Ci a ion: Melcón-Fe nandez, E.;
Ma ín-Encinas, E.; Palacios, F.;
Galli, G.; Regue a, R.M.;
Ma ínez-Vallada es, M.;
Balaña-Fouce, R.; Alonso, C.;
Pé ez-Pe ejo, Y. An ileishmanial
E ec o 1,5- and 1,8-Subs i u ed
Fused Naph hy idines. Molecules 2024,
29, 74. h ps://doi.o g/10.3390/
molecules29010074
Academic Edi o s: Ma ia Isabel
L. Soa es and Susana M. M. Lopes
Recei ed: 4 No embe 2023
Re ised: 18 Decembe 2023
Accep ed: 20 Decembe 2023
Published: 22 Decembe 2023
Copy igh : © 2023 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/).
molecules
A icle
An ileishmanial E ec o 1,5- and 1,8-Subs i u ed
Fused Naph hy idines
Es ela Melcón-Fe nandez 1, Endika Ma ín-Encinas 2, F ancisco Palacios 2, Gulio Galli 1, Rosa M. Regue a 1,
Ma ía Ma ínez-Vallada es 1, Ra ael Balaña-Fouce 1, Concepción Alonso 2,* and Yolanda Pé ez-Pe ejo 1,*
1
Depa amen o de Ciencias Biomédicas, Facul ad de Ve e ina ia, Uni e sidad de León, Campus de Vegazana s/n,
24071 León, Spain; [email p o ec ed] (G.G.)
2
Depa amen o de Química O gánica I, Facul ad de Fa macia, Lasca ay Resea ch Cen e , Uni e sidad del País
Vasco/Euskal He iko Unibe si a ea (UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain
*Co espondence: [email p o ec ed] (C.A.); [email p o ec ed] (Y.P.-P.)
Abs ac : In he absence o a accine, he e is a need o ind new d ugs o he ea men o neglec ed
opical diseases, such as leishmaniasis, ha can o e come he many d awbacks o hose cu en ly
used. These disad an ages include cos , he need o main ain a cold chain, he ou e o adminis a ion,
he associa ed ad e se e ec s and he gene a ion o esis ance. In his wo k we ha e e alua ed he
an ileishmanial e ec o 1,5- and 1,8-subs i u ed used naph hy idines h ough
in i o
and ex i o
assays, using gene ically modi ied axenic and in amac ophagic Leishmania in an um amas igo es.
The oxici y o hese compounds has been es ed in he mammalian hos cell using mu ine splenic
mac ophages, as well as in mu ine in es inal o ganoids (minigu s) in o de o assess hei po en ial
o o al adminis a ion. The 1,8- de i a i es showed g ea e leishmanicidal ac i i y and he p esence
o a ni ogen a om in he used ing o he naph hy idine was impo an o inc ease he ac i i y o
bo h ypes o molecules. The a oma iza ion o he py idine ing also had ma ked di e ences in he
ac i i y o he compounds.
Keywo ds: isce al leishmaniasis; used 1,5-naph hy idines; used 1,8-naph hy idines; in amac ophagic
Leishmania pa asi es; mouse in es inal o ganoids
1. In oduc ion
Cu en ea men o leishmaniasis, a complex o ec o -bo ne diseases caused by
pa asi ic p o is s o he genus Leishmania, emains a challenge [
1
–
3
]. The lack o a accine
makes chemo he apy he only way o manage he in ec ion when he i s symp oms appea .
Howe e , he abili y o he pa asi e o e ade he hos immune sys em and he in acellula
localiza ion o he pa asi e wi hin hos mac ophages a e pi alls ha hinde and delay he
disco e y and de elopmen o new d ugs o ea hese diseases [
4
]. Las bu no leas ,
leishmaniasis is classi ied by he WHO as a neglec ed opical disease widely sp ead in he
opical and sub opical a eas o he plane [
5
], whe e he d ugs cu en ly in use a e sca ce,
ou da ed and expensi e. In addi ion, cu en d ugs exhibi oxici y, ad e se side e ec s,
poo o al bioa ailabili y and loss o e icacy due o he inc easing eme gence o esis an
s ains [1,6,7].
Despi e hei signi ican poo ole abili y, pen a alen an imonials a e s ill he i s -
line ea men o di e en ypes o leishmaniasis. In addi ion o hei ca dio oxici y,
hey equi e p olonged and pain ul pa en e al adminis a ion, which makes adhe ence
di icul [
8
]. Fo mula ions o he polyene ungicide ampho e icin B a e s ongly e ec i e
agains he isce al o m o he disease, bu hey mus be adminis e ed by in a enous
in usions and equi e a cold chain o each he poin o ca e [
4
,
9
]. Finally, mil e osine is he
only app o ed o al an ileishmanial d ug, bu un o una ely, i canno be adminis e ed in
p egnan women due o i s e a ogenici y and easily p omo es esis ance [
9
]. Combina ions
o hese d ugs and wi h he an ibio ic pa omomycin ep esen an in e es ing scena io un il
Molecules 2024,29, 74. h ps://doi.o g/10.3390/molecules29010074 h ps://www.mdpi.com/jou nal/molecules
Molecules 2024,29, 74 2 o 16
mo e e ec i e and sa e molecules a e egis e ed [
10
,
11
]. Gi en he limi ed numbe o
d ugs cu en ly used agains leishmaniasis and hei d awbacks, i is u gen ly needed
o sea ch o new compounds ha allow sho , sa e, inexpensi e ea men s and whose
equi emen s a e in line wi h he needs o de eloping coun ies ( a ge p oduc p o ile),
whe e he disease is mos p e alen [12].
The e is a b oad consensus among medicinal chemis s ha he p esence o ni ogen
a oms in he he e ocyclic s uc u e is ele an o imp o e in e ac ions in biological sys ems
as ni ogena ed he e ocycles a e pa o he chemical s uc u e o many na u al p oduc s
and agen s wi h signi ican biological ac i i y, such as an i i al, an ibio ic and an i umo
d ugs [
13
–
20
]. In ac , he FDA da abase e eals ha a la ge numbe o accep ed d ugs
con ain a ni ogen he e ocycle in hei s uc u e [
21
]. The e o e, in he d ug disco e y
p ocess, he de elopmen o p ac ical syn he ic ou es o access hese s uc u al mo i s in
he simples way possible is an impo an goal o syn he ic and medicinal chemis s.
One o he mos s aigh o wa d and e sa ile p ocesses o he p epa a ion o he e-
ocyclic ni ogen compounds is he Po a o eac ion [
22
]. Wi h his in mind, ou g oup
has de eloped a sui able, e icien , as and e sa ile me hodology o he syn hesis o
ni ogena ed he e ocycles and good esul s we e obse ed as opoisome ase IB (TopIB)
inhibi o s o hese de i a i es, wi h an ileishmanial ac i i y [23,24].
In addi ion, i he adi ional Po a o eac ion ep esen s an excellen ool o he
p epa a ion o bicyclic ni ogena ed he e ocycles, such as he a o emen ioned quinoline
o naph hy idine de i a i es, compounds wi h a g ea e numbe o used cycles can also
be p epa ed wi h his me hodology in i s in amolecula e sion [
25
]. In hese cases, he
aza-Diels–Alde p ocess (a [4+2] cycloaddi ion eac ion) in ol es an aldehyde unc ion-
alized wi h a dienophile and an a oma ic amine. Mo eo e , i he a oma ic amine used is
he e ocyclic such as 2-aminopy idine o 3-aminopy idine, his p ocess allows he di ec
p epa a ion o used he e ocycles wi h se e al he e oa oms in hei s uc u e.
