Easy and rapid preparation of benzoylhydrazides and their diazene derivatives as inhibitors of 15-lipoxygenase

Article abstract of DOI:10.1016/j.bmcl.2017.03.017

Two series of diaza derivatives were prepared by solvent-free condensation of benzoic acid and 4-substituted phenylhydrazines in order to obtain phenylhydrazides (HYD series) and, by oxidation of these compounds, the corresponding benzoyldiazenes (DIA series). Both sets were evaluated as inhibitors of soybean 15-lipoxygenase activity and antioxidant capability in the FRAP and CUPRAC assays. The most potent inhibitors of both series exhibited IC₅₀ values in the low micromolar range. Kinetic studies showed that at least the more active compounds were competitive inhibitors. Docking results indicated that the most potent inhibitor interacts strongly with Ile-839 and iron in the active site.

Full text of DOI:10.1016/j.bmcl.2017.03.017

Accepted Manuscript
Easy and rapid preparation of benzoylhydrazides and their diazene derivatives
as inhibitors of 15-lipoxygenase
Cristian Tirapegui, Williams Acevedo-Fuentes, Pablo Dahech, Claudia Torrent,
Pablo Barrias, Macarena Rojas-Poblete, Carolina Mascayano
PII:
S0960-894X(17)30253-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.017
BMCL 24769
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2017
5 March 2017
6 March 2017
Please cite this article as: Tirapegui, C., Acevedo-Fuentes, W., Dahech, P., Torrent, C., Barrias, P., Rojas-Poblete,
M., Mascayano, C., Easy and rapid preparation of benzoylhydrazides and their diazene derivatives as inhibitors of
15-lipoxygenase, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.03.017
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Bioorganic & Medicinal Chemistry Letters
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Easy and rapid preparation of benzoylhydrazides and their diazene derivatives
as inhibitors of 15-lipoxygenase
Cristian Tirapeguia,*, Williams Acevedo-Fuentesa, Pablo Dahechb, Claudia Torrentb, Pablo Barriasb,
Macarena Rojas-Pobletec, Carolina Mascayanob
aDepartment of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile, Casilla 653, Santiago, Chile. cristian.tirapeguic@usach.cl
bFaculty of Chemistry and Biology, University of Santiago, Santiago, Chile.
cRelativistic Molecular Physics(ReMoPh) Group, Universidad Andrés Bello, Santiago, Chile.
ARTICLE INFO
ABSTRACT
Two series of diaza derivatives were prepared by solvent-free condensation of benzoic acid and
4-substituted phenylhydrazines in order to obtain phenylhydrazides (HYD series) and, by
oxidation of these compounds, the corresponding benzoyldiazenes (DIA series). Both sets were
evaluated as inhibitors of soybean 15-lipoxygenase activity and antioxidant capability in the
FRAP and CUPRAC assays. The most potent inhibitors of both series exhibited IC50 values in
the low micromolar range. Kinetic studies showed that at least the more active compounds were
competitive inhibitors. Docking results indicated that the most potent inhibitor interacts strongly
with Ile-839 and iron in the active site.
Article history:
Received
Revised
Accepted
Available online
Keywords:
15-lipoxygenase inhibition
Phenylhydrazides
Benzoyldiazene
2009 Elsevier Ltd. All rights reserved.
Antioxidant capability
Lipophilicity.
Lipoxygenases (LOXs) catalyze the production of eicosanoid
