Ci a ion: Da Cos a, G.V.; Ne o,
M.F.A.; Da Sil a, A.K.P.; De Sá,
E.M.F.; Cancela, L.C.F.; Vega, J.S.;
Loba o, C.M.; Zuliani, J.P.;
Espejo-Román, J.M.; Campos, J.M.;
e al. Iden i ica ion o Po en ial Insec
G ow h Inhibi o agains Aedes
aegyp i: A Bioin o ma ics App oach.
In . J. Mol. Sci. 2022,23, 8218.
h ps://doi.o g/10.3390/
ijms23158218
Academic Edi o : Angela Lomba di
Recei ed: 19 May 2022
Accep ed: 11 July 2022
Published: 26 July 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
Iden i ica ion o Po en ial Insec G ow h Inhibi o agains
Aedes aegyp i: A Bioin o ma ics App oach
Glaube V. Da Cos a 1,2,3 , Moysés F. A. Ne o 4, Alicia K. P. Da Sil a 3, Es e M. F. De Sá3, Luanne C. F. Cancela 3,
Jeanina S. Vega 3, Cássio M. Loba o 2,3, Juliana P. Zuliani 5, JoséM. Espejo-Román6, Joaquín M. Campos 6,
F anco H. A. Lei e 4and Cleydson B. R. San os 1,2,6,*
1G adua e P og am in Ne wo k in Pha maceu ical Inno a ion, Fede al Uni e si y o Amapá,
Macapá68902-280, AP, B azil; [email p o ec ed]
2Labo a o y o Modeling and Compu a ional Chemis y, Depa men o Biological and Heal h Sciences,
Fede al Uni e si y o Amapá, Macapá68902-280, AP, B azil; [email p o ec ed]
3Labo a o y o Bio echnology in Na u al P oduc s, Depa men o Biological and Heal h Sciences, Fede al
Uni e si y o Amapá, Macapá68902-280, AP, B azil; [email p o ec ed] (A.K.P.D.S.);
[email p o ec ed] (E.M.F.D.S.); [email p o ec ed] (L.C.F.C.); [email p o ec ed] (J.S.V.)
4
Labo a o y Molecula Modeling, S a e Uni e si y o Fei a de San ana, Fei a de San ana 44036-900, BA, B azil;
[email p o ec ed] (M.F.A.N.); [email p o ec ed] (F.H.A.L.)
5Labo a o y Cellula Immunology Applied o Heal h, Oswaldo C uz Founda ion, FIOCRUZ Rondônia,
Po o Velho 78912-000, RO, B azil; [email p o ec ed]
6
Depa men o Pha maceu ical and O ganic Chemis y, Facul y o Pha macy, Ins i u e o Biosani a y Resea ch
ibs, Uni e si y o G anada, 18071 G anada, Spain; [email p o ec ed].es (J.M.E.-R.);
jmcampos@ug .es (J.M.C.)
*Co espondence: b [email p o ec ed]; Tel.: +55-096-4009-2699
Abs ac :
Aedes aegyp i is he main ec o ha ansmi s i al diseases such as dengue, hemo hagic
dengue, u ban yellow e e , zika, and chikungunya. Wo ldwide, many cases o dengue ha e been
epo ed in ecen yea s, showing signi ican g ow h. The bes way o manage diseases ansmi ed
by Aedes aegyp i is o con ol he ec o wi h insec icides, which ha e al eady been shown o be oxic
o humans; mo eo e , insec s ha e de eloped esis ance. Thus, he de elopmen o new insec icides
is conside ed an eme gency. One way o achie e his goal is o apply compu a ional me hods based
on ligands and a ge in o ma ion. In his s udy, six een compounds wi h accep able insec icidal
ac i i ies, wi h 100% la icidal ac i i y a low concen a ions (2.0 o 0.001 mg
·
L
−1
), we e selec ed
om he li e a u e. These compounds we e used o build up and alida e pha macopho e models.
Pha macopho e model 6 (AUC = 0.78; BEDROC = 0.6) was used o il e 4793 compounds om
he subse o lead-like compounds om he ZINC da abase; 4142 compounds (dG < 0 kcal/mol)
we e hen aligned o he ac i e si e o he ju enile ho mone ecep o Aedes aegyp i (PDB: 5V13), 2240
compounds (LE <
−
0.40 kcal/mol) we e p io i ized o molecula docking om he cons uc ion o a
chi in deace ylase model o Aedes aegyp i by he homology modeling o he Bombyx mo i species (PDB:
5ZNT), which aligned 1959 compounds (dG < 0 kcal/mol), and 20 compounds (
LE < −0.4 kcal/mol
)
we e p edic ed o pha macokine ic and oxicological p edic ion in silico (P eadme , SwissADMET,
and eMolTox p og ams). Finally, he heo e ical ou es o compounds
M01
,
M02
,
M03
,
M04
, and
M05
we e p oposed. Compounds
M01
–
M05
we e selec ed, showing signi ican di e ences in pha ma-
cokine ic and oxicological pa ame e s in ela ion o posi i e con ols and in e ac ion wi h ca aly ic
esidues among key p o ein si es epo ed in he li e a u e. Fo his eason, he molecules in es iga ed
he e a e dual inhibi o s o he enzymes chi in syn hase and ju enile ho monal p o ein om insec s
and humans, cha ac e izing hem as po en ial insec icides agains he Aedes aegyp i mosqui o.
Keywo ds: Aedes aegyp i; pha macopho e; molecula docking; insec g ow h inhibi o
In . J. Mol. Sci. 2022,23, 8218. h ps://doi.o g/10.3390/ijms23158218 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 8218 2 o 30
1. In oduc ion
Mosqui oes a e one o he la ges g oups o insec s (pes s) wi h medical impo ance in
he global con ol o disease ansmission, and he highligh is he Aedes aegyp i mosqui o,
he main ansmi e o a bo i uses such as dengue, dengue hemo hagic e e , u ban yel-
low e e , and chikungunya [
1
]. Un il la ely, he ala ming g ow h o cases o hese diseases
in di e en egions o he wo ld was es ic ed o eme ging coun ies, now becoming a
global conce n. Cu en ly, he e is only one egis e ed and widely dis ibu ed accine o
yellow e e [
2
]. The way o educe he sp ead o hese i al pa hologies is h ough he
con ol o he ansmi ing mosqui o; he main means is he use o chemical insec icides in
he di e en s ages o he mosqui o’s de elopmen .
A class o hese insec icides is called insec g ow h inhibi o s (IGIs), which a e used
o con ol insec pes s in ag icul u e, o es y, s o ed p oduc s, and public heal h si ua-
ions [
3
]. IGIs ac in he ea ly s ages o de elopmen (la al and pupal) and on wo main
neu oho mones (neu opep ides), he ecdys e oids (mol ing ho mones) and he ju enile
sesqui e penoid ho mones (JHs), which ha e he unc ion o con olling mol ing and
me amo phosis [4].
Howe e , in pos -emb yonic de elopmen , bo h ho mones a e ela ed in adul hood o
he egula ion o ep oduc i e ma u a ion, elease, anspo , exc e ion, and me abolism
as he main sou ce o chi in syn hesis and insec cu icle scle o iza ion [
5
–
7
]. A cu icle
and a ached epide mis a e one o he la ges and mos impo an o gans in he insec
body, he unc ion o which is o p o ec agains physical damage, pa hogen pene a ion,
and desicca ion. Associa ed wi h g ow h ho mones, insec s need o o m new cu icles
and pe iodically elimina e old ones; his in ol es many biochemical eac ions, such as
chi iniza ion [8–11].
Chi in is a linea amino polysaccha ide polyme composed o uni s o
β
-1,4-N-ace yl-
D-glucosamine (GlcNAc). They a e ound in he cu icles o insec s, c us aceans, and ungi
and ha e he cons i u ion o chains o
α
-chi in, conside ed he mos abundan o m o
chi in since i s o m o o ganiza ion is in a e y s able an ipa allel way. This o m o
scle o iza ion, depending on he le els o o ganiza ion o he epide mis, c ea es s uc u al
and a chi ec u al di e ences whe e se e al cu icula p o eins can be c oss-linked wi h
o hodiphenols and hei quinones. In his way, he epide mis, composed o a single laye
o unde lying epide mal cells, is gene a ed and can o igina e no only he di e ences wi hin
a species o insec bu also among insec s [12,13].
Eigh enzyma ic s eps a e necessa y o con e he disaccha ide p ecu so ehalose
o chi in in he inal s ep. The UDP-N-ace yl-glucosamine uni s a e inally con e ed in o
chi in polyme s by he enzyme chi in syn hase, and his ac occu s a he limi o wo
opological spaces, om he cy osol in he lumen o he esicles o ou side he cell [10,13].
The e o e, he e is g ea in e es in insec icides ha ac speci ically in he de elopmen
o insec s since hei use on a la ge scale has p o ided esis ance and also p esen ed ad e se
eac ions in o he o ganisms, especially hose wi h chi in (c us aceans). The e o e, h ough
compu a ional me hods, i is possible o design compounds ha mee he equi emen s o
pes icides, as hey a e speci ically de eloped o mosqui o con ol.
The p esen wo k aims o sea ch o new chemical en i ies wi h po en ial insec icidal
ac i i y wi h he dual inhibi ion o ju enile ho mone p o ein and chi in deace ylases o Aedes
aegyp i, employing molecula modeling app oaches by pha macopho e and docking-based
i ual sc eening. In addi ion, compounds we e de eloped h ough he pha macokine ic
cha ac e is ics (ADME) o Aedes aegyp i physiology and oxicological p ope ies di ec ed a
o he o ganisms, mainly humans. The gene al ou line ha summa izes he me hodological
s eps in his documen is shown in Scheme 1(see mo e de ails in he Ma e ials and Me hods
sec ion) [14].
In . J. Mol. Sci. 2022,23, 8218 3 o 30
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 o 31
Scheme 1. Me hodological scheme. Fi s s age (pha macopho e models) > second s age
(compa a i e modeling) and hi d s age (hie a chical i ual sc eening—model and coupling).
2. Resul s and Discussion
2.1. Pha macopho e Model Gene a ion and E alua ion
Ligand-based app oaches can be use ul in he p io i iza ion o hi compounds wi h
he same s e eo-elec onic ea u es o known ac i e inhibi o s (e.g., pha macopho e
models). Thus, he employmen o po en compounds in pha macopho e model building
is di ec ly ela ed o a eliable 3D ep esen a ion o s e eo-elec onic ea u es and
pha macopho e quali y [15–17].
Fi s , all models p esen ed s ain ene gy o less han wo o de s o magni ude (<100
kcal/mol) and we e all conside ed accep able, complying wi h his condi ion. The non-
dominan analysis p o ided by he Pa e o index, he sco e o which is di e en om ze o,
would de e mine i he e a e dominan pa ame e s ha could a ec he s a is ical quali y
o he models; his also did no occu [18]. Rega ding he s e ic o e lap o he model
(S e ics), pha macopho e ag eemen (H_bond) and ag eemen be ween he cha ac e is ics
o he compound used in he cons uc ion o he pha macopho e model in ela ion o he
pha macopho e equi emen s (Mol_q y) e ealed ha hey we e s a is ically equi alen
(Pa e o = 0), and, he e o e, his me ic was no su icien o selec a use ul pha macopho e
model (see Table 1). In o de o ci cum en his limi a ion, he abili y o di e en ia e
ac i es compounds om alse posi i es (decoys) was employed by he a ea unde he
ROC cu e [15,19].
Table 1. In e nal s a is ical pa ame e s o pha macopho e models p o ided by GALAHAD™.
Model ENERGY
(
k
cal/mol)
STERICS H-BOND MOL_QRY
01 5.55 2662.60 627.30 365.56
02 6.83 2502.50 669.00 378.08
03 6.62 2724.20 646.60 358.13
04 6.88 2888.40 619.20 356.44
05 6.35 2496.40 650.00 362.48
Scheme 1.
Me hodological scheme. Fi s s age (pha macopho e models) > second s age (compa a i e
modeling) and hi d s age (hie a chical i ual sc eening—model and coupling).
2. Resul s and Discussion
2.1. Pha macopho e Model Gene a ion and E alua ion
Ligand-based app oaches can be use ul in he p io i iza ion o hi compounds wi h he
same s e eo-elec onic ea u es o known ac i e inhibi o s (e.g., pha macopho e models).
