Palladium-mediated synthesis and biological evaluation of C-10b substituted Dihydropyrrolo[1,2-b]isoquinolines as antileishmanial agents

Palladium-media ed syn hesis and biological e alua ion o C-10b
subs i u ed Dihyd opy olo[1,2-b]isoquinolines as an ileishmanial
agen s
I a xe Ba bolla
a
, Leidi He n
andez-Su
a ez
a
, Vi iana Que edo-Tumailli
a
,
b
,
c
,
Deyani Nocedo-Mena
a
, Sonia A asa e
a
, Ma ía Auxiliado a Dea-Ayuela
d
,
Humbe o Gonz
alez-Díaz
a
,
e
,
,
***
, Nu ia So omayo
a
,
**
, Es he Le e
a
,
*
a
Depa amen o de Química O g
anica e Ino g
anica, Facul ad de Ciencia y Tecnología, Uni e sidad Del País Vasco / Euskal He iko Unibe si a ea UPV/EHU,
Apdo. 644, 48080, Bilbao, Spain
b
RNASA-IMEDIR, Compu e Science Facul y, Uni e si y o A Co u~
na, 15071, A Co u~
na, Spain
c
Uni e sidad Es a al Amaz
onica UEA, Puyo, 160150, Pas aza, Ecuado
d
Depa amen o de Fa macia, Facul ad de Ciencias de La Salud, Uni e sidad CEU Ca denal He e a, Edificio Semina io S/n, 46113, Moncada, Valencia, Spain
e
Basque Cen e o Biophysics CSIC-UPV/EHU, Uni e si y o he Basque Coun y UPV/EHU, 48940, Bilbao, Spain
IKERBASQUE, Basque Founda ion o Science, 48011, Bilbao, Spain
a icle in o
A icle his o y:
Recei ed 15 Feb ua y 2021
Recei ed in e ised o m
12 Ma ch 2021
Accep ed 5 Ap il 2021
A ailable online 16 Ap il 2021
Keywo ds:
Leishmaniasis
Py oloisoquinoline
Palladium
Cascade eac ions
Machine lea ning
Chemin o ma ics
abs ac
The de elopmen o new molecules o he ea men o leishmaniasis is, a neglec ed pa asi ic disease, is
u gen as cu en an i-leishmanial he apeu ics a e hampe ed by d ug oxici y and esis ance. The py -
olo[1,2-b]isoquinoline co e was selec ed as s a ing poin , and palladium-ca alyzed Heck-ini ia ed
cascade eac ions we e de eloped o he syn hesis o a se ies o C-10 subs i u ed de i a i es. Thei
in i o leishmanicidal ac i i y agains isce al (L. dono ani) and cu aneous (L. amazonensis) leishmaniasis
was e alua ed. The bes ac i i y was ound, in gene al, o he 10-a ylme hyl subs i u ed py oloiso-
quinolines. In pa icula , 2ad (IC
50
¼3.30
m
M, SI >77.01) and 2bb (IC
50
¼3.93
m
M, SI >58.77) we e
app oxima ely 10- old mo e po en and selec i e han he d ug o e e ence (mil e osine), agains
L. amazonensis on in i o p omas igo e assays, while 2ae was he mo e ac i e compound in he in i o
amas igo e assays (IC
50
¼33.59
m
M, SI >8.93). No ably, almos all compounds showed low cy o oxici y,
CC
50
>100
m
g/mL in J774 cells, highes es ed dose. In addi ion, we ha e de eloped he fi s Pe u ba ion
Theo y Machine Lea ning (PTML) algo i hm able o p edic simul aneously mul iple biological ac i i y
pa ame e s (IC
50
,K
i
,e c.) s. any Leishmania species and a ge p o ein, wi h high alues o specifici y
(>98%) and sensi i i y (>90%) in bo h aining and alida ion se ies. The e o e, his model may be use ul
o educe ime and assay cos s (ma e ial and human esou ces) in he d ug disco e y p ocess.
©2021 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY
license (h p://c ea i ecommons.o g/licenses/by/4.0/).
1. In oduc ion
Leishmaniasis is a neglec ed pa asi ic disease, endemic in abou
100 coun ies, wi h mo bidi y and mo ali y inc easing daily. The
disease is caused by p o ozoan pa hogens o he Leishmania genus
ha a e ansmi ed by sandflies. Leishmania spp exis s in wo
mo phologically dis inc o ms: a mo ile flagella ed o m (p o-
mas igo es) and an in acellula non-flagella ed o m (amas i-
go es). The e a e ou main o ms o he disease: isce al
leishmaniasis (VL, also known as kala-aza ); pos -kala-aza de mal
leishmaniasis (PKDL); cu aneous leishmaniasis (CL); and mucocu-
aneous leishmaniasis (MCL). While CL is he mos common o m o
he disease, VL is he mos se ious and can be a al i un ea ed [1].
Immunosupp essed pa ien s ela ed o HIV co-in ec ion o solid
o gan ansplan a ion a e p one o in ec ion by Leishmania, which
can also p omo e cance de elopmen [2].
*Co esponding au ho .
** Co esponding au ho .
*** Co esponding au ho . Depa amen o de Química O g
anica e Ino g
anica, Fac-
ul ad de Ciencia y Tecnología, Uni e sidad del País Vasco / Euskal He iko Uni-
be si a ea UPV/EHU, Apdo. 644, 48080, Bilbao, Spain.
E-mail add esses: humbe o.g[email p o ec ed] (H. Gonz
alez-Díaz), nu ia.
so omay[email p o ec ed] (N. So omayo ), es he [email p o ec ed] (E. Le e).
Con en s lis s a ailable a ScienceDi ec
Eu opean Jou nal o Medicinal Chemis y
jou nal homepage: h p://www.else ie .com/loca e/ejmech
h ps://doi.o g/10.1016/j.ejmech.2021.113458
0223-5234/©2021 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
Cu en an ileishmanial he apeu ics a e hampe ed by d ug
oxici y, high cos , need o pa en e al adminis a ion, inc easing
ea men ailu e a es, and eme gence o Mul i-D ug Resis an
(MDR) Leishmania species o s ains. T ea men depends no only
on he e iological species and he in ec ion ype (CL, VL, PKDL, o
MCL), bu also on he geog aphical loca ion whe e he disease was
acqui ed [3]. Besides, he e a e ew well- alida ed molecula d ug
a ge s in Leishmania, bu he molecula a ge s o he cu en
clinical molecules a e unknown. VL is ea ed wi h di e en
mul id ug he apy, di e en combina ions o pen a alen an imo-
nials, pa omomycin, liposomal ampho e icin B, and mil e osine. I s
use is u he limi ed by associa ed li e- h ea ening oxici ies like
ca diac a hy hmias, p olonged QT in e al (QTc), en icula p e-
ma u e bea s, achyca dia o fib illa ion in pen a alen an imonials
o neph o oxici y, hypokalemia and myoca di is in ampho e icin B
he apy [4]. The oxici y and he na ow he apeu ic ma gin is one
o he main limi a ions o he compounds cu en ly used agains
leishmaniasis, he e o e i is impo an o sea ch o new he a-
peu ic al e na i es ha show a educed oxici y. Mo eo e , hese
d ugs can ha e significan side e ec s, e.g. mil e osine can cause
bi h de ec s i aken wi hin h ee mon hs o ge ing p egnan [5].
On he o he hand, ea men o CL should be decided by he clinical
lesions, e iological species, he di e en esponse o d ugs o spe-
cies and s ains, and he possibili y o de elop in o mucosal leish-
maniasis [6]. Bu p o en ea men s o CL a e sca ce, being
pen a alen an imonials, pa omomycin, pen amidine, and he i-
azol de i a i e fluconazole he mos e ec i e d ugs [7](Fig. 1).
Among he la es ad ances in his field s and ou no el an i-
leishmanial d ug-like chemical se ies based on ni ogen he e ocy-
clic sca olds, as py azolopy imidine GSK3186899/DDD853651 [8]
and benzabenzoxazole de i a i es GNF6702 and LXE408 [9](Fig.1).
Besides, si amaquine, an 8-aminoquinoline, is a d ug candida e o
he ea men o VL by o al ou e, al hough phase II clinical ials
poin ou some ad e se e ec s, such as me hemoglobinemia and
neph o oxici y, which ha e o be conside ed o a u he de el-
opmen decision [10](Fig. 1). Mo e ecen ly, 4-
aminos y ylquinolines, quinolone-me onidazoles, and
e ocenylquinoline-based de i a i es ha e also shown p omising
an ileishmanial p ofiles [11]. Cu en ly, iden ifica ion o new
e ec i e and sa e an ileishmanial d ugs is c ucial o ad ance in
ob aining new lead compounds and disease con ol [12].
