Synthesis, bioevaluation and docking studies of new imidamide derivatives as nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2021.116294

In search of new Nitric Oxide Synthase (NOS) inhibitor agents, two isosteric series of derivatives with an imidamide scaffold (one of them with a hydroxyl group and the other with a carbonyl one) were synthesized and evaluated on inducible (iNOS) and neuronal (nNOS) isoforms. These compounds have been designed by combining a kynurenamine framework with an amidine moiety in order to improve selectivity for the inducible isoform. In general, the in vitro inhibitory assays exhibited better inhibition values on the iNOS isoform, being the N-(3-(2-amino-5-methoxyphenyl)-3-hydroxypropyl)-4-(trifluoromethyl)benzimidamide 4i the most active inhibitor with the highest iNOS selectivity, without inhibiting eNOS. Docking studies on the two most active compounds suggest a different binding mode on both isozymes, supporting the experimentally observed selectivity towards the inducible isoform. Physicochemical in silico studies suggest that these compounds possess good drug-likeness properties.

Full text of DOI:10.1016/j.bmc.2021.116294

Bioorg. Med. Chem. 44 (2021) 116294
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, bioevaluation and docking studies of new imidamide derivatives
as nitric oxide synthase inhibitors
,
c
,
d
,
e
a,*,1
Fabio Ariasa, Francisco Franco-Montalbana, Miguel Romerob
, M. Dora Carrion
,
´
a,
*
,
1
´
M. Encarnacion Camacho
a
´
´
Departamento de Química Farmaceutica y Organica, Facultad de Farmacia, Universidad de Granada, Spain
b Departamento de Farmacología, Facultad de Farmacia, Universidad de Granada, Spain
c
´
Instituto de Investigacion Biosanitaria de Granada, ibs.GRANADA, Granada, Spain
d Department of Pharmacology, School of Pharmacy and Center for Biomedical Research (CIBM), University of Granada, 18071 Granada, Spain
e Ciber de Enfermedades Cardiovasculares (CIBERCV), Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
In search of new Nitric Oxide Synthase (NOS) inhibitor agents, two isosteric series of derivatives with an imi-
damide scaffold (one of them with a hydroxyl group and the other with a carbonyl one) were synthesized and
evaluated on inducible (iNOS) and neuronal (nNOS) isoforms. These compounds have been designed by
combining a kynurenamine framework with an amidine moiety in order to improve selectivity for the inducible
isoform. In general, the in vitro inhibitory assays exhibited better inhibition values on the iNOS isoform, being the
N-(3-(2-amino-5-methoxyphenyl)-3-hydroxypropyl)-4-(trifluoromethyl)benzimidamide 4i the most active in-
hibitor with the highest iNOS selectivity, without inhibiting eNOS. Docking studies on the two most active
compounds suggest a different binding mode on both isozymes, supporting the experimentally observed selec-
tivity towards the inducible isoform. Physicochemical in silico studies suggest that these compounds possess good
drug-likeness properties.
Drug design
Inhibitors
Imidamides
Nitric Oxide Synthase
Synthesis
1. Introduction
(RNSs). These RNSs take part in several inflammatory and/or oxidative
stress pathways, contributing in the development of various patholog-
Nitric oxide (NO) is a heterodiatomic molecule with an unpaired
electron which behaves as a free radical. Its small size, lack of charge,
and high solubility, allow NO to freely diffuse through any cellular
membrane in the organism.1
ical conditions such as Parkinson’s disease, Alzheimer’s disease,5,6
sepsis, heart failure,7 multiple sclerosis,8 rheumatoid arthritis,9 diabetes
mellitus,10 inflammatory bowel disease11 and acute lung injury.12
Nowadays, the involvement of NO in cancer is well known, specially,
how the iNOS activity affects tumour progression.13,14 Thus, the in-
crease in iNOS expression has been found in different cancer types, such
as breast,15 colon,16 pancreatic17 and lung18 cancers, as well as in head
and neck squamous carcinoma19 glioblastoma,20,21 and melanoma.22
The main role of eNOS isoform is producing NO in order to relax
vasculature, inhibit platelet and white cell adhesion, prevent smooth
muscle cell replication, promote angiogenesis, and control the expres-
sion of vascular endothelium growth factor (VEGF).23,24 In this sense,
eNOS inhibition can lead to severe hypertension or other cardiovascular
disorders.
NO is produced by the Nitric Oxide Synthase (NOS) during the
transformation of L-arginine to L-Citrulline, consuming NADPH and O2.
There are three different isoforms named according to the place where
they can be found: neurons (nNOS), endothelium (eNOS) or induced by
the immune system (iNOS), being involved, respectively, in neuro-
transmission, smooth tissue relaxation and the immune response.2
Dysregulated NO levels are implicated in different disorders.3 For
example, the overproduction of NO by nNOS has been proven as a
common factor in a wide range of neurological deficits such as Alz-
heimer’s, Huntington’s, Parkinson’s, and amyotrophic lateral sclerosis.4
Whereas, iNOS overexpression highly increases the NO levels, which
later reacts with superoxide radical to produce reactive nitrogen species
Currently, the exploration of potent and selective NOS inhibitors
could signify a step forward in future therapies for pathologies involving
* Corresponding authors.
´
E-mail addresses: dcarrion@ugr.es (M.D. Carrion), ecamacho@ugr.es (M.E. Camacho).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2021.116294
Received 11 May 2021; Received in revised form 18 June 2021; Accepted 19 June 2021
Available online 28 June 2021
0968-0896/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
NO overproduction. Thus, the development of selective nNOS inhibitors
would be important to better understand the role of NO in the central
nervous system and to deepen in the research of neurodegenerative
disorders.25 Besides, the investigation of selective iNOS inhibitors could
be crucial for the research of new treatments for diseases where the
immune and inflammatory response of the organism is involved, such as
septic shock or rheumatoid artritis.26
of target compounds 4a-i and 5a-h. 5-Methoxy-2-nitrobenzaldehyde 6c
(synthesized by methylation with MeI and K2CO3),31 along with the
commercial 2-nitrobenzaldehyde 6a and 5-chloro-2-nitrobenzaldehyde
6b, were converted into the 3-(5-substituted-2-nitrophenyl)-3-hydrox-
ypropanenitriles 7a-c by treatment with n-BuLi and CH3CN in dry THF.
The nitrile group of these derivatives was reduced selectively with BH3/
THF to the primary amines 8a-c.30 For the next step, it was necessary to
synthesize the benzyl-benzimidothioates 9b-d, starting from benzyl
bromide and the corresponding benzothioamides (Scheme 2).32
Most compounds initially investigated as NOS inhibitors were
designed mimicking the L-arginine structure, such as L-NAME (L-NG-
nitroarginine methyl ester). These molecules cannot be used clinically,
since they are able to inhibit eNOS and, therefore, induce hyperten-
sion.27 In order to find selective NOS inhibitors, we have already
investigated different kynurenamine derivatives 1 (Fig. 1).28 Afterward
the substituted acyl group in compounds 1 was modified to obtain de-
rivatives 229 with a urea or thiourea moiety finding interesting mole-
cules, such as the iNOS/nNOS selective oxopropyl-urea 2a (R1 = Cl, R2
The reaction between the amine group of the distinct 3-amino-1-(2-
nitro-5-substitutedphenyl)-propan-1-ols 8a-c and the different imidates
9a-d, which includes the commercially available ethyl acetimidate 9a,
generated the imidamide function present in the nine nitro-
phenylimidamide intermediates 10a-I, which were turned into the final
N-(3-(2-amino-5-substitutedphenyl)-3-hydroxypropyl)imidamides 4a-i
using Fe/FeSO4 as reducing agent. In addition, the hydroxyl group of the
diverse imidamides 10a-h was oxidized to a carbonyl one with Jones
reagent, to give the corresponding ketones 11a-h. Finally, the nitro
group of these intermediates were reduced to afford the final N-(3-(2-
= Et, X = O; IC50 = 100 μM in iNOS; IC50 > 1000 μM in nNOS). Then, we
have replaced the carbonyl group by a hydroxyl one, obtaining prom-
ising hydroxyproyl-ureas and thioureas 3,30 as derivative 3a (R1 = OMe,
R2 = Me, X = S; IC50 = 180
μM in iNOS; IC50 = 130
μ
M in nNOS). In the
amino-5-substitutedphenyl)-3-oxopropyl)imidamides
5a-h,
as
last years different aliphatic, aromatic and cyclic amidines have been
studied as competitive iNOS inhibitors.27 Thereby, in this paper we have
investigated different derivatives with an amidine scaffold, and set a
synthetic procedure to join two structural components, a kynurenamine
skeleton with an imidamide residue. In vitro inhibition assays on iNOS
and nNOS have been done on all synthesized compounds and the best
performing molecules selected for the determination of its IC50 values.
Docking studies have also been carried out to account for the NOS iso-
form selectivity observed.
described for 4a-i.
2.2. Biological activity evaluation
2.2.1. iNOS and nNOS inhibition
In order to elucidate the biological activity of the novel compounds
as iNOS and nNOS inhibitors, in vitro assays employing human recom-
binant isoforms have been carried out. The data presented in Table 1
show the results of the preliminary inhibition screening for the final
imidamides 4a-i and 5a-h. The tests were conducted at a terminal
concentration of 1 mM in both isoforms. The range of values obtained
oscillated between 16.22% and 99.61% in iNOS and between 7.18% and
89.16% in nNOS. Based on this preliminary evaluation, the new de-
rivatives exhibited better inhibition values on the inducible isoform than
on the neuronal one. In this sense, twelve molecules showed a
2. Results and discussion
2.1. Chemistry
Scheme 1 depicts the synthetic strategy adopted for the preparation
Fig. 1. Graphic overview of new imidamides described by our research group.
2

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
Scheme 1. Synthetic route of novel imidamides 4a-i and 5a-h. Reagents and conditions: a) CH3CN, BuLi, THF, ꢀ 78 ◦C, then RT; b) BH3-THF, 0 ◦C, then 4 h RT; c)
MeOH 0 ◦C, then 15 h RT; d) CrO3, CH3COCH3, H2SO4, DCM 0 ◦C, 10 min; e) Fe/FeSO4, H2O, 100 ◦C, 1.5 h.
compounds inhibited nNOS also with percentages of inhibition greater
than 50%. In general, molecules with a hydroxypropyl group (de-
rivatives with general structure 4) exhibited higher percentages of iNOS
inhibition, while those with a carbonyl rest (compounds type 5) better
inhibited the neuronal isoform.
Regarding the iNOS inhibition values, it can be seen that, in general,
molecules with an electron-donating substituent in R1 (OMe) show the
highest inhibition rates versus this isozyme (4g-i, 5f-h), except for 5e,
which has a high percentage of inhibition on iNOS and contains an
electron-withdrawing group (R1 = Cl).
Scheme 2. Synthesis of benzimidothioates 9b-d. Reagents and conditions: (a)
With respect to neuronal isoform, derivatives 5f-h (R1 = OMe)
behave as good nNOS inhibitors, along with 5a (R1 = H).