Thus, he employmen o po en compounds in pha macopho e model building is di ec ly
ela ed o a eliable 3D ep esen a ion o s e eo-elec onic ea u es and pha macopho e
quali y [15–17].
Fi s , all models p esen ed s ain ene gy o less han wo o de s o magni ude (<100
kcal/mol) and we e all conside ed accep able, complying wi h his condi ion. The non-
dominan analysis p o ided by he Pa e o index, he sco e o which is di e en om ze o,
would de e mine i he e a e dominan pa ame e s ha could a ec he s a is ical quali y
o he models; his also did no occu [
18
]. Rega ding he s e ic o e lap o he model
(S e ics), pha macopho e ag eemen (H_bond) and ag eemen be ween he cha ac e is ics
o he compound used in he cons uc ion o he pha macopho e model in ela ion o he
pha macopho e equi emen s (Mol_q y) e ealed ha hey we e s a is ically equi alen
(Pa e o = 0), and, he e o e, his me ic was no su icien o selec a use ul pha macopho e
model (see Table 1). In o de o ci cum en his limi a ion, he abili y o di e en ia e
ac i es compounds om alse posi i es (decoys) was employed by he a ea unde he ROC
cu e [15,19].
A es da ase wi h 7 inhibi o s (chi in syn hase—Ki < 0.001 mg
·
L
−1
and ju enile
ho mone—Ki < 0.001 mg
·
L
−1
) and 350 decoys was employed in ROC cu es buil o
calcula e he espec i e a eas unde he cu e (AUC-ROC) o each pha macopho e model
(QFIT alue; 0–100). An AUC equal o 1.0 would be ound in a model wi h he capaci y
o ecognize all ac i es be o e decoys, and an AUC less han 0.5 would be associa ed wi h
models wo se han a andom selec ion. An AUC g ea e han 0.70 is associa ed wi h a
mode a e p edic i e abili y [
19
]. Acco ding o he AUC calcula ion, all pha macopho e
models had he abili y o di e en ia e ac i e compounds om alse posi i es (AUC < 0.70;
Figu e 1).
In . J. Mol. Sci. 2022,23, 8218 4 o 30
Table 1. In e nal s a is ical pa ame e s o pha macopho e models p o ided by GALAHAD™.
Model ENERGY
(kcal/mol) STERICS H-BOND MOL_QRY
01 5.55 2662.60 627.30 365.56
02 6.83 2502.50 669.00 378.08
03 6.62 2724.20 646.60 358.13
04 6.88 2888.40 619.20 356.44
05 6.35 2496.40 650.00 362.48
06 6.73 2876.90 610.40 350.63
07 7.87 2936.50 641.60 357.15
08 6.31 2604.20 647.90 343.24
09 7.03 2638.60 674.00 335.78
10 6.82 2802.00 611.70 346.41
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 o 31
06 6.73 2876.90 610.40 350.63
07 7.87 2936.50 641.60 357.15
08 6.31 2604.20 647.90 343.24
09 7.03 2638.60 674.00 335.78
10 6.82 2802.00 611.70 346.41
A es da ase wi h 7 inhibi o s (chi in syn hase—Ki < 0.001 mg·L−1 and ju enile ho -
mone—Ki < 0.001 mg·L−1) and 350 decoys was employed in ROC cu es buil o calcula e
he espec i e a eas unde he cu e (AUC-ROC) o each pha macopho e model (QFIT
alue; 0–100). An AUC equal o 1.0 would be ound in a model wi h he capaci y o ecog-
nize all ac i es be o e decoys, and an AUC less han 0.5 would be associa ed wi h models
wo se han a andom selec ion. An AUC g ea e han 0.70 is associa ed wi h a mode a e
p edic i e abili y [19]. Acco ding o he AUC calcula ion, all pha macopho e models had
he abili y o di e en ia e ac i e compounds om alse posi i es (AUC < 0.70; Figu e 1).
Figu e 1. ROC cu e o pha macopho e models. Below he diagonal line, poo models a e ep e-
sen ed o andom selec ion (AUC < 0.5).
Al hough all pha macopho e models had good p edic i e abili y (AUC > 0.7), ROC
cu e analysis is a con inuous me ic, and hus, i is no able o classi y ac i e compounds
in hei ea ly phases [20]. Thus, he Bol zmann-enhanced disc imina ion o he ROC cu e
(BEDROC) was employed o p obe he pha macopho e models’ p edic ions o classi y ac-
i e compounds be o e decoys (Table 2).
Table 2. BEDROC alue o pha macopho e models, conside ing α = 16.10.
Models
BEDROC
01 0.36
02 0.30
04 0.41
05 0.23
06 0.60
07 0.49
09 0.52
10 0.44
Figu e 1.
ROC cu e o pha macopho e models. Below he diagonal line, poo models a e ep e-
sen ed o andom selec ion (AUC < 0.5).
Al hough all pha macopho e models had good p edic i e abili y (AUC > 0.7), ROC
cu e analysis is a con inuous me ic, and hus, i is no able o classi y ac i e compounds
in hei ea ly phases [
20
]. Thus, he Bol zmann-enhanced disc imina ion o he ROC cu e
(BEDROC) was employed o p obe he pha macopho e models’ p edic ions o classi y
ac i e compounds be o e decoys (Table 2).
Table 2. BEDROC alue o pha macopho e models, conside ing α= 16.10.
Models BEDROC
01 0.36
02 0.30
04 0.41
05 0.23
06 0.60
07 0.49
09 0.52
10 0.44
Pha macopho e models wi h BEDROC > 0.5 a e conside ed be e han a andom
assay and use ul o he ea ly and o de ed eco e y o ac i e compounds [
21
–
23
]. Al hough
In . J. Mol. Sci. 2022,23, 8218 5 o 30
wo pha macopho e models, models 06 and 09, showed sa is ac o y BEDROC alues,
pha macopho e model 6 was selec ed as i had he highes alue o pha macopho e-based
i ual sc eening.
Pha macopho e model 6, which was used in i ual sc eening, aims o ind molecules
ha ha e in hei s uc u e he s e eo-elec onic equi emen s ha a e simila o he p esen
model: wo hyd ophobic cen e s (HYs), wo hyd ogen bond dono s (HBDs), and wo
hyd ogen bond accep o s (HBAs). Compounds ha ha e simila i ies o he physicochemical
pa ame e s o model 6 we e iden i ied since he p og am used was GALAHAD
™
(T ipos
Inc., S . Louis, MO, USA) o he alignmen o e y lexible compounds and di e en
chemo ypes. The adjus men s we e pe o med using a gene ic lexible algo i hm o align
he compounds, allowing di e en con o ma ions in he ac i e si e and de ined by hei
con o me s om he adjus men s o he dihed al angles ha we e e alua ed h ough a
unc ion o he sco ing [
15
,
24
–
27
]. S uc u e–ac i i y ela ionship s udies o he mos
a o able ecogni ion o mac omolecules should consis o he a oma ic p esence, and he
hyd ophobic cha ac e is ics o hyd ogen accep ance a e an essen ial equi emen o he
ecogni ion o chi in syn hase [
28
,
29
]. Fo ju enile ho mone p o ein, he dono and accep o
hyd ogen bonds a e essen ial pa ame e s o binding o he p o ein [30,31].
In o de o cha ac e ize po en inhibi o s wi h he pha macopho e model, a po en
compound and a weak compound we e supe imposed on pha macopho e model 6. Com-
pounds we e analyzed expe imen ally by Sun e al. [
32
], and he compound wi h a alue o
0.001 mg
·
L
−1
was conside ed po en ; he weak compound was he insec g ow h inhibi o
lucycloxu on, used as a posi i e con ol (0.05 mg·L−1) (Figu e 2).
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 o 31
Pha macopho e models wi h BEDROC > 0.5 a e conside ed be e han a andom as-
say and use ul o he ea ly and o de ed eco e y o ac i e compounds [21–23]. Al hough
wo pha macopho e models, models 06 and 09, showed sa is ac o y BEDROC alues,
pha macopho e model 6 was selec ed as i had he highes alue o pha macopho e-
based i ual sc eening.
Pha macopho e model 6, which was used in i ual sc eening, aims o ind molecules
ha ha e in hei s uc u e he s e eo-elec onic equi emen s ha a e simila o he p e-
sen model: wo hyd ophobic cen e s (HYs), wo hyd ogen bond dono s (HBDs), and wo
hyd ogen bond accep o s (HBAs). Compounds ha ha e simila i ies o he physicochem-
ical pa ame e s o model 6 we e iden i ied since he p og am used was GALAHAD™
(T ipos Inc., S . Louis, MO, USA) o he alignmen o e y lexible compounds and di -
e en chemo ypes. The adjus men s we e pe o med using a gene ic lexible algo i hm o
align he compounds, allowing di e en con o ma ions in he ac i e si e and de ined by
hei con o me s om he adjus men s o he dihed al angles ha we e e alua ed h ough
a unc ion o he sco ing [15,24–27]. S uc u e–ac i i y ela ionship s udies o he mos
a o able ecogni ion o mac omolecules should consis o he a oma ic p esence, and he
hyd ophobic cha ac e is ics o hyd ogen accep ance a e an essen ial equi emen o he
ecogni ion o chi in syn hase [28,29]. Fo ju enile ho mone p o ein, he dono and accep-
o hyd ogen bonds a e essen ial pa ame e s o binding o he p o ein [30,31].
In o de o cha ac e ize po en inhibi o s wi h he pha macopho e model, a po en
compound and a weak compound we e supe imposed on pha macopho e model 6. Com-
pounds we e analyzed expe imen ally by Sun e al. [32], and he compound wi h a alue
o 0.001 mg·L−1 was conside ed po en ; he weak compound was he insec g ow h inhib-
i o lucycloxu on, used as a posi i e con ol (0.05 mg·L−1) (Figu e 2).
(
A
) (
B
)
Figu e 2. (A)—po en inhibi o (0.001 mg·L−1; QFIT = 84.94); (B)— lucycloxu on (0.05 mg·L−1; QFIT
= 58.66), supe posed o model pha macopho e 06. Sphe es ep esen H-bond dono s (g een), H-
bond accep o s (magen a) and hyd ophobic g oups (cyan). The size o sphe es a ies acco ding o
he ole ance adius calcula ed using GALAHADTM. All he dis ances a e measu ed in angs oms.
The objec i e o he sc eening was o ob ain a compound ha has a dual ac ion on
bo h chi in syn hase and ju enile ho mone ecep o s. Pha macopho e model 6 allowed us
o ep oduce he equi emen s o chi in syn hase and he ecogni ion o he ju enile ho -
mone ecep o ; he e o e, i is use ul o p io i ize he po en ial inhibi o s o Aedes aegyp i
insec g ow h egula o s.
2.2. Pha macopho e-Based Vi ual Sc eening
In o al, 214,446 compounds we e p epa ed in mul iple p o ona ion s a es and mul-
iple au ome ic o ms and we e collec ed in s uc u e da a o ma (SDF), which is a aila-
ble in mul iple con o ma ions om he Sigma-Ald ichTM subse o he ZINC [33,34] da-
abase (h ps://zinc15.docking.o g/, accessed on 12 June 2021). The da abase o p e iously
p epa ed compounds will be able o p edic compounds wi h be e p ope ies, such as
lipophilici y and wa e solubili y [35,36]. Fo each molecule in he da abase, up o 300
con o me s we e gene a ed using he MMFF94 o ce ield implemen ed wi h OMEGA
so wa e (Open Eye Scien i ic So wa e, San a Fe, NM, USA, h p://www.eyesopen.com,
accessed on 12 June 2021) [37], and he pa ial a omic cha ges we e calcula ed by means
o he empi ical Gas eige -Hückel me hod. Compounds ha comply wi h he p e ious
Figu e 2.
(
A
)—po en inhibi o (0.001 mg
·
L
−1
; QFIT = 84.94); (
B
)— lucycloxu on (0.05 mg
·
L
−1
; QFIT
= 58.66), supe posed o model pha macopho e 06. Sphe es ep esen H-bond dono s (g een), H-bond
accep o s (magen a) and hyd ophobic g oups (cyan). The size o sphe es a ies acco ding o he
ole ance adius calcula ed using GALAHADTM. All he dis ances a e measu ed in angs oms.