Unde his con ex , he objec i e o he p esen s udy was o sc een a se ies o used
1,5- and 1,8-naph hy idines o e alua e hei an ileishmanial ac i i y by using
in i o
and ex i o es s. The an ipa asi ic ac i i y o hese compounds was assessed in bo h
axenic and in amac ophagic o ms o Leishmania in an um using an in-house imp o ed
bioimaging sys em. In addi ion, he po en ial oxici y o he assayed compounds was
e alua ed in mouse splenic mac ophages and mu ine in es inal o ganoids in o de o assess
hei ole abili y as o al d ugs.
2. Resul s
2.1. Chemis y
The syn hesis o used 1,5-naph hy idines and 1,8-naph hy idines is depic ed in
Schemes 1and 2. The syn hesis o used 1,5-naph hy idines (Scheme 1) was pe o med by
an in amolecula Po a o ype [4+2] cycloaddi ion eac ion. Thus, when 3-aminopy idine
1a was eac ed wi h unc ionalized aldehydes 2in e luxing chlo o o m, in he p esence
o 2 equi alen s o BF
3·
E
2
O, e ahyd o[1,5]naph hy idine de i a i es 4,6,8(Scheme 1,
Figu e 1) we e egio- and dias e eoselec i ely ob ained as p e iously epo ed by us [
26
,
27
].
Molecules 2023, 28, x FOR PEER REVIEW 3 o 17
Scheme 1. In amolecula Po a o eac ion o 3-aminopy idine 1a wi h unc ionalized aldehydes 2.
Figu e 1. S uc u es o used 1,5-naph hy idine de i a i es 4–9 es ed as an ileishmanial agen s.
When 2-aminopy idine 1b was used e ahyd o[1,8]naph hy idines de i a i es 10,
12, 14 (Scheme 2, Figu e 2) we e isola ed as well in a egio- and dias e eoselec i e way
[28].
Scheme 2. In amolecula Po a o eac ion o 2-aminopy idine 1b and unc ionalized aldehydes 2.
Figu e 2. S uc u es o used 1,8-naph hy idine de i a i es 10–15 es ed as an ileishmanial agen s.
In addi ion, he subsequen dehyd ogena ion o e ahyd onaph hy idine de i a i es
wi h MnO2 in e luxing oluene yielded he co esponding 1,5-naph hy idines 5, 7 and 9
(Scheme 1, Figu e 1) and 1,8-naph hy idines 11, 13 and 15 (Scheme 2, Figu e 2), espec-
i ely, as p e iously epo ed [26–28]. The desc ibed me hodologies ep esen an easy and
e icien s a egy o he p epa a ion o hese compounds con aining a wide ange o elec-
on- eleasing and elec on-wi hd awing subs i uen s.
Scheme 1. In amolecula Po a o eac ion o 3-aminopy idine 1a wi h unc ionalized aldehydes 2.
Molecules 2024,29, 74 3 o 16
Molecules 2023, 28, x FOR PEER REVIEW 3 o 17
Scheme 1. In amolecula Po a o eac ion o 3-aminopy idine 1a wi h unc ionalized aldehydes 2.
Figu e 1. S uc u es o used 1,5-naph hy idine de i a i es 4–9 es ed as an ileishmanial agen s.
When 2-aminopy idine 1b was used e ahyd o[1,8]naph hy idines de i a i es 10,
12, 14 (Scheme 2, Figu e 2) we e isola ed as well in a egio- and dias e eoselec i e way
[28].
Scheme 2. In amolecula Po a o eac ion o 2-aminopy idine 1b and unc ionalized aldehydes 2.
Figu e 2. S uc u es o used 1,8-naph hy idine de i a i es 10–15 es ed as an ileishmanial agen s.
In addi ion, he subsequen dehyd ogena ion o e ahyd onaph hy idine de i a i es
wi h MnO2 in e luxing oluene yielded he co esponding 1,5-naph hy idines 5, 7 and 9
(Scheme 1, Figu e 1) and 1,8-naph hy idines 11, 13 and 15 (Scheme 2, Figu e 2), espec-
i ely, as p e iously epo ed [26–28]. The desc ibed me hodologies ep esen an easy and
e icien s a egy o he p epa a ion o hese compounds con aining a wide ange o elec-
on- eleasing and elec on-wi hd awing subs i uen s.
Scheme 2. In amolecula Po a o eac ion o 2-aminopy idine 1b and unc ionalized aldehydes 2.
Molecules 2023, 28, x FOR PEER REVIEW 3 o 17
N
X
Y
R2
N
BF3稥 2O
N
HN Y
X
R2
O
X
Y
R2
NH2
N
+
34 X = O; Y = CH2
6X = O; Y = CO
8 X = NTs; Y = CH2
MnO2
N
NY
X
R2
HH
5X = O; Y = CH2
7X = O; Y = CO
9X = NTs; Y = CH2
1a
2a X = O; Y = CH2
2b X = O; Y = CO
2c X = NTs; Y = CH2
R1
MS 4 Å
R1R1R1
R1 = H,
4-B , 6-B ,
6-OMe
Scheme 1. In amolecula Po a o eac ion o 3-aminopy idine 1a wi h unc ionalized aldehydes 2.
N
HN
O
R1
4a (R1 = H)
4c (R1 = OMe)
N
N
O
R1
N
HN
O
R1
6a (R1 = H)
6b (R1 = B )
6c (R1 = OMe)
O
N
N
O
R1
O
N
HN
NTs
8a (R1 = H)
8b (R1 = 4-B )
8c (R1 = 6-OMe)
8d (R1 = 6-B )
R1N
N
NTs
5a (R1 = H)
5b (R1 = B )
5c (R1 = OMe)
7a (R1 = H)
7b (R1 = B )
7c (R1 = OMe)
R1
9a (R1 = H)
9b (R1 = 4-B )
9c (R1 = 6-OMe)
9d (R1 = 6-B )
Figu e 1. S uc u es o used 1,5-naph hy idine de i a i es 4–9 es ed as an ileishmanial agen s.
When 2-aminopy idine 1b was used e ahyd o[1,8]naph hy idines de i a i es 10,
12, 14 (Scheme 2, Figu e 2) we e isola ed as well in a egio- and dias e eoselec i e way
[28].
N
X
Y
R2
N
R1
BF3稥 2O
N
HN Y
X
R2
O
X
Y
R2
NH2
N+
310 X = O; Y = CH2
12 X = O; Y = CO
14 X = NTs; Y = CH2
MnO2
N
NY
X
R2
HH
11 X = O; Y = CH2
13 X = O; Y = CO
15 X = NTs; Y = CH2
R3
1b
2a X = O; Y = CH2
2b X = O; Y = CO
2c X = NTs; Y = CH2
R1
R1
R3R3
R3
MS 4 Å
R1
R1= H, 5-B
Scheme 2. In amolecula Po a o eac ion o 2-aminopy idine 1b and unc ionalized aldehydes 2.
