Mul imodal an ip oli e a i e e ec s o oleanolic acid mi ocans: In i o and
in i o s udies
Ad i´
an Pue a
a
, Aday Gonz´
alez-Bakke
a
, Edua do Romanos
b,c,d
,
Inmaculada Aguila Domínguez
e
, M´
onica Ma ínez-Mon iel
e,
, Pen´
elope Me ino-Mon iel
,
Raquel P. He e a
c,*
, M. Concepci´
on Gimeno
d
, Jos´
e G. Fe n´
andez-Bola˜
nos
e
, ´
Osca L´
opez
e,*
,
Jos´
e M. Pad ´
on
a,*
a
BioLab, Ins i u o Uni e si a io de Bio-O g´
anica An onio Gonz´
alez (IUBO-AG), Uni e sidad de La Laguna, Apa ado 456, E-38200 La Laguna, Spain
b
Se icio de Imagen M´
edica y Feno ipado, Ins i u o A agon´
es de Ciencias de la Salud, Cen o de In es igaci´
on Biom´
edica de A ag´
on (CIBA), A da. San Juan Bosco, 13,
Plan a D, E-50009 Za agoza, Spain
c
Labo a o io de O ganoca ´
alisis Asim´
e ica, Depa amen o de Química O g´
anica, Ins i u o de Sín esis Química y Ca ´
alisis Homog´
enea (ISQCH), CSIC-Uni e sidad de
Za agoza, C/Ped o Ce buna 12, E-50009 Za agoza, Spain
d
Depa amen o de Química Ino g´
anica, Ins i u o de Sín esis Química y Ca ´
alisis Homog´
enea (ISQCH), CSIC-Uni e sidad de Za agoza, C/Ped o Ce buna 12, E-50009
Za agoza, Spain
e
Depa amen o de Química O g´
anica, Facul ad de Química, Uni e sidad de Se illa, Apa ado 1203, E-41071 Se ille, Spain
Facul ad de Ciencias Químicas, Benem´
e i a Uni e sidad Au ´
onoma de Puebla, Ciudad Uni e si a ia, 72570 Puebla, PUE, Mexico
ARTICLE INFO
Keywo ds:
Oleanolic acid
Mi ocan
T iphenylphosphonium
An i umo agen
Pa ap osis
Con inuous li e-cell imaging
ABSTRACT
Mi ochond ia- a ge ing d ugs (mi ocans) based on o ganic ca ions a e eme ging as powe ul and selec i e cance
he apeu ics. In his s udy, we ha e e alua ed a no el se ies o oleanolic acid-de i ed mi ocans, e ealing
nanomola - ange an ip oli e a i e e ec s agains human solid umo cells. Con inuous li e-cell imaging e ealed
ex ensi e cy oplasmic acuola ion, while mechanis ic s udies iden i ied pa ap osis as he dominan o m o cell
dea h. Rema kably, in i o expe imen s demons a ed signi ican umo g ow h inhibi ion in mice, wi h no
de ec able oxici y a he apeu ic doses. These indings highligh he po en ial o oleanolic acid-de i ed mi ocans
as p omising candida es o cance he apy.
1. In oduc ion
Mi ochond ia play a pi o al ole in he egula ion o cell dea h and
su i al in esponse o a ious s imuli [1]. The elease o p o eins
loca ed in he mi ochond ial memb ane –i.e. cy och ome c o he
cy osol– se es as an e ec o o p og ammed cell dea h. In a simila way,
mi ochond ia pa icipa e in au ophagy h ough mi ophagy. P o-
g ammed au ophagy o his o ganelle ensu es a co ec u no e ,
allowing he deg ada ion o non- unc ional mi ochond ia and acili-
a ing he syn hesis o new en i ies. Fu he mo e, al e a ions in
mi ochond ial unc ioning associa e wi h umo esis ance o d ugs ia
he de elopmen o apop osis esis ance, main enance o me abolic e-
qui emen s h ough exploi a ion o au ophagy p oduc s, and enhanced
biogenesis o he o ganelle by inc eased mi ochond ial ission [2]. The
ele ance o mi ochond ial me abolism in umo s appea s in he li e a-
u e ex ensi ely, wi h mu a ions occu ing in p53, PI3K, myc o KRAS
ha in luence he ep og amming o mi ochond ial unc ion o acili a e
umo de elopmen [3]. Gi en he ole o mi ochond ia in cell dea h and
me abolism and hei –deeply demons a ed– ele ance in umo s wi h
high ene gy demands, a ge ing mi ochond ial unc ion has gained
Abb e ia ions: 3-MA, 3-me hyladenine; BA, be ulinic acid; CCCP, ca bonyl cyanide 3-chlo ophenylhyd azone; CHX, cycloheximide; CI, con idence in e al; CPT,
camp o hecin; CQ, chlo oquine; cy a abine, A a-C; DAPI, 4
′
,6-diamidino-2-phenylindole; DCF, dichlo o luo escein; DCFDA, 2
′
,7
′
-dichlo odihyd o luo escein diac-
e a e; FDA, Food and D ug Adminis a ion; GA, glycy he inic acid; GI
50
, 50% g ow h inhibi ion; IC
50
, 50% inhibi o y concen a ion, LC3, mic o ubule-associa ed
p o ein ligh chain 3; MAPK, mi ogen-ac i a ed p o ein kinase; MDC, monodansyl cada e ine; MMP, mi ochond ial memb ane po en ial; MoA, mode o ac ion; OA,
oleanolic acid; PBS, phospha e bu e saline; PTX, pacli axel; R , esis ance ac o ; RFU, ela i e luo escence uni s; ROS, eac i e oxygen species; SDS, sodium
dodecyl sul a e; SEM, s anda d e o o he mean; TMRE, e ame hyl hodamine e hyl es e ; TPP, iphenylphosphonium; UA, u solic acid; VP, e apamil.