The objec i e o he sc eening was o ob ain a compound ha has a dual ac ion on
bo h chi in syn hase and ju enile ho mone ecep o s. Pha macopho e model 6 allowed
us o ep oduce he equi emen s o chi in syn hase and he ecogni ion o he ju enile
ho mone ecep o ; he e o e, i is use ul o p io i ize he po en ial inhibi o s o Aedes aegyp i
insec g ow h egula o s.
2.2. Pha macopho e-Based Vi ual Sc eening
In o al, 214,446 compounds we e p epa ed in mul iple p o ona ion s a es and mul iple
au ome ic o ms and we e collec ed in s uc u e da a o ma (SDF), which is a ailable in
mul iple con o ma ions om he Sigma-Ald ichTM subse o he ZINC [
33
,
34
] da abase
(h ps://zinc15.docking.o g/, accessed on 12 June 2021). The da abase o p e iously
p epa ed compounds will be able o p edic compounds wi h be e p ope ies, such as
lipophilici y and wa e solubili y [
35
,
36
]. Fo each molecule in he da abase, up o 300 con-
o me s we e gene a ed using he MMFF94 o ce ield implemen ed wi h OMEGA so wa e
(Open Eye Scien i ic So wa e, San a Fe, NM, USA, h p://www.eyesopen.com, accessed on
12 June 2021) [
37
], and he pa ial a omic cha ges we e calcula ed by means o he empi ical
Gas eige -Hückel me hod. Compounds ha comply wi h he p e ious condi ions a e be e
sui ed o molecula docking [
38
]. Then, 99,623 compounds o e lapped pha macopeia
model 06 wi h pa ial s e eo equi emen s (0.47 < QFIT > 98.12), wi h he QFIT sco e be-

In . J. Mol. Sci. 2022,23, 8218 6 o 30
ing conside ed as a pa ame e . A sco e o 100 demons a es ha a be e i be ween he
pha macopho e model and he da abase has occu ed; when his sco e is ze o, no o e lap
has occu ed [
39
]. As a pha macopho e-based i ual sc eening esul , 4793 compounds
(QFIT > 58.94) we e p io i ized wi h QFIT be ween 58.95 and 98.12 [
23
,
39
,
40
]. Howe e ,
his app oach is no use ul o e alua ing he spa ial equi emen s o he binding si e. Thus,
molecula docking was applied o he il e ed compounds om he pha macopho e-based
i ual sc eening.
2.3. Cons uc ion o he Bombyx-Aedes 3D Model and Molecula Docking
The mac omolecule deposi ed in he PDB-encoded 5ZNT is a chi in deace ylase 1 p o-
ein. Al hough he CDA1 gene o he Bombyx mo i o ganism, wi hou a mu a ion, has no
c ys allized ligand, Liu [
41
] de e mined he s uc u al and biochemical cha ac e is ics o
he ca aly ic mechanism expe imen ally since he enzyme is classi ied as a hyd olase; i s
ca aly ic ac i i y has been eco ded (EC: 3.5.1.41): [(1
→
4)-N-ace yl-
β
-D-glucosaminyl] (n)
+nH2O = n ace a e + chi osan [42].
The p ima y s uc u e o he Aedes aegyp i species was sea ched wi h he desc ip i es
(Aedes aegyp i + chi in deace ylase 1 p o ein) a h ps://www.ncbi.nlm.nih.go /p o ein,
accessed on 27 June 2021, and we ound ha he p o ein AAEL003419-PA wi h 535 amino
acids was used o he alignmen h ough BLAST. F om simila sequences ound, he
mos compa ible o ganisms (wi h he highes pe cen age o iden i ica ion, abo e 80%, and
consul a ion co e age abo e 70%) we e selec ed [43].
In he ollowing s eps, buil in he MODELLER 10.1 p og am, he compa ison, align-
men , and cons uc ion o he Bombyx-Aedes model and i s alida ion we e pe o med.
The model gene a ed by he p og am was aligned in he Pymol 2.0.7 p og am (Wa en
Ly o d DeLano, The PyMOL Molecula G aphics Sys em, Sch ödinge , LLC, New Yo k,
NY, USA) as shown in Figu e 3, in which he RMSD alignmen o e lap was 0.396, om
which he choice o he bes a omic model wi h a lowe sco e is conside ed adequa e in i s
s uc u al composi ion and eliabili y acco ding o Rahman e al. [44].
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 o 31
Figu e 3. O e lap be ween alpha-ca bons o mac omolecule 5ZNT (g een) and he Bombyx-Aedes
model ( ed); RMSD alue 0.396.
Figu e 4. Ramachand an, he Bombyx-Aedes Model e ain. The mos a o able egions a e shown
in da k g een, neu al in ligh g een, and non- a o able in whi e; each amino acid esidue is ma ked
wi h di e en colo s, gene a ed by he s uc u e assessmen p og am (h ps://swiss-
model.expasy.o g/assess/KGJV X/01, accessed on 27 June 2021).
2.3.1. Selec ion, Valida ion, and Vi ual Sc eening o Po en ial Dual Inhibi o s
The molecula adjus men was pe o med using he Au oDock Vina p og am, and
all pa ame e alues we e adjus ed h oughou he p ocess o de ine ecep o s, exhaus -
i eness, cen e _xyz, and size_xyz. To gene a e he esul in binding a ini y alues, he
Au oDock Vina p og am o esees he connec ion o he lowes ene gy s a e a he ime o
he o ma ion o he p o ein complex ( ecep o ) wi h he ligand, wi h he objec i e o a
co ela ion be ween he alues o he unc ions and he a ini y o he ligand [52–55].
Among he 4793 compounds p io i ized in he i ual pha macopho e-based sc eening o
he Zinc15 da abase, 4142 compounds (G < 0 kcal/mol) we e aligned o he binding si e
o he c ys alline s uc u e o he ju enile ho mone p o ein mac omolecules (code PDB:
5V13) o Aedes aegyp i; i s complexed binding si e has he e e ence ligand JH3 (me hyl
Figu e 3.
O e lap be ween alpha-ca bons o mac omolecule 5ZNT (g een) and he Bombyx-Aedes
model ( ed); RMSD alue 0.396.
The Bombyx-Aedes model p esen ed a pe cen age o mo e han 90% o esidues
in a o able egions, demons a ing a good s e eochemical quali y, as analyzed by he
Ramachand am plo and shown in Figu e 4. The Ramachand an plo o he Bombyx-Aedes
model shows he dis ibu ion o he o sion angles
Φ
and
Ψ
o he main chain o he model’s
buil p o ein [
45
]. The Ramachand am plo is di ided in o di e en zones: (a) hose whe e
he colo in ensi y is leas in ense because o he ac ha he e a e no in e a omic clashes;
(b) hose whe e he e a e mode a e clashes, and (c) hose whe e he clashes a e ex emely
In . J. Mol. Sci. 2022,23, 8218 7 o 30
se e e, wi h he mos in ense colo a ion [
46
]. A Global Model Quali y Es ima e GMQE
alue o 0.5 was ound, indica ing a model wi h highe eliabili y.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 o 31
Figu e 3. O e lap be ween alpha-ca bons o mac omolecule 5ZNT (g een) and he Bombyx-Aedes
model ( ed); RMSD alue 0.396.
Figu e 4. Ramachand an, he Bombyx-Aedes Model e ain. The mos a o able egions a e shown
in da k g een, neu al in ligh g een, and non- a o able in whi e; each amino acid esidue is ma ked
wi h di e en colo s, gene a ed by he s uc u e assessmen p og am (h ps://swiss-
model.expasy.o g/assess/KGJV X/01, accessed on 27 June 2021).
2.3.1. Selec ion, Valida ion, and Vi ual Sc eening o Po en ial Dual Inhibi o s
The molecula adjus men was pe o med using he Au oDock Vina p og am, and
all pa ame e alues we e adjus ed h oughou he p ocess o de ine ecep o s, exhaus -
i eness, cen e _xyz, and size_xyz. To gene a e he esul in binding a ini y alues, he
Au oDock Vina p og am o esees he connec ion o he lowes ene gy s a e a he ime o
he o ma ion o he p o ein complex ( ecep o ) wi h he ligand, wi h he objec i e o a
co ela ion be ween he alues o he unc ions and he a ini y o he ligand [52–55].
Among he 4793 compounds p io i ized in he i ual pha macopho e-based sc eening o
he Zinc15 da abase, 4142 compounds (G < 0 kcal/mol) we e aligned o he binding si e
o he c ys alline s uc u e o he ju enile ho mone p o ein mac omolecules (code PDB:
5V13) o Aedes aegyp i; i s complexed binding si e has he e e ence ligand JH3 (me hyl
Figu e 4.
Ramachand an, he Bombyx-Aedes Model e ain. The mos a o able egions a e shown
in da k g een, neu al in ligh g een, and non- a o able in whi e; each amino acid esidue is ma ked
wi h di e en colo s, gene a ed by he s uc u e assessmen p og am (h ps://swissmodel.expasy.
o g/assess/KGJV X/01, accessed on 27 June 2021).
Ca bohyd a e es e ases o amily 4 ( amily CE-4) a e me alloenzymes; p edominan ly,
hey use Co
2+
and Zn
2+
ions as di alen me al co ac o s
in i o
. These enzymes a e
cons i u ed by a s uc u e o med by a iad o zinc and His–His–Asp bonds, ac ing as a
ca aly ic domain and an acid and base. I has wo oxygena ed ace a es ha coo dina e zinc
in a dis o ed oc ahed al shape [
42
,
47
–
49
]. Acco ding o Dong [
50
], a ecombinan o m o
pep idoglycan GlcNAc-N-ace ylase om S. pneumoniae (PgdA
Sm
) binds o a zinc ion wi hin
i s ac i e si e and
in i o
and demons a es ha he p o ein PgdA
Sm
can be ully ac i a ed
by he p esence o he zinc ion. Since any modi ica ions o he CE4 mo i s can p o ide a
means o change he speci ici y o he subs a e, a p e e ence o he me al o i s eac ion
mechanism ep esen s a s ep owa ds he iden i ica ion o mechanism-based inhibi o s o
his impo an class o enzymes [51].
2.3.1. Selec ion, Valida ion, and Vi ual Sc eening o Po en ial Dual Inhibi o s
The molecula adjus men was pe o med using he Au oDock Vina p og am, and
all pa ame e alues we e adjus ed h oughou he p ocess o de ine ecep o s, exhaus-
i eness, cen e _xyz, and size_xyz. To gene a e he esul in binding a ini y alues, he
Au oDock Vina p og am o esees he connec ion o he lowes ene gy s a e a he ime
o he o ma ion o he p o ein complex ( ecep o ) wi h he ligand, wi h he objec i e o
a co ela ion be ween he alues o he unc ions and he a ini y o he ligand [
52
–
55
].
Among he 4793 compounds p io i ized in he i ual pha macopho e-based sc eening o
he Zinc15 da abase, 4142 compounds (
∆
G < 0 kcal/mol) we e aligned o he binding si e
o he c ys alline s uc u e o he ju enile ho mone p o ein mac omolecules (code PDB:
5V13) o Aedes aegyp i; i s complexed binding si e has he e e ence ligand JH3 (me hyl
(2E,6E)-9-[(2R)-3,3-dime hyloxi an-2-yl]-3,7-dime hylnona-2,6-dienoa e). The e o e, 2240
(LE <
−
0.40 kcal/mol) compounds we e chosen since he e iciency o he ligand selec s a
mo e conco dan compound o he choice o a p incipal compound [56–58].
In . J. Mol. Sci. 2022,23, 8218 8 o 30
Molecula docking was pe o med using a alida ion p o ocol o de e mine he pose
(con o ma ion + o ien a ion) o he ligands (
JH3
) wi hin he enzyme ac i e si es (PDB ID
5V13). The oo mean squa e de ia ion (RMSD) be ween he e e ence ligand and he
expe imen al
JH3
ligand (1.19 Å) was calcula ed. The RMSD analysis was de e mined
by conside ing he mos app op ia e posi ion o he ini ial s uc u e a ound he X- ay
c ys allog aphic complex h ough an expe imen o he mos s able posi ion so ha he e is
no s uc u al change be ween he p o ein and he e e ence ligand when complexed wi h
he mac omolecule unde s udy. The bes molecula i is de e mined by an RMSD less
han o equal o 1.5 [59–63], as shown in Figu e 5.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 o 31
(2E,6E)-9-[(2R)-3,3-dime hyloxi an-2-yl]-3,7-dime hylnona-2,6-dienoa e). The e o e, 2240
(LE <−0.40 kcal/mol) compounds we e chosen since he e iciency o he ligand selec s a
mo e conco dan compound o he choice o a p incipal compound [56–58].