Ou g oup has expe ience in he de elopmen o syn he ic
me hodologies o he p epa a ion o di e en benzo(he e o) used
six-membe ed he e ocycles [13], in pa icula quinoline, isoquino-
line and hei dihyd o coun e pa s, whose s uc u al co es a e
among many biologically ac i e na u al p oduc s and pha maceu-
icals [14]. In pa icula , Lyco ane- ype o Ama yllidaceae alkaloids
may p esen biological ac i i y agains opical diseases caused by
p o ozoan pa asi es [15]. On he o he hand, B ingmann and Moll
ha e epo ed ha he p esence o an a oma ic ing linked o he
ni ogen a om o he isoquinoline nucleus is c ucial o he leish-
manicidal ac i i y o hese he e ocycles [16]. The e o e, we en i-
sioned ha he change o he a yl g oup o an he e oa oma ic ing,
he py ole, bu used o he isoquinoline nucleus by he bside,
would lead o he py olo[1,2-b]isoquinoline co e, hus combining
s uc u al ea u es o bo h ypes o he e ocycles.
We ecen ly epo ed he syn hesis o C-10 (he e o)a ylme hyl
subs i u ed py olo[1,2-b]isoquinolines 2[17] ia a Heck/Suzuki
cascade eac ion (Scheme 1)[18]. The e o e, we decided o explo e
a palladium-ca alyzed in amolecula Heck/anion cap u e cascade
eac ion, which would allow o a y he na u e o he subs i uen a
Fig. 1. An ileishmanial d ugs wi h he e ocyclic mo i s and mil e osine.
Scheme 1. Syn hesis o C-10 subs i u ed py olo[1,2-b]isoquinolines 2and 3 o an i-
leishmanial assays: (a) p e iously epo ed Heck/Suzuki cascade; (b) Heck/anion
cap u e cascade.
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
2
C-10b o he s udy o s uc u e-ac i i y ela ionships. Thus, in a-
molecula Heck- ype eac ion o 2-alkenyl N-(o-iodobenzyl)py -
oles 1p oceeds ia an ini ial 6-exo cycliza ion p ocess o gi e a
s
-
alkylpalladium(II) in e media e ha would be apped by a cyanide
ion. The use o 1,1-disubs i u ed alkenes di ec s he ca bopallada-
ion o he mos subs i u ed posi ion a oiding he syn
b
-hyd ide
elimina ion. Thus, he sequen ial o ma ion o wo CeC bonds can
be achie ed o gi e py olo[1,2-b]isoquinolines 3wi h a cyano-
me hyl subs i uen a he qua e na y s e eocen e (Scheme 1).
In his con ex , Chemin o ma ics models can be use ul o ca y
ou high- h oughpu compu a ional in silico p e-sc eening o la ge
lib a ies o compounds. These s udies allow o p io i ize some
amilies o compounds (po en ial lead compounds) in he pha -
macological assays in o de o educe he ime and cos s (ma e ial
and human esou ces) o he d ug disco e y p ocess. Thus, assay o
new compounds by ial-and-e o es s a e a oided. Ne e heless,
o ou bes knowledge, he e a e no epo s o compu a ional
models o an ileishmanial compounds ha include da a o mul-
iple species and ypes o assays. The main pi alls o classic
Chemin o ma ics models a e he impossibili y o p edic simul a-
neously mul iple biological ac i i y pa ame e s o d ugs agains
di e en a ge p o eins, cell lines, o ganisms o assay, e c. They ail
o pe o m mul i-label and mul i-ou pu classifica ion o new
compounds. Ou g oup has in oduced he PTML ¼Pe u ba ion
Theo y (PT) þMachine Lea ning (ML) algo i hm o sol e simila
p oblems (mul iple s uc u es s. mul iple species and condi ions
o assay) in he d ug disco e y p ocess [19]. PTML models s a wi h
a known alue ( e e ence) o expec ed biological ac i i y o a g oup
o compounds o p ope y and add he e ec o pe u ba ions
(de ia ions) o he s uc u e and/o condi ions o assay o he new
case (que y compound) wi h espec o he e e ence. The PTML
app oach uses PT Ope a o s (PTOs) such as de ia ions, mo ing
a e ages, e c. o quan i y he e ec o hese de ia ions o pe u -
ba ions o e he final biological ac i i y. The simple PTML models
a e linea addi i e models, bu mo e complica ed and gene al PTML
models can be cons uc ed. PTML models ha e been used suc-
cess ully o p edic ing di e en pa ame e s o biological ac i i y
and/o oxici y (K
i
,IC
50
,LD
50
, Km,% inhibi ion, e c.) o he in e -
ac ion o di e en compounds wi h di e en biological a ge s
(p o eins, issues, cell lines, pa hogenic o ganisms, e c.) [20].
The e o e, PTML models a e use ul o selec compounds ou o he
se ies o be sen o pha macological assays.
We desc ibe he ein he applica ion o a ca bopallada ion ini i-
a ed domino eac ion o he syn hesis o a se ies o py olo[1,2-b]
isoquinoline sca olds, and hei in i o biological e alua ion
agains wo species o Leishmania,L. amazonensis and L. dono ani,
which cause CL and VL, espec i ely. The e ec o a ying subs i-
u ion pa e n o he a oma ic ing, as well as he na u e o he C-
10b subs i uen will be explo ed. In addi ion, we ha e de eloped
he fi s gene al-pu pose PTML model o p edic he an i-
leishmanial ac i i y o hese se ies o py oloisoquinolines in
di e en biological assays agains di e en Leishmania species.
2. Chemis y
The py olo[1,2-b]isoquinolines 3we e syn he ized h ough a
in amolecula domino Heck/nucleophile cap u e anion eac ion on
2-alkenyl N-(o-iodobenzyl)py oles 1, which we e p epa ed
ollowing p e iously epo ed p ocedu es by us [17]. These Heck/
nucleophile cap u e anion cascade eac ions ha e been mainly
applied o he cons uc ion o fi e-membe ed ings, so ou aim will
also be o expand he scope o he p ocedu e o he syn hesis o six-
membe ed ings. The main challenge was o con ol he chemo-
selec i i y o he p ocess o a o he ini ial 6-exo ca bopallada ion
o e he ea ly coupling o a oma ic ing wi h cyanide ion, so all
expe imen al condi ions should be ca e ully chosen o con ol he
na u e o he in e media e palladium(II) species ha de e mine he
selec i i y.
In hese cascade eac ions i is c ucial o choose, no only he
adequa e palladium ca alys , bu also he cyanide sou ce. Since
G igg’s seminal wo k on palladium ca alyzed alkene a ylcyana ion
using KCN as cyanide sou ce [21], he me hod has been imp o ed.
Thus, Neu ille and Zhu used K
4
Fe(CN)
6
in he in amolecula
palladium-ca alyzed domino Heck/cyana ion sequence o he
syn hesis o a 3,3
0
-disubs i u ed oxindole [22]. La e , Lau ens e-
po ed he dias e eoselec i e syn hesis o dihyd oisoquinolinones
s a ing om enan iopu e N-allylca boxamide wi h Zn(CN)
2
as
cyanide sou ce [23]. The same eagen was used in a ela ed Pd-
ca alyzed dea oma i e indole bis unc ionaliza ion eac ion o he
p epa a ion o indolines wi h conges ed s e eocen e s [24].