N
HN
O
10a (R2 = H)
10b (R2 = F)
10c (R2 = Me)
N
N
O
N
HN
O
R1
12a (R1 = H, R2 = H)
12b (R1 = B , R2 = H)
O
N
N
O
R1
O
N
HN
N
14a
N
N
N
R1
11a (R2 = H, R3 = H)
11b (R2 = F, R3 = H)
11c (R2 = H, R3 = OMe)
13a (R1 = H, R2 = H)
13b (R1 = B , R2 = H)
13c (R1 = H, R2 = F)
15a (R1 = H)
15b (R1 = B )
R2R2
R3
R2R2
Ts
Ts
Figu e 2. S uc u es o used 1,8-naph hy idine de i a i es 10–15 es ed as an ileishmanial agen s.
In addi ion, he subsequen dehyd ogena ion o e ahyd onaph hy idine de i a i es
wi h MnO2 in e luxing oluene yielded he co esponding 1,5-naph hy idines 5, 7 and 9
(Scheme 1, Figu e 1) and 1,8-naph hy idines 11, 13 and 15 (Scheme 2, Figu e 2), espec-
i ely, as p e iously epo ed [26–28]. The desc ibed me hodologies ep esen an easy and
efficien s a egy o he p epa a ion o hese compounds con aining a wide ange o elec-
on- eleasing and elec on-wi hd awing subs i uen s.
Figu e 1. S uc u es o used 1,5-naph hy idine de i a i es 4–9 es ed as an ileishmanial agen s.
When 2-aminopy idine 1b was used e ahyd o[1,8]naph hy idines de i a i es 10,12,
14 (Scheme 2, Figu e 2) we e isola ed as well in a egio- and dias e eoselec i e way [28].
Molecules 2023, 28, x FOR PEER REVIEW 3 o 17
Scheme 1. In amolecula Po a o eac ion o 3-aminopy idine 1a wi h unc ionalized aldehydes 2.
Figu e 1. S uc u es o used 1,5-naph hy idine de i a i es 4–9 es ed as an ileishmanial agen s.
When 2-aminopy idine 1b was used e ahyd o[1,8]naph hy idines de i a i es 10,
12, 14 (Scheme 2, Figu e 2) we e isola ed as well in a egio- and dias e eoselec i e way
[28].
Scheme 2. In amolecula Po a o eac ion o 2-aminopy idine 1b and unc ionalized aldehydes 2.
Figu e 2. S uc u es o used 1,8-naph hy idine de i a i es 10–15 es ed as an ileishmanial agen s.
In addi ion, he subsequen dehyd ogena ion o e ahyd onaph hy idine de i a i es
wi h MnO2 in e luxing oluene yielded he co esponding 1,5-naph hy idines 5, 7 and 9
(Scheme 1, Figu e 1) and 1,8-naph hy idines 11, 13 and 15 (Scheme 2, Figu e 2), espec-
i ely, as p e iously epo ed [26–28]. The desc ibed me hodologies ep esen an easy and
e icien s a egy o he p epa a ion o hese compounds con aining a wide ange o elec-
on- eleasing and elec on-wi hd awing subs i uen s.
Figu e 2. S uc u es o used 1,8-naph hy idine de i a i es 10–15 es ed as an ileishmanial agen s.
In addi ion, he subsequen dehyd ogena ion o e ahyd onaph hy idine de i a i es
wi h MnO
2
in e luxing oluene yielded he co esponding 1,5-naph hy idines 5,7and 9
(Scheme 1, Figu e 1) and 1,8-naph hy idines 11,13 and 15 (Scheme 2, Figu e 2), espec i ely,
as p e iously epo ed [
26
–
28
]. The desc ibed me hodologies ep esen an easy and e icien
s a egy o he p epa a ion o hese compounds con aining a wide ange o elec on-
eleasing and elec on-wi hd awing subs i uen s.
2.2. In Silico ADME
In silico ADME s udies, also known as compu e -based s udies, [
29
–
32
] play a c ucial
ole in he d ug disco e y and de elopmen p ocess. ADME s ands o abso p ion, dis ibu-
ion, me abolism and exc e ion, which a e key ac o s ha de e mine he pha macokine ics
and e icacy o a d ug. In silico ADME s udies in ol e he use o compu a ional models
and algo i hms o p edic he ADME p ope ies o a d ug which no only sa es ime and
esou ces, bu also helps in he iden i ica ion o po en ial sa e y conce ns.
Molecula p ope ies such as he pa i ion coe icien (Log P o/w), molecula weigh ,
hyd ogen bond dono s and accep o s, opological pola su ace a ea (TPSA) and iola ion
o Lipinski’s ule o i e we e assessed (Table 1).
Molecules 2024,29, 74 4 o 16
Table 1. Physicochemical p ope ies and d ug-likeness o ch omeno[4,3-b][1,5]naph hy idine and
ch omeno[4,3-b][1,8]naph hy idine de i a i es. #-numbe .
Compound LogP Mol. W . H-Dono H-Accep o TPSA Lipinski #Viola ions
4a 2.71 314.38 1 2 34.15 0
4c 2.76 344.41 1 3 43.38 0
6a 2.51 328.36 1 3 51.22 0
6b 2.69 407.26 1 3 51.22 0
6c 3.02 358.39 1 4 60.45 0
8a 3.11 467.58 1 3 70.68 0
8b 3.73 546.48 1 3 70.68 2
8c 3.81 497.61 1 4 79.91 0
8d 3.81 546.48 1 3 70.68 2
5a 3.26 310.35 0 3 35.01 0
5b 3.60 389.24 0 3 35.01 0
5c 3.60 340.37 0 4 44.24 0
7a 2.80 324.33 0 4 55.99 0
7b 3.12 403.23 0 4 55.99 0
7c 3.19 354.36 0 5 65.22 0
9a 3.70 463.55 0 4 71.54 0
9b 3.87 542.45 0 4 71.54 2
9c 3.97 493.58 0 5 80.77 0
9d 3.52 542.45 0 4 71.54 2
10a 3.05 314.38 1 2 34.15 0
10b 2.76 332.37 1 3 34.15 0
10c 2.92 328.41 1 2 34.15 0
12a 2.56 328.36 1 3 51.22 0
12b 2.94 407.26 1 3 51.22 0
14a 3.38 467.58 1 3 70.68 0
11a 2.99 310.35 0 3 35.01 0
11b 3.08 328.34 0 4 35.01 0
11c 3.27 340.37 0 4 44.24 0
13a 2.61 324.33 0 4 55.99 0
13b 3.00 403.23 0 4 55.99 1
13c 2.69 342.32 0 5 55.99 0
15a 3.48 463.55 0 4 71.54 0
15b 3.70 542.45 0 4 71.54 2
In gene al, a d ug candida e wi h a LogP alue be ween 0 and 5 is conside ed o ha e
a o able ADME p ope ies. A LogP alue ha is oo low may indica e poo lipid solubili y,
which can a ec he abso p ion and dis ibu ion o he d ug in he body. A LogP alue ha
is oo high may indica e poo aqueous solubili y, which can lead o poo bioa ailabili y
and po en ial oxici y due o he accumula ion o he d ug in a y issues. In he case o
ou compounds, hey ha e a LogP alue be ween 2.5 and 4 (wi h a mean o 3.12), so hey
would all wi hin he a o able ange men ioned abo e.
TPSA, o he opological pola su ace a ea, is ano he commonly used pa ame e in
d ug disco e y and de elopmen o p edic he ADME p ope ies o a d ug candida e.