Molecula docking was pe o med using a alida ion p o ocol o de e mine he pose
(con o ma ion + o ien a ion) o he ligands (JH3) wi hin he enzyme ac i e si es (PDB ID
5V13). The oo mean squa e de ia ion (RMSD) be ween he e e ence ligand and he ex-
pe imen al JH3 ligand (1.19 Å) was calcula ed. The RMSD analysis was de e mined by
conside ing he mos app op ia e posi ion o he ini ial s uc u e a ound he X- ay c ys-
allog aphic complex h ough an expe imen o he mos s able posi ion so ha he e is no
s uc u al change be ween he p o ein and he e e ence ligand when complexed wi h he
mac omolecule unde s udy. The bes molecula i is de e mined by an RMSD less han
o equal o 1.5 [59–63], as shown in Figu e 5.
Figu e 5. O e lays o c ys allog aphic ligand poses in JH3 (in ed) wi h he calcula ed pose (in blue).
Kenny e al. [64] s a ed ha i does no seem possible o quan i y he absolu e binding
e iciency o compounds objec i ely. The e iciency can s ill be de ined in a ela i e way
by scaling he a ini y di e ences by he co esponding molecula size di e ences, and
he di e ences we e p io i ized o molecula docking o he chi in deace ylase model A.
aegyp i.
Molecula docking o he binding si e o he c ys alline s uc u e o he mac omole-
cules o he ju enile ho mone p o ein (PDB code: 5V13) was ca ied ou on all compounds,
and, a e il e ing he ligand e icien ly, 2240 compounds wen on o he nex s age o
molecula docking wi h he model Bombyx-Aedes enzyme insec chi in deace ylase buil
om he Aedes aegyp i mosqui o.
2.3.2. In e ac ion Analysis o Inhibi o s and Ju enile Ho mone (PDB 5V13)
The ju enile ho mone p o ein p esen s in i s binding si e he main in e ac ion wi h
he epoxy g oup and o ms a hyd ogen bond wi h he phenolic hyd oxy g oup o Ty 129;
he es o he isop enoid chain is su ounded by hyd ophobic side chains, including hose
o Phe144, Ty 64, T p53, Val65, Val68, Leu72, Leu74, Val51, and Ty 33 [37,65].
When we analyzed ou ligands, impo an in e ac ions we e ca ied ou wi h he
main amino acid esidues o he binding si e o he Aedes aegyp i ju enile ho mone mac o-
molecule (COD: 5V13). All selec ed ligands (M01–M05) and he e e ence ligand com-
plexed wi h he mac omolecule (JH3) in e ac ed wi h esidues Ty 64 (excep M03), T p53,
T p50 (excep M04), T y33 (excep M03 and M05), and Val51 (excep M03 and M04). M03
was he only one ha in e ac ed wi h he amino acid esidues Val65 and Val68, as shown
in Figu e 6.
Figu e 5.
O e lays o c ys allog aphic ligand poses in
JH3
(in ed) wi h he calcula ed pose (in blue).
Kenny e al. [
64
] s a ed ha i does no seem possible o quan i y he absolu e binding
e iciency o compounds objec i ely. The e iciency can s ill be de ined in a ela i e way
by scaling he a ini y di e ences by he co esponding molecula size di e ences, and
he di e ences we e p io i ized o molecula docking o he chi in deace ylase model
A. aegyp i.
Molecula docking o he binding si e o he c ys alline s uc u e o he mac omolecules
o he ju enile ho mone p o ein (PDB code: 5V13) was ca ied ou on all compounds, and,
a e il e ing he ligand e icien ly, 2240 compounds wen on o he nex s age o molecula
docking wi h he model Bombyx-Aedes enzyme insec chi in deace ylase buil om he
Aedes aegyp i mosqui o.
2.3.2. In e ac ion Analysis o Inhibi o s and Ju enile Ho mone (PDB 5V13)
The ju enile ho mone p o ein p esen s in i s binding si e he main in e ac ion wi h he
epoxy g oup and o ms a hyd ogen bond wi h he phenolic hyd oxy g oup o Ty 129; he
es o he isop enoid chain is su ounded by hyd ophobic side chains, including hose o
Phe144, Ty 64, T p53, Val65, Val68, Leu72, Leu74, Val51, and Ty 33 [37,65].
When we analyzed ou ligands, impo an in e ac ions we e ca ied ou wi h he main
amino acid esidues o he binding si e o he Aedes aegyp i ju enile ho mone mac omolecule
(COD: 5V13). All selec ed ligands (
M01
–
M05
) and he e e ence ligand complexed wi h
he mac omolecule (
JH3
) in e ac ed wi h esidues Ty 64 (excep
M03
), T p53, T p50 (excep
M04
), T y33 (excep
M03
and
M05
), and Val51 (excep
M03
and
M04
).
M03
was he only
one ha in e ac ed wi h he amino acid esidues Val65 and Val68, as shown in Figu e 6.
In . J. Mol. Sci. 2022,23, 8218 9 o 30
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 o 31
Figu e 6. In e ac ion o selec ed molecules, in yellow, wi h he amino acid esidues, in g een, o he
mac omolecule 5V13 (ju enile ho mone), in g een. (A) M01 (ZINC13514543); (B) M02
(ZINC14413017); (C) M03 (ZINC05680928); (D) M04 (ZINC04691148); (E) M05 (ZINC95851150); (F)
JH3.
2.3.3. In e ac ion Analysis o Inhibi o s and Enzyme Model Insec Chi in Deace ylase om
Aedes aegyp i
The selec ion o compounds ook place in a double inhibi ion o mac omolecules,
whe e he pa ame e o which hey we e associa ed pe o med mo e sa is ac o ily in il-
e ing he choice o he mos app op ia e ligand ha would, in pa icula , unc ion as bo h
an inhibi o o he ju enile ho mone and a g ow h egula o o he Aedes aegyp i mosqui o.
In iew o his app oach, 2240 (LE < −0.40 kcal/mol) compounds we e selec ed in he i s
s age wi h he ju enile ho mone mac omolecule. Fo he nex s age, he e was, again, a
selec ion o he molecula docking based on he insec model Aedes aegyp i chi in deace y-
lase, cons uc ed om he enzyme insec chi in deace ylase deposi ed in he PDB (PDB
code: 5ZNT); 1959 compounds (G < 0 kcal/mol) we e aligned and 20 compounds (LE <
−0.4 kcal/mol) we e p io i ized o pha macokine ic and oxicological p edic ion.
When we analyzed he in e ac ions desc ibed by Liu [41], he 5ZNT p o ein, wi h he
p esence o he amino acid esidues obse ed expe imen ally (Asp205, His 261, and
A g306; Figu e 7A), and he in e ac ions in he Bombyx-Aedes model (Figu e 7B; Asp201
and 202, His257 and 262 and A g302) main ained he same egions a ound zinc. This
demons a es he conse a ion o impo an amino acids o ca ying ou ca aly ic ac i i y
and, in Figu e 7C, he o e lap o he 5ZNT p o ein wi h he Bombyx-Aedes model, wi h
he p esence o amino acid esidues close o he me al (Asp206, His261, and 265). Figu e
7D shows he o e lapping o he Bombyx-Aedes model p o ein wi h he 5ZNT p o ein,
wi h he p esence o amino acid esidues close o he me al (His257 and 261).
Figu e 6.
In e ac ion o selec ed molecules, in yellow, wi h he amino acid esidues, in g een,
o he mac omolecule 5V13 (ju enile ho mone), in g een. (
A
)
M01
(ZINC13514543); (
B
)
M02
(ZINC14413017); (
C
)
M03
(ZINC05680928); (
D
)
M04
(ZINC04691148); (
E
)
M05
(ZINC95851150);
(F)JH3.
2.3.3. In e ac ion Analysis o Inhibi o s and Enzyme Model Insec Chi in Deace ylase om
Aedes aegyp i
The selec ion o compounds ook place in a double inhibi ion o mac omolecules,
whe e he pa ame e o which hey we e associa ed pe o med mo e sa is ac o ily in
il e ing he choice o he mos app op ia e ligand ha would, in pa icula , unc ion as
bo h an inhibi o o he ju enile ho mone and a g ow h egula o o he Aedes aegyp i
mosqui o. In iew o his app oach, 2240 (LE <
−
0.40 kcal/mol) compounds we e selec ed
in he i s s age wi h he ju enile ho mone mac omolecule. Fo he nex s age, he e was,
again, a selec ion o he molecula docking based on he insec model Aedes aegyp i chi in
deace ylase, cons uc ed om he enzyme insec chi in deace ylase deposi ed in he PDB
(PDB code: 5ZNT); 1959 compounds (
∆
G < 0 kcal/mol) we e aligned and 20 compounds
(LE < −0.4 kcal/mol) we e p io i ized o pha macokine ic and oxicological p edic ion.
When we analyzed he in e ac ions desc ibed by Liu [
41
], he 5ZNT p o ein, wi h
he p esence o he amino acid esidues obse ed expe imen ally (Asp205, His 261, and
A g306; Figu e 7A), and he in e ac ions in he Bombyx-Aedes model (Figu e 7B; Asp201
and 202, His257 and 262 and A g302) main ained he same egions a ound zinc. This
demons a es he conse a ion o impo an amino acids o ca ying ou ca aly ic ac i i y
and, in Figu e 7C, he o e lap o he 5ZNT p o ein wi h he Bombyx-Aedes model, wi h he
p esence o amino acid esidues close o he me al (Asp206, His261, and 265). Figu e 7D
shows he o e lapping o he Bombyx-Aedes model p o ein wi h he 5ZNT p o ein, wi h
he p esence o amino acid esidues close o he me al (His257 and 261).
In addi ion, he chi in deace ylase enzyme ca alyzes he emo al o ace yl g oups om
chi in and modi ies his polyme du ing i s syn hesis and eo ganiza ion. The enzyme
has a s uc u e o wo egions: a subdomain o NodB homology conse ed in CE4 and
C- e minal loops. The homology domain o NodB is a ba el (
β
/
α
)
7
( esidues 162–471)
composed o se en pa allel
β
s ips a anged in a ba el su ounded by six
α
helices. The
ac i e si e and he subs a e connec ion gap a e loca ed h ough he uppe cen e o he
ba el (
β
/
α
)
7
, which con ains a me al bond iad conse ed h oughou he CE4 amily,
In . J. Mol. Sci. 2022,23, 8218 16 o 30
addi ion o inju y o he li e and endoc ine, whose ac ions a e in some pa hway signaling
ha is simila o o eheads
in i o
. As o he DFB and TEF posi i e con ols, he ale s
o inju ies we e in he li e , ne ous sys em, immune sys em, and kidney, and hei
ac ions we e in modula o s; all con idence le els we e highe han 0.981. On he o he
hand, only he
M04
compound was on ale o skin inju y, oge he wi h ale s in he
li e o he ac ion o blocking he (OATP1) B1 anspo e , which is he same o he
M02
and
M03
compounds. The o ganic anion anspo e polypep ide (OATP) 1B is a
de oxi ying limi ing p o ein ha media es he up ake and elimina ion o many endogenous
and xenobio ic compounds (d ugs o insec icides) h ough he sinusoidal memb ane o
li e cells
[111–115]
;
M02
,
M03
, and
M04
a e possibly blocking his anspo e , making
hem molecula s uc u es o be elimina ed by li ing beings.
M01
and
M05
did no show
any ac ions o inju ies (Table 4).