To es he iabili y o he me hod ou lined abo e, we ca ied ou
an op imiza ion o he Heck/cyana ion eac ion condi ions on N-(o-
iodobenzyl)py ole 1a.Wefi s explo ed he eac ion wi h
phosphane- ee ca aly ic sys ems o economical and en i on-
men al easons, es ing se e al cyana ion agen s. Thus, a e
examining a ious p e-ca alys s [Pd(OAc)
2
, Pd(PPh
3
)
4
, Pd(dba)
2
,
Pd
2
(dba)
3
$CHCl
3
,e c.], o ganic (NE
3
, PMP) and ino ganic bases
(K
2
CO
3
,Cs
2
CO
3
, NaHCO
3
), pola and non-pola sol en s, addi i es
and eac ion empe a u es, he op imal esul s we e ob ained using
Pd(OAc)
2
(10 mol%) as ca alys , K
4
Fe(CN)
6
$3H
2
O (0.22 equi .) as
cyanide sou ce, Na
2
CO
3
(1.3 equi .) as ino ganic base and nBu
4
NCl
(1 equi .) as addi i e, using a mix u e o DMF:H
2
O (8:2) as sol en
a 120

C (See Supplemen a y Ma e ial o he de ails o he op i-
miza ion o he eac ion condi ions). I is no ewo hy ha
K
4
Fe(CN)
6
$3H
2
O is he bes cyana ion agen , as i is non- oxic
compa ed wi h adi ional cyana ion eagen s [KCN, NaCN,
Zn(CN)
2
, TMSCN] and i can easily be handled wi hou special
p ecau ions [25]. In addi ion, subs oichiome ic amoun s o eagen
can be used, as all cyanide ions bound o he i on (II) cen e can be
eleased in he cyana ion eac ion. In ac , he eac ion was slowed
(24 h s. 1 h) when using highe amoun s o K
4
Fe(CN)
6
$3H
2
O (0.44
s. 0.22 equi .), p obably because highe concen a ion o ee cy-
anide ions in he eac ion mix u e can o m ca aly ically inac i e
palladium(II)-cyano complexes ha lead o pa ial ca alys deac i-
a ion [26]. No ably, he p esence o nBu
4
NCl as phase- ans e
ca alys was essen ial o sho en he eac ion ime and o
enhance he o ma ion o py oloisoquinoline 3a. In ac , i is
known ha he addi ion o e abu ylammonium halides inc eases
he a es o some s eps o he ca aly ic cycle o he Heck eac ion,
a o ing he 6-exo- ig cycliza ion s ep [27]. Howe e , he yield is
mode a e (65%), as he compe i i e di ec cyana ion coupling e-
ac ion o gi e 4a (26%) could no be comple ely supp essed. The use
o a se ies o phosphane ligands [PPh
3
,P Bu
3
, P(2- u yl)
3
] did no
imp o e he e ficiency o he cascade cycliza ion o 1a. Selec ed
examples a e shown on Table 1 (See Supplemen a y Ma e ial o
mo e de ails). The e o e, ei he using condi ions ha a o ca ionic
o neu al mechanisms o he syn inse ion o he A ePd(II)-X
complex o he alkene, whe e he selec i i y o he Heck eac ion is
con olled [28], we could achie e he selec i e syn hesis o he
py oloisoquinolines 3.
Wi h an op imized se o condi ions in hand, he scope o he
Heck/cyana ion cascade was examined. Gene ally, di e en sub-
s i u ion pa e ns on he a oma ic ing a e ole a ed, a o ding he
py oloisoquinolines 3in mode a e yields, as he di ec cyana ion
p ocess was always compe i i e (Table 2). Specifically, s ongly
elec on-dona ing g oups as me hoxy and me hylenedioxy g oups
(Table 2, en ies 3e4 and 7e9) and elec on-wi hd awing g oups
(F) (en y 6), as well as unsubs i u ed de i a i es (Table 2, en y 2)
we e iable unde he op imized eac ion condi ions.
Finally, he cyano g oup o he py olo[1,2-b]isoquinolines 3
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
3
ob ained h ough he domino Heck/cyana ion eac ion has been
e ficien ly de i a ized o di e en unc ional g oups, such as alde-
hyde, amide and amine, showing he e sa ili y o he p ocedu e
(see Supplemen a y Ma e ial).
3. An ileishmanial assays
The new syn hesized 10-cyanome hyl subs i u ed 5,10-
dihyd opy olo[1,2-b]isoquinoline de i a i es 3, oge he wi h he
p e iously ob ained C-10 (he e o)a ylme hyl subs i u ed de-
i a i es 2[17], we e sc eened agains wo species o Leishmania,
L. amazonensis and L. dono ani, which cause CL and VL, espec i ely
(Fig. 2). In i o p omas igo e suscep ibili y assays and in i o
in acellula amas igo e suscep ibili y assays ha e been ca ied ou ,
as well as cy o oxici y assay on J774 cell line o mac ophages, a line
o mac ophages used o es cy o oxici y o d ugs in i o p io o
animal es s (see Expe imen al Sec ion). Mil e osine was he d ug
o e e ence, as i can be used o he ea men o di e en o ms o
he disease. The ini ial sc eening on he in i o p omas igo e assays
e ealed ha some 5,10-dihyd opy olo[1,2-b]isoquinolines a o -
ably compa e o mil e osine in e ms o ac i i y and selec i i y
agains L. amazonensis. In gene al, bes ac i i y was ound o he
10-a ylme hyl subs i u ed de i a i es 2aa,2ab, 2ad,2ae,2ag,2ah,
2bb, and 2db. In pa icula , he mos ac i e and selec i e com-
pounds 2ad (IC
50
¼3.30 ±2.80
m
M, SI >77.01) and 2bb
(IC
50
¼3.93 ±0.23
m
M, SI >58.77) we e app oxima ely 10- old
mo e po en and selec i e han he d ug o e e ence (mil e o-
sine), ollowed by compound 2db (IC
50
¼8.00 ±0.28
m
M, SI >34.4).
In con as , he p esence o a cyanome hyl g oup a C-10 esul ed in
compounds wi h weake an ileishmanial ac i i y agains
L. amazonensis (Table 3, en ies 12e19). Only compounds 3h and 3i,
which ha e an O-benzyl g oup a he 7-posi ion o he py oloi-
soquinoline co e, ha e an ac i i y agains L. amazonensis compa-
able o ha o mil e osine (Table 3, en ies 18e19 s. en y 20).
The e o e, he p esence o a benzyl g oup in he py oloisoquino-
line co e seems o be c ucial o he an ileishmanial ac i i y.
The a oma ic subs i u ion pa e n o he py oloisoquinoline
co e o his se ies 2also plays an impo an ole, ha ing he
elec on-dona ing subs i uen s a posi i e impac on an i-
leishmanial ac i i y agains L. amazonensis. Thus, compound 2cb,
wi h an unsubs i u ed a oma ic ing, is 4-47- old less po en han
o he compounds o se ies 2(Table 3, en y 10 s. en ies 1 and 11).
Besides, as shown in Table 3, eplacemen o he me hyl g oup a
he 10-posi ion o he py oloisoquinoline ing sys em by a i-
fluo ome hyl g oup esul ed in mo e ac i e compounds, being 2bb
o e 4- old mo e ac i e agains L. amazonensis han 2ab (Table 3,
en y 2 s. en y 9). Rega ding he subs i u ion pa e n o he benzyl
moie y, he p esence o wo ifluo ome hyl g oups was de i-
men al o ac i i y and selec i i y, being 2ac he less ac i e and
mo e oxic de i a i e o bo h se ies (Table 3, en y 3). Howe e , i
should be poin ed ou ha he in oduc ion o his CF
3
g oup a C-
10 has less e ec (Table 3, en y 2 s. en y 9).
Con e sely, all py oloisoquinolines es ed a e no ably less
ac i e han mil e osine o he ea men o L. dono ani, being again
compound 2bb one o he mo e ac i e and selec i e o bo h se ies
wi h IC
50
¼16.41 ±4.90
m
M and SI >14.09. In addi ion, and e y
no ably, almos all py oloisoquinolines a e much less oxic han
he d ug o e e ence wi h alues o concen a ion o he compound
ha p oduces 50% educ ion o cell iabili y (Cy o oxic Concen-
a ion, CC
50
) in he ange 195e416
m
M in J774 cells. In ac , he
Selec i i y Index (SI ¼CC
50
/IC
50
) is highe o almos all py oloi-
soquinolines han o mil e osine whose SI is only 4.43. I also
should be poin ed ou ha al hough be e IC
50
alues ha e been
epo ed o di e en ypes o molecules agains di e en Leish-
mania species [29], hey usually ha e lowe CC
50
alues han mos
Table 1
Op imiza ion o he ca bopallada ion/cyana ion cascade eac ion o 1a.
En y [Pd] Ligand Time (h) Yield 3a (%)
a
Yield 4a (%)
a
1 Pd(OAc)
2b
e154 32
2 Pd(OAc)
2c
e165 26
3 Pd(TFA)
2b
e446 30
4 Pd(dba)
2b
e48 37
d
48
5Pd
2
(dba)
3
$CHCl
3b
e48 53
e
27
6 Pd(OAc)
2c
L1 644 40
7 Pd(OAc)
2c
L2 152 35
8 Pd(OAc)
2c
L3 24 24 56
9 Pd(TFA)
2c
L2 24 33 31
10 Pd(dba)
2c
L2 642 33
11 Pd
2
(dba)
3
$CHCl
3c
L2 136 39
a
Isola ed yield.
b
Na
2
CO
3
(1 equi .).
c
Na
2
CO
3
(1.3 equi .).
d
Con e sion 88%.
e
Con e sion 86%.
Table 2
Ca bopallada ion/cyana ion cascade eac ion. Syn hesis o 3a-i.