TPSA is a measu e o he pola su ace a ea o a compound, which is impo an o i s
in e ac ion wi h biological a ge s and i s abili y o c oss biological memb anes. In gene al,
a d ug candida e wi h a TPSA alue be ween 20 and 140 Å
2
is conside ed o ha e a o able
ADME p ope ies. In Table 1, all he compounds es ed in his wo k all wi hin he a o able
ange o good o al bioa ailabili y.
Finally, Lipinski’s Rule o Fi e [
33
], a widely used ule in d ug disco e y o assess
he d ug-like p ope ies o a compound, was pe o med o s udy ou compounds. I
is ecommended ha he o ally ac i e d ug candida e should no ha e mo e han one
iola ion o Lipinski’s ule. All compounds es ed excep compounds 8b,8d,9b,9d and
15b mee he es ablished c i e ia (Table 1).
The abso p ion o a d ug is a c i ical ac o in de e mining i s e icacy and sa e y.
I e e s o he p ocess by which a d ug en e s he bloods eam and eaches i s a ge
si e o ac ion. Unde s anding he ac o s ha in luence d ug abso p ion is essen ial o
op imizing d ug he apy and minimizing ad e se e ec s. Because o his, he abso p ion o
Molecules 2024,29, 74 5 o 16
d ug was e alua ed based on aqueous solubili y, in es inal abso p ion and pe meabili y
(Table 2). Thus, he aqueous solubili y (Log S) o all compounds anges om
−
7.48
o
−4.40 log mol/L
, which shows he mode a e solubili y in wa e o he syn hesized
compounds. In addi ion, all o hem show in es inal abso p ion abo e 93%, which is
in e es ing because mos o ally adminis e ed d ugs a e abso bed mainly h ough he small
in es ine due o i s la ge su ace a ea. Finally, he Caco-2 pe meabili y, he loga i hm o he
appa en pe meabili y coe icien (log Papp > 8
×
10
−6
cm/s), was p edic ed. Since he
compound is conside ed o ha e high Caco-2 pe meabili y i he p edic ed alue is >0.90,
in ou case, all he syn hesized compounds ha e high Caco-2 pe meabili y.
Finally, he olume o dis ibu ion (VDss), blood–b ain ba ie pe meabili y (BBB
pe meabili y) [
34
,
35
] and he ac ion o unbound o he syn hesized compounds we e
u he assessed. Conside ing ha a Log VDss > 0.45 L/kg indica es a high olume o
d ug dis ibu ion, we can say ha all syn hesized compounds ha e low o mode a e
olume o dis ibu ion in issues, excep o compound 5a. On he o he hand, a com-
pound is said o be eadily pe meable ac oss he BBB i he p edic ed alue o log BBB
is >0.3 and poo ly dis ibu ed i he alue is <
−
1. In ou es ima ions i can be seen ha
ch omeno[4,3-b][1,5]naph hy idines (compounds 4,5,6and 7) and ch omeno[4,3-b][1,8]
naph hy idines (compounds 10,11,12 and 13) show excellen BBB pa ame e s bu hose
used o quinolines (compounds 8,9,14 and 15) a e no easily pe meable h ough he BBB.
Addi ional ac o s o conside in d ug de elopmen include me aboli e o ma ion medi-
a ed by cy och ome P450 (CYP) enzyme ac i i ies [
36
] and d ug clea ance (CL) [
37
]. CYP450
enzymes a e a amily o heme-con aining enzymes ha a e in ol ed in he me abolism o
a wide ange o endogenous compounds and xenobio ics. These enzymes play a c ucial
ole in d ug me abolism, and hei ac i i y can in luence he e icacy and sa e y o many
he apeu ic agen s. Compounds we e s udied as possible CYP2D6, CYP3A4, CYP1A2,
CYP2C19 and CYP2C9 enzyme inhibi o s. No ewo hy, all compounds show he abili y
o inhibi he CYP2C19 enzyme. Compounds wi h a me hoxy g oup in hei s uc u e
(5c,9c and 11c) show inhibi ion o CYP2C9, which is p ima ily exp essed in he li e and
plays a c i ical ole in he me abolism o nume ous d ugs. Con e sely, he o al clea ance
is p ima ily a combina ion o hepa ic as well as enal clea ance and is measu ed by he
p opo ionali y cons an CL o in log(mL/min/kg). The p edic ed alue o all he syn-
hesized compounds shows low CL o anging om
−
0.154 o 1.91. These esul s clea ly
indica e ha all hese compounds show app op ia e pha macokine ic p ope ies and can
be conside ed an ileishmanial agen s.
The boiled egg model [
38
] isually ep esen s some o he ADME pa ame e s o he
e alua ed 1,5- and 1,8-naph hy idines (Figu e 3). Compounds ha would c oss bo h he
blood–b ain ba ie (BBB) and human gas oin es inal ac (HIA) a e placed in he yolk and
hose wi h only HIA-abso p ion in he whi e. Mos o he es ed compounds ha e posi i e
BBB and HIA pe meabili y, excep compounds 8,9a,14a and 15a which only show posi i e
HIA and compounds 9b–dand 15b which do no show esul s o ei he pa ame e .
P-glycop o ein (PGP) is a anspo e p o ein ound in he memb ane o cells in a ious
issues, such as he gas oin es inal ac , li e and kidneys [
39
,
40
]. I s main unc ion is o
anspo o eign subs ances, such as d ugs, ou o cells. The e o e, i may be desi able o
a d ug o be a PGP subs a e, as i can help p o ec agains oxici y o unwan ed side e ec s.
Do s colo ed blue co espond o molecules ha a e p edic ed o be P-glycop o ein subs a es
(PGP+) and a e he e o e ac i ely pumped om he b ain o in o he gas oin es inal
lumen (Figu e 3). I hey a e p edic ed no o be P-glycop o ein subs a es (PGP
−
), he
co esponding do appea s in ed. In he case o ou es ed compounds, i should be no ed
ha quinolino naph hy idine de i a i es 8,9,14 and 15 and ch omeno naph hy idine
de i a i es 7and 13 do no quali y as PGP-subs a e candida es. In con as , all he o he
compounds, de i a i es 4,5,6,10,11 and 12, a e sui able as PGP subs a e candida es.
Molecules 2024,29, 74 6 o 16
Table 2. Resul s o he ADME p edic i e s udy o ch omeno[4,3-b][1,5]naph hy idine and ch omeno[4,3-b][1,8]naph hy idine de i a i es.
Compound Log S
(Log mol/L)
Caco-2 Pe m.
(Log Paap in
10−6cm/s)
In . Abs.
(% abs) VDss (L/kg) F ac . Unb.
(Fu.)