The possible oxici y o insec g ow h inhibi o s speci ic o se e al species has been
analyzed in he li e a u e. I can be ound in he Scien i ic Opinion o he Panel on Plan
P o ec ion P oduc s and Thei Residues (PPR Panel), a he eques o he PRASA Uni o
he Eu opean Food Sa e y Au ho i y (EFSA), on he geno oxic and ca cinogenic po en ial
o bup o ezin in he con ex o human isk assessmen . I was e alua ed in a wo-yea
s udy ha , in mice and a s, nei he he na u e no he incidence o umo s was a ec ed
by i s adminis a ion [
116
]. Howe e , wi h e lubenzu on, he main a ge o gan o which
is he li e , a e sho o long- e m exposu e in species o a s, mice, o dogs, hepa o-
oxici y was obse ed in an 18-mon h oncogenici y s udy in mice. Benign hepa ocellula
adenomas, obse ed only in his s udy wi h mice, we e no conside ed ele an o human
exposu e [117].
In one s udy, BUP and o ganophospha es we e es ed o ch omosomal abe a ions in
soma ic and ge m cells in mice, induced by high doses. The g ow h inhibi o was a ela i ely
sa e compound han con en ional o ganophospha e insec icides [
118
]. I is undoub edly
common ha he oxici y o BUPs is ela ed o hei me aboli es because hey a e deg aded
by hyd olysis and pho odeg ada ion, gene a ing me aboli es 2,6-di luo obenzamide, 4-
chlo ophenylu ea, 4-chlo oace anilide, 4-chlo oaniline, and N-me hyl-4-chlo oaniline, o
which he las h ee a e classi ied as mu agenic [
119
,
120
]. I was obse ed in blue-g een
algae (Plec onema bo yanum) ha when he phenylu ea de i a i es we e exposed and
became highly esis an , he main me aboli e was p-chlo ophenylu ea [
1
,
119
,
121
]. None
o ou
M01
–
M05
compounds (Figu e 9) we e planned wi hou he chlo ine a om, and,
he e o e, hey ha e p omising insec icide po en ial wi hou he mu agenic e ec .
The compound ha showed excellen esul s o he s anda ds es ablished by he
p og am when analyzing Table 4was compound M05, which did no p esen any oxici y.
Rega ding hepa oxici y [
122
,
123
] and cy o oxici y, all posi i e con ols we e ale ed as
ac i e, while
M02
and
M03
compounds we e ale ed as a anspo a ion block in he li e ;
oge he wi h
M04
(ale ed o skin sensi i i y), hese compounds could gi e ise o a ew
ad e se eac ions. Te lubenzu on (TEF) is used in he delousing o salmon a ms; analyses
in non- a ge species such as lobs e (Homa us gamma us) and sh imp (Palaemon elegans)
ha e shown exoskele al abno mali ies (de o mi ies o he claws o igid legs); de o mi ies
o ju enile lobs e s ha e been obse ed in se e al o gans [124–126].
2.5. S uc u e–Ac i i y Rela ionship o he P omising Molecule
Insec g ow h inhibi o s possess he basic chemical s uc u e o benzoylphenylu ean
compounds, which consis o h ee egions o SAR analysis: he benzoyl ing (A), he
u ea b idge (B), and he aniline (C), as shown in Figu e 10. The bes -known insec icide is
di lunzu on, which has a s uc u e composed o benzoyl u ea, simila o sul onylu eas used
as o al an idiabe ics. Vila-Ca iles e al. [
127
] ound ha he subs i u ion o a oma ic o
lipophilic he e ocyclic g oups inc eased he po ency o compounds such as glibenclamide
by 100–1000 imes and ha he inc eased a ini y was he esul o in e ac ions wi h wo
o e lapping binding si es ha we e analogous o BPU. This can lead o new insec icides

In . J. Mol. Sci. 2022,23, 8218 17 o 30
h ough he sc eening o bioassays, and in his s udy, he compound LY 131215, which does
no con ain a u ea b idge, had he bes esul conce ning insec icidal ac i i y [128].
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 17 o 31
exposed and became highly esis an , he main me aboli e was p-chlo ophenylu ea
[1,119,121]. None o ou M01–M05 compounds (Figu e 9) we e planned wi hou he chlo-
ine a om, and, he e o e, hey ha e p omising insec icide po en ial wi hou he mu a-
genic e ec .
The compound ha showed excellen esul s o he s anda ds es ablished by he
p og am when analyzing Table 4 was compound M05, which did no p esen any oxici y.
Rega ding hepa oxici y [122,123] and cy o oxici y, all posi i e con ols we e ale ed as ac-
i e, while M02 and M03 compounds we e ale ed as a anspo a ion block in he li e ;
oge he wi h M04 (ale ed o skin sensi i i y), hese compounds could gi e ise o a ew
ad e se eac ions. Te lubenzu on (TEF) is used in he delousing o salmon a ms; analyses
in non- a ge species such as lobs e (Homa us gamma us) and sh imp (Palaemon elegans)
ha e shown exoskele al abno mali ies (de o mi ies o he claws o igid legs); de o mi ies
o ju enile lobs e s ha e been obse ed in se e al o gans [124–126].
2.5. S uc u e–Ac i i y Rela ionship o he P omising Molecule
Insec g ow h inhibi o s possess he basic chemical s uc u e o benzoylphenylu ean
compounds, which consis o h ee egions o SAR analysis: he benzoyl ing (A), he u ea
b idge (B), and he aniline (C), as shown in Figu e 10. The bes -known insec icide is
di lunzu on, which has a s uc u e composed o benzoyl u ea, simila o sul onylu eas
used as o al an idiabe ics. Vila-Ca iles e al. [127] ound ha he subs i u ion o a oma ic
o lipophilic he e ocyclic g oups inc eased he po ency o compounds such as
glibenclamide by 100–1000 imes and ha he inc eased a ini y was he esul o in e ac-
ions wi h wo o e lapping binding si es ha we e analogous o BPU. This can lead o
new insec icides h ough he sc eening o bioassays, and in his s udy, he compound LY
131215, which does no con ain a u ea b idge, had he bes esul conce ning insec icidal
ac i i y [128].
Figu e 10. The basic chemical s uc u e o benzoylphenylu ean compounds consis s o h ee egions
o SAR analysis: he benzoyl ing (A), he u ea b idge (B), and he aniline (C).
A e he high-pe o mance pha macopho ic sc eening o he s udied compounds,
only M01 p esen ed u ea in i s molecula s uc u e. M01 and i s de i a i es ha e 19 s ud-
ies and pa en s ha a e mainly ela ed o an ibac e ial es s, ni ic oxide, DNA inhibi ion,
neu oin lamma o y ea men and au oimmune diseases, ibonucleo ide educ ase, y o-
sinase inhibi o s, and sickle cell anemia ea men s [116,117,129–134].
Ano he widely s udied compound, M05, has 26 s udies ela ed mainly o his one
deace ylase, u ease, me allo-β-lac amase inhibi o FEZ-1, an i umo ac i i y, DNA syn-
hesis, hyd oxamic β-glucosyl-phenolic acid inhibi o , ibonucleo ide educ ase, 5-lipox-
ygenase inhibi o s, and ac i i y agains ypanosome [59,135–143]. On he o he hand, he
M02 compound has only one s udy analyzing an ibac e ial ac i i y [144]; in ela ion o
M03 and M04 compounds, he e a e no biological s udies; none o ou compound cells
we e es ed agains insec -g ow h-inhibi ing ac i i y o ans o m hem in o a new insec-
icide a e addi ional es s in i o and in i o.
2.6. P edic ion o Syn he ic Accessibili y (SA) and Theo e ical Syn he ic Rou es
The selec ed compounds M01–M05, a e hie a chical i ual sc eening, we e sub-
mi ed o e alua ion o syn he ic accessibili y (Table 5); all i e compounds we e easily
Figu e 10.
The basic chemical s uc u e o benzoylphenylu ean compounds consis s o h ee egions
o SAR analysis: he benzoyl ing (A), he u ea b idge (B), and he aniline (C).
A e he high-pe o mance pha macopho ic sc eening o he s udied compounds,
only
M01
p esen ed u ea in i s molecula s uc u e.
M01
and i s de i a i es ha e 19 s udies
and pa en s ha a e mainly ela ed o an ibac e ial es s, ni ic oxide, DNA inhibi ion, neu-
oin lamma o y ea men and au oimmune diseases, ibonucleo ide educ ase, y osinase
inhibi o s, and sickle cell anemia ea men s [116,117,129–134].
Ano he widely s udied compound,
M05
, has 26 s udies ela ed mainly o his one
deace ylase, u ease, me allo-
β
-lac amase inhibi o FEZ-1, an i umo ac i i y, DNA syn he-
sis, hyd oxamic
β
-glucosyl-phenolic acid inhibi o , ibonucleo ide educ ase, 5-lipoxygenase
inhibi o s, and ac i i y agains ypanosome [
59
,
135
–
143
]. On he o he hand, he
M02
compound has only one s udy analyzing an ibac e ial ac i i y [
144
]; in ela ion o
M03
and
M04
compounds, he e a e no biological s udies; none o ou compound cells we e
es ed agains insec -g ow h-inhibi ing ac i i y o ans o m hem in o a new insec icide
a e addi ional es s in i o and in i o.
2.6. P edic ion o Syn he ic Accessibili y (SA) and Theo e ical Syn he ic Rou es
The selec ed compounds
M01
–
M05
, a e hie a chical i ual sc eening, we e sub-
mi ed o e alua ion o syn he ic accessibili y (Table 5); all i e compounds we e easily
syn hesized, wi h sco es abo e 89 [
145
–
147
]. Acco ding o classi ica ion by sco e, he alue
o 100 is he maximum syn he ic accessibili y; ha is, he molecule is mo e easily syn he-
sized i i has a highe sco e (see Table 5). In his s udy, we p opose he heo e ical syn he ic
ou e o p omising compounds, as can be seen in Figu es 10–16.
Table 5. P edic ion o syn he ic accessibili y (SA).
COD COMPOUNDS SA
M01 ZINC13514543 96.096
M02 ZINC14413017 92.731
M03 ZINC05680928 89.776
M04 ZINC04691148 92.951
M05 ZINC95851150 91.439
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 18 o 31
syn hesized, wi h sco es abo e 89 [145–147]. Acco ding o classi ica ion by sco e, he alue
o 100 is he maximum syn he ic accessibili y; ha is, he molecule is mo e easily syn he-
sized i i has a highe sco e (see Table 5). In his s udy, we p opose he heo e ical syn-
he ic ou e o p omising compounds, as can be seen in Figu es 10–16.
Table 5. P edic ion o syn he ic accessibili y (SA).
COD COMPOUNDS SA
M01
ZINC13514543 96.096
M02
ZINC14413017 92.731
M03
ZINC05680928 89.776
M04 ZINC04691148 92.951
M05
ZINC95851150 91.439
2.6.1. Theo e ical Syn he ic Rou e (Compound M01)
Compound M01 can be p epa ed acco ding o he esea ch ca ied ou by B. Bue el-
man e al. [148]. Con e sion o bu y aldehyde I wi h hyd oxylamine hyd ochlo ide
(NH2OH·HCl) in E OH and H2O as sol en s and using NaOH as a base will allow us o
ob ain he inal bu y aldehyde oxime—M01 (Figu e 11).
Figu e 11. Syn he ic ou e o he p epa a ion o M01. DCM (dichlo ome hane). S a ing ma e ial I
is comme cially a ailable.
2.6.2. Theo e ical Syn he ic Rou e (Compound M02)
We p opose a heo e ical syn he ic ou e o M02 based on he p epa a ion o oxime
de i a i e V as an in e media e, wi h IV and NH2OH·HCl as ini ial eac an s and NaOAc
and E OH/H2O as sol en s unde e lux condi ions [149]. By a educ ion eac ion wi h
sodium cyanobo ohyd ide (NaBH3CN) o in e media e V, inal hyd oxylamine M02 will
be ob ained [150] (Figu e 12).
Figu e 12. Theo e ical syn he ic ou e o he p epa a ion o M02. S a ing ma e ial IV is comme -
cially a ailable.
Fu he mo e, we designed a seconda y s a egy based on he p e ious wo k o C.
Be ini e al. [151]. Fi s ly, ni one VII could be u nished by condensa ion be ween hy-
d oxylamine VI and ace aldehyde wi h magnesium sul a e in DCM. E en ually, he nu-
cleophilic addi ion o py ole o in e media e VII using HCl in MeOH would o igina e
he a ge compound (Figu e 13).
