N-Phenylbenzamide derivatives as alternative oxidase inhibitors: Synthesis, molecular properties, 1H-STD NMR, and QSAR

Article abstract of DOI:10.1016/j.molstruc.2020.127903

In the present work, 117 N-phenylbenzamides (NPDs) were prepared and evaluated against recombinant AOX from the fungal pathogen Moniliophthora perniciosa. ¹H, ¹³C NMR, FTIR, and mass spectra provided structural information on NPDs. The library compounds were tested as Alternative Oxidase inhibitors in two different assays using the model yeast Pichia pastoris: cell growth and oxygen consumption assays. The most active compound, 3_FH, was further characterized by DRX and 1H-NMR-STD. Single crystal X-ray diffraction showed intra- and intermolecular interactions of 3_FH in solid-state and elucidated its 3D structural configuration. 1H-NMR-STD allowed us to derive protein-ligand interactions in a membrane-mimetic system and evidenced an outstanding interaction of 3_FH with this enzyme. Results of both biological assays were used as input to Quantitative Structure-Activity Relationship models, which highlighted the more important molecular fragments contributions for protein-ligand interaction.

Full text of DOI:10.1016/j.molstruc.2020.127903

Journal of Molecular Structure 1208 (2020) 127903
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
N-Phenylbenzamide derivatives as alternative oxidase inhibitors:
Synthesis, molecular properties, 1H-STD NMR, and QSAR
,
,
c
,
,
1
b
Paulo C.S. Costa a 1, Mario R.O. Barsottini b 1, Maria L.L. Vieira d , Barbara A. Pires ,
Joel S. Evangelista a, Ana C.M. Zeri e, Andrey F.Z. Nascimento e, Jaqueline S. Silva d,
Marcelo F. Carazzolle b, Gonçalo A.G. Pereira b, Maurício L. Sforça d,
ꢀ
Paulo C.M.L. Miranda a 2, Silvana A. Rocco d
,
*
,
, **, 2
a Institute of Chemistry, University of Campinas e UNICAMP, Zip Code 13083-970, Campinas, SP, Brazil
b Laboratory of Genomics and BioEnergy, Institute of Biology, University of Campinas e UNICAMP, Zip Code 13083-862, Campinas, SP, Brazil
c Biochemistry & Biomedicine, University of Sussex, Falmer, BN1 9QG, Brighton, United Kingdom
d Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Zip Code 13083-970, Campinas, SP,
Brazil
e Brazilian Synchrotron Light Laboratory (LNLS) - Brazilian Center for Research in Energy and Materials (CNPEM), Zip Code 13083-970, Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work, 117 N-phenylbenzamides (NPDs) were prepared and evaluated against recombinant
AOX from the fungal pathogen Moniliophthora perniciosa. 1H, 13C NMR, FTIR, and mass spectra provided
structural information on NPDs. The library compounds were tested as Alternative Oxidase inhibitors in
two different assays using the model yeast Pichia pastoris: cell growth and oxygen consumption assays.
The most active compound, 3FH, was further characterized by DRX and 1H-NMR-STD. Single crystal X-ray
diffraction showed intra- and intermolecular interactions of 3FH in solid-state and elucidated its 3D
structural configuration. 1H-NMR-STD allowed us to derive protein-ligand interactions in a membrane-
mimetic system and evidenced an outstanding interaction of 3FH with this enzyme. Results of both
biological assays were used as input to Quantitative Structure-Activity Relationship models, which
highlighted the more important molecular fragments contributions for protein-ligand interaction.
© 2020 Elsevier B.V. All rights reserved.
Received 7 December 2019
Received in revised form
12 February 2020
Accepted 13 February 2020
Available online 19 February 2020
Keywords:
1H-STD NMR
NMR-based ligand screening
DRX
Enzyme inhibition
Alternative Oxidase (AOX)
QSAR
1. Introduction
treatment of other human [2e6] and plant [7e9] pathogens, such
as Trypanosoma brucei, Paracoccidioides brasiliensis, Cryptococcus
We have recently described the biological activity of NPDs
against the mitochondrial enzyme alternative oxidase (AOX) from
the cocoa pathogen Moniliophthora perniciosa. The compound N-(3-
bromophenyl)-3-fluorobenzamide inhibited the germination of
M. perniciosa spores and reduced the ability of the fungus to infect
cocoa plants [1]. AOX plays a critical role in M. perniciosa patho-
genesis, and the same enzyme is also a desirable target for the
neoformans, Aspergillus fumigatus, Candida albicans, Septoria tritici,
Sclerotinia sclerotiorum and Venturia inaequalis.
AOX is found in plants, many microorganisms, and some ani-
mals [10]. It is a membrane protein located in the matrix-side of the
inner mitochondrial membrane, and its biological role is associated,
among other things, with the resistance of a variety of biotic and
abiotic stress conditions [11]. Therefore, increasing interest has
been given to the AOX in terms of biotechnological application,
such as drug development [1,2,12,13], crop improvement [14e17]
and human gene therapy [18,19]. In this regard, the development of
new inhibitors, alongside crystallographic and kinetic studies, have
contributed to a better understanding of AOX [20,21].
* Corresponding author.
** Corresponding author.
E-mail addresses: pmiranda@unicamp.br (P.C.M.L. Miranda), silvana.rocco@
lnbio.cnpem.br (S.A. Rocco).
1
Here, our main goal was to shorten the knowledge gap on a
fungal AOX enzyme and its inhibition, which can be useful for
ligand-based drug design [22]. We present an expanded library of
These authors contributed equally to this work: Paulo C. S. Costa, Mario R. O.
Barsottini, and Maria L. L. Vieira.