CHCl3, reflux 2 h.
Despite presenting such a large range of percentages, iNOS selec-
tivity prevails in most of these compounds, with the exception of the last
members of this family 5f-h, where a greater inhibition in nNOS is
observed.
Table 1
In vitro iNOS and nNOS inhibition (%) observed in the presence of 1 mM con-
centration of compounds 4a-i and 5a-h.
Among all candidates, the best performing in terms of percentage of
inhibition were selected for the evaluation of their IC50 values on the
inducible isoform and on the neuronal one (see Table 2). The IC50 values
observed for iNOS range between 20.0 and 59.7 µM. The most active
iNOS inhibitors of all tested compounds are the 3-hydroxypropyl de-
rivatives with a methoxy group in R1, 4i (R2 = p-CF3Ph, IC50 = 20.0 µM)
and 4g (R2 = Ph, IC50 = 22.5 µM), also presenting selectivity toward
nNOS. In addition, the oxopropyl derivative 5e (R1 = Cl, R2 = Me)
Compound
R1
R2
% iNOS inhibitiona
% nNOS inhibitiona
4a
4b
4c
4d
4e
4f
H
Me
16.22 ± 1.89
64.09 ± 2.63
73.17 ± 1.38
60.50 ± 2.33
49.11 ± 1.25
57.39 ± 1.42
95.91 ± 0.27
81.02 ± 1.80
99.61 ± 0.55
22.25 ± 0.84
35.63 ± 2.25
33.42 ± 2.48
68.61 ± 1.28
99.14 ± 0.69
97.59 ± 1.24
94.73 ± 0.50
75.12 ± 1.46
78.63 ± 1.34
76.55 ± 0.33
28.26 ± 2.20
23.19 ± 1.88
31.57 ± 0.82
36.45 ± 1.49
66.11 ± 1.17
19.01 ± 2.75
7.18 ± 2.81
58.47 ± 0.69
12.50 ± 1.06
88.83 ± 0.45
66.47 ± 1.02
37.43 ± 1.67
55.98 ± 0.23
19.88 ± 0.57
89.16 ± 0.61
85.86 ± 1.62
86.44 ± 0.99
9.86 ± 3.17
80.55 ± 2.29
H
Ph
H
p-ClPh
p-CF3Ph
Me
H
Cl
OMe
OMe
OMe
OMe
H
Me
4g
4h
4i
Ph
p-ClPh
p-CF3Ph
Me
5a
5b
5c
5d
5e
5f
Table 2
H
Ph
IC50 values (μM) for the inhibition of iNOS and nNOS activities by the most
H
p-ClPh
p-CF3Ph
Me
potent imidamide derivatives 4g, 4i, 5a and 5e-g.
Compound
H
iNOSa
nNOSa
Cl
OMe
OMe
OMe
Cl
Me
4g
4i
22.5
20.0
>1000
25.6
59.7
45.2
100
>1000
>1000
324.7
>1000
223.3
193.7
>1000
130
5g
5h
2ab
3ab
Ph
p-ClPh
Et
5a
5e
5f
OMe
Me
a
Values are the mean ± SEM of the percentage of iNOS and nNOS inhibition
produced by 1 mM concentration of each compound. Each value is the mean of
three experiments performed by triplicate using recombinant iNOS and nNOS
enzymes. b2a29 and 3a30 were used as reference.
5g
2ab
3ab
180
a
Data obtained by measuring the percentage of inhibition on at least six
concentrations of each compound.
b
2a29 and 3a30 were used as reference.
percentage of inhibition greater than 50% on iNOS while eight
3

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
exhibited an iNOS inhibition value almost comparable to its previous
isosters (IC50 = 25.6 µM), maintaining selectivity for this isoform.
In nNOS, the IC50 values obtained are significantly higher than on
iNOS, being the molecules with a carbonyl rest the best inhibitors,
ranging from 193.7 µM in 5g to 324.7 µM for 5a, the latter being the
compound presenting the best selectivity on nNOS. In this way, the
replacement of the hydroxyl moiety for a carbonyl one decreases the
iNOS potency and the selectivity versus nNOS.
weakly (approximately 20%) reduced (Fig. 3).
2.3. Molecular docking study
Docking studies on human proteins iNOS and nNOS (pdb IDs 4CX7
and 6AV2) were performed to shed some light on the binding mode of
these imidamide derivatives, and to account for the selectivity differ-
ences observed experimentally on both enzymes. Compounds were
selected for the docking analysis as they perform best on the inhibition
assays. Before any calculations were made, redocking of the cognate
ligands on crystal structures 4CX7 and 6AV2 were carried out to account
for the validity of docking protocol. In both cases, the preferred poses for
the cognate ligands matched those of the crystal structures (results not
shown). Next, compounds 4g and 4i were docked on human iNOS (pdb
4CX7) and nNOS (pdb 6AV2). Fig. 4a shows the preferred binding poses
of both compounds on the catalytic active site of iNOS.
These two series of imidamide derivatives have been designed by
isosteric replacement from the oxopropyl-ureas and tioureas of general
structure 229 and the hydroxypropyl-ureas and thioureas 330, being the
most active molecules in each series the oxopropylurea 2a (R1 = Cl, R2
= Et, X = O; IC50 = 100 µM in iNOS and selectivity versus nNOS) and the
hydroxypropyl-thiourea 3a (R1 = OMe, R2 = Me, X = S; IC50 = 180 µM
versus iNOS and 130 µM in nNOS) (Table 2). In this regard, the amidine
residue in imidamides 4 and 5 improve de iNOS inhibition values when
compared to their previous isosters 3 and 2, in addition to the iNOS/
nNOS selectivity regarding the hydroxypropyl-thioureas.
4g displays its phenyl ring underneath the Fe atom of the heme group
favoring a π–cation interaction (Fig. 4a). Its imidamide group is dis-
played toward the carboxylate residues of the heme group forming a H-
bond. Similarly, 4i orients its p-CF3Ph moiety directly underneath the Fe
atom of the heme group, and because of its bulkier p-trifluorophenyl
imidamide group, is forced to display the imidamide group away from
the catalytic site and toward the carboxylate residues of the heme group.
Unlike 4g, 4i can establish a H-bond with Gln263 at the entrance of the
catalytic pocket. As these poses show, the imidamide groups of 4g and 4i
are not binding the catalytic residue Glu377 as expected, however it
should be noted the disposition of the p-methoxyaniline ring. In both
compounds the ring is displayed toward a catalytic pocket generated by
Gln263, Tyr347, Arg388, Asp382 and Tyr373, H-bonding the important
catalytic residues Arg388, Asp382 and Tyr373.
2.2.2. eNOS inhibition activity
As indicated above, the undesirable side effects for NOS inhibitors
derive from the alteration of the endothelial isoform. Hence, in order to
prove that our most potent inhibitor, does not affect the cardiovascular
system, a functional test with 4i was carried out. In this way,
acetylcholine-induced endothelium-dependent relaxation has been
studied using endothelium intact rat aortic rings. This classic cholinergic
agonist activates eNOS by a calcium-dependent mechanism.33 The
endothelium-dependent relaxation to acetylcholine was not affected by
4i (Fig. 2), confirming the absence of eNOS inhibition of this compound,
whereas the non-selective NOS inhibitor L-NAME almost abolished this
response.
These poses are of significance if we compare them with those
adopted by NPA (Nω-propyl-L-arginine), and the natural substrate L-
arginine, on the catalytic site of iNOS. Both compounds, 4g and 4i, show
binding interactions with at least two of the catalytically important
residues in the active site region.34
2.2.3. Cell viability (cytitixicity determination)
We investigated the cell viability activity of compounds 4g, 4i, 5e
and 5g using HUVECs. The tested concentrations were within the range
of IC50 values for the inhibition of iNOS activity by the most potent
imidamide derivatives. We observed that these compounds had little
toxicity, but not significant as compared with the control conditions,
since only at higher concentration tested (500 µM) the cell viability is
As for the binding poses of 4g and 4i on the nNOS they are shown in
Fig. 4b. Like the poses seen on iNOS, in the neuronal model both com-
pounds display their phenylimidamide moiety under the heme group,
although with significant differences. 4i and 4g display its imidamide
group toward Glu597 (Glu377 on iNOS) but no H-bonds are established
with this residue. As for the p-methoxyaniline ring, and unlike the poses
with the iNOS isozyme, both ligands display this moiety toward the
carboxylic acids of the heme group, although only 4i can H-bond the
4i
4i
Fig. 2. Effect of compound 4i on eNOS activity. Acetylcholine-evoked relaxa-
tion in aortic rings with endothelium contracted with 1 mol/L noradrenaline,
μ
Fig. 3. Effect of compounds 4g, 4i, 5e and 5g on cell viability assessed by MTT
reduction in HUVECs. Results were calculated as percentage of control (un-
treated cells) and data are presented as mean ± standard error of the mean
(SEM) (n = 3 per group).
after incubation with L-NAME (100 μM), 4i (20 μM), or its vehicle (DMSO, 1/
106) for 30 min. Data are expressed as mean ± SEM of n experiments. **P <
0.01 vs. rings incubated with vehicle.
4

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
and 5g) using the free access website: https://www.molsoft.com/server
s.html. These results are shown in Table 3. All compounds comply the
Lipinski and Veber rules, so it could be predicted that they will present a
high oral bioavailability.
2.4.2. ADME properties
The evaluation of the pharmacokinetic properties of absorption,
distribution, metabolism and excretion are very important for devel-
oping a new therapeutic agent, to save time and costs. In order to
calculate these properties in the most active compounds we have used
the free access website: https://preadmet.bmdrc.kr/. These results are
shown in Table 4.
Human intestinal absorption is one of the most important parameters
in drug discovery. It is considered high absorption: 70–100%, medium
absorption: 20–70% and low absorption: 0–20%.37 These compounds
have a high intestinal absorption which reinforces the theory that they
could be good candidates for the design of oral drugs. Penetration
through the blood–brain barrier can be advantageous if the drug exerts
its effect on the central nervous system, but harmful if the drug acts only
at the peripheral level. High penetration through the blood–brain bar-
rier is considered >2, medium absorption 2-0.1, and low absorption
<0.1.37 The imidamide derivatives have shown moderate values of BBB
penetration, in the case of the compounds 4g and 4i these values could
be improved making a prodrug by the esterification of the hydroxyl
group.
Fig. 4. (a) Superposition of predicted binding poses of 4g (green) and 4i
(yelow) in iNOS (pdb ID 4CX7, tan). Hydrogen bonds are represented by dashed
purple lines. (b) Superposition of predicted binding poses of 4g (green) and 4i
(yelow) in nNOS (pdb ID 6AV2, blue). Hydrogen bonds are represented by
dashed purple lines. (c). Superposition of the predicted binding poses of 4g
(green) and 4i (yelow) in iNOS depicted as a Van der Waals surface. Red
(negative charge), blue (positive charge), white (neutral). Heme group and BH4
cofactor are represented as tan sticks and hydrogen bonds as dashed purple
lines. (d) Superposition of the predicted binding poses of 4g (green) and 4i
(yelow) in nNOS depicted as a Van der Waals surface. Red (negative charge),
blue (positive charge), white (neutral). Heme group and BH4 cofactor are
represented as blue sticks and hydrogen bonds as dashed purple lines.