BBB
Pe meabili y
(log BB)
CYP1A2
Inhibi o
CYP2C19
Inhibi o
CYP2C9
Inhibi o
CYP2D6
Inhibi o
CYP3A4
Inhibi o
To al Clea ence
Nume ic (Log
mL/min/kg)
4a −4.73 1.779 96.038 0.312 0.027 Yes No Yes No Yes Yes 0.180
4c −5.00 1.195 94.748 −0.121 0.109 Yes No Yes No Yes Yes 0.493
6a −4.47 1.268 98,408 0.190 0.003 Yes No Yes No Yes No 0.003
6b −5.59 1.210 93.336 −0.277 0.122 Yes No Yes No No No −0.024
6c −4.75 1.182 95.066 −0.303 0.112 Yes No Yes No Yes Yes 0.379
8a −6.21 1.153 98.895 −0.719 0.081 No No Yes No Yes Yes 0.286
8b −7.32 1.136 97.026 −0.669 0.073 No No Yes No No No −0.154
8c −6.49 1.063 95.572 −0.961 0.238 No No Yes No Yes Yes 0.392
8d −7.12 1.140 97.414 −0.810 0.087 No No Yes No Yes No −0.103
5a −4.92 1.660 100 0.525 0.26 Yes Yes Yes No Yes Yes 0.834
5b −6.03 1.143 96.994 0.099 0.354 Yes Yes Yes No No Yes 0.135
5c −5.18 1.122 98.725 0.025 0.343 Yes Yes Yes Yes Yes Yes 0.881
7a −5.00 1.410 100 0.403 0.368 Yes Yes Yes No No No 0.921
7b −6.11 1.137 98.66 0.193 0.363 Yes Yes Yes No No No 0.363
7c −5.27 1.098 100 0.108 0.352 Yes Yes Yes Yes No Yes 1.091
9a −6.36 1.119 100 −0.312 0.334 No Yes Yes No No Yes 0.819
9b −7.48 1.114 100 −0.293 0.333 No Yes Yes No No No 0.137
9c −6.64 1.167 99.946 −0.574 0.393 No No Yes Yes No Yes 0.949
9d −7.27 1.106 100 −0.386 0.338 No Yes Yes No No No 0.123
10a −4.92 1.765 96.706 0.142 0.035 Yes No Yes No Yes No 0.577
10b −5.07 1.803 94.633 −0.151 0.031 Yes No Yes No Yes No 0.268
10c −5.21 1.054 95.189 0.014 0.027 Yes No Yes No Yes No 0.601
12a −4.66 1.247 97.879 −0.184 0 Yes No Yes No Yes No 0.467
12b −5.56 1.044 94.333 −0.230 0.007 Yes No Yes No No No −0.092
14a −6.40 0.431 98.124 −0.935 0.086 No No Yes No Yes Yes 0.384
11a −5.07 1.550 100 0.297 0.285 Yes Yes Yes No Yes Yes 0.928
11b −5.23 1.659 98.699 −0.046 0.306 Yes Yes Yes No No Yes 0.777
11c −5.13 1.121 100 0.131 0.294 Yes Yes Yes Yes Yes Yes 0.905
13a −5.16 1.393 100 0.273 0.344 Yes Yes Yes No No No 1.068
13b −6.06 1.101 99.395 0.046 0.349 Yes Yes Yes No No No 0.046
13c −5.31 1.430 100 0.028 0.346 Yes Yes Yes No No No 1.006
15a −6.51 1.000 100 −0.464 0.335 No Yes Yes No No Yes 0.851
15b −7.43 0.974 100 −0.596 0.341 No No Yes No No No −0.026
Molecules 2024,29, 74 7 o 16
Molecules 2023, 28, x FOR PEER REVIEW 7 o 17
Addi ional ac o s o conside in d ug de elopmen include me aboli e o ma ion
media ed by cy och ome P450 (CYP) enzyme ac i i ies [36] and d ug clea ance (CL) [37].
CYP450 enzymes a e a amily o heme-con aining enzymes ha a e in ol ed in he me-
abolism o a wide ange o endogenous compounds and xenobio ics. These enzymes play
a c ucial ole in d ug me abolism, and hei ac i i y can in luence he e icacy and sa e y
o many he apeu ic agen s. Compounds we e s udied as possible CYP2D6, CYP3A4,
CYP1A2, CYP2C19 and CYP2C9 enzyme inhibi o s. No ewo hy, all compounds show he
abili y o inhibi he CYP2C19 enzyme. Compounds wi h a me hoxy g oup in hei s uc-
u e (5c, 9c and 11c) show inhibi ion o CYP2C9, which is p ima ily exp essed in he li e
and plays a c i ical ole in he me abolism o nume ous d ugs. Con e sely, he o al clea -
ance is p ima ily a combina ion o hepa ic as well as enal clea ance and is measu ed by
he p opo ionali y cons an CL o in log(mL/min/kg). The p edic ed alue o all he syn-
hesized compounds shows low CL o anging om −0.154 o 1.91. These esul s clea ly
indica e ha all hese compounds show app op ia e pha macokine ic p ope ies and can
be conside ed an ileishmanial agen s.
The boiled egg model [38] isually ep esen s some o he ADME pa ame e s o he
e alua ed 1,5- and 1,8-naph hy idines (Figu e 3). Compounds ha would c oss bo h he
blood–b ain ba ie (BBB) and human gas oin es inal ac (HIA) a e placed in he yolk
and hose wi h only HIA-abso p ion in he whi e. Mos o he es ed compounds ha e
posi i e BBB and HIA pe meabili y, excep compounds 8, 9a, 14a and 15a which only
show posi i e HIA and compounds 9b–d and 15b which do no show esul s o ei he
pa ame e .
P-glycop o ein (PGP) is a anspo e p o ein ound in he memb ane o cells in a -
ious issues, such as he gas oin es inal ac , li e and kidneys [39,40]. I s main unc ion
is o anspo o eign subs ances, such as d ugs, ou o cells. The e o e, i may be desi able
o a d ug o be a PGP subs a e, as i can help p o ec agains oxici y o unwan ed side
e ec s. Do s colo ed blue co espond o molecules ha a e p edic ed o be P-glycop o ein
subs a es (PGP+) and a e he e o e ac i ely pumped om he b ain o in o he gas oin-
es inal lumen (Figu e 3). I hey a e p edic ed no o be P-glycop o ein subs a es (PGP−),
he co esponding do appea s in ed. In he case o ou es ed compounds, i should be
no ed ha quinolino naph hy idine de i a i es 8, 9, 14 and 15 and ch omeno naph hy-
idine de i a i es 7 and 13 do no quali y as PGP-subs a e candida es. In con as , all he
o he compounds, de i a i es 4, 5, 6, 10, 11 and 12, a e sui able as PGP subs a e candi-
da es.
Figu e 3. Boiled egg illus a ion o 1,5-naph hy idines (A) and 1,8-naph hy idines (B). The whi e
egion is he physicochemical space o molecules wi h he highes p obabili y o being abso bed by
he gas oin es inal ac , and he yellow egion (yolk) is he physicochemical space o molecules
wi h he highes p obabili y o pe mea ing in o he b ain. In he illus a ion, se e al compounds a e
ep esen ed wi h he same spo .
Figu e 3. Boiled egg illus a ion o 1,5-naph hy idines (A) and 1,8-naph hy idines (B). The whi e
egion is he physicochemical space o molecules wi h he highes p obabili y o being abso bed by
he gas oin es inal ac , and he yellow egion (yolk) is he physicochemical space o molecules
wi h he highes p obabili y o pe mea ing in o he b ain. In he illus a ion, se e al compounds a e
ep esen ed wi h he same spo .
2.3. An ileishmanial E ec
The 1,5- and 1,8- used naph hy idine de i a i es we e e alua ed as an ileishmanial agen s
in assays using wo o ms o L. in an um amas igo es: axenic amas igo es eco e ed om bone
ma ow cells o in ec ed BALB/c mice and in amac ophagic amas igo es ob ained om splenic
explan s o he same subjec s. The L. in an um s ain used is a gene ically modi ied s ain
p e iously enginee ed by ou g oup o cons i u i ely exp ess he in a ed p o ein iRFP om
Rhodopseudomonas palus is bac e iophy och ome (iRFP L. in an um) [
41
,
42
]. This s ain allows
moni o ing he iabili y o bo h o ms o he pa asi e by quan i ying he luo escence emi ed a
700 nm by he iRFP p o ein p oduced by li ing amas igo es [42].