2
These authors jointly supervised this work: Paulo C. M. L. Miranda and Silvana
A. Rocco.
https://doi.org/10.1016/j.molstruc.2020.127903
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
easily synthesized NPDs, alongside their physicochemical and
functional characterization. 1H and 13C NMR spectra were acquired
for each NPD. The lead compound was also characterized by
infrared spectroscopy, mass spectrometry, crystallographic studies,
and saturation-transfer difference (STD) NMR to determine the
epitope of ligand/protein binding. The NPD bioactivities were
determined in the model yeast Pichia pastoris, as we previously
reported [1], which were used as input to quantitative structure-
activity relationship models. We envisage that our results will be
over MgSO4, filtered, and evaporated to dryness under reduced
pressure to give the N-phenylbenzamides in 19e90% yields. One
hundred and seventeen compounds were synthesized following
this procedure. Detailed physicochemical properties, 1H and 13C
NMR data, FT-IR data, and yield are presented for each N-phenyl-
benzamide in the supplementary data.
2.3. Recombinant MpAOX expression and purification
useful for the development of
M. perniciosa and other microorganisms that depend on the AOX.
a
novel treatment against
The His-tagged rMpAOX
E. coli have been described elsewhere [1]. BL21 (DE3) Rosetta 2 E.
coli cells (Novagen) were transformed with pET28a-rMpAOX 40
vector and plated on selective LB medium with 25
g mLꢀ1 kana-
mycin and 50
g mLꢀ1 chloramphenicol. Individual colonies were
D40 cloning and transformation in
D
m
2. Material and methods
m
picked and inoculated into liquid LB medium with antibiotics and
grown at 37 ꢁC and 250 rpm for 16 h. The absorbance at 600 nm was
measured and aliquots were diluted in 1 L of fresh LB medium with
2.1. General information for physicochemical characterization
ATR-FTIR spectra were recorded on a Bomem spectrophotom-
eter, model MB100 and are reported in wavenumbers (cmꢀ1). The
spectral window was between 4000 and 400 cmꢀ1 using 64 scans
for sample and background characterization. Mass fragmentograms
were acquired using a GC coupled to a mass spectrometer (70 eV EI/
MSD), Agilent 5973A. A 30 m DB-5 fused-silica capillary column
antibiotics plus 50
2 h at 30 ꢁC and agitation, expression of the recombinant protein
was induced with 25 M IPTG. The culture was kept in the same
mM FeSO4 to a final optical density of 0.01. After
m
conditions for another 8e10 h and cells were harvested by centri-
fugation and stored at ꢀ80 ꢁC.
rMpAOX purification was performed essentially as described for
other AOXs, with some modifications [26,27]. E. coli cells were
resuspended in 25 mL of 50 mM Tris-HCl, pH 7.5 per initial litre of
cell culture, which was supplemented with protease inhibitors
(J&W Scientific) was used with a film thickness of 0.25 mm and an
internal diameter of 0.25 mm. The following oven temperature
settings wꢀer1e adopted: 60 ꢁC as initial temperature, an increase of
20 ꢁC min up to 290 ꢁC, and 28.5 min at 290 ꢁC. Temperatures
were set at 250 ꢁC in the injector and 300 ꢁC in the interface to the
detector.
(1 mM phenylmethylsulfonyl fluoride, 1
mg
mLꢀ1 leupeptin,
1
m
g mLꢀ1 aprotinin and 1
m
M pepstatin). The cell suspension was
passed two times through a pre-cooled cell disruption system
(Constant Systems Ltd) at 25 kPa and centrifuged for 15 min at
16,000 rcf to decant unbroken cells and large cell debris. The su-
pernatant was then centrifuged for 60 min at 200,000 rcf to collect
the cell membranes fraction which contains rMpAOX. The mem-
brane pellet was thoroughly resuspended with 50 mM Tris-HCl pH
7.5, 10 mM imidazole, 20% glycerol, 200 mM MgSO4 and 1% Fos-
Choline-12 (m/v). The absorbance at 600 nm was measured and
adjusted to 1 with the same buffer. The sample was left under
gentle rocking for 1 h at 4 ꢁC, after which it was centrifuged at
200,000 rcf for 30 min.
The supernatant containing the solubilized rMpAOX was sub-
jected to immobilized metal affinity chromatography with the Ni-
NTA Superflow resin (1 mL per cell culture litre; Qiagen) loaded
with Ni2þ, and was performed gravimetrically at 4 ꢁC. The resin was
pre-equilibrated with 50 mM Tris-HCl pH 7.5. The solubilized
protein sample was passed through the resin, which was then
washed with 10 vol of buffer A [20 mM Tris-HCl pH7.5, 50 mM
MgSO4, 60 mM NaCl, 20% glycerol (v/v) and 0.05% (m/v) n-dodecyl
Melting points (mp) were measured on GEHAKA digital PF1500
capillary melting point apparatus and were uncorrected. Analytical
thin-layer chromatography (TLC) was performed on Merck
analytical plates pre-coated with silica gel 60 F254 (0.25 mm thick).
TLC plates were visualized by exposure to ultraviolet light (UV) and/
or exposure to phosphomolybdic acid solution followed by brief
heating on a hot plate. The purity of compounds was confirmed to
be higher than 95% by analytical HPLC. The HPLC system consisted
of a Waters Alliance equipment series 2695 (Milford, MA, USA)
equipped with a quaternary pump, a sampler manager, a degasser
and a Waters 2996 UVeVis spectrophotometric detector model.