Plasma proteins binding could influence the half-life of a drug, since
the protein-bound portion could act as a deposit of the drug that will be
released slowly. A high union is estimated: >90%, and a weak union:
<90.37 According to theoretical calculations, the imidamide 4i is the
compound that shows the highest binding to plasma proteins.
CYP3A4 and CYP2C19 are cytochrome P450 enzymes responsible for
drug metabolism, the inhibition of these proteins could cause an in-
crease in plasma levels and toxicity. The in silico calculations indicate
that these compounds are not inhibitors of these cytochromes.
A drug-like score is a value that indicates theoretically the similarity
of a compound to a known therapeutic agent. A score of 1 shows that the
compound is a good candidate to be a therapeutic agent.38 Derivatives
4g and 4i present predicted values very close to 1, which is a good
theoretical data in the development of new drugs.
carboxylic moieties as well as the tetrahydrobiopterin (BH4) cofactor. 4g
on the other hand, H-bond residue Asn574 (Asn354 on iNOS) at the
entrance of the catalytic channel.
Although both poses are similar in terms of orientations, the reduced
number of binding interactions in nNOS, and the tendency to bind the
catalytically important pocket generated by residues Gln263, Tyr347,
Arg388, Asp382 and Tyr373 in iNOS, might explain the low selectivity
of both compounds toward nNOS found on the IC50 assays.
3. Conclusions
To account for the differences in orientation of 4g and 4i in both
isozymes, the predicted binding poses are shown in Fig. 4c-d where the
enzymes are represented as a Van der Waals surface. These pictures
show the preference of the docked compounds to bid the region un-
derneath the BH4 cofactor and near the catalytic residues on iNOS,
suggesting a higher selectivity toward this isozyme.
Along this paper, the successful synthetic route for seventeen novel
molecules has been described. In this process, two well-known struc-
tures in NOS inhibition: the imidamide core and kynurenamine skeleton
have been fused, introducing at the same time, several moieties with
different electronic characteristics. These modifications allowed us to
study the way each one affected the activity. All final compounds were
subjected to an inhibition screening on the neuronal and inducible NOS
isoforms, selecting those with highest inhibition percentages for further
2.4. In silico prediction of physicochemical parameters and ADME
properties
Table 3
2.4.1. Physicochemical parameters
Calculated Lipisnki and Veber parameters for compounds 4g, 4i, 5e, 5f and 5g.
After potency and selectivity, another key aspect in the design of
drugs is optimizing the administration route. A good strategy for pre-
dicting the oral bioavailability of a drug is to check if the compound
meets the Lipinski and Veber rules. Lipinski’s rules state that for a
compound to be administered orally, it should meet at least three of the
following conditions: (a) have a molecular weight below 500; (b) have
an octanol–water partition coefficient below 5; (c) have no more than 5
hydrogen bond donor atoms; and (d) have no more than 10 hydrogen
bond acceptor atoms.35 Afterwards, Veber establishes additional rules
for drug bioavailability: (a) the number of rotatable bonds must be less
than 10, and (b) the polar surface area must be no greater than 140 Å2.36
In this article the Lipinski and Veber rules have been calculated for the
compounds with the most interesting biological activity (4g, 4i, 5e, 5f
Comp.
MW
LogP
HBD
HBA
nVs
nRB
MolPSA
Lipinski*
≤500
–
299.16
367.15
239.08
235.13
297.15
≤5
≤5
–
≤10
–
–
–
Veber**
4g
–
–
≤10
≤140
1.65
2.71
1.49
0.84
2.16
5
3
0
0
0
0
0
7
72.40 Å2
72.40 Å2
72.40 Å2
70.08 Å2
69.49 Å2
4i
5
3
8
5e
4
2
5
5f
4
3
6
5g
4
3
7
*Lipinski reference values; ** Veber reference values; MW, Molecular weight;
LogP, lipophilicity (O/W); HBD, Number of hydrogen bond donors; HBA,
Number of hydrogen bond acceptors; nVs, Number of Lipinski rule violations;
nRB, Number of rotatable bonds; MolPSA, molecular polar surface area (PSA)
(Å2).
5

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
substitutedphenyl)-propan-1-ols 8a-c.30 A solution of 1 M BH3 (10 mL) in
THF under argon was cooled to 0 ◦C and was added to the different 3-(2-
nitro-5-substitutedphenyl)3-hydroxypropanenitriles 7a-c (2.0 mmol)
dropwise. The mixture was stirred for 5 h at RT. Afterward, the reaction
mixture was cooled to 0 ◦C and an ice-cold solution of 6 N HCl (8.5 mL)
was added carefully. The THF was evaporated, and the aqueous phase
was basified with 10 M NaOH to pH = 8–9 and extracted with EtOAc (3
× 15 mL). The organic phase was washed with brine, dried over Na2SO4
and evaporated. The crude was used for the next step without
purification.
Table 4
ADME properties of compounds 4g, 4i, 5e, 5f and 5g.
Comp.
HIA
BBB
PPB
CYP3A4
CYP2C19
inhibition
Drug-
inhibition
likeness
score
4g
4i
78.52
79.83
79.77
68.28
81.52
0.29
0.59
0.74
0.20
0.36
51.65
71.58
3.38
Non
Non
Non
Non
Non
Non
Non
Non
Non
Non
0.93
0.91
5e
5f
ꢀ 0.16
ꢀ 0.13
0.28
10.40
55.23
5g
HIA, Human Intestinal Absorption (%); BBB, Blood-Brain Barrier penetration;
PPB, plasma protein binding; CYP3A4, Cytochrome P450 3A4; CYP2C19, Cy-
tochrome P4502C19.
General method for the synthesis of the benzyl-benzimidothioates 9b-d.
Commercially available benzothioamides (benzothioamide, 4-chloro-
benzothioamide or 4-(trifluoromethyl)benzothioamide) (5 mmol) were
dissolved in CHCl3 (15 mL); then, benzyl bromide (5.5 mmol, 634 µl)
were added and the mixture was refluxed for 2 h. After cooling, the
resulting white solid was filtered under vacuum.
analysis in form of IC50. The chosen ones were (4h, 4i, 5e-g) for iNOS,
and (5a, 5f-g) for nNOS, showing better inhibition values in the
Benzylbenzimidothioate hydrobromide (9b).32 White solid; mp:
194–196 ◦C; 94% yield.
inducible isoenzyme, being 4g (IC50 = 22.5 μM) and 4i (IC50 = 20.0 μM)
the best inhibitors. Using in silico models these compounds have shown
an interesting oral bioavailability. Data obtained from docking studies
on 4g and 4i reveal that these derivatives show their p-methoxyaniline
ring toward an important catalytic pocket on iNOS establishing H-bond
interactions with three vital residues, Gln263, Tyr373 and Asp382,
meanwhile on nNOS the same scaffold shifts toward Asn574 (Asn354 on
iNOS) at the entrance of the catalytic channel which could explain their
iNOS selectivity. Furthermore, the cell viability test proved the absence
of cytotoxicity in the most potent inhibitors at their IC50 values.
Regarding the pharmacological experiments with aortic rat tissue, we
can conclude that 4i does not modify the relaxation associated to ACh
making this compound an effective iNOS inhibitor. Moreover, this
compound could be considered a new selective iNOS inhibitor for the
development of new therapies for the treatment of inflammatory dis-
orders, such as septic shock or rheumatoid arthritis, among others.
Benzyl 4-chlorobenzimidothioate hydrobromide (9c).39 White solid;
mp: 182–184 ◦C; 90% yield.
Benzyl 4-(trifluoromethyl)benzimidothioate hydrobromide (9d). White
solid; mp: 147–145 ◦C; 79% yield. 1H NMR (400 MHz, DMSO d6): δ
11.02 (sa, 1H), 8.08 (d, J = 7.7 Hz, 2H), 8.00 (d, J = 7.7 Hz, 2H), 7.53 (d,
J = 7.0 Hz, 2H), 7.39 (m, 3H), 4.75 (s, 2H). 13C NMR (100 MHz,
DMSO d6) δ 166.68, 137.32, 135.01, 132.72, 129.39, 128.41, 128.34,
127.31, 125.26 (q, J = 3.4 Hz), 123.40 (q, J = 272.4 Hz), 41.58. HRMS
(LSIMS): m/z calcd for C15H13NSF3: 296.0721 [M + H]+; found:
296.0733.
General method for the synthesis of N-(3-(2-nitro-5-substitutedphenyl)-
3-hydroxypropyl)imidamides 10a-i. 3-Amino-1-(2-nitro-5-substituted-
phenyl)propan-1-ol derivatives 8a-c (2 mmol) were solved in MeOH
(10 mL) and cooled to 0 ◦C; then, the different imidates 9a-d (3 mmol)
were added. The mixture was stirred for 15 h at room temperature.
Afterward, the reaction mixture was evaporated and was purified by
flash chromatography (DCM: MeOH, 9: 1 saturating with NH4OH).
N-(3-Hydroxy-3-(2-nitrophenyl)propyl)acetimidamide (10a). Orange
oil; 88% yield. 1H NMR (400 MHz, CD3OD) δ 7.90 (dd, J = 7.5 Hz, J =
4.7 Hz, 2H), 7.71 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 5.24 (d, J
= 9.2 Hz, 1H), 3.60–3.42 (m, 2H), 2.26 (s, 3H), 2.19–2.07 (m, 1H),
1.95–1.84 (m, 1H). 13C NMR (100 MHz, CD3OD) δ 166.08, 148.74,
141.51, 134.70, 129.40, 129.11, 125.22, 67.34, 40.75, 37.29, 19.08.
HRMS (LSIMS): m/z calcd for C11H15N3O3: 238.1192 [M + H]+; found:
238.1178.
4. Experimental section
4.1. Chemistry
All starting materials, reagents and solvents, were commercially
available. 1H NMR and 13C NMR spectra were obtained using Bruker
Avance NEO spectrometers with Smart Probe BBFO equipped, operating
at 400.57 MHz for 1H and 100.73 MHz for 13C, at 499.79 MHz for 1H and
125.68 MHz for 13C, or 600.25 MHz for 1H and 150.95 MHz for 13C, in
the deuterated solvents. Chemical shifts are reported in ppm (δ ppm) and
are referenced to the residual solvent peak. HRMS was conducted on a
Waters LCT Premier Mass Spectrometer. Melting points were deter-
mined on an electrothermal melting point apparatus and were
uncorrected.