leukotrienes and lipoxins, both biosynthesized from arachidonic
acid (AA)1 derived from the cell membrane, and important
biological mediators of inflammatory processes.2 This family of
enzymes is well conserved among mammalian species3 and
catalyzes stereo- and regiospecific introduction of dioxygen into
the polyunsaturated chain of AA as hydroperoxide, which finally
is converted to the hydroxyl group.4 Due to this activity, the
name of this enzyme is often preceded by the number of the
carbon atom in the AA chain which undergoes oxidation. Thus,
using the usual acronym, 5-LOX, 12-LOX and 15-LOX isoforms
introduce an oxygen atom at positions C-5, C-12 and C-15
respectively.5
Hashimoto,12 where a phtalocyanine was used as the oxidant.
Another example of a green strategy applicable to our work was
reported by Metro,13 who prepared different amide derivatives by
mechanochemical means. Specifically, the latter procedure was
used to obtain the first set of compounds in this research.
The aim of the present work was to study the effects of two
different structural features on the inhibitory behavior of two
families of inhibitors: the influence of the oxidation of the central
nitrogen-nitrogen single bond of phenylhydrazides, and the
systematic substitution of the para position of the N-phenyl
moiety. Both series proposed here were prepared using short and
clean procedures, and some of the resulting compounds proved to
be fairly potent inhibitors of 15-LOX.
Some benzothiophene derivatives and esters of caffeic acid
have been reported to be potent inhibitors of 5-LOX. Examples
of these are the commercial Zileuton (IC50 = 0.4 µM)6 and CAPE
(IC50 = 0.13 µM).7 Likewise, some phenylhydrazones inhibit the
dual cyclooxygenase/peroxidase activity of prostaglandin
synthase,8 phenylhydrazide derivatives selectively inhibit
cycloxygenase-2,9 and pyrazole carboxamides inhibit 15-
lipoxygenase-110,11 but until now we are not aware of any
systematic studies of these compounds as 15-LOX inhibitors.
The same is true for the related diazene derivatives.
Scheme I shows the synthesis of both series. The first step is
the preparation of phenylhydrazide derivatives (HYD series)
where both moieties, hydrazine and benzoic acid, where
connected by a solvent-free reaction carried out by grinding the
reactants in a simple mortar. The oily material prepared by this
method was rinsed with pure water affording a white or
yellowish white solid that was crystallized in ethanol to obtain
fine needles in almost every case. This procedure is an
environmentally friendly coupling reaction, with easy recovery of
the desired compounds and negligible formation of side products.
The second step in Scheme I is the oxidation of the former
substances with potassium ferricyanide14. This reaction was
carried out by shaking two immiscible solutions, one containing
the substituted hydrazide dissolved in dichloromethane, and the
other, the ferricyanide in a strongly basic aqueous solution. Using
During the last decades a growing interest has arisen to
develop new, greener synthetic strategies whereby the amount of
residual waste becomes progressively smaller, and a many such
reactions can be found in the literature. An interesting example of
a
green oxidation of arylhydrazides was proposed by