Chromatographic separation of the N-phenylbenzamide derivatives
was achieved at room temperature, using
a
reversed-phase
OmniSpher C18 column (250 ꢂ 4.6 mm I.D.; 5
mm particle size)
protected with a ChromoSep guard column SS (10 ꢂ 3 mm) (Agi-
lent, CA USA). The mobile phase consisted of a mixture of aceto-
nitrile and 100 mM ammonium acetate buffer, pH 7.2 (70:30, v/v)
freshly prepared and filtered through a 0.45 mm filter (Millipore,
Milford, Mass., USA). All samples were eluted under isocratic con-
ditions, at 1.0 mL minꢀ1 and detected spectrophotometrically be-
tween 200 and 400 nm. The control, acquisition, and data
processing were performed using Empower 2002.
b-D-maltoside (DDM)] containing 100 mM imidazole. rMpAOX
elution was carried out with 3 washes (one resin bed volume each)
of buffer A with 250 mM imidazole.
2.4. NMR and saturation-transfer difference (STD)
2.2. General protocol for the synthesis of N-phenylbenzamide
derivatives (3xy
)
To record NMR spectra of each sample, approximately 10 mg
was dissolved in 0.6 mL of deuterated solvent [CDCl3 or DMSO‑d6].
The NMR spectra were recorded on an Agilent DD2 spectrometer
from the Brazilian Biosciences National Laboratory (Brazilian
Centre for Research in Energy and Materials - CNPEM) operating in
Larmor frequency of 499.726 MHz. Chemical shifts for protons were
reported in parts per million (ppm) downfield from tetrame-
All materials, reagents and solvents were purchased from
Sigma-Aldrich, Merck, and Tedia Brazil and were used as received.
The classical Schotten-Baumann reaction was used for the syn-
thesis of amides [23e25]. Typical procedure: A solution of the acyl
chloride (0.5 mmol) in dichloromethane (2 mL) was added drop-
wise to a well-stirred mixture of the aniline (0.5 mmol) in
dichloromethane (2 mL) and 2 mL of aqueous sodium carbonate
(10%) at room temperature. The reaction media was stirred for
90 min and then the organic phase was separated and extracted
with brine (3 ꢂ 2 mL). The combined organic fractions were dried
€
thylsilane (TMS, 0 d) and were referenced to the residual proton in
the NMR solvent (CDCl3: 7.27 and DMSO‑d6: 2.50 ppm). Coupling
constants (J) are given in hertz (Hz). Splitting patterns were
designated as s, singlet; d, doublet; t, triplet; q, quadruplet; m,
multiplet; dd, double doublet; ddd, double double doublet; dt,

P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
3
doublet of triplets. Carbon nuclear magnetic resonance spectra
were recorded at 125.655 MHz and chemical shifts for carbon were
reported in parts per million (ppm) downfield from tetrame-
Hydrogens atoms were positioned geometrically in their idealized
positions [34]. The general-purpose crystallographic tool PLATON
[36] was used for the structure validation and MERCURY [37] was
used for molecular graphics representations.
thylsilane (TMS, 0 d) and were referenced to the carbon resonance
of the solvent (CDCl3: 77.2 and DMSO‑d6: 39.5 ppm). The structures
of the synthesized compounds were elucidated through 1D NMR
(1H and 13C) and 2D NMR (COSY 1He1H, HSQC and HMBC 1He13C)
spectroscopy.
Crystallographic data of compound 3FH reported in this manu-
script have been deposited with the Cambridge Crystallographic
Data Centre under the CCDC number: 1968438. The data can be
obtained free of charge from http://www.ccdc.cam.ac.uk/data_
request/cif.
The purified rMpAOX was concentrated by ultrafiltration
(Amicon, Millipore; NCO of 30 kDa) to approx. 300 mM, as measured
spectrophotometrically at 280 nm (Nanodrop 2000c spectropho-
tometer; Thermo Fisher Scientific) and the molar extinction coef-
ficient calculated by the ProtParam online tool [28]. The sample was
dialyzed against buffer A (used for protein purification) with
0.0125% (v/v) dodecyl octaethylene glycol ether (C12E8) instead of
DDM, then centrifuged at 20,000 rcf for 10 min at 4 ꢁC to remove
aggregates. A 20 mM 3FH stock solution was prepared in DMSO‑d6.
2.6. Biological activity
NPD biological activity was evaluated as described elsewhere
[1]. Briefly, the respiration rate and the growth rate of Pichia pas-
toris were measured in the presence of each NPD (control condi-
tion) or each NPD plus azoxystrobin (AOX induction condition).
Respiration and growth rates were converted to a percentage
relative to P. pastoris cultured without NPDs), and each experiment
was repeated at least three times. The identification of compounds
with a low correlation between AOX inhibition and antifungal ac-
tivity was performed as follows. An expected correlation of 1 be-
tween respiration and growth measurements where f(x) ¼ x was
assumed and the residual value for each NPD was determined.
Then, rMpAOX and compound 3FH were diluted to 1
mM and
400 M, respectively, in 600 L D2O containing 20 mM Tris-HCl, pH
m
m
7.5 and 0.0125% C12E8. 3FH without rMpAOX was used as negative
control.