M02
M02
Figu e 11.
Syn he ic ou e o he p epa a ion o
M01
. DCM (dichlo ome hane). S a ing ma e ial
I
is
comme cially a ailable.
In . J. Mol. Sci. 2022,23, 8218 18 o 30
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 18 o 31
syn hesized, wi h sco es abo e 89 [145–147]. Acco ding o classi ica ion by sco e, he alue
o 100 is he maximum syn he ic accessibili y; ha is, he molecule is mo e easily syn he-
sized i i has a highe sco e (see Table 5). In his s udy, we p opose he heo e ical syn-
he ic ou e o p omising compounds, as can be seen in Figu es 10–16.
Table 5. P edic ion o syn he ic accessibili y (SA).
COD COMPOUNDS SA
M01
ZINC13514543 96.096
M02
ZINC14413017 92.731
M03
ZINC05680928 89.776
M04 ZINC04691148 92.951
M05
ZINC95851150 91.439
2.6.1. Theo e ical Syn he ic Rou e (Compound M01)
Compound M01 can be p epa ed acco ding o he esea ch ca ied ou by B. Bue el-
man e al. [148]. Con e sion o bu y aldehyde I wi h hyd oxylamine hyd ochlo ide
(NH2OH·HCl) in E OH and H2O as sol en s and using NaOH as a base will allow us o
ob ain he inal bu y aldehyde oxime—M01 (Figu e 11).
Figu e 11. Syn he ic ou e o he p epa a ion o M01. DCM (dichlo ome hane). S a ing ma e ial I
is comme cially a ailable.
2.6.2. Theo e ical Syn he ic Rou e (Compound M02)
We p opose a heo e ical syn he ic ou e o M02 based on he p epa a ion o oxime
de i a i e V as an in e media e, wi h IV and NH2OH·HCl as ini ial eac an s and NaOAc
and E OH/H2O as sol en s unde e lux condi ions [149]. By a educ ion eac ion wi h
sodium cyanobo ohyd ide (NaBH3CN) o in e media e V, inal hyd oxylamine M02 will
be ob ained [150] (Figu e 12).
Figu e 12. Theo e ical syn he ic ou e o he p epa a ion o M02. S a ing ma e ial IV is comme -
cially a ailable.
Fu he mo e, we designed a seconda y s a egy based on he p e ious wo k o C.
Be ini e al. [151]. Fi s ly, ni one VII could be u nished by condensa ion be ween hy-
d oxylamine VI and ace aldehyde wi h magnesium sul a e in DCM. E en ually, he nu-
cleophilic addi ion o py ole o in e media e VII using HCl in MeOH would o igina e
he a ge compound (Figu e 13).
M02
M02
Figu e 12.
Theo e ical syn he ic ou e o he p epa a ion o
M02
. S a ing ma e ial
IV
is comme cially
a ailable.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 18 o 31
syn hesized, wi h sco es abo e 89 [145–147]. Acco ding o classi ica ion by sco e, he alue
o 100 is he maximum syn he ic accessibili y; ha is, he molecule is mo e easily syn he-
sized i i has a highe sco e (see Table 5). In his s udy, we p opose he heo e ical syn-
he ic ou e o p omising compounds, as can be seen in Figu es 10–16.
Table 5. P edic ion o syn he ic accessibili y (SA).
COD COMPOUNDS SA
M01
ZINC13514543 96.096
M02
ZINC14413017 92.731
M03
ZINC05680928 89.776
M04 ZINC04691148 92.951
M05
ZINC95851150 91.439
2.6.1. Theo e ical Syn he ic Rou e (Compound M01)
Compound M01 can be p epa ed acco ding o he esea ch ca ied ou by B. Bue el-
man e al. [148]. Con e sion o bu y aldehyde I wi h hyd oxylamine hyd ochlo ide
(NH2OH·HCl) in E OH and H2O as sol en s and using NaOH as a base will allow us o
ob ain he inal bu y aldehyde oxime—M01 (Figu e 11).
Figu e 11. Syn he ic ou e o he p epa a ion o M01. DCM (dichlo ome hane). S a ing ma e ial I
is comme cially a ailable.
2.6.2. Theo e ical Syn he ic Rou e (Compound M02)
We p opose a heo e ical syn he ic ou e o M02 based on he p epa a ion o oxime
de i a i e V as an in e media e, wi h IV and NH2OH·HCl as ini ial eac an s and NaOAc
and E OH/H2O as sol en s unde e lux condi ions [149]. By a educ ion eac ion wi h
sodium cyanobo ohyd ide (NaBH3CN) o in e media e V, inal hyd oxylamine M02 will
be ob ained [150] (Figu e 12).
Figu e 12. Theo e ical syn he ic ou e o he p epa a ion o M02. S a ing ma e ial IV is comme -
cially a ailable.
Fu he mo e, we designed a seconda y s a egy based on he p e ious wo k o C.
Be ini e al. [151]. Fi s ly, ni one VII could be u nished by condensa ion be ween hy-
d oxylamine VI and ace aldehyde wi h magnesium sul a e in DCM. E en ually, he nu-
cleophilic addi ion o py ole o in e media e VII using HCl in MeOH would o igina e
he a ge compound (Figu e 13).
M02
M02
Figu e 13.
Al e na i e syn he ic ou e o he p epa a ion o
M02
. S a ing ma e ial
VI
is comme cially
a ailable.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 19 o 31
Figu e 13. Al e na i e syn he ic ou e o he p epa a ion o M02. S a ing ma e ial VI is comme -
cially a ailable.
2.6.3. Theo e ical Syn he ic Rou e (Compound M03)
Oxime de i a i e M03 can be syn hesized by a condensa ion eac ion (Figu e 14) be-
ween aldehyde VIII and hyd oxylamine hyd ochlo ide in a 30% MeOH aqueous solu ion
[152].
Figu e 14. T adi ional me hod o he p epa a ion o compound M03. S a ing ma e ial VIII is com-
me cially a ailable.
In o de o imp o e he yield, we sugges he use o a g een chemis y p ocedu e by
J. Yu e al. [153]. The inal compound M03 will be o med h ough an ae obic oxida ion o
amine IX wi h (2,2,6,6- e ame hylpipe idin-1-yl)oxyl (TEMPO), InCl3, and ace aldoxime
as ca alys s, using oluene as a sol en (Figu e 15).
Figu e 15. Al e na i e syn he ic ou e o he p epa a ion o compound M03. S a ing ma e ial IX is
comme cially a ailable.
2.6.4. Theo e ical Syn he ic Rou e (Compound M04)
We p esen a syn he ic ou e o syn hesize compound M04 (Figu e 16) based on he
o ma ion o iodobenzaldehyde XI om iodobenzyl alcohol X using py idinium dich o-
ma e as an oxidizing agen and DCM and E 2O as sol en s. The condensa ion eac ion
be ween in e media e XI and hyd oxylamine hyd ochlo ide and he subsequen educ-
ion wi h sodium cyanobo ohyd ide will u nish he desi ed N-(iodobenzyl) hyd oxyla-
mine M04 [154].
Figu e 16. Syn he ic ou e o he p epa a ion o MO4. THF ( e ahyd o u an), Po assium hyd ox-
ide, S a ing ma e ial X is comme cially a ailable.
2.6.5. Theo e ical Syn he ic Rou e (Compound M05)
We p o e a heo e ical syn he ic ou e o compound M05 using in e media e XIV
as he key subs a e. The ini ial s a egy o ob ain XIV consis s o wo s eps [155]. Fi s ly,
he coupling eac ion o acid de i a i e XII wi h o-( e ahyd o-2H-py an-2-yl) hyd oxyla-
mine (NH2OTHP) in DMF wi h 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC)
and N,N-diisop opyle hylamine (DIPEA) will p o ide de i a i e XIII. Then, he clea age
o he THP-p o ec ing g oup in acidic condi ions will p oduce benzamide XIV. The
M03
M03
M04
Figu e 14.
T adi ional me hod o he p epa a ion o compound
M03
. S a ing ma e ial
VIII
is
comme cially a ailable.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 19 o 31
Figu e 13. Al e na i e syn he ic ou e o he p epa a ion o M02. S a ing ma e ial VI is comme -
cially a ailable.
2.6.3. Theo e ical Syn he ic Rou e (Compound M03)
Oxime de i a i e M03 can be syn hesized by a condensa ion eac ion (Figu e 14) be-
ween aldehyde VIII and hyd oxylamine hyd ochlo ide in a 30% MeOH aqueous solu ion
[152].
Figu e 14. T adi ional me hod o he p epa a ion o compound M03. S a ing ma e ial VIII is com-
me cially a ailable.
In o de o imp o e he yield, we sugges he use o a g een chemis y p ocedu e by
J. Yu e al. [153]. The inal compound M03 will be o med h ough an ae obic oxida ion o
amine IX wi h (2,2,6,6- e ame hylpipe idin-1-yl)oxyl (TEMPO), InCl3, and ace aldoxime
as ca alys s, using oluene as a sol en (Figu e 15).
Figu e 15. Al e na i e syn he ic ou e o he p epa a ion o compound M03. S a ing ma e ial IX is
comme cially a ailable.
2.6.4. Theo e ical Syn he ic Rou e (Compound M04)
We p esen a syn he ic ou e o syn hesize compound M04 (Figu e 16) based on he
o ma ion o iodobenzaldehyde XI om iodobenzyl alcohol X using py idinium dich o-
ma e as an oxidizing agen and DCM and E 2O as sol en s. The condensa ion eac ion
be ween in e media e XI and hyd oxylamine hyd ochlo ide and he subsequen educ-
ion wi h sodium cyanobo ohyd ide will u nish he desi ed N-(iodobenzyl) hyd oxyla-
mine M04 [154].
Figu e 16. Syn he ic ou e o he p epa a ion o MO4. THF ( e ahyd o u an), Po assium hyd ox-
ide, S a ing ma e ial X is comme cially a ailable.
2.6.5. Theo e ical Syn he ic Rou e (Compound M05)
We p o e a heo e ical syn he ic ou e o compound M05 using in e media e XIV
as he key subs a e. The ini ial s a egy o ob ain XIV consis s o wo s eps [155]. Fi s ly,
he coupling eac ion o acid de i a i e XII wi h o-( e ahyd o-2H-py an-2-yl) hyd oxyla-
mine (NH2OTHP) in DMF wi h 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC)
and N,N-diisop opyle hylamine (DIPEA) will p o ide de i a i e XIII. Then, he clea age
o he THP-p o ec ing g oup in acidic condi ions will p oduce benzamide XIV. The
M03
M03
M04
Figu e 15.
Al e na i e syn he ic ou e o he p epa a ion o compound
M03
. S a ing ma e ial
IX
is
comme cially a ailable.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 19 o 31
Figu e 13. Al e na i e syn he ic ou e o he p epa a ion o M02. S a ing ma e ial VI is comme -
cially a ailable.
2.6.3. Theo e ical Syn he ic Rou e (Compound M03)
Oxime de i a i e M03 can be syn hesized by a condensa ion eac ion (Figu e 14) be-
ween aldehyde VIII and hyd oxylamine hyd ochlo ide in a 30% MeOH aqueous solu ion
[152].
Figu e 14. T adi ional me hod o he p epa a ion o compound M03. S a ing ma e ial VIII is com-
me cially a ailable.
In o de o imp o e he yield, we sugges he use o a g een chemis y p ocedu e by
J. Yu e al. [153]. The inal compound M03 will be o med h ough an ae obic oxida ion o
amine IX wi h (2,2,6,6- e ame hylpipe idin-1-yl)oxyl (TEMPO), InCl3, and ace aldoxime
as ca alys s, using oluene as a sol en (Figu e 15).
Figu e 15. Al e na i e syn he ic ou e o he p epa a ion o compound M03. S a ing ma e ial IX is
comme cially a ailable.
2.6.4. Theo e ical Syn he ic Rou e (Compound M04)
We p esen a syn he ic ou e o syn hesize compound M04 (Figu e 16) based on he
o ma ion o iodobenzaldehyde XI om iodobenzyl alcohol X using py idinium dich o-
ma e as an oxidizing agen and DCM and E 2O as sol en s. The condensa ion eac ion
be ween in e media e XI and hyd oxylamine hyd ochlo ide and he subsequen educ-
ion wi h sodium cyanobo ohyd ide will u nish he desi ed N-(iodobenzyl) hyd oxyla-
mine M04 [154].