N-(3-Hydroxy-3-(2-nitrophenyl)propyl)benzimidamide (10b). Brown
oil; 71% yield. 1H NMR (400 MHz, DMSO d6) δ 9.77 (sa, 1H), 9.48, 9.05
(sa, 1H), 7.91 (dd, J = 8.3 Hz, J = 1.1 Hz, 1H), 7.87 (dd, J = 7.9 Hz, J’ =
1.3 Hz, 1H), 7.81–7.67 (m, 4H), 7.61 (t, J = 7.6 Hz, 2H), 7.53 (dt, J =
8.3 Hz, J = 1.3 Hz, 1H), 5.78 (d, J = 4.5 Hz, 1H), 5.14 (m, 1H),
3.68–3.55 (m, 2H), 2.15–2.04 (m, 1H), 1.98–1.87 (m, 1H). 13C NMR
(100 MHz, DMSO d6) δ 163.17, 147.36, 140.19, 133.49, 133.22,
129.14, 128.88, 128.34, 128.22, 128.15, 123.89, 65.40, 36.02, 30.70.
HRMS (LSIMS): m/z calcd for C16H18N3O3: 300.1348 [M + H]+; found:
300.1343.
General method for the synthesis of 3-(2-nitro-5-substitutedphenyl)3-
hydroxypropanenitriles 7a-c. n-BuLi (1.6 M/hexane, 12.5 mL) was added
to dry THF (20 mL) cooled to ꢀ 78 ◦C under argon; then, acetonitrile
(1.572 mL) was added dropwise. The mixture was stirred at ꢀ 78 ◦C for 1
h. Afterward, a solution of the corresponding 2-nitrobenzaldehydes 6a-c
(10 mmol) in dry THF (10 mL) was added dropwise. The mixture was
stirred again at ꢀ 78 ◦C for 30 min and then warmed to RT. The mixture
reaction was quenched with cold water (25 mL) and concentrated under
vacuum. The crude was extracted with EtOAc (3 × 30 mL) and the
organic phase was washed with NaHCO3 saturated solution (15 mL) and
brine (15 mL), dried over anhydrous Na2SO4, filtered and evaporated.
The crude was purified by flash chromatography (EtOAc/hexane, 1: 4).
3-(2-Nitrophenyl)3-hydroxypropanenitrile (7a).30 Yellow solid: mp:
82–85 ◦C; 93% yield.
4-Chloro-N-(3-hydroxy-3-(2-nitrophenyl)propyl)benzimidamide (10c).
Orange oil; 70% yield. 1H NMR (500 MHz, CD3OD) δ 8.02 (ddd, J = 8.2
Hz, J = 2.9 Hz, J’ = 1.2 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.82 (dt, J =
7.4 Hz, J = 1.2H, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.58 (dt, J = 7.5 Hz, J =
1.5 Hz, 1H), 5.42 (dd, J = 9.6 Hz, J = 2.4 Hz, 1H), 3.90–3.84 (m, 1H),
3.80–3.75 (m, 1H), 2.38–2.31 (m, 1H), 2.12–2.05 (m, 1H). 13C NMR
(125 MHz, CD3OD) δ 165.01, 148.81, 141.67, 140.74, 134.81, 130.83,
130.80, 130.58, 129.50, 129.19, 129.23, 125.30, 67.50, 41.50, 37.29.
HRMS (LSIMS): m/z calcd for C16H17N3O3Cl: 334.0958 [M + H]+;
found: 334.0987.
3-(5-Chloro-2-nitrophenyl)3-hydroxypropanenitrile (7b).30 Yellow
solid; mp: 63–43 ◦C; 75% yield.
3-(5-Methoxy-2-nitrophenyl)3-hydroxypropanenitrile (7c).30 Yellow
solid: mp: 88–90 ◦C; 86% yield.
N-(3-Hydroxy-3-(2-nitrophenyl)propyl)-4-(trifluoromethyl)benzimida-
mide (10d). Yellow oil; 96% yield. 1H NMR (400 MHz, DMSO d6) δ 9.98
(sa, 1H), 9.60, 9.35 (sa, 1H), 7.98 (dd, J = 26.1 z Hz, J = 8.3 Hz, 4H),
General method for the synthesis of 3-amino-1-(2-nitro-5-
6

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
7.91 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 7.5 Hz, 1H), 7.76 (t, J = 7.5 Hz,
1H), 7.52 (t, J = 7.5 Hz, 1H), 5.82 (sa, 1H), 5.13 (dd, J = 9.2 Hz, J = 2.1
Hz, 1H), 3.62 (m, 2H), 2.15–2.05 (m, 1H), 1.99–1.88 (m, 2H). 13C NMR
(100 MHz, DMSO d6) δ 162.20, 147.36, 140.19, 133.52, 133.14 (d, J =
1.0 Hz), 132.72 (q, J = 32.5 Hz), 129.40, 128.37, 128.22, 125.77 (q, J =
3.7 Hz), 123.92, 123.56 (q, J = 272.8 Hz), 65.45, 40.02, 35.94. HRMS
(LSIMS): m/z calcd for C17H17N3O3F3: 368.1222 [M + H]+; found:
368.1218.
7.70 (d, J = 7.3 Hz, 1H), 3.74 (t, J = 5.9 Hz, 2H), 3.31–3.25 (m, 2H),
2.28 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 201.04, 166.23, 147.16,
137.68, 135.62, 132.48, 128.84, 125.52, 41.14, 37.94, 19.05. HRMS
(LSIMS): m/z calcd for: C11H14N3O3 236.1035 [M + H]+; found:
236.1043.
N-(3-(2-Nitrophenyl)-3-oxopropyl)benzimidamide (11b). Yellow oil;
88% yield. 1H NMR (400 MHz, CD3OD) δ 8.22 (dd, J = 8.2 Hz, J = 0.8
Hz, 1H), 7.91 (dt, J = 7.6 Hz, J = 3.8 Hz, 1H), 7.85–7.79 (m, 3H),
7.79–7.73 (m, 2H), 7.71–7.63 (m, 2H), 4.03–3.95 (m, 2H), 3.50–3.32
(m, 2H). 13C NMR (100 MHz, CD3OD) δ 201.25, 166.11, 147.24, 137.76,
135.69, 134.63, 132.53, 130.70, 130.35, 129.02, 128.89, 125.60, 38.58,
37.57. HRMS (LSIMS): m/z calcd for C16H16N3O3: 298.1192 [M + H]+;
found: 298.1187.
N-(3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl)acetimidamide (10e).
Orange oil; 67% yield. 1H NMR (500 MHz, CD3OD) δ 8.00 (d, J = 8.7 Hz,
1H), 7.91 (d, J = 2.3 Hz, 1H), 7.51 (dd, 1H, J = 8.7 Hz, J = 2.3 Hz, 1H),
5.27 (dd, J = 9.5 Hz, J = 2.3 Hz, 1H), 3.60–3.45 (m, 2H), 2.41–2.29 (m,
1H), 2.26 (s, 3H), 1.91–1.78 (m, 1H). 13C NMR (125 MHz, CD3OD) δ
166.20, 146.99, 144.35, 141.08, 129.51, 129.18, 127.46, 67.24, 40.55,
37.15, 19.04. HRMS (LSIMS): m/z calcd for C11H15N3O3Cl: 272.0796
[M + H]+; found: 272.0789.
4-Chloro-N-(3-(2-nitrophenyl)-3-oxopropyl)benzimidamide
(11c).
White solid; mp: 177–179 ◦C; 67% yield. 1H NMR (400 MHz, CD3OD) δ
8.20 (dd, J = 8.2 Hz, J = 0.9 Hz, 1H), 7.90 (td, J = 7.6 Hz, J = 0.9 Hz,
1H), 7.82 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.42 (dd, J = 7.6
Hz, J = 1.5 Hz, 1H), 7.31–7.27 (m, 1H), 3.96 (t, J = 5.8 Hz, 2H), 3.44 (t,
J = 5.8 Hz, 2H). 13C NMR (100 MHz, CD3OD) δ 201.17, 165.02, 147.17,
140.75, 137.69, 135.68, 132.50, 130.82, 130.53, 129.19, 128.91,
125.56, 41.17, 38.72. HRMS (LSIMS): m/z calcd for C16H15N3O3Cl:
332.0802 [M + H]+; found: 332.0791.
N-(3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl)acetimidamide
(10f). Brown oil; 68% yield. 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J =
9.1 Hz, 1H), 7.44 (d, J = 2.7 Hz, 1H), 7.00 (dd, J’ = 9.1 Hz, J = 2.7 Hz,
1H), 5.42 (dd, J = 9.3 Hz, J = 2.0 Hz, 1H), 3.93 (s, 3H), 3.60–3.50 (m,
2H), 2.28 (s, 3H), 2.21–2.08 (m, 1H), 1.90–1.76 (m, 1H). 13C NMR (100
MHz, CD3OD) 166.14, 165.50, 145.66, 141.24, 128.65, 114.16, 113.65,
67.68, 56.59, 40.71, 37.29, 19.07. HRMS (LSIMS): m/z calcd for
N-(3-(2-nitrophenyl)-3-oxopropyl)-4-(trifluoromethyl)benzimidamide
(11d). Orange oil; 71% yield. 1H NMR (500 MHz, CD3OD): δ 8.21 (d, J
= 8.2 Hz, 1H), 7.98 (q, J = 8.7 Hz, 4H), 7.90 (t, J = 7.5 Hz, 1H), 7.79 (t,
J = 7.8 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 3.98 (m, 2H), 3.35 (m, 2H). 13C
NMR (125 MHz, CD3OD) δ 201.20, 165.08, 147.19, 137.74, 135.74,
135.71(q, J = 32.9 Hz), 134.47, 132.54, 130.13, 128.89, 127.22 (q, J =
3.5 Hz), 125.61, 124.90 (q, J = 271.8 Hz), 43.92, 38.73. HRMS (LSIMS):
m/z calcd for C17H15N3O3F3: 366.1066 [M + H]+; found: 366.1082.
N-(3-(5-Chloro-2-nitrophenyl)-3-oxopropyl)acetimidamide (11e). Or-
ange oil; mp: 153–155 ◦C; 65% yield. 1H NMR (400 MHz, CD3OD) δ 8.23
(d, J = 8.6 Hz, 1H), 7.79 (dd, J = 8.6 Hz, J = 2.2 Hz, 1H), 7.77 (d, J =
2.2 Hz, 1H), 3.76 (t, J = 5.8 Hz, 2H), 3.31 (t, J = 5.8 Hz, 2H), 2.31 (s,
3H). 13C NMR (125 MHz, CD3OD) δ 198.31, 164.88, 143.97, 140.62,
138.12, 130.85, 127.40, 126.04, 39.84, 36.36, 17.65. HRMS (LSIMS):
m/z calcd for C11H13N3O3Cl: 270.0645 [M + H]+; found: 270.0629.
C
12H18N3O4: 268.1297 [M + H]+; found: 268.1292.