1H-STD NMR spectra were acquired on a 600 MHz Agilent
INOVA spectrometer equipped with a triple-resonance (15 N/13C/
1H) cold probe at 298 K. 1H-STD NMR spectra were obtained by
subtraction of saturated spectra (on-resonance) from the reference
spectra (off-resonance) performed simultaneously by phase cycling
using a sequence dpfgse_satxfer implemented on the biopack
package of the VNMRJ software. 1H-STD-NMR spectra were recor-
ded with 8192 scans and selective saturation of protein resonances
at ꢀ0.5 ppm and 30 ppm for off-resonance spectra using a series of
40 Gaussian-shaped pulses (50 ms, 1 ms delay between pulses), for
a total saturation time of 2.04 s. The reference spectra (off-reso-
nance) were recorded with 4096 scans. Measurement of enhance-
ment intensities was performed by direct comparison of 1H-STD-
NMR spectra and reference spectra (off-resonance). The fractional
STD effect was calculated by (I0 e Isat)/I0, where (I0 e Isat) is the peak
intensity measured directly in the 1H-STD-NMR spectrum and I0 is
the peak intensity of an unsaturated reference spectrum (off-
resonance).
Inactive NPDs were used to estimate
were considered outliers (i.e., low correlation between both bio-
logical assays) if their residual value was higher than 1.96
s and AOX-selective NPDs
s
.
2.7. Quantitative structure-activity relationship (QSAR) studies
Respiration and growth rates from the yeast Pichia pastoris were
used to perform a QSAR analysis with the test compounds. We
chose the ISIDA/QSPR software to perform this study because it
uses a “Substructural Molecular Fragments” (SMF) approach during
QSAR modeling [38,39]. The SMF method splits the molecular co-
ordinates into smaller fragments and quantifies the contribution of
each small substructure to a given property. Chemical database files
(”.sdf”) were generated from ChemBioOffice 14, with fragment sizes
between 2 and 15 atoms (hydrogen not included) using “atoms and
bonds” as fragment descriptor. The results were submitted to a
linear fitting with a fragment-independent term [38,39]. The
external validation of the model consisted of 20% of the molecules
that were left out of the training set, and we considered only pre-
dictive correlation coefficient values (Q2) greater than 0.5. For each
analysis, 1120 models were generated including fragments and
equations.
2.5. Single crystal X-ray studies
For crystallization assays, 0.20 g of 3FH was treated with 2 mL of
water and the suspension was stirred manually. Approximately
1 mL of methanol was slowly added to the sample until its complete
solubilization. After that, volatiles were gently evaporated under
nitrogen flow at room temperature for 5 h, which led to the for-
mation of white needle-like crystals at the bottom of the mother
liquor.
3. Results and discussion
3.1. NPD synthesis and structural characterization
Crystallographic data for 3FH were collected at 100 K on the MX2
beamline (Brazilian Synchrotron Light Laboratory, Campinas,
Brazil) [29] with a PILATUS2M detector using monochromatic X-ray
Here, our main goal was to expand and further characterize our
NPD library of putative AOX inhibitors [1]. NPDs were identified
with the serial code 3XY, where X descriptor varies from A to I, and
the Y descriptor varies from E to S (Scheme 1). In total, one hundred
and seventeen N-phenylbenzamide derivatives (3XY) were pre-
(0.82104 Å). Data were recorded in the rotation mode using the
u
scan technique with 2
q
max ¼ 55.5ꢁ using the data collection
software MXCuBE [30]. MX2 is a macromolecular crystallography
beamline equipped with a mini-kappa goniometer head [31] and,
to avoid the lack of completeness at high angles, 6 kappa positions
(0, 30, 60, 90, 120 and 150ꢁ) were scanned from two 3FH crystals.
Data reduction and merging were performed using the XDS fol-
lowed by correction for absorption effects using an empirical
method implemented in XDS [32]. The structure was solved by
direct methods (intrinsic phasing) using SHELXT [33]. The refine-
ment was carried out by the full-matrix least-squares method with
anisotropic displacement parameters for all non-hydrogen atoms
based on F2 using SHELXL [34] through OLEX2 interface [35].
€
pared using traditional SchotteneBaumann conditions (Scheme 1)
[23e25] and yields varied from 19 to 90%. All compounds were
characterized by 1H NMR, 13C NMR, FT-IR, and EM (Supplementary
material).
The NPD structural assignment was performed by 1H and 13C
NMR spectroscopy, and the general trends are presented below. All
NPDs exhibited similar results, with characteristic coupling pat-
terns and variations depending on the particular substituent
(Supplementary material).

4
P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
Scheme 1. Preparation of N-phenylbenzamide derivatives (3XY) from benzoyl chlorides (1A-I)and amines (2E-S). The X descriptor varies from A to I, and the Y descriptor varies
from E to S.
In meta-substituted NPDs, the signals from hydrogens H-2 and
of the benzamide. This occurs due to an intramolecular interaction
that leaves H-14 oriented towards the benzamide carbonyl group,
that is, a hydrogen bonding between the NH of N-phenylbenzamide
and the carboxamide at the position 10. H-11 signal is less shielded
because of resonance and inductive effects of carboxamide. H-12
and H-13 signals are triplets. However, H-12 is more shielded and
presents a lower chemical shift than H-13. The shielding of H-12 is
related to the resonance effect on the nitrogen electron pair, which
increases the electron density at positions 10, 12 and 14.
H-10 at R1 and R2 were presented as triplets with second-order
4
distortion with, on average, JH-H of 2.0 Hz. The signal of those
hydrogens appears shifted to higher fields. The chemical shift of H-
2 has two important contributions, that is, the inductive effect of
the substituent at position 3 and the resonance effect of the
carbonyl group present in the structure. H-10 suffers the inductive
effect of the nitrogen and an eventual anisotropic deprotection by
the nonbonding electrons of the HNC(O) group. Hydrogens H-4, H-
6, H-12, and H-14 were presented as doublets (3JH-H ~8.0 Hz), as
double doublets (3JH-H ~8.0 Hz; 4JH-H ~ 2.0 Hz) or as double double
doublets (3JH-H ~8.0 Hz; 4JH-H ~ 2.0 Hz, 4JH-H ~ 1.0 Hz). Hydrogens H-
5 and H-13 were seen as triplets with a coupling constant of 8.0 Hz
(3JH-H). Their relative positions in the spectra were strongly
dependent on the substituents.