En y A Time (h) P oduc Yield (%)
a
P oduc Yield (%)
a
1 1 3a 65 4a 26
243b 25 4b 41
313c 37 4c 38
423d 39 4d 29
548 3e 21
b
4e -
c
6 48 3 16 4 18
763g 23
d
4g 28
813h 23 4h 15
924 3i 16 4i 30
a
Yield (%) o isola ed pu e compound.
b
Con e sion: 63%.
c
4e was de ec ed by
1
H NMR in eac ion c ude, bu i could no be isola ed.
d
Con e sion: 94%.
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
4
o ou py oloisoquinolines, which a e no oxic a he highes
es ed dose (CC
50
>100
m
g/mL). The e o e, his is a e y in e es ing
esul , conside ing ha cu en ly a ailable chemo he apy o
leishmaniasis is hampe ed by oxici y and d ug esis ance.
Then, he mos ac i e compounds o he p omas igo e assays
we e sc eened agains amas igo es o L. amazonensis and
L. dono ani (Table 4). All py oloisoquinolines es ed we e less
ac i e han mil e osine when es ed agains L. dono ani amas i-
go es.Howe e , hese compounds ha e IC
50
alues in he ange
33.59e77.12
m
M, which a e simila o e en be e han mil e osine
(IC
50
¼47.60 ±7.04
m
M) in he es agains L. amazonensis. All o
hem ha e SI alues be ween 3.58 and 8.93, which a e 2- o 4- old
highe han mil e osine (SI ¼2.85). In his case, py oloisoquinoline
2ae showed he bes ac i i y wi h IC
50
¼33.59 ±2.64
m
M and
highe selec i i y wi h SI >8.93.
We can obse e ha 10-a ylme hyl subs i u ed dihy-
d opy oloisoquinoline se ies seems o be mo e selec i e and p e-
sen s simila o e en be e ac i i y han he d ug o e e ence o
he ea men o L. amazonensis amas igo es.The esul is p omising
because in he p esen model o expe imen al assay he amas i-
go es a e inside he human mac ophage cells. The e o e, i seems
ha hese py oloisoquinolines could be able o c oss/a ached hos
and pa asi e ba ie s o exe hei ac i i y wi hou damaging he
hos memb ane (mac ophage memb ane). Fi s , hey need o c oss
he memb ane o he hos cells (mac ophages), nex hey need o
c oss Pa asi opho ous Vacuole Memb ane (PVM). Las , i he ac-
i i y is no o e PVM di ec ly, he compounds should each/c oss
he pa asi e memb ane o each hei molecula a ge in he
memb ane o inside he pa asi e. The PVM p e en s he acidifica-
ion o he media by lysosomes o he hos cell o des oy an
Fig. 2. C-10 subs i u ed 5,10-dihyd opy olo[1,2-b]isoquinolines 2and 3sc eened agains L. amazonensis and L. dono ani.
Table 3
IC
50
leishmanicidal and cy o oxic e ec s om py oloisoquinoline de i a i es (exp essed as
m
M) on in i o p omas igo e assay.
En y Comp. L. amazonensis L. dono ani Mac ophages J774
IC
50
±SD (
m
M)
a
SI
b
IC
50
±SD (
m
M)
a
SI
b
CC
50
±SD (
m
M)
c
12aa 26.41 ±2.20 >10.40 47.40 ±1.79 >5.80 275.13
e
22ab 18.00 ±0.79 >14.68 79.93 ±4.36 >3.31 264.24
e
32ac 39.62 ±2.13 1.18 111.34 ±10.8 0.42 46.80 ±4.00
42ad 3.30 ±2.80 >77.01 38.30 ±3.36 >6.63 254.14
e
52ae 17.09 ±0.60 >17.54 30.29 ±2.01 >10.09 299.91
e
62a 36.71 ±0.44 >5.95 86.01 ±1.60 >2.54 218.54
e
72ag 23.29 ±1.41 >15.15 124.43 ±14.70 >2.84 352.89
e
82ah 12.24 ±0.56 >23.00 33.66 ±2.06 >8.27 278.20
e
92bb 3.93 ±0.23 >58.77 16.41 ±4.90 >14.09 231.30
e
10 2cb 157.35 ±6.28 >2.00 159.40 ±26.20 >1.97 314.09
e
11 2db 8.00 ±0.28 >34.49 30.35 ±3.92 >9.09 275.95
e
12 3a 309.20 ±41.10 >1.14 226.21 ±11.60 >1.57 354.18
e
13 3c 77.35 ±1.13 >4.85 120.12 ±12.01 >3.13 375.51
e
14 3d 171.07 ±6.38 >2.07 nd
d
nd
d
354.18
e
15 3e 93.85 ±6.02 >3.77 nd
d
nd
d
354.20
e
16 3 96.55 ±1.66 >4.31 192.19 ±7.66 >2.17 416.18
e
17 3g 123.25 ±10.70 >3.21 359.99 ±34.44 >1.10 396.32
e
18 3h 15.83 ±0.06 14.04 60.25 ±0.34 3.69 222.30 ±42.70
19 3i 24.82 ±0.28 7.86 34.37 ±5.30 5.68 195.30 ±27.30
20 mil e osine 30.70 ±0.98 4.43 0.15 ±0.02 906.00 135.90 ±10.30
a
IC
50
: Concen a ion o he compound ha p oduced a 50% educ ion in pa asi es; SD: S anda d De ia ion.
b
SI: Selec i i y Index, SI ¼CC
50
/IC
50
.
c
CC
50
: Concen a ion o he compound ha p oduced a 50% educ ion o cell iabili y in ea ed cul u e cells wi h espec o un ea ed ones.
d
nd: no de e mined.
e
CC
50
alues, exp essed as
m
M, co espond o 100
m
g/mL, which was he highe doses es ed.
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
5

in ading pa asi e. PVM is shaped by he pa asi e using pa s o he
memb ane o he hos cell. The PVM su ounds he in acellula
pa asi e, c ea ing a sepa a e bubble o cy oplasm-filled plasma
memb ane wi hin he hos cell [30].
4. Compu a ional model
As desc ibed abo e, we ha e measu ed he expe imen al
IC
50
(
m
M) alues o he se ies o py oloisoquinolines 2and 3 om
he es s agains wo species o Leishmania,L. amazonensis and
L. dono ani, a wo di e en s ages (S) o de elopmen o he
pa asi e: amas igo es (A) and p omas igo es (P). We could obse e
a ce ain endency in he beha io o hese py oloisoquinolines
agains he p omas igo es o he wo di e en species o Leishmania
s udied. In ac , we ound a eg ession coe ficien o R ¼0.67 o he
IC
50
o py oloisoquinoline de i a i es in L. amazonensis s.
L. dono ani. Howe e , we ha e no ye clues abou he possible
a ge p o eins o hese compounds. In addi ion, he e a e many
o he species o Leishmania (including MDR s ains) ha could no
be assayed and may p esen di e en suscep ibili ies o he same
d ugs.
In o de o pu ou esul s in o con ex , a ChEMBL da ase o
>145,000 p eclinical assays o pu a i e an ileishmanial compounds
was downloaded and explo ed, as de ailed in Expe imen al Sec ion
and he Supplemen a y Ma e ial. The expe imen s include up o 10
di e en condi ions o assay c
j
¼[c
0
,c
1
,c
2
,…c
10
], which we e spli
in o wo pa i ions o subse s o expe imen al condi ions c
I
and c
II
.
The p incipal condi ions c
I
a e hose ha cha ac e ize he biological
expe imen pe se (pa ame e measu ed, a ge p o ein, pa asi e
s age, o ganism o assay, e c.). The da ase includes alues o
n(c
0
)>50 di e en biological pa ame e s measu ed o compounds
s. a leas one ou o n(c
1
)¼32 a ge p o eins, n(c
2
)¼29 cell lines,
n(c
3
)¼40 o ganisms o assay (no all a e pa asi es), and n(c
4
)¼37
Leishmania pa asi e species o s ains, e c. Ins ead o ou comes o
all possible expe imen s, he da ase includes n(c
I
)>240 combi-
na ions o hese p incipal condi ions c
I
(di e en expe imen s) o
nume ous compounds. In addi ion, he da ase includes n(c
II
)¼80
combina ions o seconda y c
II
condi ions ela ed o he biological
na u e and/o accu acy o da a ( a ge ype, a ge mapping, alue
exac i ude, e c.). This s udy e ealed ha abo e 70 assays in ol ing
20 species/s ains o Leishmania a e mos commonly used. Un o -
una ely, e en limi ing he analysis o hese assays i may be cos ly
in e ms o esou ces and ime. Tes ing a sho se ies o only 10
compounds would equi e n
assay
¼10$70 ¼700 expe imen al as-
says. The e o e, we decided o ca y ou a p elimina y
compu a ional s udy o he suscep ibili y o o he species o his
se ies o compounds. As s a ed abo e, he e a e no epo s on a
compu a ional model o pe o m such s udy. The e o e, we fi s
de eloped a new PTML model o ca y ou his ype o p edic ions.