Amas igo es ob ained om mouse bone ma ow cells we e used o pe o m a i s
sc eening wi h he comple e se ies o used 1,5- and 1,8-naph hy idines. Molecules ha
did no inhibi amas igo e g ow h a a concen a ion o 25
µ
M we e conside ed o be
poo ly ac i e and we e disca ded o subsequen assays on in amac ophagic amas igo es
ex i o. In he case o he es s ca ied ou wi h in amac ophage amas igo es, he com-
pounds ha showed ac i i y a a concen a ion lowe han 20
µ
M we e selec ed o pe o m
dose– esponse cu es, ob aining hei co esponding EC
50
alues. The oxici y o hese
selec ed compounds was assessed in non-in ec ed splenic cells and he cy o oxic con-
cen a ion (CC
50
) alue ob ained was used o calcula e he selec i e index (SI). The o al
ole abili y o hese same compounds was es ed by calcula ing he iabili y o mu ine
in es inal o ganoids exposed o wo concen a ions (50 and 25 µM).
Table 3shows he esul s ob ained wi h he ch omeno[4,3-b][1,5]naph hy idines and
ch omeno[4,3-b][1,5]naph hy idines-6-one de i a i es.
O hese compounds, only wo we e candida es o be es ed in in amac ophagic amas ig-
o es (6b and 7c), ob aining EC
50
alues o 12.86
±
0.82
µ
M and 36.99
±
2.87
µ
M, espec i ely.
Howe e , he SI o bo h molecules was no e y high (4.3 and 2.6), and he oxici y in mu ine
in es inal o ganoids was simila o ha obse ed in mouse splenic mac ophages cells.
The eplacemen o he oxygen a om by ni ogen in hese ypes o molecules p o-
duced mo e ac i e compounds (Table 4). In his g oup, we ound he mo e ac i e and
selec i e compounds such as molecules 8b and 8c, wi h EC
50
alues o 5.53
±
0.26
µ
M and
4.93 ±0.35 µM
, espec i ely, and SI alues o >18.1 and >20.2. In he case o compound 8c
he iabili y o mu ine in es inal o ganoids was o 100% a 50
µ
M. Compound 9b has he
peculia i y o being less oxic in mu ine in es inal o ganoids han in splenic cells, ob aining
an SI o only 4.6 while he iabili y in o ganoids was o 100% a 50
µ
M. This esul could be
ela ed o he nega i e HIA pe meabili y (Figu e 3) o his compound. Ano he poin o
highligh among he compounds o his g oup is he g ea e ac i i y o compounds ha
lack he a oma ic naph hy idine ing e sus hose ha ha e he a oma ici y.
Molecules 2024,29, 74 8 o 16
Table 3. Biological ac i i y o ch omeno[4,3-b][1,5]naph hy idines and ch omeno[4,3-b][1,5]naph hy idine-
6-ones. The an ileishmanial e ec was e alua ed in axenic IRFP-L.in an um amas igo es om bone
ma ow cells and subsequen ly in in amac ophagic IRFP-L.in an um om mu ine spleens. Cy o oxic-
i y was assessed in unin ec ed splenic mac ophages and in es inal ole ance in
mu ine gu o ganoids.
Molecules 2023, 28, x FOR PEER REVIEW 8 o 17
2.3. An ileishmanial E ec
The 1,5- and 1,8- used naph hy idine de i a i es we e e alua ed as an ileishmanial
agen s in assays using wo o ms o L. in an um amas igo es: axenic amas igo es eco e ed
om bone ma ow cells o in ec ed BALB/c mice and in amac ophagic amas igo es ob-
ained om splenic explan s o he same subjec s. The L. in an um s ain used is a gene -
ically modi ied s ain p e iously enginee ed by ou g oup o cons i u i ely exp ess he
in a ed p o ein iRFP om Rhodopseudomonas palus is bac e iophy och ome (iRFP L. in-
an um) [41,42]. This s ain allows moni o ing he iabili y o bo h o ms o he pa asi e by
quan i ying he luo escence emi ed a 700 nm by he iRFP p o ein p oduced by li ing
amas igo es [42].
Amas igo es ob ained om mouse bone ma ow cells we e used o pe o m a i s
sc eening wi h he comple e se ies o used 1,5- and 1,8-naph hy idines. Molecules ha
did no inhibi amas igo e g ow h a a concen a ion o 25 µM we e conside ed o be
poo ly ac i e and we e disca ded o subsequen assays on in amac ophagic amas igo es
ex i o. In he case o he es s ca ied ou wi h in amac ophage amas igo es, he com-
pounds ha showed ac i i y a a concen a ion lowe han 20 µM we e selec ed o pe o m
dose– esponse cu es, ob aining hei co esponding EC50 alues. The oxici y o hese se-
lec ed compounds was assessed in non-in ec ed splenic cells and he cy o oxic concen a-
ion (CC50) alue ob ained was used o calcula e he selec i e index (SI). The o al ole abil-
i y o hese same compounds was es ed by calcula ing he iabili y o mu ine in es inal
o ganoids exposed o wo concen a ions (50 and 25 µM).
Table 3 shows he esul s ob ained wi h he ch omeno[4,3-b][1,5]naph hy idines and
ch omeno[4,3-b][1,5]naph hy idines-6-one de i a i es.
Table 3. Biological ac i i y o ch omeno[4,3-b][1,5]naph hy idines and ch omeno[4,3-b][1,5]naph-
hy idine-6-ones. The an ileishmanial e ec was e alua ed in axenic IRFP-L.in an um amas igo es
om bone ma ow cells and subsequen ly in in amac ophagic IRFP-L.in an um om mu ine
spleens. Cy o oxici y was assessed in unin ec ed splenic mac ophages and in es inal ole ance in
mu ine gu o ganoids.
Compound
R1
Axenic
Amas igo es a
In acellula
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM)
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
4a
H
>25
n.d.*
n.d
-
4c
OMe
>25
n.d.
n.d.
-
5a
H
>25
n.d.
n.d.
-
5b
B
>25
n.d.
n.d.
-
5c
OMe
>25
n.d.
n.d.
-
6a
H
>25
n.d.
n.d.
-
6b
B
<25
12.86 ± 0.82
55.66 ± 6.42
4.3
60 ± 7
72 ± 5
6c
OMe
>25
n.d.
n.d.
-
7a
H
>25
n.d.
n.d.
-
7b
B
>25
n.d.
n.d.
-
7c
OMe
<25
36.99 ± 2.87
97.68 ± 1.77
2.6
85 ± 13
99 ± 4
* n.d. no de e mined. a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e con-
side ed no e y ac i e and we e disca ded o subsequen assays.
Compound R1Axenic
Amas igo es a
In acellula
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM)
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
4a H >25 n.d.* n.d -
4c OMe >25 n.d. n.d. -
5a H >25 n.d. n.d. -
5b B >25 n.d. n.d. -
5c OMe >25 n.d. n.d. -
6a H >25 n.d. n.d. -
6b B <25 12.86 ±0.82 55.66 ±6.42 4.3 60 ±7 72 ±5
6c OMe >25 n.d. n.d. -
7a H >25 n.d. n.d. -
7b B >25 n.d. n.d. -
7c OMe <25 36.99 ±2.87 97.68 ±1.77 2.6 85 ±13 99 ±4
* n.d. no de e mined.
a
Compounds wi h inhibi ion a concen a ions highe han 25
µ
M we e conside ed no
e y ac i e and we e disca ded o subsequen assays.