Figu e 16. Syn he ic ou e o he p epa a ion o MO4. THF ( e ahyd o u an), Po assium hyd ox-
ide, S a ing ma e ial X is comme cially a ailable.
2.6.5. Theo e ical Syn he ic Rou e (Compound M05)
We p o e a heo e ical syn he ic ou e o compound M05 using in e media e XIV
as he key subs a e. The ini ial s a egy o ob ain XIV consis s o wo s eps [155]. Fi s ly,
he coupling eac ion o acid de i a i e XII wi h o-( e ahyd o-2H-py an-2-yl) hyd oxyla-
mine (NH2OTHP) in DMF wi h 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC)
and N,N-diisop opyle hylamine (DIPEA) will p o ide de i a i e XIII. Then, he clea age
o he THP-p o ec ing g oup in acidic condi ions will p oduce benzamide XIV. The
M03
M03
M04
Figu e 16.
Syn he ic ou e o he p epa a ion o
MO4
. THF ( e ahyd o u an), Po assium hyd oxide,
S a ing ma e ial Xis comme cially a ailable.
2.6.1. Theo e ical Syn he ic Rou e (Compound M01)
Compound M01 can be p epa ed acco ding o he esea ch ca ied ou by B. Bue -
elman e al. [
148
]. Con e sion o bu y aldehyde
I
wi h hyd oxylamine hyd ochlo ide
(NH
2
OH
·
HCl) in E OH and H
2
O as sol en s and using NaOH as a base will allow us o
ob ain he inal bu y aldehyde oxime—M01 (Figu e 11).
2.6.2. Theo e ical Syn he ic Rou e (Compound M02)
We p opose a heo e ical syn he ic ou e o
M02
based on he p epa a ion o oxime
de i a i e
V
as an in e media e, wi h
IV
and NH
2
OH
·
HCl as ini ial eac an s and NaOAc
and E OH/H
2
O as sol en s unde e lux condi ions [
149
]. By a educ ion eac ion wi h
sodium cyanobo ohyd ide (NaBH
3
CN) o in e media e
V
, inal hyd oxylamine
M02
will
be ob ained [150] (Figu e 12).
In . J. Mol. Sci. 2022,23, 8218 19 o 30
Fu he mo e, we designed a seconda y s a egy based on he p e ious wo k o C.
Be ini e al. [
151
]. Fi s ly, ni one
VII
could be u nished by condensa ion be ween hyd oxy-
lamine
VI
and ace aldehyde wi h magnesium sul a e in DCM. E en ually, he nucleophilic
addi ion o py ole o in e media e
VII
using HCl in MeOH would o igina e he a ge
compound (Figu e 13).
2.6.3. Theo e ical Syn he ic Rou e (Compound M03)
Oxime de i a i e
M03
can be syn hesized by a condensa ion eac ion (Figu e 14)
be ween aldehyde
VIII
and hyd oxylamine hyd ochlo ide in a 30% MeOH aqueous solu-
ion [152].
In o de o imp o e he yield, we sugges he use o a g een chemis y p ocedu e by J.
Yu e al. [
153
]. The inal compound
M03
will be o med h ough an ae obic oxida ion o
amine
IX
wi h (2,2,6,6- e ame hylpipe idin-1-yl)oxyl (TEMPO), InCl
3
, and ace aldoxime
as ca alys s, using oluene as a sol en (Figu e 15).
2.6.4. Theo e ical Syn he ic Rou e (Compound M04)
We p esen a syn he ic ou e o syn hesize compound
M04
(Figu e 16) based on he
o ma ion o iodobenzaldehyde
XI
om iodobenzyl alcohol
X
using py idinium dich o-
ma e as an oxidizing agen and DCM and E
2
O as sol en s. The condensa ion eac ion
be ween in e media e
XI
and hyd oxylamine hyd ochlo ide and he subsequen educ ion
wi h sodium cyanobo ohyd ide will u nish he desi ed N-(iodobenzyl) hyd oxylamine
M04 [154].
2.6.5. Theo e ical Syn he ic Rou e (Compound M05)
We p o e a heo e ical syn he ic ou e o compound
M05
using in e media e
XIV
as
he key subs a e. The ini ial s a egy o ob ain
XIV
consis s o wo s eps [
155
]. Fi s ly, he
coupling eac ion o acid de i a i e
XII
wi h o-( e ahyd o-2H-py an-2-yl) hyd oxylamine
(NH
2
OTHP) in DMF wi h 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC) and N,N-
diisop opyle hylamine (DIPEA) will p o ide de i a i e
XIII.
Then, he clea age o he THP-
p o ec ing g oup in acidic condi ions will p oduce benzamide
XIV
. The second possibili y o
syn hesizing he key in e media e can be h ough palladium-ca alyzed hyd ogena ion [
156
],
emo ing he benzyl g oup o s a ing ma e ial
XV
. Finally, he a ge compound
M05
can
be u nished om in e media e
XIV
by a hyd ogena ion eac ion using pla inum/ce ia-
zi conia mixed oxide (P /CeO2-Z O2) as a ca aly ic sys em [157] (Figu e 17).
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 20 o 31
second possibili y o syn hesizing he key in e media e can be h ough palladium-ca a-
lyzed hyd ogena ion [156], emo ing he benzyl g oup o s a ing ma e ial XV. Finally,
he a ge compound M05 can be u nished om in e media e XIV by a hyd ogena ion
eac ion using pla inum/ce ia-zi conia mixed oxide (P /CeO2-Z O2) as a ca aly ic sys em
[157] (Figu e 17).
Figu e 17. Theo e ical syn he ic ou e o he p epa a ion o M05. S a ing ma e ials XII and XV a e
comme cially a ailable.
3. Ma e ials and Me hods
3.1. Da ase
A da ase o 14 benzoylphenylu ean analogs was chosen o de e mine he se s o
aining and es ing as hey we e designed and syn hesized om he e e ence s uc u e,
wi h modi ica ions a speci ic poin s o gene a e he model om a single base s uc u e
(Figu e 18); he da ase had expe imen al da a wi h excellen la icidal ac i i y [32], po-
en ially o he chi in syn hase and he ju enile ho mone ecep o .
Figu e 18. The 2D s uc u e o benzoylphenylu ean de i a i es. R1 and R2 a e adicals, and X =
me hyl o halogens.
GALAHADTM (T ipos Inc.), was employed o build en pha macopho e models om
a aining se (see Figu e 19) o p e iously ela ed inhibi ion ac i i y o chi in syn hase
and ju enile ho mone ecep o s [32,120].
M
0
5
Figu e 17.
Theo e ical syn he ic ou e o he p epa a ion o
M05
. S a ing ma e ials
XII
and
XV
a e
comme cially a ailable.
In . J. Mol. Sci. 2022,23, 8218 20 o 30
3. Ma e ials and Me hods
3.1. Da ase
A da ase o 14 benzoylphenylu ean analogs was chosen o de e mine he se s o
aining and es ing as hey we e designed and syn hesized om he e e ence s uc u e,
wi h modi ica ions a speci ic poin s o gene a e he model om a single base s uc u e (Fig-
u e 18); he da ase had expe imen al da a wi h excellen la icidal ac i i y [
32
], po en ially
o he chi in syn hase and he ju enile ho mone ecep o .
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 20 o 31
second possibili y o syn hesizing he key in e media e can be h ough palladium-ca a-
lyzed hyd ogena ion [156], emo ing he benzyl g oup o s a ing ma e ial XV. Finally,
he a ge compound M05 can be u nished om in e media e XIV by a hyd ogena ion
eac ion using pla inum/ce ia-zi conia mixed oxide (P /CeO2-Z O2) as a ca aly ic sys em
[157] (Figu e 17).
Figu e 17. Theo e ical syn he ic ou e o he p epa a ion o M05. S a ing ma e ials XII and XV a e
comme cially a ailable.
3. Ma e ials and Me hods
3.1. Da ase
A da ase o 14 benzoylphenylu ean analogs was chosen o de e mine he se s o
aining and es ing as hey we e designed and syn hesized om he e e ence s uc u e,
wi h modi ica ions a speci ic poin s o gene a e he model om a single base s uc u e
(Figu e 18); he da ase had expe imen al da a wi h excellen la icidal ac i i y [32], po-
en ially o he chi in syn hase and he ju enile ho mone ecep o .
Figu e 18. The 2D s uc u e o benzoylphenylu ean de i a i es. R1 and R2 a e adicals, and X =
me hyl o halogens.
GALAHADTM (T ipos Inc.), was employed o build en pha macopho e models om
a aining se (see Figu e 19) o p e iously ela ed inhibi ion ac i i y o chi in syn hase
and ju enile ho mone ecep o s [32,120].
M
0
5
Figu e 18.
The 2D s uc u e o benzoylphenylu ean de i a i es. R1 and R2 a e adicals, and X =
me hyl o halogens.
GALAHAD
TM
(T ipos Inc.), was employed o build en pha macopho e models om
a aining se (see Figu e 19) o p e iously ela ed inhibi ion ac i i y o chi in syn hase and
ju enile ho mone ecep o s [32,120].
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 21 o 31
Figu e 19. S uc u es o selec ed aining se compounds, wi h canonical smiles and concen a ions
([ ]) wi h 100% la icidal ac i i y (mg·L−1). (A)—Compound 01; (B)—Compound 05; (C)—Com-
pound 06; (D)—Compound 07; (E)—Compound 12; (F)—Compound 13; (G)—Compound 16.
Fi s , hey we e d awn using Ma inTM Ske ch 16.9.5 so wa e (CHEMAXON, Buda-
pes , Hunga y, h ps://www.chemaxon.com/, accessed on 15 Ma ch 2021), om which we
selec ed he mos eliable au ome s (pH = 6.5) o all compounds [158]. Nex , 2D s uc-
u es we e con e ed o a 3D o ma by CONCORD implemen ed in he SYBYL-XTM 2.0
package (T ipos Inc.). All s uc u es we e ene gy-minimized using a conjuga e g adien
(CG) wi h a con e gence c i e ion o 0.001 kcal/mol and a T ipos o ce ield (dielec ic
cons an ε = 80.0 and maximum numbe o i e a ions = 50,000) [159]. Pa ial a omic cha ges
we e calcula ed using he Gas eige -Hückel me hod [160], a ailable on SYBYL-X 2.0TM
(T ipos Inc.).
The s udy o la icidal ac i i y was ca ied ou by Sun [32] e al., om which he
ou een compounds wi h 100% la icidal ac i i y a low concen a ions (2 o 0.001 mg·L−1)
we e selec ed, se en o which we e used o de e mine he aining se and se en o he
es se . They we e used andomly in he cons uc ion o he pha macopho e models,
while he o he compounds wi h he same c i e ia we e used in he alida ion o he pha -
macopho e model.
3.2. Pha macopho e Model Gene a ion
The aining da ase s we e lexibly aligned o each o he in o de o build hype mo-
lecula alignmen s ha map common pha macopho e ea u es. The gene ic algo i hm em-
ployed in his s ep s a s wi h 45 con o ma ions (popula ion size) o each
Figu e 19.
S uc u es o selec ed aining se compounds, wi h canonical smiles and concen a ions (
[ ]
)
wi h 100% la icidal ac i i y (mg
·
L
−1
). (
A
)—Compound
01
; (
B
)—Compound
05
; (
C
)—Compound
06
;
(D)—Compound 07; (E)—Compound 12; (F)—Compound 13; (G)—Compound 16.
In . J. Mol. Sci. 2022,23, 8218 21 o 30
Fi s , hey we e d awn using Ma in
TM
Ske ch 16.9.5 so wa e (CHEMAXON, Bu-
dapes , Hunga y, h ps://www.chemaxon.com/, accessed on 15 Ma ch 2021), om which
we selec ed he mos eliable au ome s (pH = 6.5) o all compounds [
158
]. Nex , 2D
s uc u es we e con e ed o a 3D o ma by CONCORD implemen ed in he SYBYL-X
TM
2.0 package (T ipos Inc.). All s uc u es we e ene gy-minimized using a conjuga e g adien
(CG) wi h a con e gence c i e ion o 0.001 kcal/mol and a T ipos o ce ield (dielec ic
cons an
ε
= 80.0 and maximum numbe o i e a ions = 50,000) [
159
]. Pa ial a omic cha ges
we e calcula ed using he Gas eige -Hückel me hod [
160
], a ailable on SYBYL-X 2.0
TM
(T ipos Inc.).