The equa ion o he bes PTML model ound is he ollowing:
ð
n
ijÞcalc ¼59:918599 $ ð
n
ijÞ e þ1:631537$
D
D1ðcIÞ
þ0:041494$
D
D2ðcIÞ2:675709 $
D
D3ðcIÞ
1:562187$
D
D1ðcIIÞe0:041886$
D
D2ðcIIÞ
þ2:649511$
D
D3ðcIIÞ25:182671
n¼109389
c
2¼135169:7p<0:05
The idea o his PTML model is o s a using as inpu a unc ion
o e e ence (
ij
)
e
, which is ob ained om he expe imen al assays
o a se o compounds o e e ence ca ied ou unde specific con-
di ions c
j
. This unc ion quan ifies he expec ed p io p obabili y o
gi e a posi i e esul in he specific assays o a compound selec ed
a andom. Nex , he alues o he PT Ope a o s (PTOs) wi h he
gene al o m
D
D
k
(c
j
) we e added o he (
ij
)
e
. These PTOs quan i y
he de ia ion (
D
) o he molecula desc ip o s D
k
(s uc u e) o ou
que y compound wi h espec o he g oup o e e ence compounds
(see Expe imen al Sec ion). The desc ip o s D
k
a e D
1
¼ALOGP, he
n-oc anol/wa e pa i ion coe ficien , D
2
¼TPSA, he Topological
Pola Su ace A ea, and D
3
¼NRV, he Numbe o Viola ions o Rule
o V (Lipinski’so Pfize ’s ule) we e used o iden i y each com-
pound in he equa ion. The desc ip o s ALOGP and TPSA quan i y
he molecula s uc u e o he d ug by means o a weig hed sum o
di e en molecula agmen s in he molecules. NRV ule quan ifies
he likeness/simila i y o one compound wi h espec o known
d ugs based on molecula weigh , hyd ogen bonds, e c.[31]. The
alues o D
k
we e ex ac ed om ChEMBL da ase and/o calcula ed
wi h he so wa e DRAGON o new compounds. Finally, he ou pu
o he model (
ij
)
calc
is a sco ing unc ion used o calcula e he
p obabili y o ac i i y p( (
ij
)
p ed
¼1) o di e en compounds. In
o de o ain his model, we selec ed a andom a la ge aining
se ies o n ¼109,389 p eclinical assays downloaded om ChEMBL
da abase. The alues o Specifici y (Sp) and Sensi i i y (Sn) a e in
he ange z90e98% o aining se ies (see Supplemen a y Ma-
e ial), which a e excellen alues o his ype o ML classifica ion
models. Mo eo e , he p-le el <0.05 o he Chi-squa e es wi h
c
2
¼135,169.7 poin s o a s a is ically significan disc imina ion
Table 4
IC
50
Leishmanicidal and cy o oxic e ec s om py oloisoquinoline de i a i es (exp essed as
m
M) on in i o amas igo e assay.
En y Compound L. amazonensis L. dono ani Mac ophages J774
IC
50
±SD (
m
M)
a
SI
b
IC
50
±SD (
m
M)
a
SI
b
CC
50
±SD (
m
M)
c
12aa 51.56 ±12.00 >5.34 45.00 ±7.51 >6.12 275.13
e
22ab 56.12 ±13.50 >4.70 29.62 ±6.87 >8.92 264.24
e
32ad 60.79 ±5.74 >4.18 46.30 ±0.33 >5.49 254.14
e
42ae 33.59 ±2.64 >8.93 55.03 ±3.96 >5.45 299.91
e
52ag 43.54 ±9.53 >8.10 255.88 ±42.10 >1.38 352.89
e
62ah 69.26 ±6.96 >4.02 16.74 ±0.14 >16.61 278.20
e
72bb 56.72 ±4.58 >4.08 22.17 ±4.16 >10.43 231.30
e
82db 77.12 ±19.10 >3.58 68.65 ±9.63 >4.02 275.95
e
93h nd
d
e44.75 ±8.53 4.97 222.30 ±42.70
10 mil e osine 47.60 ±7.04 2.85 0.37 ±0.05 369.30 135.90 ±10.30
a
IC
50
: Concen a ion o he compound ha p oduced a 50% educ ion in pa asi es; SD: S anda d De ia ion.
b
SI: Selec i i y Index, SI ¼CC
50
/IC
50
.
c
CC
50
: Concen a ion o he compound ha p oduced a 50% educ ion o cell iabili y in ea ed cul u e cells wi h espec o un ea ed ones.
d
nd: no de e mined.
e
CC
50
alues, exp essed as
m
M, co espond o 100
m
g/mL, which was he highe doses es ed.
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
6
be ween ac i e ( (
ij
)
obs
¼1) and non-ac i e compounds
( (
ij
)
obs
¼0) in all hese assays. This model may be used o p edic
new compounds no included in aining se ies. Fi s , he alues o
he (
ij
)
e
and PTOs (con aining D
k
o d ug and <D
k
(c
j
)>o assay)
we e subs i u ed in he equa ion o calcula e he ou pu unc ion
(
ij
)
calc
. Nex , he alues o (
ij
)
calc
we e ans o med in o pos e io
p obabili ies o success p( (
ij
)
p ed
¼1) o each compound in
di e en assays using a sigmoid unc ion. Once he alues o
p obabili ies we e calcula ed, he compounds could be classified.
Thus, hose ou comes in he ange o p obabili y
p( (
ij
)
p ed
¼1) >0.5 a e conside ed in e es ing o assay
(
ij
)
p ed
¼1. Then, he p esen PTML model was es ed wi h a e y
la ge alida ion se ies, ob aining alues o Sp and Sn also in he
ange z90e98% o he ex e nal alida ion se ies. In conclusion,
his simple bu powe ul PTML model p edic s e y well (o e all
Accu acy ¼97.8%) a la ge da ase ( aining þ alida ion) o an i-
leishmanial ac i i y p eclinical assays (n
assay
>145,000) in ol ing
96,800 unique compounds.
5. P edic i e s udy
As men ioned abo e, some compounds o ou se ies show
in e es ing IC
50
alues and can be conside ed ac i e ( (
ij
)¼1))
using he cu o o IC
50
¼10
m
M in he ange o mo e ypical
expe imen al s udies. Then, we decided o use his new model o
ca y ou a compu a ional p edic ion o he ou comes o he 19
py oloisoquinolines o ou se ies s. di e en Leishmania species
(>20) in mo e han 160 di e en p eclinical assays. This PTML
model is able o p edic he pos e io p obabili y p( (
ij
)
p ed
¼1) o
ge ing he desi ed le el o mo e han 50 di e en biological
p ope ies (IC
50
,K
i
,K
m
,e c.). In his p elimina y s udy, IC
50
was
selec ed as unique p ope y, as i was he fi s p ope y expe i-
men ally measu ed in he ea ly s ages o sc eening, using a e y low
cu -o alue o IC
50
¼0.01
m
M, in an e o o educe he numbe o
alse posi i e cases om he fi s s eps o sc eening. Consequen ly,
he model was used o calcula e he alues o p obabili y
p(IC
50
(
m
M) <0.01)
calc
o which a compound would show an
IC
50
(
m
M) <0.01 in di e en assays. The o al numbe o calcula ions
o in i o assays ha do no speci y he a ge p o ein was
n
calc1
¼n
cmpd
$n
assay
¼19$162 ¼3078. The ull esul s o his p e-
dic i e s udy a e compiled in Table S9 o he Supplemen a y Ma-
e ial. The alues o p(IC
50
(
m
M) <0.01)
calc
o assays wi h known
p o ein a ge s we e also p edic ed, being he numbe o calcula-
ions n
calc2
¼n
cmpd
$n
p o
¼19$32 ¼608 o he 19 compounds s.