In monosubstituted compounds in R1, H-13 and H-11 signals
were overlaid in the NMR spectra, as well as H-10 and H-14 signals.
In this case, each hydrogen pair is magnetically and chemically
equivalent. On these compounds, H-2 was shifted to higher fields
when compared to H-10, except when R1 is CH3. Likewise, the same
effect happened in hydrogens H-2, H-3, H-5, and H-6 in R2 mono-
substituted compounds. On these compounds, H-10 has greater
chemical shift than H-2, because of a mesomeric effect of the amide
group. All hydrogen signal assignments were confirmed by COSY
(nJH-H) correlations.
In general, chemical shift values on 13C NMR for C-1, C-7, and C-9
signals showed low intensity. These quaternary carbons are bonded
to nitrogen atoms and undergo 14N quadrupole effects [40]. In
compound 3EE (Markush structure) the pairs C-3/C-5, C-2/C-6, C-
10/C-14 and C-11/C-13 have their signals overlaid since each carbon
pair and their corresponding hydrogens are magnetically and
chemically equivalent. The carboxylic carbon C-7 showed a less
shielded signal above 163 ppm. Carbon C-1 presented chemical
shifts ranging from 130 to 140 ppm, which can be explained by less
shielding due to ipso effects related to carboxamide groups. On the
other hand, C-9 (above 135 ppm) was less shielded because of a
nitrogen atom bonded to it.
Monosubstituted compounds at the benzoyl moiety have C-2, C-
3, C-5 and C-6 signals overlaid due to their chemical and magnetic
equivalence, similarly to C-10, C-11, C-13 and C-14 in R2 mono-
substituted NPDs at the aniline moiety. For meta-substituted
compounds, C-3 and C-11 signals exhibit higher chemical shifts
when R ¼ F, Cl, NO2, HN(CO) or OCH3 due to the electronegativity of
these groups. Their chemical shift reflects the inductive, electronic
effects, and shielding capacity of these substituents.
H-3 and H-6 signals in ortho-substituted compounds in the
3
benzamide aromatic ring are double doublets with JH-H between
4
7.0 and 9.0 Hz and JH-H between 1.0 and 2.0 Hz. In those com-
pounds, H-6 presents higher chemical shifts, due to the inductive,
electronic and mesomeric effects of the carbonyl group. The H-3
chemical shift is dependent on the nature of the substituent effects
and its magnitude. H-4 and H-5 signals are triplets and H-4 is less
shielded. This phenomenon occurs due to resonance effects of the
carbonyl group and the substituent at position 2.
The carbonyl carbon atom of a carboxamide placed at position
10 presents higher chemical shift than the carbonyl carbon C-7,
which is due to the formation of an intramolecular hydrogen bond.
The presence of this substituent increases the chemical shift of C-9
and C-10 to above 138 ppm. H-10 and H-14 signals in N-(3,4,5-
H-11 and H-14 in ortho-substituted compounds in the N-phenyl
ring are double doublets, and both hydrogens are shifted to higher
fields. H-14 undergoes an inductive effect of the nitrogen atom and
a mesomeric effect of nonbonding electrons of the carbonyl group
trimethoxyphenyl)benzamide (3EN) are overlaid and with a
chemical shift around 6.9 ppm. This is summed result of resonance
and inductive effects of electron-donor methoxy groups. For the
same reasons, C-10 and C-14 have a chemical shift around 90 ppm,

P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
5
although C-11, C-12 and C-13 display chemical shifts above
130 ppm due to the strong inductive effect of oxygen. H-10 and H-
14, as well as H-11 and H-13 signals are doublets in the N-phenyl
ring of para-substituted compounds and their 1H NMR signals
overlay. H-10 and H-14 are less shielded than H-11 and H-13,
because of the amide group effect. C-12 and C-9 signals show low
intensity and chemical shift above 130 ppm. In general, the
chemical shifts of the remaining carbons were strongly dependent
on the substituents and could be assigned by HSQC (1JC-H) corre-
lations. The assignment for the quaternary carbon signals was
confirmed by HMBC (nJC-H) correlations.
regions of the ligand (NMR peaks) that are closer to the protein
upon binding with a dissociation constant (Kd) ranging from
100 nM to 100 mM. The signal intensity of each hydrogen peak
reflects not only the relative amount of saturation received by each
particular hydrogen from the macromolecule through magnetiza-
tion transfer but also the relative proximity to the macromolecule.
Thus, a relative STD value for each hydrogen in the fragment can be
obtained based on their peak area integral value [41e44].
We used 1H-STD NMR experiments to define the binding
epitope of the 3FH inhibitor (Scheme 2E) in presence of pure AOX
from the fungal pathogen Moniliophthora perniciosa. To evaluate
possible nonspecific binding of the ligand to the mimetic mem-
brane (C12E8 micelle) itself, 1H-STD-NMR spectra was acquired
with the micelle lacking rMpAOX as a negative control, which must
be subtracted from the protein-containing sample to yield the true
STD difference spectrum for the remaining signals [45]. However,
the 1H-STD-NMR spectrum of 3FH with the membrane mimetic
showed no signals indicating no interaction between them
(Scheme 2A and 2B).