N-(3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl)benzimidamide
(10g). Brown oil; 74% yield. 1H NMR (500 MHz, CD3OD) δ 8.14 (d, J =
9.1 Hz, 1H), 7.88 (d, J = 7.3 Hz, 3H), 7.78 (t, J = 7.5 Hz, 1H), 7.68 (t, J
= 7.8 Hz, 2H), 7.53 (d, J = 2.8 Hz, 1H), 7.05 (dd, J = 9.1 Hz, J = 2.8 Hz,
1H), 5.59 (dd, J = 9.4 Hz, J = 2.1 Hz, 1H), 3.98 (s, 3H), 3.93–3.87 (m,
1H), 3.83–3.77 (m, 1H), 2.39––2.31 (m, 1H), 2.06–1.98 (m, 1H). 13C
NMR (125 MHz, CD3OD) δ 165.84, 165.51, 145.67, 141.13, 134.61,
130.57, 130.36, 128.96, 128.70, 114.21, 113.70, 67.80, 56.66, 41.43,
37.24. HRMS (LSIMS): m/z calcd for C17H20N3O4: 330.1454 [M + H]+;
found: 330.1449.
4-Chloro-N-(3-hydroxy-3-(5-methoxy-2-nitrophenyl)propyl)benzimida-
mide (10h). Orange oil; 76% yield. 1H NMR (500 MHz, CD3OD) 8.14 (d,
J = 9.1 Hz, 1H), 7.85 (d, J = = 8.7 Hz, 2H), 7.68 (d, J = 8.7 Hz, 2H), 7.51
(d, J = 2.8 Hz, 1H), 7.04 (dd, J = 9.1 Hz, J = 2.8 Hz, 1H), 5.55 (dd, J =
10.4 Hz, J = 2.1 Hz, 1H), 3.97 (s, 3H), 3.92–3.82 (m, 1H), 3.79–3.72 (m,
1H), 2.37–2.27 (m, 1H), 2.04–1.94 (m, 1H). 13C NMR (125 MHz,
CD3OD) δ 165.58, 165.02, 145.74, 141.21, 140.76, 130.78, 130.57,
129.27, 128.73, 114.21, 113.71, 67.79, 56.62, 41.50, 37.22. MS
(LSIMS): m/z calcd for C17H18N3O4Cl: 364.1059 [M + H]+; found:
364.1051.
N-(3-(5-methoxy-2-nitrophenyl)-3-oxopropyl)acetimidamide
(11f).
Yellow oil; 83% yield. 1H NMR (500 MHz, CD3OD): δ 8.22 (d, J = 9.2 Hz,
1H), 7.20 (dd, J = 9.2 Hz, J = 2.7 Hz, 1H), 7.11 (d, J = 2.7 Hz, 1H), 3.98
(s, 3H), 3.75 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 6.0 Hz, 2H), 2.29 (s, 3H). 13
C
NMR (125 MHz, CD3OD): δ 201.20, 166.27, 166.16, 141.22, 139.28,
128.32, 116.51, 113.57, 57.21, 41.44, 37.92, 19.06. HRMS (LSIMS): m/z
calcd for C12H16N3O4: 266.1141 [M + H]+; found: 266.1147.
N-(3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl)-4-(trifluoromethyl)
benzimidamide (10i). Orange oil; 68% yield. 1H NMR (500 MHz, CD3OD)
δ 8.14 (d, J = 9.1 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.97 (d, J’ = 8.3 Hz,
2H), 7.51 (d, J = 2.8 Hz, 1H), 7.04 (dd, J = 9.1 Hz, J = 2.8 Hz, 1H), 5.54
(dd, J = 9.4 Hz, J = 2.1 Hz, 1H), 3.97 (s, 3H), 3.91–3.84 (m, 1H),
3.79–3.73 (m, 1H), 2.36–2.28 (m, 1H), 2.01–1.94 (m, 1H). 13C NMR
(125 MHz, CD3OD) δ 165.66, 165.07, 145.80, 141.29, 135.75 (q, J =
33.0 Hz), 134.56 (d, J = 1.2 Hz), 130.09, 128.78, 127.28 (q, J = 3.8 Hz),
124.92 (q, J = 272.1 Hz), 114.20, 113.74, 67.80, 56.59, 41.60, 37.20.
HRMS (LSIMS): m/z calcd for C18H19N3O4F3: 392.1328 [M + H]+;
found: 398.1332.
N-(3-(5-methoxy-2-nitrophenyl)-3-oxopropyl)benzimidamide
(11g).
Brown oil; 74% yield. 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 9.1 Hz,
1H), 7.83 (d, J = 7.6 Hz, 2H), 7.76 (t, J = 7.3 Hz, 1H), 7.64 (t, J = 7.5 Hz,
2H), 7.21 (dd, J’ = 9.1 Hz, J = 2.0 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 3.98
(s, 3H), 3.43–3.29 (m, 4H). 13C NMR (100 MHz, CD3OD) δ 201.35,
168.49, 166.16, 140.82, 139.18, 135.22, 128.90, 128.85, 128.62,
128.29, 116.71, 113.52, 57.30, 45.93, 35.80. HRMS (LSIMS): m/z calcd
for C17H18N3O4: 328.1297 [M + H]+; found: 328.1293.
4-Chloro-N-(3-(5-methoxy-2-nitrophenyl)-3-oxopropyl)benzimidamide
(11h). Brown oil; 76% yield. 1H NMR (400 MHz, CD3OD): δ 8.26 (d, J =
9.2 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.24 (dd,
J = 9.2, J = 2.7 Hz, 1H), 7.16 (d, J = 2.7 Hz, 1H), 4.01 (s, 3H), 3.97 (t, J
= 5.6 Hz, 2H), 3.38–3.32 (m, 2H). 13C NMR (100 MHz, CD3OD) δ
201.28, 166.19, 165.15, 141.19, 140.79, 139.30, 130.83, 130.75,
129.32, 128.36, 116.51, 113.64, 57.21, 47.94, 38.60. HRMS (LSIMS):
m/z calcd for C17H17N3O4Cl: 362.0908 [M + H]+; found: 362.0919.
General method for the synthesis of N-(3-(2-amino-5-substitutedphenyl)-
3-hydroxypropyl)imidamides 4a-i and N-(3-(2-amino-5-substitutedphenyl)-
3-oxopropyl)imidamides 5a-h. A mixture of the nitro derivatives 10a-i or
11a-h (1 mmol) was disolved in water (10 mL), then Fe powder (10
mmol) and FeSO4 (1 mmol) were added. The reaction was heated under
reflux for 1.5 h. Next, the suspension was filtered through Celite. The
General method for the synthesis of N-(3-(2-nitro-5-substitutedphenyl)-
3-oxopropyl)imidamides 11a-h. On a solution of the corresponding al-
cohols 10a-h (1.5 mmol) in acetone (15 mL) and DCM (15 mL), cooled
to 0 ◦C, freshly prepared Jones reagent (8 mL, 2.67 M) was added
dropwise. After 20 min, MeOH (10 mL) was added. Then, the green
precipitate was removed by filtration. The reaction mixture was evap-
orated and was purified by flash chromatography (DCM: MeOH, 10:1
saturating with NH4OH).
N-(3-(2-Nitrophenyl)-3-oxopropyl)acetimidamide (11a). Orange oil;
72% yield. 1H NMR (400 MHz, CD3OD): δ 8.16 (d, J = 8.2 Hz, 1H), 7.87
(dd, J = 7.8 Hz, J = 7.3 Hz, 1H), 7.76 (dd, J = 8.2 Hz, J = 7.8 Hz, 1H),
7

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
aqueous solution was evaporated under vacuum and purified by flash
chromatography (DCM: MeOH, 8:1 saturating with NH4OH).
1H), 6.77 (d, J = 8.6 Hz, 1H), 6.71 (dd, J = 8.6 Hz, J = 2.8 Hz, 1H), 4.95
(dd, J = 7.8 Hz, J = 5.4 Hz, 1H), 3.75 (s, 3H), 3.65 (t, J = 6.9 Hz, 2H),
2.32–2.22 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 164.75, 154.44,
140.74, 138.87, 131.27, 130.74, 130.53, 129.14, 119.67, 114.80,
113.73, 70.74, 56.13, 41.79, 34.64. HRMS (LSIMS): m/z calcd for
N-(3-(2-Aminophenyl)-3-hydroxypropyl)acetimidamide (4a). Orange
oil; 68% yield. 1H NMR (400 MHz, CD3OD) δ 7.14 (dd, J = 7.9 Hz, J =
1.1 Hz, 1H), 7.03 (dt, J = 7.9 Hz, J = 1.1 Hz, 1H), 6.74 (d, J = 7.9 Hz,
1H), 6.69 (dt, J = 7.9 Hz, J = 0.8 Hz, 1H), 4.64–4.59 (m, 1H) 3.47–3.32
(m, 2H), 2.22 (s, 3H), 2.20–2.05 (m, 2H). 13C NMR (100 MHz, CD3OD) δ
166.02, 146.12, 129.25, 129.12, 128.01, 119.27, 118.07, 70.82, 40.94,
34.49, 18.99. HRMS (LSIMS): m/z calcd for C11H17N3O: 208.1450 [M +
H]+; found: 208.1435.
C
17H21N3O2Cl: 334.1322 [M + H]+; found: 334.1313.
N-(3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl)-4-(tri-
fluoromethyl)benzimidamide (4i). Brown oil; 75% yield. 1H NMR (500
MHz, CD3OD) δ 7.96–7.91 (m, 4H), 6.89 (d, J’ = 2.9 Hz, 1H), 6.78 (d, J
= 8.6 Hz, 1H), 6.72 (dd, J = 8.6 Hz, J = 2.9 Hz, 1H), 4.95 (dd, J = 8.0 Hz,
J = 5.3 Hz, 1H), 3.75 (s, 3H), 3.67 (t, J = 6.9 Hz, 2H), 2.32–2.24 (m, 2H).
13C NMR (125 MHz, CD3OD) δ 164.73, 154.58, 138.67, 135.70 (q, J =
33.0 Hz), 134.37, 131.40, 130.08, 127.21 (q, J = 3.7 Hz), 124.90 (q, J =
272.4 Hz), 119.79, 114.81, 113.80, 70.81, 56.13, 41.89, 34.61 (C-2′).
HRMS (LSIMS): m/z calcd for C18H21N3O2F3: 368.1586 [M + H]+;
found: 368.1573.
N-(3-(2-Aminophenyl)-3-hydroxypropyl)benzimidamide (4b). Yellow
oil; 71% yield. 1H NMR (500 MHz, CD3OD) δ 7.75–7.72 (m, 1H), 7.71 (d,
J = 7.8 Hz, 2H), 7.60 (t, J = 7.8 Hz, 2H), 7.18 (d, J = 7.4 Hz, 1H), 7.05
(td, J = 8.0, J = 1.5 Hz, 1H), 6.76 (dd, J = 8.0 Hz, J = 1.0 Hz, 1H), 6.71
(td, J = 7.4 Hz, J = 1.0 Hz, 1H), 4.91 (dd, J = 8.4 Hz, J = 5.0 Hz, 1H),
3.74–3.60 (m, 2H), 2.35–2.17 (m, 2H). 13C NMR (125 MHz, CD3OD) δ
165.79, 146.19, 134.62, 130.38, 129.34, 129.14, 128.90, 128.61,
128.07, 119.36, 118.14, 71.44, 41.79, 34.36. MS (LSIMS): m/z calcd for
N-(3-(2-Aminophenyl)-3-oxopropyl)acetimidamide (5a). Orange solid;
mp: 130–133 ◦C; 88% yield. 1H NMR (500 MHz, CD3OD): δ 7.79 (dd, J =
8.2 Hz, J = 1.5 Hz, 1H), 7.30 (ddd, J = 8.4 Hz, J = 6.9 Hz, J = 1.5 Hz,
1H), 6.80 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 6.65 (ddd, J = 8.2 Hz, J = 6.9
Hz, J = 1.2 Hz, 1H), 3.67 (t, J = 6.1 Hz, 2H), 3.40 (t, J = 6.1 Hz, 2H),
2.24 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 199.90, 166.03, 152.81,
135.75, 131.97, 118.42, 118.05, 116.27, 38.72, 37.40, 18.87. HRMS
(LSIMS): m/z calcd for C11H14N3O3: 206.1293 [M + H]+; found:
206.1291.