this simple experimental setup, the formation of diazene is easily
detectable by the change of color in the organic phase from
colorless to red, keeping the inorganic iron in aqueous solution.
Further purification of the oily red products was not needed. Both
reactions represent simple and clean methods for the rapid
preparation of phenylhydrazides and their phenylcarbonylazo
analogs in high yields.
inhibitors boswellic acid (IC50 = 1 µM)15 and baicalein (IC50
=
1.6 µM),16 respectively, but it is 3.4 times more potent than the
commercial 15-LOX inhibitor 4-methyl-2-(4-methylpiperazinyl)-
pyrimido[4,5b]benzothiazine.17 The diazene derivatives DIA-4H
and DIA-4CN inhibit 15-LOX to a somewhat lesser extent but in
the same range as HYD-4Me. Even though the IC50 values of
these compounds lie far from the desirable nanomolar range, they
incorporate good molecular scaffolds that could be used to
introduce a broad variety of structural modifications taking
advantage of their simple synthesis and the ready availability of
many differently substituted benzoic acids and arylhydrazines.
Scheme I. Synthesis of phenylhydrazide derivatives (HYD
series) and azocarbonyl derivatives (DIA series).
To evaluate the ability of both sets of compounds to inhibit the
activity of 15-LOX an initial screen was performed, whose
results are shown in Table 1.
Table 1. Screening of 15-LOX inhibition by HYD and DIA
series at 10
µM inhibitor. *DIA-4SA (sulfamide) not evaluated
due to poor solubility.
Inhibitor
HYD-4OMe
HYD-4Me
HYD-4H
Inhibition (%)
Inhibitor
DIA-4OMe
DIA-4Me
DIA-4H
Inhibition (%)
19 4.6
Figure 1. Lineweaver-Burk plots for two representative 15-
LOX inhibitors of the HYD and DIA series. Left, HYD-4Me and
right, DIA-4CN.
73 3.3
93 5.3
33 5.5
44 7.8
87 7.6
HYD-4Br
HYD-4SA
HIYD-4CN
HYD-4NO2
28 2.2
10 2.2
DIA-4Br
DIA-4SA
DIA-4CN
DIA-4NO2
44 3.8
*
80 4.1
To study the type of inhibition induced by both series of
compounds, HYD-4Me and DIA-4CN were selected (Figure 1).
The Lineweaver-Burk plots indicate that the inhibition was
competitive for both derivatives, suggesting that these inhibitors
compete with arachidonic acid to reach the active site without
affecting the theoretical maximum rate of conversion to product.
The active site of this oxidoreductase enzyme has a non-heme
iron atom which plays a key role in the introduction of a
hydroxyl group at the C-15 position of the fatty acid by
generating the allyl radical in the fatty acid chain and
subsequently stabilizing the hydroperoxide radical formed by
addition of molecular oxygen.19 With these results, it is possible
to hypothesize that the inhibitor blocks the access of arachidonic
acid to the active site by generating specific interactions with the
iron and/or an amino acid residue present in the active site of 15-
LOX, lowering the Km. As an approach to understand the likely
molecular interactions in the environment of the metal we built
molecular models of possible complexes with the inhibitors
(Figure 2). Our results indicate that both inhibitors are able to
reach the active site of 15-LOX, which is in agreement with the
competitive inhibition found by kinetics experiments. A detailed
analysis of the models shows HYD-4Me establishing basically
two different interactions. One of these arises between the
aromatic amine NH group and the iron atom (3.6 Å). The second
interaction found is a hydrogen bond (2.3 Å) between the
hydrogen of the amide NH group and the carboxyl of the C-
terminal amino acid residue Ile-839. It is interesting to note that
if hydrogen bonding contributes to the strength of binding in the
active site, and/or if subsequently this capability is enhanced by
electron donor substituents at the para position of the phenyl
ring, it would be possible to determine some grade of correlation
between the presence of the substituents and the effective acidity
9
1.6
17 2.1
34 4.6
The comparison of inhibition values would seem to indicate
that, in the HYD series, an electron donor group at the para
position increases and an electron attractor decreases activity, as
is particulary noticeable with methyl and methoxyl substitution.
When the central nitrogen-nitrogen bond is oxidized to diazene,
this trend seems to be reversed, although the electron-
withdrawing para-cyano group has no apparent effect on the
inhibitory activity. This behavior is more evident when the values
of inhibition for each HYD and its DIA counterpart are
compared. If the inhibitory activity is mediated by hydrogen
bond formation in the enzyme’s active site, it may be supposed
that there is a correlation between the effect of the electron donor
group and the basicity of the secondary amine in the inhibitor’s
dinitrogen bridge. This tendency indicates that arylhydrazide
derivatives with electron donating aryl groups are better
candidates for the design of 15-LOX inhibitors. In contrast,
although DIA-4H and DIA-4CN are practically as potent as
HYD-4Me and HYD-4OMe, the screening data do not suggest a
design strategy for additional diazene analogs, although the
smaller size of the cyano group (Es = -0.51) as compared to
methyl and bromine (Es = -1.24 and -1-34 respectively)18 might
explain the strong inhibition by DIA-4CN.
The most potent 15-LOX inhibitors in our series were chosen
to obtain their IC50 values and kinetic parameters (Table 2).
Table 2. Inhibition and kinetic constants for 15-LOX
inhibition by HYD and DIA compounds.
parameter Σαͼ of Abraham,20 which indicates precisely the
Inhibitor
Ki
Km
(µM)
89.6
N.D
139.8
Vmax
(µM/s)
0.46
N.D
0.57
Type of
Inhibition
competitive
-
IC50 (µM)
ͦ
(µM)
0.81
N.D
3.89
ability to donate a hydrogen atom toward a hydrogen acceptor. In
order to evaluate which interaction would be mainly responsible
for the inhibition, the series of 4-substituted anilines proposed by
Abraham was taken as a model due to their structural analogy
with the phenylhydrazide derivatives of this work. In the aniline
series, higher values of acidity are observed for 4-chloroaniline
(0.30) and lower values for 4-methoxy- and 4-methylaniline
HYD-4Me
DIA-4H
DIA-4CN
5.35 0.80
6.97 0.59
9.78 0.72
competitive
The most potent inhibitor of the series (HYD-4Me), shows an
IC50 value five and three times higher than the well-known