Fluorinated compounds exhibited 1H and 13C NMR spectra with
4
a HeF coupling 3JH-F at the order of 8.0e10.0 Hz, JH-F between 1.0
1
and 2.5 Hz, and a CeF coupling at the order of 247.0 Hz for JC-F
,
22.3 Hz for 2JC-F, 7.0 Hz for JC-F, and 2.9 Hz for JC-F, respectively.
3
4
N-(3-Bromophenyl)-3-fluorobenzamide (3FH was the most
)
potent, selective AOX inhibitor in our NPD library, while N,N’-(1,3-
phenylene)bis(3-methoxybenzamide) (3AQ) was biologically inert
[1]. Therefore, NPDs 3FH and 3AQ were further characterized by IR
spectrum and mass spectrometry analysis. The 3FH mass spec-
trometry shows molecular ions with m/z 292.9 and 294.9, and an
ion with m/z 123.0 corresponding to the cation 3-fluoro-acyl-ben-
zene produced after the break of the C7eN bond. The molecular
ions of 3FH reveal the bromine isotopic profile. The mass spec-
trometry of compound 3AQ shows the molecular ion with m/z 446.1
and another ion with m/z 134.9, which are related to the ion 3-
methoxy-acyl-benzene produced from the break of C7eN or
In the presence of rMpAOX, 3FH 1H NMR signals were readily
observed. The most intense STD effect in the spectrum was set as
100% and the remaining peaks were relatively normalized. The
strongest enhancement was observed for signals from H-12/H-13
(100%) followed by H-4 (~98%), H-14 (~90%) and H5 (~90%). A
weaker enhancement was observed for H-10 (~40%), and H-2
(~60%) and H6 (~52%) signals yielded moderate enhancements
(Scheme 2-C and 2D). This suggests an epitope near to protons H-
12/H-13/H-14 and H-4/H-5, indicative of a hydrophobic driving
model.
Those results agree with in silico simulations of NPD-AOX in-
teractions [1], according to which 3FH, especially H-12 and H-13,
interacts with hydrophobic residues in the enzyme active site, and
with the bromine group facing the metallic catalytic centre. Simi-
larly, H-4 and H-5 were placed close to the fluorine and interact
with the catalytic site. Those results corroborate with the experi-
mental evidence that 3FH interacts predominantly with the AOX
catalytic site and not with other cell components or enzymes.
C
23eN bonds.
The infrared spectra of both compounds were similar. 3FH pre-
sented an NeH stretching at 3261 cmꢀ1, and a carbonyl stretching
at 1646 cmꢀ1. The CeN stretching bands were assigned at
1260 cmꢀ1 and 1247 cmꢀ1. In compound 3AQ the NeH stretching
appeared at 3306 cmꢀ1 and the band related to the carbonyl group
at 1644 cmꢀ1. The CeN stretching bands appeared at 1270 cmꢀ1
and 1233 cmꢀ1
.
3.2. STD-NMR
1H-STD-NMR is a screening technique for identifying lead
structures or a tool useful for elucidating ligand moieties important
for ligand binding (epitope). The 1H-STD-NMR spectrum identifies
3.3. Single crystal X-ray studies
Since it was possible to generate
a mimetic model for
Scheme 2. 1H-STD-NMR of 3FH with AOX: (A) 3FH þ C12E8 micelle off-resonance spectrum; (B) 3FH þ C12E8 micelle difference spectrum (off resonance minus on-resonance); (C)
3FH þ C12E8 micelle þ AOX off-resonance spectrum; (D) 3FH þ C12E8 micelle þ AOX difference spectrum (off resonance minus on-resonance); (E) 3FH chemical structure and
Table 1 representing the relative degree of saturation of the individual hydrogens mapped into the structure and normalized to the most intense incremented proton (STD%).

6
P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
Table 2
Crystal data and refinement details for 3FH compound.
3FH
Molecular formula
Mr
C13H9BrFNO
294.12
Crystal system, space group
a, b, c (Å)
Monoclinic, P21/n
14.872(2), 4.8860(18),
15.501(2)
90, 94.158(2), 90
1123.4(5)
a
,
b,
g
(ꢁ)
V (Å3)
Z
4
Radiation source, wavelength (Å)
Synchrotron, 0.82104
5.323
m
(mmꢀ1
)
Crystal size (mm)
Tmin, Tmax
1.5 ꢂ 0.075 x 0.01
0.9, 1.0
No. of measured, independent and observed
138401, 2383, 2241
[I > 2.0
(sin
R [F2 > 2
s
(I)] reflections
q
/l)
(Åꢀ1
)
0.637
0.037, 0.095, 1.069
2241
max
Fig. 1. ORTEP representation of compound 3FH showing atom-numbering. Thermal
ellipsoids are drawn at the 50% probability level and H atoms are shown as small
spheres of arbitrary radii.
s
(F2)], wR(F2), S
No. of reflections
No. of parameters
154
Drmax
,
Drmin (eÅ3)
1.6, ꢀ0.49
CCDC number
1968438
monitoring the ligand binding on the anchored protein using STD, it
was necessary to compare the preferred conformations of the
ligand in solution and in solid state. Compound 3FH has two halo-
gens, therefore it was interesting to evaluate the influence of non-
classical hydrogen or halogen bonding in molecular conformation.
Fig. 1 shows an ORTEP diagram for the 3FH crystal structure and its
crystal packing. The asymmetric unit comprises one molecule.