C
16H19N3O: 270.1606 [M + H]+; found: 270.1597.
N-(3-(2-Aminophenyl)-3-hydroxypropyl)-4-chlorobenzimidamide (4c).
Yellow oil; 69% yield. 1H NMR (500 MHz, CD3OD) δ 7.70 (dt, J = 8.7 Hz,
J = 2.1 Hz, 2H), 7.64 (dt, J = 8.7 Hz, J = 2.4 Hz, 2H), 7.18 (dd, J = 7.4
Hz, J = 1.4 Hz, 1H), 7.06 (td, J = 7.8 Hz, J = 1.4 Hz, 1H), 6.77 (dd, J =
7.8 Hz, J = 1.1 Hz, 1H), 6.72 (td, J = 7.4 Hz, J = 1.1 Hz, 1H), 4.93–4.89
(m, 1H), 3.61 (t, J = 7.0 Hz, 2H), 2.36–2.20 (m, 2H). 13C NMR (125
MHz, CD3OD) δ 164.89, 146.34, 140.82, 130.69, 130.56, 129.38,
129.24, 129.03, 128.09, 119.30, 118.11, 71.53, 41.84, 34.24. HRMS
(LSIMS): m/z calcd for C16H19N3OCl: 304.1217 [M + H]+; found:
304.1189.
N-(3-(2-Aminophenyl)-3-oxopropyl)benzimidamide (5b). Yellow oil;
69% yield. 1H NMR (400 MHz, CD3OD): δ 7.72 (dd, J = 8.2 Hz, J = 1.3
Hz, 1H), 7.65–7.60 (m, 2H), 7.59–7.56 (m, 1H), 7.48 (dd, J = 10.8 Hz, J
= 4.4 Hz, 2H), 7.18 (ddd, J = 8.4 Hz, J = 7.0 Hz, J = 1.3 Hz, 1H), 6.70
(dd, J = 8.4 Hz, J = 1.1 Hz, 1H), 6.54 (ddd, J = 8.2 Hz, J = 7.0 Hz, J =
1.1 Hz, 1H), 3.76 –3.69 (m, 2H), 3.31–3.25 (m, 2H). 13C NMR (100,
CD3OD) δ 200.56, 165.62, 152.67, 135.62, 133.99, 132.10, 131.87,
130.18, 128.71, 118.38, 117.59, 116.30, 43.45, 39.72. HRMS (LSIMS):
m/z calcd for C16H18N3O: 268.1450 [M + H]+; found: 268.1440.
N-(3-(2-Aminophenyl)-3-hydroxypropyl)-4-(trifluoromethyl)benzimi-
damide (4d). Yellow oil; 65% yield. 1H NMR (500 MHz, CD3OD) δ
7.94–7.88 (m, 4H), 7.19 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.04 (td, J =
7.9 Hz, J = 1.5 Hz, 1H), 6.76 (dd, J = 7.9 Hz, J = 1.1 Hz, 1H), 6.71 (td, J
= 7.5 Hz, J = 1.1 Hz, 1H), 4.93 (dd, J = 8.4 Hz, J = 5.2 Hz, 1H), 3.64 (t,
J = 6.9 Hz, 2H), 2.35–2.22 (m, 2H). 13C NMR (125 MHz, CD3OD) δ
164.70, 146.14, 135.66 (q, J = 33.4 Hz), 134.33 (d, J = 1.4 Hz), 130.07,
129.32, 129.13, 128.08, 127.19 (q, J = 3.8 Hz), 124.88 (q, J = 271.6
Hz), 119.36, 118.13, 71.32, 41.97, 34.29. HRMS (LSIMS): m/z calcd for
N-(3-(2-Aminophenyl)-3-oxopropyl)-4-chlorobenzimidamide
(5c).
Brown oil; 75% yield. 1H NMR (600 MHz, CD3OD) δ 7.73 (d, J = 5.8 Hz,
1H), 7.54 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.18 (t, J = 8.0
Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H), 6.54 (t, J = 8.0 Hz, 1H), 3.47 (t, J =
7.5 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H). 13C NMR (150 MHz, CD3OD) δ
202.69, 165.03, 152.54, 137.20, 136.76, 134.25, 132.23, 129.60,
129.56, 127.31, 118.04, 115.56, 40.53, 34.90. HRMS (LSIMS): m/z
calcd for C16H17N3OCl: 302.1060 [M + H]+; found: 302.1036.
N-(3-(2-Aminophenyl)-3-oxopropyl)-4-(trifluoromethyl)benzimidamide
(5d). Orange oil; 78% yield. 1H NMR (400 MHz, CD3OD): δ 8.22 (d, J =
7.6 Hz, 1H), 7.99 (q, J = 8.8 Hz, 4H), 7.91 (t, J = 7.6 Hz, 1H), 7.80 (t, J
= 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 4.03–3.94 (m, 2H), 3.36–3.33
(m, 2H). 13C NMR (100 MHz, CD3OD) δ 201.18, 165.16, 147.24, 137.81,
135.78 (q, J = 33.2 Hz), 135.76, 134.53, 132.57, 130.13, 128.84,
127.23 (q, J = 3.5 Hz), 125.66, 124.92 (q, J = 271.7 Hz), 38.68, 37.78.
HRMS (LSIMS): m/z calcd for C17H17N3OF3: 336.1324 [M + H]+; found:
336.1308.
C
17H19N3OF3: 338.1480 [M + H]+; found: 338.1504.
N-(3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl)acetimidamide (4e).
Orange oil; 67% yield. 1H NMR (400 MHz, CD3OD) δ 7.06 (d, J = 2.5 Hz,
1H), 6.89 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H), 6.61 (d, J = 8.5 Hz, 1H), 4.73
(t, J = 7.0 Hz, 1H), 3.41–3.24 (m, 2H), 2.14 (s, 3H), 2.03–1.95 (m, 2H).
13C NMR (100 MHz, CD3OD) δ 166.04, 145.08, 130.81, 128.75, 127.46,
123.30, 118.90, 69.82, 40.82, 34.38, 19.03. HRMS (LSIMS): m/z calcd
for C11H17N3OCl: 242.1060 [M + H]+; found: 242.1055.
N-(3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl)acetimidamide
(4f). Red oil; 68% yield. 1H NMR (400 MHz, CD3OD) δ 6.81 (d, J = 2.8
Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.70 (dd, J = 8.7 Hz, J = 2.8 Hz, 1H),
4.96 (d, J = 3.4 Hz, 1H), 3.71 (s, 3H), 3.41–3.33 (m, 2H), 2.19 (s, 3H),
2.12–2.04 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 166.07, 155.42,
136.55, 132.46, 120.65, 114.80, 113.89, 70.57, 56.14, 40.83, 35.01,
18.95. HRMS (LSIMS): m/z calcd for C12H20N3O2: 238.1556 [M + H]+;
found: 238.1555.
N-(3-(2-Amino-5-chlorophenyl)-3-oxopropyl)acetimidamide (5e). Or-
ange oil; 78% yield. 1H NMR (500 MHz, CD3OD): δ 7.73 (d, J = 2.4 Hz,
1H), 7.21 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H), 6.79 (d, J = 8.9 Hz, 1H), 3.64
(t, J = 6.1 Hz, 2H), 3.35 (t, J = 6.1 Hz, 2H), 2.22 (s, 3H). 13C NMR (125
MHz, CD3OD) δ 199.04, 166.01, 151.38, 135.53, 130.86, 120.16,
120.09, 118.36, 38.59, 37.46, 18.95. HRMS (LSIMS): m/z calcd for
N-(3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl)benzimidamide
(4g). Brown oil; 70% yield. 1H NMR (500 MHz, CD3OD) δ 7.77–7.72 (m,
3H), 7.62 (t, J = 7.8 Hz, 2H), 6.88 (d, J = 2.8 Hz, 1H), 6.78 (d, J = 8.6
Hz, 1H), 6.71 (dd, J = 8.6 Hz, J = 2.8 Hz, 1H), 4.95 (dd, J = 8.0 Hz, J =
C
11H15N3OCl: 240.0904 [M + H]+; found: 240.0886.
5.3 Hz, 1H), 3.74 (s, 3H), 3.65 (t, J = 7.0 Hz, 2H), 2.30–2.23 (m, 2H). 13
C
N-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)acetimidamide
(5f).
NMR (125 MHz, CD3OD) δ 165.71, 154.55, 138.64, 134.61, 131.41,
130.56, 130.36, 128.90, 119.78, 114.81, 113.73, 70.80, 56.13, 41.71,
34.71. HRMS (LSIMS): m/z calcd for C17H22N3O2: 300.1712 [M + H]+;
found: 300.1730.
Brown oil; 69% yield. 1H NMR (400 MHz, CD3OD) δ 7.23 (d, J = 2.8 Hz,
1H), 7.01 (dd, J = 9.0 Hz, J = 2.8 Hz, 1H), 6.78 (d, J = 9.0 Hz, 1H), 3.78
(s, 3H), 3.66 (t, J = 6.1 Hz, 2H), 3.39 (t, J = 6.1 Hz, 2H), 2.23 (s, 3H). 13
C
NMR (125 MHz, CD3OD) δ 199.50, 166.01, 151.24, 147.48, 125.13,
120.01, 117.82, 113.87, 56.45, 38.73, 37.58, 18.90. HRMS (LSIMS): m/z
calcd for C12H18N3O2: 236.1399 [M + H]+; found: 236.1388.
N-(3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl)-4-chlor-
obenzimidamide (4h). Brown oil; 71% yield. 1H NMR (500 MHz, CD3OD)
δ 7.74 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 2.8 Hz,
N-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)benzimidamide (5g).
8

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
Orange oil; 84% yield. 1H NMR (400 MHz, CD3OD): δ 7.80–7.74 (m,
3H), 7.64 (dd, J = 10.9 Hz, J = 4.5 Hz, 2H), 7.30 (d, J = 2.9 Hz, 1H),
7.05 (dd, J = 9.0 Hz, J = 2.9 Hz, 1H), 6.81 (d, J = 9.0 Hz, 1H), 3.91 (t, J
= 6.2 Hz, 2H), 3.82 (s, 3H), 3.55 (t, J = 6.2 Hz, 2H). 13C NMR (125 MHz,
CD3OD) δ 199.81, 165.88, 151.27, 147.48, 134.60, 130.62, 130.34,
128.94, 125.20, 120.05, 117.91, 113.94, 56.51, 39.74, 37.66. HRMS
(LSIMS): m/z calcd for C17H20N3O2: 298.1556 [M + H]+; found:
298.1553.