Table 4. Biological ac i i y o quinolino[4,3-b][1,5]naph hy idines. The an ileishmanial e ec was
e alua ed in axenic IRFP-L.in an um amas igo es om bone ma ow cells and subsequen ly in in a-
mac ophagic IRFP-L. in an um om mu ine spleens. Cy o oxici y was assessed in unin ec ed splenic
mac ophages and in es inal ole ance in mu ine gu o ganoids.
Molecules 2023, 28, x FOR PEER REVIEW 9 o 17
O hese compounds, only wo we e candida es o be es ed in in amac ophagic
amas igo es (6b and 7c), ob aining EC50 alues o 12.86 ± 0.82 µM and 36.99 ± 2.87 µM,
espec i ely. Howe e , he SI o bo h molecules was no e y high (4.3 and 2.6), and he
oxici y in mu ine in es inal o ganoids was simila o ha obse ed in mouse splenic mac-
ophages cells.
The eplacemen o he oxygen a om by ni ogen in hese ypes o molecules p o-
duced mo e ac i e compounds (Table 4). In his g oup, we ound he mo e ac i e and se-
lec i e compounds such as molecules 8b and 8c, wi h EC50 alues o 5.53 ± 0.26 µM and
4.93 ± 0.35 µM, espec i ely, and SI alues o >18.1 and >20.2. In he case o compound 8c
he iabili y o mu ine in es inal o ganoids was o 100% a 50 µM. Compound 9b has he
peculia i y o being less oxic in mu ine in es inal o ganoids han in splenic cells, ob ain-
ing an SI o only 4.6 while he iabili y in o ganoids was o 100% a 50 µM. This esul
could be ela ed o he nega i e HIA pe meabili y (Figu e 3) o his compound. Ano he
poin o highligh among he compounds o his g oup is he g ea e ac i i y o com-
pounds ha lack he a oma ic naph hy idine ing e sus hose ha ha e he a oma ici y.
Table 4. Biological ac i i y o quinolino[4,3-b][1,5]naph hy idines. The an ileishmanial e ec was
e alua ed in axenic IRFP-L.in an um amas igo es om bone ma ow cells and subsequen ly in in a-
mac ophagic IRFP-L. in an um om mu ine spleens. Cy o oxici y was assessed in unin ec ed splenic
mac ophages and in es inal ole ance in mu ine gu o ganoids.
Compound
R1
Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM) *
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
8a
H
<25
5.30 ± 0.71
45.03 ± 3.91
8.5
89 ± 10
93 ± 7
8b
4-B
<25
5.53 ± 0.26
>100
>18.1
98 ± 13
>100
8c
6-OMe
<25
4.93 ± 0.35
>100
>20.28
>100
>100
8d
6-B
<25
34.36 ± 9.70
>50
1.5
3 ± 1
4 ± 1
9a
H
>25
n.d.
n.d.
-
9b
4-B
<25
3.63 ± 0.15
16.89 ± 0.86
4.6
>100
>100
9c
6-OMe
>25
>20
n.d.
-
9d
6-B
>25
>20
n.d.
-
* n.d. no de e mined. a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e con-
side ed no e y ac i e and we e disca ded o subsequen assays.
Fou ch omeno[4,3-b][1,8]naph hy idines and ch omeno[4,3-b][1,8]naph hy idine-6-
one de i a i es we e es ed in in amac ophagic amas igo es (10b, 11a, 13a and 13b, Table
5). O hese ou , he highes SI was ob ained wi h 11a (EC50 = 7.70 ± 0.29 µM; SI ≥ 13).
Compound 10b was less oxic in in es inal o ganoids han in mouse splenic mac ophages,
showing 90.04% o iabili y in o ganoids a 50 µM agains o a CC50 alue o 50.72 ± 0.99
µM in mouse splenic mac ophages. The in silico p edic ions o his compound ha e no
iden i ied p oblems ela i e o i s HIA abso p ion. Con a y o 1,5-naph hy idines (Table
4) compounds in his g oup we e mo e ac i e wi h he a oma ic naph hy idine ing.
Compound
R1Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM) *
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
8a H <25 5.30 ±0.71 45.03 ±3.91 8.5 89 ±10 93 ±7
8b 4-B <25 5.53 ±0.26 >100 >18.1 98 ±13 >100
8c 6-OMe <25 4.93 ±0.35 >100 >20.28 >100 >100
8d 6-B <25 34.36 ±9.70 >50 1.5 3 ±1 4 ±1
9a H >25 n.d. n.d. -
9b 4-B <25 3.63 ±0.15 16.89 ±0.86 4.6 >100 >100
9c 6-OMe >25 >20 n.d. -
9d 6-B >25 >20 n.d. -
* n.d. no de e mined.
a
Compounds wi h inhibi ion a concen a ions highe han 25
µ
M we e conside ed no
e y ac i e and we e disca ded o subsequen assays.
Fou ch omeno[4,3-b][1,8]naph hy idines and ch omeno[4,3-b][1,8]naph hy idine-6-
one de i a i es we e es ed in in amac ophagic amas igo es (10b,11a,13a and 13b, Table 5).
O hese ou , he highes SI was ob ained wi h 11a (EC
50
= 7.70
±
0.29
µ
M; SI
≥
13). Com-
pound 10b was less oxic in in es inal o ganoids han in mouse splenic mac ophages, show-
Molecules 2024,29, 74 9 o 16
ing 90.04% o iabili y in o ganoids a 50
µ
M agains o a CC
50
alue o
50.72 ±0.99 µM
in mouse splenic mac ophages. The in silico p edic ions o his compound ha e no
iden i ied p oblems ela i e o i s HIA abso p ion. Con a y o 1,5-naph hy idines (Table 4)
compounds in his g oup we e mo e ac i e wi h he a oma ic naph hy idine ing.
Table 5. Biological ac i i y o ch omeno[4,3-b][1,8]naph hy idines and ch omeno[4,3-b][1,8]naph hy idine-
6-ones. The an ileishmanial e ec was e alua ed in axenic IRFP-L.in an um amas igo es om bone
ma ow cells and subsequen ly in in amac ophagic IRFP-L.in an um om mu ine spleens. Cy o oxic-
i y was assessed in unin ec ed splenic mac ophages and in es inal ole ance in
mu ine gu o ganoids.
Molecules 2023, 28, x FOR PEER REVIEW 10 o 17
Table 5. Biological ac i i y o ch omeno[4,3-b][1,8]naph hy idines and ch omeno[4,3-b][1,8]naph-
hy idine-6-ones. The an ileishmanial e ec was e alua ed in axenic IRFP-L.in an um amas igo es
om bone ma ow cells and subsequen ly in in amac ophagic IRFP-L.in an um om mu ine
spleens. Cy o oxici y was assessed in unin ec ed splenic mac ophages and in es inal ole ance in
mu ine gu o ganoids.
Compound
R1
R2
R3
Axenic
Amas igo
es a
In acelulla
Amas igo es
EC50 (µM)
Mouse splenic
Mac ophages
CC50 (µM) *
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
10a
-
H
H
>25
>20
n.d.