The s udy o la icidal ac i i y was ca ied ou by Sun [
32
] e al., om which he
ou een compounds wi h 100% la icidal ac i i y a low concen a ions (2 o 0.001 mg
·
L
−1
)
we e selec ed, se en o which we e used o de e mine he aining se and se en o he
es se . They we e used andomly in he cons uc ion o he pha macopho e models,
while he o he compounds wi h he same c i e ia we e used in he alida ion o he
pha macopho e model.
3.2. Pha macopho e Model Gene a ion
The aining da ase s we e lexibly aligned o each o he in o de o build hype molecu-
la alignmen s ha map common pha macopho e ea u es. The gene ic algo i hm employed
in his s ep s a s wi h 45 con o ma ions (popula ion size) o each benzoylphenylu ean
de i a i e and e ol es h ough a maximum o 90 gene a ions by means o de aul gene ic
ope a o s (mu a ion a e = 0.2 and c osso e a e = 0.2) a ailable in he GALAHAD
TM
module in SYBYL-XTM 2.0 [161] (T ipos Inc.).
3.3. Pha macopho e Model E alua ion
Pha macopho e models wi h he lowes ene gy (<100.0 kcal/mol) we e selec ed and
analyzed o he esul s o he Pa e o sco e. To de e mine he bes pha macopho e model
and i s disc imina o y powe , he models we e used o build decoys ha we e emo ed om
he DUD-E se e , Ma ch (h p://dude.docking.o g/, accessed on 15 Ma ch 2021), [
162
]
and execu ed in he SigmaPlo
TM
p og am . 12.0 (SYSTAT, Leland Wilkinson, Chicago, IL,
USA) by calcula ing he a ea unde he ecei e ope a ing cha ac e is ics cu e (AUC-ROC
cu e); i was possible o isualize he ecogni ion o asse s and decoys o be e alida ion
o he models h ough he ue-posi i e a e e sus he alse-posi i e a e.
The Bol zmann-enhanced disc imina ion o he ROC cu e (BEDROC) was employed
o disc imina e models wi h AUC > 0.7 acco ding o ea ly ecogni ion o ac i es using
he ROCKER se e [
163
] (www.jyu. i/ ocke , accessed on 15 Ma ch 2021). Addi ionally,
BEDROC is o ien ed by a sea ch algo i hm (
α
= 16.10) ha co esponds o he ini ial 10% o
he da abase. The pha macopho e model ha demons a ed ea ly ecogni ion (BEDROC >
0.5) was employed in he i ual sc eening s ep.
3.4. Pha macopho e-Based Vi ual Sc eening
The bes pha macopho e model was au oma ically con e ed o he UNITY
TM
3D
que y ha was employed o i ually sc een he Sigma-Ald ich subse om he ZINC
da abase [
34
] (h ps://zinc15.docking.o g/, accessed on 15 Ma ch 2021). Compounds we e
anked acco ding o QFIT alues, and hose wi h alues highe han mean plus wo imes
s anda d de ia ion (Equa ion (1)) we e selec ed o molecula docking.
X=x+2×σ(1)
X = QFIT alue
x = A e age
σ= S anda d de ia ion
Equa ion (1): Ma hema ical equa ion o he mean plus wo imes he s anda d de ia-
ion.

In . J. Mol. Sci. 2022,23, 8218 22 o 30
3.5. Homology Modeling, Molecula Docking, and Vi ual Sc eening o Po en ial Dual
Modula o s
The p ima y sequence o Aedes aegyp i species was collec ed in he Na ional Cen e o
Bio echnology In o ma ion (NCBI), wi h he desc ip i es (Aedes aegyp i + chi in deace ylase
1 p o ein) a h ps://www.ncbi.nlm.nih.go /p o ein, accessed on 27 June 2021. We ound
ha he p o ein AAEL003419-PA was used as a p ima y p o ein; he BLAST
TM
,h ps:
//blas .ncbi.nlm.nih.go /Blas .cgi, accessed on 27 June 2021 [
164
] pe o med and compa ed
he p ima y biological sequences and ound he mos simila and s a is ically signi ican
ones in he PDB (P o ein Da a Bank).
The mos simila mac omolecules we e in oduced and analyzed in he MODELLER
10.1 p og am, which, wi h he p ima y s uc u e o Aedes aegyp i, pe o med he compa ison,
alignmen , cons uc ion, and alida ion o he model, called Bombyx-Aedes.
Using he SWISS-MODEL se e [
165
–
167
], s uc u e assessmen (h ps://swissmodel.
expasy.o g/assess, accessed on 27 June 2021) was employed o analyze he model c ea ed
o A. aegyp i chi in deace ylase using he global model quali y es ima ion (GMQE) sco e on
he accu acy o he cons uc ed model and he model used [
168
]; he p io i ized model had
a GMQE sco e o <0.50 [
169
]. Fu he mo e, Ramachand an plo s we e used o e alua e he
ene ge ically a o ed egions o he model [
170
]. In he Pymol p og am, he 3D alignmen
be ween he s uc u es was pe o med, which collec ed he coo dina es o he alpha-ca bons
o each o he esidues in his analysis.
The con o ma ional sea ch and sco ing e alua ion we e pe o med using Au oDock
Vina 1.1.2 (Sc ipps Resea ch Ins i u e, Olson Labo a o y, La Jolla, CA, USA). The abili y o
gene a e sa is ac o y solu ions o sco ing unc ions was p obed acco ding o he oo mean
squa e de ia ion alue (RMSD < 2Å) [171,172].
The compounds p io i ized in pha macopho e-based i ual sc eening we e employed
in molecula docking, and in silico app oaches o he Aedes aegyp i ju enile ho mone
ecep o (PDB: 5V13) we e pe o med acco ding o p e ious s udies [
173
,
174
]. A e he
molecula docking, ligand e iciency (LE) was calcula ed h ough he binding a ini y o
compounds employed in docking s udies (Equa ion (2)), and he compounds wi h LE <
−
0.4
kcal/mol we e p io i ized o dockig wi h he A. aegyp i chi in deace ylase ecep o model.
LE = −∆G/N (2)
LE = Ligand e iciency
∆G = Binding a ini y
N = Numbe o hea y a oms
Equa ion (2). Ma hema ical equa ion o ligand e iciency.
The ligand e iciency (LE) was calcula ed o compounds employed in docking wi h
he A. aegyp i chi in deace ylase ecep o , and compounds wi h LE <
−
0.4 kcal/mol we e
p io i ized o pha macokine ic and oxicological analysis.
3.6. In Silico Pha macokine ic and Toxicological P ope ies
Fo he analysis o pha macokine ic p ope ies, he bes compounds om he p e ious
s ep we e selec ed, and we used he P eadme (h ps://p eadme .bmd c.k /, accessed
on 27 June 2021) and SwissADME ools [
79
,
175
] and he p o icien me hod o ce eb al o
in es inal pe mea ion Es ima ion D (BOILED-Egg) [
78
] (h p://www.swissadme.ch/index.
php, accessed on 27 June 2021). The analysis o he oxici y p o ile o he compounds was
e alua ed using he online pla o m eMolTox (h p://xund ug.cn/mol ox, accessed on 27
June 2021).
3.7. Syn he ic Accessibili y (SA) P edic ion
The p edic ion o he syn he ic accessibili y (SA) o he compounds selec ed by he
hie a chical i ual sc eening was pe o med using he AMBIT p og am (h p://ambi .
sou ce o ge.ne / eac o .h ml, accessed on 10 July 2021), which implemen s he au ome
In . J. Mol. Sci. 2022,23, 8218 23 o 30
gene a ions by he algo i hm me hod o combina o ics, exhaus i ely calcula ing all au-
ome ic o ms o a gi en o ganic compound by molecula and s e eochemical complexi y
and conside ing he p esence o used and b idged sys ems o sugges he canonical
au ome by an ene gy base o classi ica ion by di e en s uc u al and opological cha -
ac e is ics combined wi h an addi i e scheme using weigh ed molecula desc ip o s. By
gene a ing a sco e anging om 0 o 100 (maximum syn he ic accessibili y), he molecule is
mo e easily syn hesized when he sco e is close o 100 [176–179].
4. Conclusions
This s udy has es ablished he planning o insec -g ow h-inhibi ing compounds wi h
po en ial insec icidal ac i i y ha ha e he possible mechanism o ac ion o dual inhibi ion
o he ju enile ho mone p o ein; he s udy has also es ablished a chi in deace ylase model
o Aedes aegyp i h ough molecula modeling app oaches ia pha macopho e and i ual
sc eening based on molecula docking by p edic ing molecula a ge binding. In addi ion,
compounds we e de eloped h ough he pha macokine ic cha ac e is ics (ADME) o he
physiology o he Aedes aegyp i mosqui o, and oxicological p ope ies di ec ed a o he
o ganisms, mainly humans, as well as he syn he ic heo e ical ou es o compounds
M01
,
M02,M03,M04, and M05 ha e been p oposed.
Finally, compounds
M01
–
M05
we e selec ed, showing pa ame e s simila and supe io
o hose obse ed o posi i e con ols and also signi ican di e ences in he in e ac ion
wi h key esidues in each ca aly ic p o ein si e epo ed in he li e a u e. Acco dingly, he
molecules in es iga ed he e a e dual inhibi o s o he enzymes chi in syn hase and ju enile
ho monal p o ein ( om insec s), cha ac e izing hem as po en ial insec icides agains he
mosqui o Aedes aegyp i. Wi h hese p omising esul s, ou esea ch g oup in ends o ca y
ou biological es s
in i o
wi h he compounds ob ained in his wo k o con i m he in
silico p edic ions.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/ijms23158218/s1.
Au ho Con ibu ions:
Concep ualiza ion, G.V.D.C., J.M.E.-R., F.H.A.L. and C.B.R.S.; Da a cu a ion,
A.K.P.D.S. and C.M.L.; Fo mal analysis, M.F.A.N., A.K.P.D.S., E.M.F.D.S., L.C.F.C., C.M.L., J.M.C.
and C.B.R.S.; Funding acquisi ion, G.V.D.C., J.P.Z., J.M.C. and C.B.R.S.; In es iga ion, A.K.P.D.S.,
E.M.F.D.S., L.C.F.C., J.S.V., C.M.L., J.M.E.-R. and F.H.A.L.; Me hodology, G.V.D.C., M.F.A.N., E.M.F.D.S.,
L.C.F.C., J.S.V., J.M.E.-R., J.M.C. and F.H.A.L.; P ojec adminis a ion, C.B.R.S.; So wa e, M.F.A.N.;
Supe ision, F.H.A.L. and C.B.R.S.; Valida ion, J.P.Z.; Visualiza ion, A.K.P.D.S., J.S.V., J.P.Z. and J.M.C.;
W i ing—o iginal d a , G.V.D.C., M.F.A.N., J.M.E.-R. and F.H.A.L.; W i ing— e iew and edi ing,
J.P.Z. and C.B.R.S. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch ecei ed no ex e nal unding.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Acknowledgmen s:
We g a e ully acknowledge he compu a ional suppo p o ided by he Labo a-
o y o Modeling and Compu a ional Chemis y (LMQC) and he G adua e P og am in-Ne wo k
in Pha maceu ical Inno a ion o he Depa men o Biological Sciences a he Fede al Uni e si y
o Amapá(UNIFAP/Macapá-B azil) and hank he Labo a o y o Molecula Modeling o he S a e
Uni e si y o Fei a de San ana (Bahia-B azil). We would also like o hank he Labo a o y o Cellula
Immunology Applied o Heal h o he Oswaldo C uz Founda ion (FIOCRUZ); he Depa men o
Pha maceu ical and O ganic Chemis y, Facul y o Pha macy o he Uni e si y o G anada (Spain);
and he Resea che Assis ance P og am—PAPESQ/UNIFAP o inancial suppo .
Con lic s o In e es :
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o
pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .
In . J. Mol. Sci. 2022,23, 8218 24 o 30
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