4.3. In vitro eNOS activity determination
In order to evaluate the eNOS activity, it has been necessary working
with tissue from experimental animals. Regulations for the protection of
animals used for scientific purposes of the European Union were fol-
lowed to carry out this study. All experimental protocols were approved
by the Animal Care and Ethics Committee of the University of Granada
(Spain; permit no: 12/11/2017/164). Male Wistar rats (250–300 g),
obtained from Harlam Laboratories SA (Barcelona, Spain), were eutha-
nized by a quick blow on the head followed by exsanguination.
The descending thoracic aortic rings were dissected. Then, they were
mounted in organ chambers filled with Krebs solution (composition in
mM: NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4,
1.2; and glucose, 11) at 37 ◦C and gassed with 95% O2 and 5% CO2 and
were stretched to 2 g of resting tension by means of two L-shaped
stainless-steel wires inserted into the lumen and attached to the chamber
and to an isometric force–displacement transducer (UF-1, Cibertec,
Madrid, Spain), and recorded in a recording and analysis system
(MacLab ADInstruments), as described previously.39
N-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)-4-chlor-
obenzimidamide (5h). Yellow oil; 83% yield. 1H NMR (400 MHz,
CD3OD): δ 7.76 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 7.29 (d, J =
2.7 Hz, 1H), 7.08 (dd, J = 9.0 Hz, J = 2.7 Hz, 1H), 6.85 (d, J = 9.0 Hz,
1H), 3.89 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H), 3.35 (t, J = 6.0 Hz, 2H). 13
C
NMR (100 MHz, CD3OD) δ 199.73, 164.97, 151.28, 147.51, 140.77,
130.76, 130.65, 130.55, 125.22, 120.04, 117.89, 113.96, 56.50, 47.94,
39.70. HRMS (LSIMS): m/z calcd for C17H19N3O2Cl: 332.1166 [M +
H]+; found: 332.1179.
Rings were then mounted in organ chambers filled with Krebs so-
lution and were stretched to 2 g of resting tension by means of two L-
shaped stainless-steel wires inserted into the lumen and attached to the
chamber and to an isometric force–displacement transducer (UF-1,
Cibertec, Madrid, Spain), and recorded in a recording and analysis sys-
tem (MacLab ADInstruments), as described previously.41
4.2. In vitro nNOS and iNOS activity determination
Enzymatic inhibition determination of the final products was carried
out following different procedures depending on each isoform of NOS.
Most of the used compounds were supplied by Sigma-Aldrich (Merk),
except the tritium-labeled L-[3H]-arginine, from Amersham Biosciences
(Perkin Elmer) and the human enzymes iNOS and nNOS recombinants,
from Enzo Life Sciences (Taper).
After stabilization of the medium conditions, these rings were incu-
bated with a known eNOS inhibitor: L-NAME (100 μM), the compound 4i
(20
μ
M), or its vehicle (DMSO, 1/106) for 30 min. The contraction was
In these two isoforms, the in vitro enzymatic activity tests were
performed in triplicate, following the Bredt & Snyder methodology.40
The basic foundation of this methodology is the control of the con-
version of L-[3H]-arginine to L-[3H]-citrulline, due to the use of a
Beckman LS 6500 multi-purpouse scintillation counter detector.
then induced by a [1 µM] norepinephrine solution. Once a plateau
contraction was reached, a concentration–response curve was con-
structed by cumulative addition of acetylcholine. Results are expressed
as percentage of norepinephrine-evoked contraction. Data are expressed
as means ± standard error mean and n reflects the number of aortic rings
from different rats. Statistically significant differences among groups
were calculated by two-way ANOVA followed by a Dunnett’s multiple
comparison test. P < 0.05 was considered statistically significant.
The final volume for each one of the replicates is 100 μl, of which: 50
µl correspond to a buffer solution containing 25 mM tris-(hydrox-
ymethyl)-aminomethane hydrochloride (Tris-HCl), 1 mM DL-dithio-
threitol (DTT), 4 µM 5,6,7,8-tetrahydro-L-biopterin dihydrochloride
(BH4), 10 µM flavin-adenine dinucleotide (FAD), 0.5 mM hypoxanthine-
9-ribofuranoside (inosine), 0.5 mg/ml bovine serum albumin (BSA), 0.1
mM CaCl2, 10 µM L-arginine, 10 µg/ml calmodulin (CaM) (only in
nNOS) and 0.05 µM L-[3H]-arginine, always maintaining a pH of 7.6; 10
µl of an aliquot of the NOS isoform correspondent to the study per-
formed; 10 µl of a solution of the product to be evaluated; 10 µl of 7.5
mM NADPH (except in control wells); and, finally, enough H2O MiliQ to
reach 100 µl.
4.4. Cell viability assay
To examine the cytotoxicity induced by compounds 4g, 4i, 5e and 5f,
changes in the viability of HUVECs, after incubation with these com-
pounds, were evaluated by assessing mitochondrial activity by the MTT
reduction assay. Briefly, HUVECs were seeded at 1x104 cells per well in a
96-well microtiter plate and incubated at 37 ◦C for up to 24 h. Then, the
cells supernatants were removed and replaced by fresh medium serum
free for at least two hours before the cells were exposed to serial di-
lutions of compounds (10 - 500 µM) for 30 min. At the selected time, 20
uL of 5 mg/ml MTT in PBS was added to the cells and further incubated
at 37 ◦C for 3 h. After washing, 100 µl of DMSO were added into each
well, and the spectrophotometric analysis was run at 570 nm using a
multi-well plate reader (Fluorostart; BMG Labtechnologies, Offenburg,
Germany) with background subtraction at 630 nm. Cell viability was
calculated as the percentage of the viable cells compared with untreated
controls. The viability experiments were conducted in at least three in-
dependent times, each run in triplicate. Results are expressed as per-
centage relatively to the untreated condition.
All samples are incubated for 30 min at 37 ◦C. After that, 400 µl of a
low-temperature buffer is added to stop the enzymatic process. This
solution contains 0.1 M N-(2-hydroxymethyl)piperazine-N’-(2-ethane-
sulfonic) (HEPES) acid, 10 mM ethylene glycol-bis-(2-aminoethyl
ether)-N,N,N’,N’-tetraacetic acid (EGTA) and 0.175 mg/ml L-citrulline
at pH = 5.5.
The sudden drop in temperature, the pH change and the dilution of
all the components found in the reaction well, are the main factors that
cause the stop of the enzymatic reaction; in addition, EGTA is also
capable of chelating the Ca2+ ions, essential for the dimerization and
functioning of the enzyme.
After this stage, each sample is passed through a column with
Dowex-50 W ion exchange resin (Na+) and then washed with H2O MiliQ
(1.2 mL), thus losing about 98% of the radioactivity.
4.5. Docking studies
Finally, 50 µl of each replica are diluted in Eco Lite (+) scintillation
liquid to take measurements the next day, once other factors capable of
exciting the scintillation liquid beyond the β emissions are eliminated.
NOS activity is expressed in picomoles of L-citrulline produced per
mg of protein per minute (pM/mg min).
4.5.1. Docking protocol
Docking studies were carried out with Autodock 4.2.6 (AD4)42 on the
iNOS and nNOS pdb IDs 4CX7 and 6AV2, respectively. Ligands struc-
tures were built on Avogadro43 and optimized using Gaussian44 (HF/6-
31G(d,p)). Compounds presenting tertiary amines, prone to protonation
at physiological pH, were also considered. Once optimized, ligands PDBs
were prepared for docking using the prepare_ligand4.py script included
For the statistical treatment of data, the Prism8 program of GraphPad
was used, where the IC50 calculation was carried out by means of non-
linear regression ELISA studies with variable slope of four factors.
9

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
MGLTools 1.5.4.42 Protein structures, on the other hand, were prepared
for docking using the PDB2PQR tools.45 Water and ligand molecules
were removed and charges and non-polar hydrogen atoms were added at
pH 7.0. The produced structures were saved as a pdb files and prepared
for docking using the prepare_receptor4.py script from MGLTools. The
Fe atom of heme was assigned a charge of +3. AD4 was used to auto-
matically dock the ligands into the iNOS and nNOS binding sites. For
both enzymes, the docking grid was centered on the ligand binding site
and set with the following grid parameters: 60 Å × 60 Å × 60 Å with
0.375 Å spacing. In all calculations, AD4 parameter file was set to 100
GA runs, 2.500.000 energy evaluations and a population size of 150. The
Lamarckian genetic algorithm local search (GALS) method was used for
the docking calculations. All dockings were performed with a population
size of 250 and a Solis and Wets local search of 300 rounds was applied
with a probability of 0.06. A mutation rate of 0.02 and a crossover rate of
0.8 were used. The docking results from each of the 100 calculations
were clustered based on root-mean square deviation (RMSD) solutions
differing by less than 2.0 Å between the Cartesian coordinates of the
atoms and ranked on the basis of free energy of binding.
13 Wang H, Wang L, Xie Z, et al. Nitric Oxide (NO) and NO Synthases (NOS)-Based
Targeted Therapy for Colon Cancer. Cancers. 2020;12:1–25. https://doi.org/
10.3390/cancers12071881.
14 Belgorosky D, Girouard J, Langle YV, et al. Relevance of iNOS expression in tumor
growth and maintenance of cancer stem cells in a bladder cancer model. J Mol Med.
2020;98:1615–1627. https://doi.org/10.1007/s00109-020-01973-0.
15 Vahora H, Munawwar AK, Alalami U, Hussain A. The potential role of nitric oxide in
halting cancer progression through chemoprevention. J Cancer Prev. 2016;21:1–12.
https://doi.org/10.15430/JCP.2016.21.1.1.
16 Puglisi MA, Cenciarelli C, Tesori V, et al. High nitric oxide production, secondary to
inducible nitric oxide synthase expression, is essential for regulation of the
tumourinitiating properties of colon cancer stem cells. J Pathol. 2015;236:479–490.
https://doi.org/10.1002/path.4545.
17 Singh S, Gupta AK. Nitric oxide: role in tumour biology and iNOS/NO-based
anticancer therapies. Cancer Chemother Pharmacol. 2011;67:1211–1224. https://doi.
org/10.1007/s00280-011-1654-4.
18 Prueitt RL, Boersma BJ, Howe TM, et al. Inflammation and IGF-I activate the Akt
pathway in breast cancer. Int J Cancer. 2007;120:796–805. https://doi.org/10.1002/
ijc.22336.