Generally, the geometrical parameters of the studied compounds
are comparable with each other and with structures having the
same moieties [46]. The planarity of the molecule is disturbed
mainly by C7, O1, N8 (amide group) and Br1, with maximum r.m.s.
deviations of ꢀ0.257, ꢀ0.834, ꢀ0.204 and 0.364 Å, respectively, and
a dihedral angle between the plane of the two rings of 175.12ꢁ. This
planarity disturbance seems to be related to the absence of
hydrogen bond acceptor atoms in the vicinity of the N(8)eH(8)
group. The addition of a hydrogen bond acceptor atom close to the
NeH group could lead to an increase in the planarity of the mole-
cule, as observed for 5-chloro-2-methoxy-N-phenylbenzamide
[46]. Details of the data collection and refinement are given in
Table 2.
the AOX pathway. The most active compound, 3CH, inhibited 89.6%
of the azoxystrobin-insensitive oxygen consumption. Concerning
P. pastoris growth assay, 16 of 43 NPDs displayed some level of
activity. Nine of those compounds were nonspecific for AOX, as they
inhibited P. pastoris growth on control conditions (without azox-
ystrobin), and 7 NPDs were selective for the AOX-dependent
growth. From the selective growth-inhibitors, compound 3CK was
the most active and inhibited P. pastoris growth rate by 85.4%.
Within the expanded set of 117 NPDs, 3FH remained as the most
potent, selective AOX inhibitor both in oxygen consumption mea-
surements and antifungal activity assays and is therefore regarded
as the lead compound in our library. The complete dataset can be
found at the PubChem database (PubChem AID: 1259412).
Notably, some compounds were more effective as AOX in-
hibitors (oxygen consumption assay) than as antifungal agents
(growth assay). For instance, compound 3CH inhibited 89.9% of the
AOX activity, but displayed only mild antifungal effect with 42.3%
inhibition of P. pastoris growth. On the contrary, compound 3CK
displayed similar activity levels with 85% inhibition in both assays.
This suggests that the cellular environment influences NPD activity,
such as P. pastoris detoxification machinery that may be more
effective on 3CH than 3CK. This has also been observed for other
NPDs but has not been fully explored [1].
After excluding toxic NPDs, i.e. those that have any effect on
P. pastoris in the absence of azoxystrobin, 66 compounds were
either inactive (17) or active specifically on AOX (49). From the 49
active compounds, 25 show a good correlation between AOX in-
hibition and growth inhibition (Fig. 2). On average, they inhibited
39.1% of the AOX activity and 29.4% of P. pastoris growth. On the
other hand, 24 NPDs were considered as outliers, indicating that
factors other than the affinity towards the AOX are relevant for the
antifungal activity. In general, a lower antifungal activity was found
(24.8% growth rate reduction) than expected from oxygen con-
sumption measurements (75% AOX inhibition). These results
corroborate the hypothesis that those NPD are susceptible to the
cell detoxification machinery and are, therefore, less active on a
cellular environment. This information can be useful for the design
of new AOX inhibitors that have a higher chance of being effective
in a cellular environment. Notably, two NPDs (3GF and 3FF) were
more active on growth assays than expected, indicating a syner-
gistic effect with azoxystrobin through a yet unknown cellular
target.
3.4. Biological activity
Pichia pastoris is a robust model for the evaluation of NPD bio-
logical activity and has been previously used to characterize 74
NPDs from our library of 117 compounds [1]. The alternative res-
piratory pathway (AOX) is induced by azoxystrobin or other in-
hibitors of the main respiration. Moreover, AOX activity can be
detected directly by oxygen consumption measurements, whereas
the antifungal activity of test compounds is assessed through the
measurement of P. pastoris growth rates [1].
Among the 43 newly tested NPDs presented here, 17 were active
on oxygen consumption measurements e all of them specific for
Table 1
1H-STD-Hydrogen relationship.
d
(ppm)
1H-STD (%)
H
7.87
7.67
7.61
7.55
7.50
7.43
7.21
39.5
52.3
90.2
59.8
98.1
89.7
100.0
10
6
14
2
4
5
13 and 12

P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
7
Fig. 2. Correlation between oxygen consumption and growth assays with P. pastoris. The continuous line represents the assumed linear correlation between AOX inhibition and
growth inhibition where f(x) ¼ x. While 25 NPDs follow the expected distribution (red circles), 24 NPDs do not (green triangles), which is likely explained by additional effects that
occur in a cellular environment and influence their antifungal activity.
3.5. QSAR studies
were also submitted to the QSAR in a separate analysis. Both bio-
assays yielded similar linear correlation coefficients (R2 of 0.91 and
0.93). Usually, ISIDA/QSPR builds up to 1276 structure-property
models involving four linear and non-linear fitting equations and
315 molecular descriptors to perform the analysis. All those 315
descriptors along with their brief meaning, cumulative incidence,
and average regression coefficients (which will serve as a measure
of their estimate across these models) are considered during the
calculus. These models were able to describe up to 90.64% variance
(R2 ¼ 0.9064) in the oxygen consumption bioassay and 92.57%
(R2 ¼ 0.9257) in the growth inhibition (Figs. 3 and 4, respectively).
Both biological assays e respiration and growth e allowed us to
group the NPDs in three different groups: (i) inactive, (ii) active, but
nonspecific for AOX (also inhibiting the main respiratory pathway
or other cellular processes); and (iii) active and specific for AOX. The
last group was used in QSAR-2D analysis for each biological assay
separately. From the 117 assessed NPDs, 58 inhibited O2 con-
sumption exclusively via AOX pathway and were submitted to the
QSAR analysis. Conversely, from the same molecular set, 36 com-
pounds inhibited cell growth exclusively via AOX pathway and
Fig. 3. Plot showing the correlation between observed versus calculated valuesfor AOX inhibition in oxygen consumption bioassay using the selected compounds.

8
P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
Fig. 4. Plot showing the comparison between observed versus calculatedvalues for AOX inhibition in growth bioassay using the selected compounds.