(0.23), which means that electron donor groups weaken the
hydrogen donating ability. This tendency is opposite to the
behavior observed in the HYD series, which seems to suggest
that the higher affinity for the active site of the phenylhydrazides
with electron donor groups obeys mainly to complex formation
between the NH group and iron rather than hydrogen bond
formation between -N´H- and Ile-839.
beyond doubt that the HYDs are much better able to reduce the
active Fe(III) to the inactive Fe(II). Secondly, the differences of
antioxidant capacity values for different compounds of both
series are not significant when the CUPRAC and FRAP methods
are analyzed. In spite of this, it is interesting to point out the
greater antioxidant capacity exhibited by the unsubstituted
phenylhydrazide (HID-4H) in both assays.
Since metal reductive capability is not related to the enzyme
inhibitory potency, it seems that some other property should
enhance activity in the DIA series. So far we have considered the
electronic nature of the substituent at the para position, azo bond
formation, the hydrogen bonding ability of the -NH- group, and
now the antioxidant capacity of the inhibitors. Due to the
hydrophobic nature of the catalytic domain, it would come as no
surprise if the hydrophobicity of the inhibitor plays a crucial role
in accessing the iron-histidine complex. To evaluate this effect,
both series were subjected to a preliminary assessment using the
ALOGPS 2.1 software,24 The results are shown in Figure 4. From
these, it is noteworthy that dehydrogenation of the central
CONH-NH- moiety of the HYD inhibitors enhances their
lipophilicity by about two log units. This remarkable difference
might explain the behavior of DIA-4CN found by docking
calculations, where this compound is unable to reach the metallic
center of catalytic domain due to hydrophobic interactions
between its extended conjugated system and the lipophilic helix
in the active site. Although the 15-LOX inhibition by both
families of compounds is competitive, the molecular interactions
involved occur at different loci in the active site of the enzyme.
For the sake of comparison, log P values were calculated with the
same software for competitive inhibitors of similar molecular
weight, ebselen,25 zileuton,26 baicalein,16 and ML351.27 The
figures obtained were 1.91, 2.01, 3.19 and 4.57 respectively, in
the same range as the lipophilicities of the inhibitors studied here
(log P ca. 2-4), suggesting that such values are appropriate for the
design of new 15-LOX inhibitors.
Figure 2. Enzyme-inhibitor complexes and main molecular
interactions in the 15-LOX binding site obtained by docking
calculations with HYD-4Me (left) and DIA-4CN (right).
In the case of DIA-4CN the docking results do not show any
interaction between the iron atom and the inhibitor, presumably
due to the reduced ability to form a complex involving the -
CON=N- moiety versus the bidentate -CONHNH- central group
of HYD-4Me. This results in a considerable gap between the
isoleucine-histidine-iron complex and the diazene inhibitor,
associated with the benzoyl moiety’s approach to the
hydrophobic loop in the catalytic domain of LOX.21 This seems
reasonable based on the increased hydrophobic character of the
diazene. On this basis we can suggest that iron complexation by
HYD-4Me is important for its 15-LOX inhibitory activity, while
DIA-4CN acts primarily by blocking the access of the substrate
to the α-helical domain of the catalytic site. The docking energies
support this notion, as the interaction with HYD-4Me (-4.67
kcal/mol) is stronger than with DIA-4CN (-3.66 kcal/mol).
Because the active site of this oxidoreductase contains a redox
active iron atom which is the key to the initial radical generation
from the at least doubly unsaturated arachidonic (or linolenic)
acid, two different antioxidant capability assays were carried out.
These are based on the change of oxidation state of iron and
copper, namely, the Ferric Reducing Antioxidant Power
(FRAP)22 and Cupric Reducing Antioxidant Capacity
(CUPRAC)23 assays respectively. The results for the HYD and
DIA series are shown in Figure 3.
Figure 4. Lipophilicity of the HYD and DIA series calculated
using ALOGPS 2.1 software.
In conclusion, two sets of dinitrogen derivatives, the HYD and
DIA series, were prepared by "one pot", two-phase reactions by
clean and easy procedures. This methodology is environmentally
friendly and provides the desired products in high yields. Both
series of compounds were shown to be low micromolar
competitive 15-LOX inhibitors. The biochemical results show
that the more potent HYD derivatives are those that bear an
electron donor para-substituent, but no such relationship is clear
in the DIA series. These differences were interpreted tentatively
on the basis of docking calculations for two representative
molecules (HID-4Me and DIA-4CN) indicating that compounds
belonging to each of the two series may interact with different
loci of the same active site. Besides, the combination of moderate
lipophilicity with a greater propensity of the HYD series to
reduce the active Fe(III), suggests the synthesis of new analogs
that could be good leads for the design of useful 15-LOX
inhibitors.
Figure 3. Antioxidant capacity of the HYD and DIA series
evaluated by: left, Ferric Reducing Antioxidant Power (FRAP)
assay, and right, Cupric Reducing Antioxidant Capacity
(CUPRAC) assay. *Not evaluated due to poor solubility.
The same marked difference of antioxidant capacity of the
HYD and DIA series shown by different methods indicates

Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors are grateful to FONDECYT for postdoctoral grant
3130557, MECESUP USA1498 project for
a
research
assistantship, CONICYT FONDEQUIP/UHPLC MS/MS EQM
120065 project for the UHPLC-MS/MS characterization, and
South American Phytochemical (SAPHYCHEM) for FRAP and
CUPRAC experiments.
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Graphical Abstract
Easy and rapid preparation of
benzoylhydrazides and their diazene
derivatives as inhibitors of 15-lipoxygenase
Leave this area blank for abstract info.
Cristian Tirapegui, Williams Acevedo-Fuentes, Pablo Dahech, Claudia Torrent, Pablo Barrias,
Macarena Rojas-Poblete, Carolina Mascayano