19 Pervin S, Singh R, Hernandez E, Wu G, Chaudhuri G. Nitric oxide in physiologic
concentrations targets the translational machinery to increase the proliferation of
human breast cancer cells: Involvement of mammalian target of rapamycin/eIF4E
pathway. Cancer Res. 2007;67:289–299. https://doi.org/10.1158/0008-5472.CAN-
05-4623.
20 Thomas DD, Ridnour LA, Espey MG, et al. Superoxide fluxes limit nitric oxide-
induced signaling. J Biol Chem. 2006;281:25984–25993. https://doi.org/10.1074/
jbc.M602242200.
Declaration of Competing Interest
Authors declare no interest conflict.
Acknowledgements
21 Maccallini C, Arias F, Gallorini M, et al. Antiglioma Activity of Aryl and Amido-Aryl
Acetamidine Derivatives Targeting iNOS: Synthesis and Biological Evaluation. ACS
Med Chem Lett. 2020;11:1470–1475. https://doi.org/10.1021/
acsmedchemlett.5b00149.
22 Thomas DD, Espey MG, Ridnour LA, et al. Hypoxic inducible factor 1α, extracellular
signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of
nitric oxide. Proc Natl Acad Sci USA. 2004;101:8894–8899. https://doi.org/10.1073/
pnas.0400453101.
´
This work was supported by the grant from Comision Interministerial
23 Garcia V, Sessa WC. Endothelial NOS: perspective and recent developments. Br J
Pharmacol. 2019;176:189–196. https://doi.org/10.1111/bph.14522.
24 Maccallini C, Mollica A, Amoroso R. The positive regulation of eNOS signaling by
PPAR agonists in cardiovascular diseases. Am J Cardiovasc Drugs. 2017;17:273–281.
https://doi.org/10.1007/s40256-017-0220-9.
de Ciencia y Tecnología, Ministerio de Economía y competitividad
(MINECO) (SAF2017-84894-R), with funds from the European Union,
and by the Ministerio de Economia y Competitividad, Instituto de Salud
Carlos III (CIBER-CV).
25 Li H, Xue F, Kraus II JM, et al. In search of potent and selective inhibitors of neuronal
nitric oxide synthase with more simple structures. Bioorg Med Chem. 2013;21:
1333–1343. https://doi.org/10.1016/j.bmc.2013.06.014.
´
The authors also thank the Centro de Supercomputacion de la Uni-
versidad de Granada (CSIRC) for the computing resources and the
Granada University Library for the financial support to the APC.
26 Liu ZC, Hai YS, Jing W, et al. Discovery and development of novel pyrimidine and
pyrazolo/thieno-fused pyrimidine derivatives as potent and orally active inducible
nitric oxide synthase dimerization inhibitor with efficacy for arthritis. Eur J Med
Chem. 2021;213, 113174. https://doi.org/10.1016/j.ejmech.2021.113174.
27 Cinelli MA, Do HT, Miley GP, Silverman RB. Inducible nitric oxide synthase:
Regulation, structure, and inhibition. Med Res Rev. 2020;40:158–189. https://doi.
org/10.1002/med.21599.
References
1
2
Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat Rev Drug
Discov. 2002;1:939–950. https://doi.org/10.1038/nrd960.
´
28 Entrena A, Camacho ME, Carrion MD, et al. Kynurenamines as Neural Nitric Oxide
Mohamed MFA, Marzouk AA, Nafady A, et al. Design, synthesis and molecular
modeling of novel aryl carboximidamides and 3-aryl-1,2,4-oxadiazoles derived from
indomethacin as potent anti-inflammatory iNOS/PGE2 inhibitors. Bioorg Chem.
2020;105, 104439. https://doi.org/10.1016/j.bioorg.2020.104439.
Grottelli S, Amoroso R, Macchioni L, et al. Acetamidine-Based iNOS Inhibitors as
Molecular Tools to Counteract Inflammation in BV2 Microglial Cells. Molecules.
2020;25:1–18. https://doi.org/10.3390/molecules25112646.
Cinelli MA, Reidl CT, Li H, Chreifi G, Poulos TL, Silverman RB. First Contact: 7-
Phenyl-2-Aminoquinolines, Potent and Selective Neuronal Nitric Oxide Synthase
Inhibitors That Target an Isoform-Specific Aspartate. J Med Chem. 2020;63:
4528–4554. https://doi.org/10.1021/acs.jmedchem.9b01573.
Akiyama H, Barger S, Barnum S, et al. Inflammation and Alzheimer’s disease.
Neurobiol Aging. 2000;21:383–421. https://doi.org/10.1016/S0197-4580(00)00124-
X.
Synthase Inhibitors. J Med Chem. 2005;48:8174–8181. https://doi.org/10.1021/
jm050740o.
´
´
29 Chayah M, Carrion MD, Gallo MA, Jimenez R, Duarte J, Camacho ME. Development
of urea and thiourea kynurenamine derivatives: Synthesis, molecular modeling and
biological evaluation as nitric oxide synthase inhibitors. ChemMedChem. 2015;10:
874–882. https://doi.org/10.1002/cmdc.201500007.
3
4
´
30 Chayah M, Camacho ME, Carrion MD, Gallo MA, Romero M, Duarte J. N, N’-
disubstituted thiourea and urea derivatives: Design, synthesis, docking studies and
biological evaluation against nitric oxide synthase. MedChemComm. 2016;7:
667–678. https://doi.org/10.1039/C5MD00477B.
31 Fürstner A, Jumbam DN, Seidel G. Syntheses of Zindoxifene and Analogues by
Titanium-Induced Oxo-Amide Coupling. Chem Ber. 1994;127:1125–1130. https://
doi.org/10.1002/cber.19941270624.
5
6
7
32 Shearer BG, Oplinger JA, Lee S. S-2-Naphthylmethyl Thioacetimidate Hydrobromide:
A New Odorless Reagent for the Mild Synthesis of Substituted Acetamidines. Tetrah
Lett. 1997;38:179–182. https://doi.org/10.1016/S0040-4039(96)02268-X.
33 Ungvari Z, Sun D, Huang A, Kaley G, Koller A. Role of endothelial [Ca2+]i in
activation of eNOS in pressurized arterioles by agonists and wall shear stress. Am J
Physiol Heart Circ Physiol. 2001;281(2):H606–H612. https://doi.org/10.1152/
ajpheart.2001.281.2.H606.
Trippier PC, Labby KJ, Hawker DD, Mataka JJ, Silverman RB. Target- and
mechanism-based therapeutics for neurodegenerative diseases: strength in numbers.
J Med Chem. 2013;56:3121–3147. https://doi.org/10.1021/jm301592.
Minhas R, Bansal Y, Bansal G. Inducible nitric oxide synthase inhibitors: A
comprehensive update. Med Res Rev. 2020;40:823–855. https://doi.org/10.1002/
med.21636.
8
9
Smith KJ, Lassmann H. The role of nitric oxide in multiple sclerosis. Lancet Neurol.
2002;1:232–241. https://doi.org/10.1016/S1474-4422(02)00102-3.
Mateen S, Moin S, Zafar A, Khan AQ. Redox signaling in rheumatoid arthritis and the
preventive role of polyphenols. Clin Chim Acta. 2016;463:4–10. https://doi.org/
10.1016/j.cca.2016.10.007.
34 Fedorov R, Hartmann E, Ghosh DK, Schlichting I. Structural Basis for the Specificity
of the Nitric-oxide Synthase Inhibitors W1400 and Nω-Propyl-l-Arg for the Inducible
and Neuronal Isoforms. Structural Basis for the Specificity of the Nitric-oxide
Synthase Inhibitors W1400 and Nω-Propyl-l-Arg for the Inducible and Neuronal
Isoforms. J Biol Chem. 2003;278:45818–45825. https://doi.org/10.1074/jbc.
M306030200.
´
10 Frances DE, Ingaramo PI, Ronco MT, Carnovale CE. Diabetes, an inflammatory
process: oxidative stress and TNFalpha involved in hepatic complication. J Biomed
Sci Eng. 2013;(6):645–653. https://doi.org/10.4236/jbise.2013.66079.
11 Dijkstra G, Zandvoort AJH, Muller Kobold AC, et al. Increased expression of
inducible nitric oxide synthase in circulating monocytes from patients with active
inflammatory bowel disease. Scand J Gastroenterol. 2002;37:546–554. https://doi.
org/10.1080/00365520252903099.
35 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and
development settings. Adv Drug Deliv Rev. 1997;46(23):3–25. https://doi.org/
10.1016/S0169-409X(96)00423-1.
36 Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular
properties that influence the oral bioavailability of drug candidates. J Med Chem.
2002;45:2615–2623. https://doi.org/10.1021/jm020017n.
12 Tang L, Gao X, Zhao B, et al. Design and synthesis of new disubstituted
benzoxazolone derivatives that act as iNOS inhibitors with potent anti-inflammatory
activity against LPS-induced acute lung injury (ALI). Bioorg Med Chem. 2020;28,
115733. https://doi.org/10.1016/j.bmc.2020.115733.
37 Gurung AB, Bhattacharjee A, Ali MA. Exploring the physicochemical profile and the
binding patterns of selected novel anticancer Himalayan plant derived active
10

F. Arias et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116294
compounds with macromolecular targets. Inform Med Unlocked. 2016;5:1–14.
43 Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR.
Avogadro: an advanced semantic chemical editor, visualization, and analysis
platform. J Cheminf. 2012;4:17. https://doi.org/10.1186/1758-2946-4-17.
44 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani
G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP,
Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda
R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T,
Montgomery Jr JA., Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin N,
Staroverov VN, Keit T, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant
JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB,
Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ,
Cammi R, Pomelli C, Ochterski JW, Martin L, Morokuma K, Zakrzewski VG, Voth GA,
Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz
JV, Cioslowski J, Fox DJ. GAUSSIAN 09 (Revision D.1), GAUSSIAN 09 (Revision
B.01); 2010.
https://doi.org/10.1016/j.imu.2016.09.004.
38 Fukunishi Y, Nakamura H. Definition of Drug-Likeness for compound affinity. J Chem
Inf Model. 2011;51:1012–1016. https://doi.org/10.1021/ci200035q.
39 Varoli L, Burnelli S, Garuti L, Vitali B. Synthesis and antimicrobial activity of new
diazoimidazole derivatives containing an N-acylpyrrolidine ring. Il Farmaco. 2011;56
(11):885–890.
40 Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring
enzyme. Proc Natl Acad Sci U S A. 1990;87(2):682–685. https://doi.org/10.1073/
pnas.87.2.682.
´
41 Robles-Vera I, Toral M, de la Visitacion N, et al. The Probiotic Lactobacillus
fermentum Prevents Dysbiosis and Vascular Oxidative Stress in Rats with
Hypertension Induced by Chronic Nitric Oxide Blockade. Mol Nutr Food Res. 2018;62,
e1800298. https://doi.org/10.1002/mnfr.201800298.
42 Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: Automated
docking with selective receptor flexibility. J Comput Chem. 2009;30:2785–2791.
https://doi.org/10.1002/jcc.21256.
45 Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated
pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids
Res. 2004;32:W665–W667. https://doi.org/10.1093/nar/gkh381.
11