The root-mean-squared error (RMSE) of 5.8636 and mean absolute
error (MAE) of 4.5621 were observed for the oxygen consumption
bioassay, while the values 5.2635 (RMSE) and 4.0192 (MAE) were
observed for the observed growth inhibition bioassay.
to the compound. Consequently, the oxygen consumption assay is
not influenced by xenobiotic metabolization and efflux mecha-
nisms. Fig. 5 shows the more effective fragment descriptors gov-
erning AOX inhibition in oxygen consumption bioassay. In this
Figure, each fragment is associated to its respective Carhart’s no-
tation [47], and weight where red-colored substructures increase
NPD’s efficacy (negative values), while, blue-colored substructures
decrease the NPD’s efficacy (positive values). Negative values
indicate better AOX inhibitors, hence fragments with negative
values are desired. The values inside the parenthesis is the standard
deviation of the contribution.
Although both linear regressions gave almost the same results,
the set of “Substructural Molecular Fragments” (SMF) generated for
each assay was different, which arose from the fact that the input
set of molecules was different in each case. The apparent contra-
diction between the data of growing and oxygen consumption
assay can be justified by the duration of the assays. While the cell
growth is measured for 3 days, the oxygen consumption mea-
surement is almost instantaneous, lasting not more than 4 min in
total. Therefore, in the oxygen consumption assay there is enough
time for NPDs to pass through cell barriers and reach the AOX, but
not time enough for P. pastoris cell machinery to respond and adapt
4. Conclusion
One hundred and seventeen compounds were synthesized and
Fig. 5. Main fragment structures responsible for oxygen consumption inhibition: fragments correlated with IC50 decreasing are red-colored and fragments correlated with IC50
increasing are blue-colored. Contribution weight and standard deviation, in parenthesis, for each fragment is depicted below them. Fig. 6 shows the fragment descriptors governing
AOX inhibition in growth bioassay. Once again, each fragment is associated to its respective Carhart’s notation [47], and weight where red-colored substructures increase NPD’s
efficacy (negative values), while, blue-colored substructures decrease the NPD’s efficacy (positive values). Negative values indicate better AOX inhibitors, hence fragments with
negative values are desired. The values inside the parenthesis is the standard deviation of the contribution.

P.C.S. Costa et al. / Journal of Molecular Structure 1208 (2020) 127903
9
Fig. 6. Main fragment structures responsible for growth inhibition: fragments correlated with IC50 decreasing are red-colored and fragments correlated with IC50 increasing are
blue-colored. Contribution weight and standard deviation, in parenthesis, for each fragment is depicted below them. It was observed that NPDs containing larger and polarizable
substituents at the aniline moiety have higher activity in both bioassays. Bromine, chlorine, trifluoromethyl, and methoxy at meta position showed negative values (reduce IC50
values) for both bioassays. It is noteworthy that the NPDs with carboxy-amide at ortho or bromine at meta position on the aromatic ring of N-phenyl have greater biological activity.
Notwithstanding, large substituents such as iodine, chlorine, methoxy group on the benzamide aromatic ring decrease NPD biological activity. This fact reveals that the substitution
at one aromatic chain is preferable than on the other. The fact that halogens are good substituents agrees with the common use of chlorine and fluorine as bioisosteres to prevent
drug metabolization [48].
assayed against the Alternative Oxidase enzyme (AOX) using the
model yeast Pichia pastoris. Compound 3FH showed a superior
bioactivity in selective inhibition of oxygen consumption and
fungal growth. 1H-STD-NMR results evidenced an outstanding
interaction of 3FH with AOX enzyme anchored in a membrane
mimetic system. Results of both biological assays were used as
input to quantitative structure-activity relationship models, which
elucidated the most important molecular fragments contributions.
We envisage that our results, alongside the facile synthesis of NPDs,
will be useful for the development of novel antifungal agents based
on AOX inhibitors.
Acknowledgements
~
This work was supported by The Sao Paulo Research Foundation
(FAPESP) through research grant 2016/10498-4 and scholarships
2014/15339-6, 2015/09870-3 and 2018/03130-6. This work was
also supported by the National Council for Scientific and Techno-
logical Development (CNPq) grant 424949/2018-0 and scholarships
142358/2014-2, 120294/2016-8, 153823/2016-0, 131264/2017-6
and 140312/2019-6. J. S. E. thanks SAE/UNICAMP for his scholarship.
The authors also acknowledge the Brazilian Biosciences National
Laboratory (LNBio-CNPEM/MCTI), specifically the Chemistry and
Natural Products Laboratory. This research used resources of the
Brazilian Synchrotron Light Laboratory (LNLS), an open national
facility operated by the Brazilian Centre for Research in Energy and
Materials (CNPEM) for the Brazilian Ministry for Science, Technol-
ogy, Innovations and Communications (MCTIC). The MX2 beamline
staff is acknowledged for the assistance during the experiments.
Author contribution
Wrote the manuscript: P.C.S.C., M.R.O.B., M.L.S., S.A.R., and
P.C.M.L.M.; NPD synthesis and structural characterization: P.C.S.C.,
M.L.L.V., J.S., S.A.R, and P.C.M.L.M.; crystallographic studies: A.F.Z.N.
and A.C.M.Z. P. pastoris and antifungal assays in vitro: M.R.O.B.,
B.A.P., M.F.C., and G.A.G.P.; Quantitative Structure Activity-
Relationship determination: P.C.S.C., J.S.E., M.R.O.B., and
P.C.M.L.M.; 1H-STD NMR: M.L.S., S.A.R, P.C.S.C., and M.R.O.B.
Conceptualization: S.A.R and P.C.M.L.M.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2020.127903.
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CRediT authorship contribution statement
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