32 a ge p o eins o di e en species. I makes a o al o
n
calc
¼n
calc1
þn
calc2
¼3686 calcula ions o ou 19 compounds in
di e en assays. Fo he sake o simplici y, we only changed he
condi ions c
I
(assay pe se) using a fix sub-se o condi ions c
II
o
he 3686 calcula ions. Consequen ly, he o al numbe o p edic ed
alues o p(IC
50
(
m
M) <0.01)
calc
was 3686. In o de o summa ize
he esul s and wi hd aw conclusions, we calcula ed he a e age
alue o he p obabili y p(IC
50
(
m
M) <0.01)
a g
, which is he a e age
p obabili y ha he compound is p edic ed o ha e an
IC
50
(
m
M) <0.01 in mul iple assays s. he same species. The PTML
model p edic s a cohe en beha io o his se ies o compounds as
a homologous se ies. I means ha species esis an /suscep ible o
one compound a e p edic ed o be esis an o he ac ion o almos
all compounds o he whole se ies. The model does no de ec
o e all significan di e ences on he beha io o bo h subse ies
(2aa-2db s.3a-3i subse ies) o compounds, which could be
cohe en wi h he ac ha bo h subse ies o compounds ha e he
py oloisoquinoline co e. The a e age alue o p obabili y
p(IC
50
(
m
M) <0.01)
a g
o almos all compounds o he se ies a e in
he ange 0.1e0.4, p(IC
50
(
m
M) <0.01) <0.5, o many species o
Leishmania, which is in ag eemen wi h he expe imen al findings.
Table 5 shows selec ed esul s o he p edic i e s udy s. di e en
species o he wo op lead compounds o each subse ies (2ad,2bb,
3h, and 3i) expe imen ally es ed in his wo k (see Supplemen a y
Ma e ial o ull de ails). In e es ingly, he PTML model p edic s
alues o p(IC
50
(
m
M) <0.01)
a g
>0.8 o some species/s ains no
p e iously es ed, despi e o he demanding compu a ional
h eshold alue used in he compu a ional s udy. See, o example,
he alues o p(IC
50
(
m
M) <0.01)
a g
o he ou op hi compounds
o L. b aziliensis s ain M2904 (L. b m.), and L. majo s ain F iendlin
(L. ma .), and L. mexicana (L. mex.), shown in Table 5.Rega ding he
possible a ge p o ein, he model p edic s alues o p(IC
50
(
m
M) <
0.01)
a g
>0.7 o bo h subse ies o py oloisoquinolines e sus
some p o eins. The s udy poin s o he T ypano hione educ ase o
L. dono ani (P39050) [32],
L
-isoaspa a e(
D
-aspa a e) O-me hyl-
ans e ase (P22061) o L. dono ani [33] and he O ni hine deca -
boxylase o L. dono ani (P27116) [34] as plausible a ge s o be
es ed, al hough mo e candida es a e also collec ed in Table 5.I is
in e es ing ha some o hese p o eins a e enzymes wi h amino
acid de i a i es (T ypano hione,
D
-aspa a e, and O ni hine) as
subs a es. The e o e, his compu a ional s udy opens he doo o a
u he es ing o hese compounds o hei de i a i es s. o he
species o Leishmania and specific es s s. p obable p o ein a ge s.
6. Conclusions
In conclusion, he palladium-ca alyzed Heck-ini ia ed cascade
eac ions a e e ec i e p ocedu es o he cons uc ion o he py -
olo[1,2-b]isoquinoline sca olds wi h a qua e na y cen e a C-10b.
The in i o e alua ion o hei leishmanicidal ac i i y agains
isce al (L. dono ani) and cu aneous (L. amazonensis) leishmaniasis
e elead ha almos all compounds showed e y low cy o oxici y,
CC
50
>100
m
g/mL in J774 cells (highes es ed dose). This is an
impo an ea u e, as d ug oxici y is one o he main limi a ions o
cu en chemo he apy o leishmaniasis. In gene al, 10-a ylme hyl
subs i u ed py oloisoquinolines showed bes ac i i y agains
L. amazonensis on in i o p omas igo e assays. In pa icula , 2ad
(IC
50
¼3.30
m
M, SI >77.01) and 2bb (IC
50
¼3.93
m
M, SI >58.77)
we e app oxima ely 10- old mo e po en and selec i e han he
d ug o e e ence (mil e osine). On he o he hand, 2ae was he
mo e ac i e compound in he in i o amas igo e assays
(IC
50
¼33.59
m
M, SI >8.93). In addi ion, i has been demons a ed
ha Pe u ba ion Theo y Machine Lea ning (PTML) algo i hms a e
use ul o model la ge (>145,000 cases) ChEMBL da ase s o an i-
leishmanial p eclinical assays. I is possible o use he de eloped
PTML model o educe assay cos s by p edic ing he p obabili y
wi h which a que y compound o his o o he se ies o compounds
eaches a desi ed le el o mul iple pa ame e s (IC
50
,K
i
,e c.) s.
di e en Leishmania species and a ge p o eins, wi h high alues o
specifici y (>98%) and sensi i i y (>90%) in bo h aining and
alida ion se ies.
7. Expe imen al sec ion
7.1. Chemis y
Gene al. All comme cial chemicals we e eagen g ade and we e
used wi hou u he pu ifica ion unless o he wise specified.
Palladium ca alys s we e comme cially a ailable, and we e used
wi hou u he pu ifica ion: Pd(OAc)
2
: 98% pu i y; Pd(TFA)
2
:97%
pu i y; Pd(PPh
3
)
4
: 99% pu i y.; Pd(dba)
2
: 99.9% pu i y;
Pd
2
(dba)
3
$CHCl
3
: 97% pu i y. The 1-(o-iodobenzyl)-2-
alkenylpy oles 1we e syn he ized acco ding o he p ocedu es
p e iously epo ed by us [17]. All sol en s used in eac ions we e
anhyd ous and pu ified acco ding o s anda d p ocedu es. All ai -
o mois u e-sensi i e eac ions we e pe o med unde a gon; he
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
7
glasswa e was d ied (130

C) and pu ged wi h a gon. TLC was
ca ied ou wi h 0.2 mm- hick silica gel Me ck F254 pla es. Visu-
aliza ion was accomplished by UV ligh (
l
¼254 nm and 360 nm).
Flash column ch oma og aphic sepa a ions and pu ifica ions we e
pe o med on silica Flash P60 (Silicycle), 230e400 mesh ASTM.
Final compounds we e pu ified o 95% pu i y as assessed by
1
H
NMR spec a and analy ical liquid ch oma og aphy. Mel ing poin s
we e measu ed in a Büchi B-540 appa a us in unsealed capilla y
ubes. IR spec a we e ob ained using A enua ed To al Reflec ion
(ATR) in a JASCO FT/IR 4100 in he in e al be ween 4000 and
400 cm
1
wi h a 4 cm
1
esolu ion. Only cha ac e is ic bands a e
gi en in each case.
1
H and
13
C NMR spec a we e eco ded a
20e25

C on ei he a B uke AC-300 spec ome e (300 MHz o
1
H
and 75.5 MHz o
13
C) and on a B uke AC-500 spec ome e
(500 MHz o
1
H and 125.7 MHz o
13
C). Chemical shi s a e e-
po ed in pa s pe million (ppm) ela i e o an in e nal sol en
e e ence. Reco ded peaks a e lis ed in he o de mul iplici y (s,
single ; d, double ; dd, double o double s; m, mul iple ), coupling
cons an s, and numbe o p o ons. Assignmen s o indi idual
13
C
and
1
H esonances a e suppo ed by DEPT expe imen s and 2D
co ela ion expe imen s (COSY, HSQCed o HMBC) when necessa y.
High esolu ion mass spec a (HRMS) we e pe o med by he Mass
Spec ome y Gene al Se ice a he Uni e si y o he Basque
Coun y using an ul a pe o mance liquid ch oma og aph (Acqui y
UPLC, Wa e s Ch oma og aphy.), in andem wi h a QTOF mass
spec ome e (SYNAPT G2 HDMS, Wa e s Ch oma og aphy), wi h
an elec osp ay ioniza ion sou ce in a posi i e mode.
Table 5
PTML p edic ion o a e age alue o p obabili y p(IC
50
(
m
M) <0.01)
a g
o py oloisoquinolines 2ad,2bb,3h, and 3i agains >20 di e en Leishmania species.