-
10b
-
F
H
<25
7.57 ± 0.44
50.72 ± 0.99
6.7
90 ± 22
99 ± 8
10c
-
Me
H
>25
>20
n.d.
-
11a
-
H
H
<25
7.70 ± 0.29
>100
>13
96 ± 1
92 ± 1
11b
-
F
H
>25
>20
n.d.
-
11c
-
H
OMe
>25
>20
n.d.
-
12a
H
H
-
>25
>20
n.d.
-
12b
B
H
-
>25
>20
n.d.
-
13a
H
H
-
<25
10.58 ± 1.21
34.65 ± 1.79
3.3
53 ± 5
87 ± 15
13b
B
H
-
<25
7.53 ± 0.97
>50
>6.6
61 ± 19
90 ± 5
13c
H
F
-
>25
>20
n.d.
-
* n.d. no de e mined. a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e con-
side ed no e y ac i e and we e disca ded o subsequen assays.
The h ee a ailable 1,8-de i a i es wi h ni ogen a oms ins ead o oxygen we e es ed
in in amac ophagic amas igo es (Table 6). Howe e , hei SI was low and he oxici y in
o ganoids was simila han ob ained wi h splenic cells.
Table 6. Biological ac i i y o quinolino[4,3-b][1,8] naph hy idines. The an ileishmanial e ec was
e alua ed in axenic IRFP-L.in an um amas igo es om bone ma ow cells and subsequen ly in in a-
mac ophagic IRFP-L.in an um om mu ine spleens. Cy o oxici y was assessed in unin ec ed splenic
mac ophages and in es inal ole ance in mu ine gu o ganoids.
Compound
R1
Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM)
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
14a
H
<25
9.34 ± 0.77
27.19 ± 1.55
2.9
56 ± 14
90 ± 9
15a
H
<25
67.43 ± 9.78
8.78 ± 2.21
0.1
15b
B
<25
6.16 ± 0.54
24.64 ± 3.59
4.0
13 ± 3
58 ± 16
a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e conside ed no e y ac i e
and we e disca ded o subsequen assays.
Compound
R1R2R3Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM) *
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
10a - H H >25 >20 n.d. -
10b - F H <25 7.57 ±0.44 50.72 ±0.99 6.7 90 ±22 99 ±8
10c - Me H >25 >20 n.d. -
11a - H H <25 7.70 ±0.29 >100 >13 96 ±1 92 ±1
11b - F H >25 >20 n.d. -
11c - H OMe >25 >20 n.d. -
12a H H - >25 >20 n.d. -
12b B H - >25 >20 n.d. -
13a H H - <25 10.58 ±1.21 34.65 ±1.79 3.3 53 ±5 87 ±15
13b B H - <25 7.53 ±0.97 >50 >6.6 61 ±19 90 ±5
13c H F - >25 >20 n.d. -
* n.d. no de e mined.
a
Compounds wi h inhibi ion a concen a ions highe han 25
µ
M we e conside ed no
e y ac i e and we e disca ded o subsequen assays.
The h ee a ailable 1,8-de i a i es wi h ni ogen a oms ins ead o oxygen we e es ed
in in amac ophagic amas igo es (Table 6). Howe e , hei SI was low and he oxici y in
o ganoids was simila han ob ained wi h splenic cells.
Table 6. Biological ac i i y o quinolino[4,3-b][1,8] naph hy idines. The an ileishmanial e ec was
e alua ed in axenic IRFP-L.in an um amas igo es om bone ma ow cells and subsequen ly in in a-
mac ophagic IRFP-L.in an um om mu ine spleens. Cy o oxici y was assessed in unin ec ed splenic
mac ophages and in es inal ole ance in mu ine gu o ganoids.
Molecules 2023, 28, x FOR PEER REVIEW 10 o 17
Table 5. Biological ac i i y o ch omeno[4,3-b][1,8]naph hy idines and ch omeno[4,3-b][1,8]naph-
hy idine-6-ones. The an ileishmanial e ec was e alua ed in axenic IRFP-L.in an um amas igo es
om bone ma ow cells and subsequen ly in in amac ophagic IRFP-L.in an um om mu ine
spleens. Cy o oxici y was assessed in unin ec ed splenic mac ophages and in es inal ole ance in
mu ine gu o ganoids.
Compound
R1
R2
R3
Axenic
Amas igo
es a
In acelulla
Amas igo es
EC50 (µM)
Mouse splenic
Mac ophages
CC50 (µM) *
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
10a
-
H
H
>25
>20
n.d.
-
10b
-
F
H
<25
7.57 ± 0.44
50.72 ± 0.99
6.7
90 ± 22
99 ± 8
10c
-
Me
H
>25
>20
n.d.
-
11a
-
H
H
<25
7.70 ± 0.29
>100
>13
96 ± 1
92 ± 1
11b
-
F
H
>25
>20
n.d.
-
11c
-
H
OMe
>25
>20
n.d.
-
12a
H
H
-
>25
>20
n.d.
-
12b
B
H
-
>25
>20
n.d.
-
13a
H
H
-
<25
10.58 ± 1.21
34.65 ± 1.79
3.3
53 ± 5
87 ± 15
13b
B
H
-
<25
7.53 ± 0.97
>50
>6.6
61 ± 19
90 ± 5
13c
H
F
-
>25
>20
n.d.
-
* n.d. no de e mined. a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e con-
side ed no e y ac i e and we e disca ded o subsequen assays.
The h ee a ailable 1,8-de i a i es wi h ni ogen a oms ins ead o oxygen we e es ed
in in amac ophagic amas igo es (Table 6). Howe e , hei SI was low and he oxici y in
o ganoids was simila han ob ained wi h splenic cells.
Table 6. Biological ac i i y o quinolino[4,3-b][1,8] naph hy idines. The an ileishmanial e ec was
e alua ed in axenic IRFP-L.in an um amas igo es om bone ma ow cells and subsequen ly in in a-
mac ophagic IRFP-L.in an um om mu ine spleens. Cy o oxici y was assessed in unin ec ed splenic
mac ophages and in es inal ole ance in mu ine gu o ganoids.
Compound
R1
Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM)
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
14a
H
<25
9.34 ± 0.77
27.19 ± 1.55
2.9
56 ± 14
90 ± 9
15a
H
<25
67.43 ± 9.78
8.78 ± 2.21
0.1
15b
B
<25
6.16 ± 0.54
24.64 ± 3.59
4.0
13 ± 3
58 ± 16
a Compounds wi h inhibi ion a concen a ions highe han 25 µM we e conside ed no e y ac i e
and we e disca ded o subsequen assays.
Compound R1Axenic
Amas igo es a
In acelulla
Amas igo es
EC50 (µM)
Mouse Splenic
Mac ophages
CC50 (µM)
SI
In es inal O ganoids
50 µM
(%Viabili y)
25 µM
(%Viabili y)
14a H <25 9.34 ±0.77 27.19 ±1.55 2.9 56 ±14 90 ±9
15a H <25 67.43 ±9.78 8.78 ±2.21 0.1
15b B <25 6.16 ±0.54 24.64 ±3.59 4.0 13 ±3 58 ±16
a
Compounds wi h inhibi ion a concen a ions highe han 25
µ
M we e conside ed no e y ac i e and we e
disca ded o subsequen assays.
Molecules 2024,29, 74 16 o 16
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au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .
