30 μL of resazurin solution (0.02%) was added to each well, incubated overnight
and assessed for color development. A change from blue to pink indicated the reduction
of resazurin and bacterial growth. The minimum inhibitory concentration (MIC) was
24

JOURNAL PRE-PROOF
defined as the lowest drug concentration capable of inhibiting bacterial growth. Those
compounds with a MIC > 200 μg/mL were considered inactive.
Cytotoxicity assay: In a 96-well plate, 200 µL of a suspension containing adhered
J774.A1 cells (ATCC TIB-67) cultivated in DMEM (Sigma-Aldrich) supplemented
5
with 10% fetal bovine serum at a concentration of 1 x 10 cells/mL was added. The cells
were maintained for 24 hours at 37 °C in a humid atmosphere with 5% CO2, and after
this period the adherent cells were exposed to the compounds at the same concentrations
evaluated in the antimycobacterial activity assay. After 24 hours of exposure, 30 μL of
0
.01% resazurin was added to each well, 56 and the plate was reincubated overnight until
the optical density (OD) was measured to determine the half-maximal inhibitory
concentration (IC50), which is the concentration of the compound capable of
maintaining the viability of 50% of the cells (% Inhibition = 100 − (Treated
OD/Nontreated OD) x 100).
Structure-activity relationship (SAR) and selectivity index (SI): The SAR analysis
was performed based on the MIC and IC50 values and the chemical structures of the
compounds described in Schemes 1-5. In addition, the SI was calculated according to
the following formula: IC50/MIC. An SI value equal to or greater than 10 indicated that
the compound was pharmacologically safe.57
Conflicts of interest
There are no conflicts to declare.
25

JOURNAL PRE-PROOF
Acknowledgements
E.N. da Silva Júnior acknowledges funding from CNPq 404466/2016-8, PQ
3
05741/2017-9, FAPEMIG (PPM-00638-16 and Rede de Pesquisa e Inovação para
Bioengenharia de Nanossistemas-RED-00282-16), Capes-Humboldt research
fellowship programme for experienced researchers (Proc. Nº 88881.145517/2017-01).
D.F. Ramos acknowledges funding from CNPq 474767∕2013-2. P.E.A. da Silva
acknowledges funding from Capes-AUX PE-PNPD-2929/2011. Prof. CAS would like
to thank the Institute of Physics of University of São Paulo (São Carlos), for kindly
allowing the use of the KappaCCD diffractometer.
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2
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2
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019;130:408.
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Ferreira SB, da Silva FC, Bezerra FAFM, Loureno MCS, Kaiser CR, Pinto AC,
Ferreira VF. Arch Pharm Chem Life Sci. 2010;343:81.
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(a) Dias GG, Rogge T, Kuniyil R, Jacob RC, Menna-Barreto RFS, da Silva
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Jardim GAM, Santos ACC, Araujo MH, Oliveira WXC, Bombaça ACS, Menna-
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018;24:15227; (c) Gontijo TB, de Freitas RP, de Lima GF, de Rezende LCD,
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JR, Emery FS, da Silva Júnior EN. Chem Commun. 2016;52:13281.
27

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Rodrigues T, Werner M, Roth J, da Cruz EHG, Marques MC, Akkapeddi P,
Lobo SA, Koeberle A, Corzana AF, da Silva Júnior EN, Werz O, Bernardes GJL.
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de Castro SL, Emery FS, da Silva Júnior EN. Eur J Med Chem. 2013;69:678.
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