Leish. S
b
Ta ge Compound Leish. S
b
Ta ge Compound
Species P o ein 2ad 2bb 3h 3i Species
a
P o ein 2ad 2bb 3h 3i
L. ae . Ae0.02 0.01 0.01 0.01 L. maj. eP37268 0.41 0.34 0.30 0.32
L. ae . Pe0.02 0.01 0.01 0.01 L. maj. eQ01782 0.58 0.51 0.47 0.49
L. ama. Ae0.10 0.08 0.06 0.07 L. maj. eQ0GKD7 0.43 0.36 0.32 0.34
L. ama. Pe0.21 0.17 0.15 0.15 L. maj. eQ4Q5S8 0.04 0.03 0.03 0.03
L. ama. eO96394 0.64 0.58 0.53 0.55 L. maj. eQ4Q5W4 0.78 0.73 0.69 0.71
L. a i. ee 0.21 0.17 0.15 0.16 L. maj. eQ4QBL1 0.52 0.46 0.41 0.43
L. b a. Ae0.12 0.09 0.08 0.09 L. maj. eQ4QE15 0.02 0.02 0.01 0.02
L. b a. Pe0.20 0.16 0.14 0.15 L. maj. eQ6S996 0.17 0.13 0.11 0.12
L. b m. Ae0.93 0.91 0.89 0.90 L. maj. eQ9LM02 0.04 0.03 0.02 0.03
L. b m. Pe0.11 0.09 0.07 0.08 L. maj. Pe0.06 0.05 0.04 0.05
L. cha. Ae0.18 0.15 0.13 0.14 L. maj. P Q01782 0.41 0.35 0.31 0.33
L. cha. Pe0.25 0.21 0.18 0.19 L. ma . Pe0.94 0.92 0.90 0.91
L. don. Ae0.30 0.27 0.25 0.25 L. mex. Ae0.30 0.26 0.24 0.25
L. don. eP39050 0.83 0.79 0.76 0.77 L. mex. eP04406 0.73 0.67 0.63 0.65
L. don. eQ95WR6 0.16 0.13 0.11 0.12 L. mex. eP36400 0.35 0.29 0.26 0.27
L. don. eQ95Z89 0.29 0.24 0.21 0.22 L. mex. eQ01558 0.60 0.54 0.50 0.51
L. don. eQ9NJG8 0.07 0.05 0.04 0.05 L. mex. eQ27686 0.96 0.95 0.95 0.95
L. don. Pe0.25 0.23 0.22 0.23 L. mex. eQ4U254 0.41 0.35 0.31 0.32
L. don. P P22061 1.00 1.00 1.00 1.00 L. mex. eQ9U5N6 0.34 0.28 0.25 0.26
L. don. P P27116 1.00 0.99 0.99 0.99 L. mex. Pe0.27 0.22 0.20 0.21
L. dod. ee 0.21 0.17 0.15 0.16 L. mex. P P11166 0.09 0.07 0.06 0.06
L. en . Pe0.13 0.10 0.09 0.09 L. mem. eQ27686 0.29 0.24 0.21 0.22
L. ga . ee 0.21 0.17 0.15 0.16 L. mem. Pe0.02 0.01 0.01 0.01
L. guy. Ae0.08 0.06 0.05 0.06 L. me . ee 0.21 0.17 0.15 0.16
L. guy. Pe0.28 0.23 0.20 0.21 L. pan. Ae0.10 0.08 0.07 0.07
L. in . Ae0.32 0.28 0.26 0.27 L. pan. Pe0.42 0.38 0.35 0.36
L. in . eQ8I6E4 0.28 0.23 0.20 0.21 L. pe . Pe0.23 0.19 0.16 0.17
L. in . Pe0.20 0.17 0.15 0.15 L. pi . Ae0.11 0.09 0.07 0.08
L. maj. Ae0.23 0.20 0.18 0.18 L. pi . Pe0.21 0.17 0.14 0.15
L. maj. eO15826 0.76 0.70 0.67 0.68 L. p o. Pe0.11 0.09 0.07 0.08
L. maj. eO96526 0.07 0.06 0.05 0.05 L. a . ee 0.10 0.08 0.07 0.07
L. maj. eP00374 0.21 0.17 0.15 0.16 L. o. Pe0.18 0.14 0.12 0.13
L. maj. eP07382 0.22 0.18 0.16 0.16 L. u . ee 0.08 0.06 0.05 0.05
a
Leishmania species: L. ae hiopica ¼L. ae ., L. amazonensis ¼L. ama., L. a is idesi ¼L. a i.,L. b aziliensis ¼L. b a., L. b aziliensis M2904 ¼L. b m,L. chagasi ¼L. cha., L. dono ani ¼
L. don.,L. dono ani dono ani ¼L. dod.,L. en ie ii ¼L. en .,L. ga nhami ¼L. ga ., L. guyanensis ¼L. guy,L. in an um ¼L. in .,L. majo ¼L. maj., L. Mexicana ¼L. mex.,L. mexicana
mexicana ¼L. mem.,L. mexicana enezuelensis ¼L. me .,L. panamensis ¼L. pan.,L. pe u iana ¼L. pe .,L. pi anoi ¼L. pi .,L. a en olae ¼L. a ., L. opica ¼L. o., L. u anica ¼L. u .,
L. majo s ain F iendlin ¼L. ma .
b
S¼S age: A ¼Amas igo es, P ¼P omas igo es.
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
8
In amolecula Heck/cyanide cap u e cascade eac ion on 1.
Syn hesis o py olo[1,2-b]isoquinolines 3. Gene al p ocedu e.
Pd(OAc)
2
(0.1 mmol) was added o a mix u e o N-(o-iodobenzyl)
py ole 1(1 mmol), po assium hexacyano e a e(II) ihyd a e
(0.22 mmol), sodium ca bona e (1.3 mmol), and e abu ylammo-
nium chlo ide (1 mmol) in an 8/2 mix u e o DMF/H
2
O (3 mL). The
mix u e was s i ed a 120

C o he ime indica ed in each case.
H
2
O (15 mL) was added and he esul ing aqueous phase was
ex ac ed wi h E OAc (3 20 mL). The combined o ganic ex ac s
we e washed wi h b ine (3 20 mL), d ied o e anhyd ous Na
2
SO
4
and concen a ed in acuo. Pu ifica ion by column ch oma og aphy
(silica gel) o he esul ing esidue a o ded he co esponding
py oloisoquinoline 3and di ec a yl halide cyana ion p oduc 4
(see Supplemen a y Ma e ial).
2-(7,8-Dime hoxy-10-me hyl-5,10-dihyd opy olo[1,2-b]iso-
quinolin-10-yl)ace oni ile (3a). Acco ding o Gene al P ocedu e,
N-(o-iodobenzyl)py ole 1a (115 mg, 0.30 mmol) was ea ed wi h
Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) i-
hyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
0.30 mmol) in a 8/2 mix u e o DMF/H
2
O (1 mL) o 1 h. A e
wo kup, pu ifica ion by column ch oma og aphy (silica gel, pe o-
leum e he /E OAc 8/2) a o ded 3a (54.9 mg, 65% yield) as a yellow
solid: m. p. (pe oleum e he /E OAc): 132e134

C. IR (ATR): 2935,
2245, 1515 cm
1
.
1
H NMR (300 MHz, CDCl
3
):
d
7.05 (s, 1H),
6.75e6.74 (m, 2H), 6.26 (dd, J¼3.6, 2.7 Hz, 1H), 6.20 (dd, J¼3.6,
1.7 Hz, 1H), 5.18 (d, J¼15.9 Hz, 1H), 5.07 (d, J¼15.9 Hz, 1H), 3.95 (s,
3H), 3.90 (s, 3H), 2.72 (s, 2H), 1.88 (s, 3H).
13
C{
1
H} NMR (75.5 MHz,
CDCl
3
):
d
148.6, 148.4, 132.5, 129.8, 124.0, 119.3, 117.8, 109.2, 108.8,
108.4, 103.5, 56.2, 56.0, 47.1, 37.5, 34.2, 26.4. MS (ESI) m/z ( el in-
ensi y): 283 (MH
þ
, 100), 242 (46). HRMS (ESI-TOF): calcd o
C
17
H
19
N
2
O
2
[MH
þ
] 283.1447; ound, 283.1450.
2-(10-Me hyl-5,10-dihyd opy olo[1,2-b]isoquinolin-10-yl)
ace oni ile (3b). Acco ding o Gene al P ocedu e, N-(o-iodoben-
zyl)py ole 1b (101 mg, 0.30 mmol) was ea ed wi h Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) ihyd a e
(27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg, 0.39 mmol) and
e abu ylammonium chlo ide (83.4 mg, 0.30 mmol) in an 8/2
mix u e o DMF/H
2
O (1 mL) o 4 h. A e wo kup, pu ifica ion by
column ch oma og aphy (silica gel, pe oleum e he /E OAc 95/5)
a o ded 3b (17.4 mg, 23% yield) as a colo less oil: IR (ATR): 2925,
2250, 1515 cm
1
.
1
H NMR (300 MHz, CDCl
3
):
d
7.59e7.56 (m, 1H),
7.40e7.28 (m, 3H), 6.76 (dd, J¼2.7, 1.7 Hz, 1H), 6.26 (dd, J¼3.6,
2.7 Hz,1H), 6.21 (dd, J¼3.6, 1.7 Hz,1H), 5.24 (d, J¼16.1 Hz,1H), 5.14
(d, J¼16.1 Hz,1H), 2.74 (s, 2H),1.89 (s, 3H).
13
C{
1
H} NMR (75.5 MHz,
CDCl
3
):
d
137.9, 132.4, 131.8, 128.1, 127.5, 126.6, 125.2, 119.4, 117.5,
108.8, 103.7, 47.5, 37.8, 33.8, 26.0. MS (ESI) m/z ( el in ensi y): 223
(MH
þ
, 65), 182 (100). HRMS (ESI-TOF): calcd o C
15
H
15
N
2
[MH
þ
]
223.1235; ound, 223.1237.
2-(10-Me hyl-5,10-dihyd o- [1,3]dioxolo[4,5-g]py olo[1,2-b]
isoquinolin-10-yl)ace oni ile (3c). Acco ding o Gene al P oced-
u e, N-(o-iodobenzyl)py ole 1c (110 mg, 0.30 mmol) was ea ed
wi h Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II)
ihyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
0.30 mmol) in a 8/2 mix u e o DMF/H
2
O (1 mL) o 1 h. A e
wo kup, pu ifica ion by column ch oma og aphy (silica gel, pe o-
leum e he /E OAc 8/2) a o ded 3c (29.5 mg, 37% yield) as a yellow
oil: IR (ATR): 2915, 2250, 1485 cm
1
.
1
H NMR (300 MHz, CDCl
3
):
d
7.01 (s,1H), 6.74e6.70 (m, 2H), 6.26e6.25 (m,1H), 6.18 (dd, J¼3.7,
1.7 Hz, 1H), 6.00 (s, 2H), 5.15 (d, J¼15.9 Hz,1H), 5.03 (d, J¼15.9 Hz,
1H), 2.73 (d, J¼16.4 Hz, 1H), 2.66 (d, J¼16.4 Hz, 1H), 1.84 (s, 3H).
13
C{
1
H} NMR (75.5 MHz, CDCl
3
):
d
147.6, 147.0, 132.3, 131.3, 125.3,
119.2, 117.5, 108.8,106.4, 105.4, 103.6,101.5, 47.5, 37.8, 34.0, 26.3. MS
(ESI) m/z ( el in ensi y): 267 (MH
þ
, 100), 226 (76). HRMS (ESI-TOF):
calcd o C
16
H
15
N
2
O
2
[MH
þ
] 267.1134; ound, 267.1137.
2-(6,7-Dime hoxy-10-me hyl-5,10-dihyd opy olo[1,2-b]iso-
quinolin-10-yl)ace oni ile (3d). Acco ding o Gene al P ocedu e,
N-(o-iodobenzyl)py ole 1d (113 mg, 0.30 mmol) was ea ed wi h
Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) i-
hyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
0.30 mmol) in a 8/2 mix u e o DMF/H
2
O (1 mL) o 2 h. A e
wo kup, pu ifica ion by column ch oma og aphy (silica gel, pe o-
leum e he /E OAc 9/1) a o ded 3d (32.4 mg, 39% yield) as a b own
oil: IR (ATR): 2935, 2250, 1495 cm
1
.
1
H NMR (300 MHz, CDCl
3
):
d
7.26 (d, J¼8.7 Hz, 1H), 6.96 (d, J¼8.7 Hz, 1H), 6.79e6.78 (m, 1H),
6.26e6.24 (m, 1H), 6.17 (dd, J¼3.6, 1.7 Hz, 1H), 5.35 (d, J¼16.9 Hz,
1H), 5.05 (d, J¼16.9 Hz, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 2.70 (s, 2H),
1.86 (s, 3H).
13
C{
1
H} NMR (75.5 MHz, CDCl
3
):
d
151.2, 144.8, 132.5,
130.8, 126.1, 120.6, 119.7, 117.6, 111.7, 108.7, 103.4, 60.5, 55.8, 42.7,
37.4, 34.2, 26.1. MS (ESI) m/z ( el in ensi y): 283 (MH
þ
, 100), 242
(30). HRMS (ESI-TOF): calcd o C
17
H
19
N
2
O
2
[MH
þ
] 283.1447; ound,
283.1453.
2-(7,9-Dime hoxy-10-me hyl-5,10-dihyd opy olo[1,2-b]iso-
quinolin-10-yl)ace oni ile (3e). Acco ding o Gene al P ocedu e,
N-(o-iodobenzyl)py ole 1e (115 mg, 0.30 mmol) was ea ed wi h
Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) i-
hyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
0.30 mmol) in a 8/2 mix u e o DMF/H
2
O (1 mL) o 48 h. A e
wo kup, pu ifica ion by column ch oma og aphy (silica gel, pe o-
leum e he /E OAc 8/2) a o ded 3e (18.1 mg, 21% yield) (63% con-
e sion) as a yellow solid: m. p. (pe oleum e he /E OAc):
167e169

C. IR (ATR): 2935, 2250, 1460 cm
1
.
1
H NMR (300 MHz,
CDCl
3
):
d
6.68 (dd, J¼2.7,1.7 Hz,1H), 6.47 (d, J¼2.5 Hz, 1H), 6.35 (d,
J¼2.5 Hz, 1H), 6.33 (dd, J¼3.7, 2.7 Hz, 1H), 6.20 (dd, J¼3.7, 1.7 Hz,
1H), 5.25 (d, J¼16.3 Hz, 1H), 5.13 (d, J¼16.3 Hz, 1H), 3.90 (s, 3H),
3.83 (s, 3H), 3.54 (d, J¼16.3 Hz,1H), 2.97 (d, J¼16.3 Hz,1H),1.80 (s,
3H).
13
C{
1
H} NMR (75.5 MHz, CDCl
3
):
d
159.7, 159.2, 134.6, 133.6,
118.8,117.9, 117.2, 109.3,103.2, 102.2, 98.8, 55.4, 55.3, 47.2, 37.8, 31.5,
28.9. MS (ESI) m/z ( el in ensi y): 283 (MH
þ
, 100), 242 (33). HRMS
(ESI-TOF): calcd o C
17
H
19
N
2
O
2
[MH
þ
] 283.1447; ound, 283.1440.
2-(7-Fluo o-10-me hyl-5,10-dihyd opy olo[1,2-b]iso-
quinolin-10-yl)ace oni ile (3 ). Acco ding o Gene al P ocedu e,
N-(o-iodobenzyl)py ole 1 (102 mg, 0.30 mmol) was ea ed wi h
Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) i-
hyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
0.30 mmol) in a 8/2 mix u e o DMF/H
2
O (1 mL) o 48 h. A e
wo kup, pu ifica ion by column ch oma og aphy (silica gel, pe o-
leum e he /E OAc 9/1) a o ded 3 (11.8 mg, 16% yield) as a yellow
oil: IR (ATR): 2925, 2250, 1500 cm
1
.
1
H NMR (300 MHz, CDCl
3
):
d
7.57 (dd, J¼8.8, 5.3 Hz, 1H), 7.11 ( d, J¼8.8, 2.7 Hz, 1H), 7.01 (dd,
J¼8.8, 2.7 Hz, 1H), 6.78e6.77 (m, 1H), 6.29e6.21 (m, 2H), 5.25 (d,
J¼16.3 Hz, 1H), 5.14 (d, J¼16.3 Hz, 1H), 2.80e2.68 (m, 2H), 1.90 (s,
3H).
13
C{
1
H} NMR (75.5 MHz, CDCl
3
):
d
161.6 (d, J¼247.5 Hz), 134.1
(d, J¼7.7 Hz), 133.7 (d, J¼3.3 Hz), 132.2, 127.2 (d, J¼8.3 Hz), 119.4,
117.4,115.1 (d, J¼21.4 Hz), 113.3 (d, J¼22.4 Hz),109.1,103.9, 47.4 (d,
J¼2.2 Hz), 37.6, 34.0, 26.2. MS (ESI) m/z ( el in ensi y): 241 (MH
þ
,
59), 200 (100). HRMS (ESI-TOF): calcd o C
15
H
14
FN
2
[MH
þ
]
241.1141; ound, 241.1145.
2-(7-Me hoxy-10-me hyl-5,10-dihyd opy olo[1,2-b]iso-
quinolin-10-yl)ace oni ile (3g). Acco ding o Gene al P ocedu e,
N-(o-iodobenzyl)py ole 1g (106 mg, 0.30 mmol) was ea ed wi h
Pd(OAc)
2
(6.7 mg, 0.03 mmol), po assium hexacyano e a e(II) i-
hyd a e (27.9 mg, 0.07 mmol), sodium ca bona e (41.3 mg,
0.39 mmol) and e abu ylammonium chlo ide (83.4 mg,
I. Ba bolla, L. He n
andez-Su
a ez, V. Que edo-Tumailli e al. Eu opean Jou nal o Medicinal Chemis y 220 (2021) 113458
9