* Co esponding au ho s a : Ins i u o Uni e si a io de Bio-O g´
anica An onio Gonz´
alez, Uni e sidad de La Laguna, PO Box 456, 38200 La Laguna, Spain.
E-mail add esses: [email p o ec ed] (R.P. He e a), [email p o ec ed] (´
O. L´
opez), [email p o ec ed] (J.M. Pad ´
on).
Con en s lis s a ailable a ScienceDi ec
Biochemical Pha macology
jou nal homepage: www.else ie .com/loca e/biochempha m
h ps://doi.o g/10.1016/j.bcp.2025.116807
Recei ed 25 Oc obe 2024; Recei ed in e ised o m 11 Feb ua y 2025; Accep ed 13 Feb ua y 2025
Biochemical Pha macology 234 (2025) 116807
A ailable online 18 Feb ua y 2025
0006-2952/© 2025 The Au ho s. Published by Else ie Inc. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
impo ance and cen e ed e o s du ing he las ew yea s in cance
ea men esea ch [1–3], ep esen ing a way o selec i ely a ge umo
cells.
Focusing on chemo he apy agains mi ochond ia, he e a e mul iple
s a egies ha can be used o di ec ly and/o indi ec ly inhibi i s
unc ion. In e en ion o e me abolic p ocesses ha occu in he mi o-
chond ia is one o hem. To impede he no mal elec on anspo chain,
one s a egy is o inhibi he syn hesis o biomolecules h ough he
al e a ion o he ica boxylic acid cycle, hus educing cance cell
g ow h. The an idiabe ic d ug me o min has been widely s udied p e-
clinical and clinically, bo h alone and in combina ion wi h o he d ugs,
wi h p omising esul s [4]. O he s a egies explo ed include di ec in-
hibi ion o he ica boxylic acid cycle (i.e. inhibi ion o mi ochond ial
glu aminase by elaglenas a o dis up ion o inhibi ion o py u a e
dehyd ogenase kinase by dichlo oace a e) as well as he inhibi ion o
a y acid syn hesis [5]. Ta ge ing mi ochond ial me abolism is an
e ec i e o m o combina ion he apy. Coupling inhibi o s o he
me abolic pa hways occu ing in his o ganelle wi h known FDA-
Fig. 1. Mi ochond ia- a ge ing sca olds: (A) Chemical s uc u e o mi ochond ia- a ge ing ec o s. Compiled om [9]; (B) Na u ally occu ing pen acyclic i-
e penoids and ep esen a i e TPP de i a i es. The numbe e e s o he numbe ing used in he co esponding o iginal wo k. IC
50
alues epo ed agains human
solid umo cells.
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
2
app o ed d ugs (e.g. mTOR inhibi o e e olimus) ha e esul ed in be e
e icacy [4]. Ano he mi ochond ia- a ge ing s a egy is he induc ion o
mi ochond ial ou e memb ane pe meabiliza ion. This p oduces he
elease o he p o eins loca ed in he in e memb ane space o he
cy osol, which include ac i a o s o caspases and cy och ome c, subse-
quen ly inducing cell dea h [6].
The mi ochond ial memb ane consis s o a double laye . Ex e nally,
and in con ac wi h he cy oplasm, he e is a po ous memb ane. In e -
nally, i is a p o ein- ich memb ane. This s uc u e igh ly egula es
mi ochond ial me abolism and p e en s xenobio ics om c ossing he
mi ochond ial memb ane. The e o e, mi ocans (i.e. d ugs a ge ing he
mi ochond ia) mus be in e nalized in he o ganelles c ossing he
double-laye ed memb ane. To achie e success, d ug deli e y o he
mi ochond ia is o pi o al impo ance. The mi ochond ial memb ane
po en ial o no mal cells is se a 150–180 mV so lipophilic ca ions end
o accumula e in he mi ochond ial ma ix [7]. The mi ochond ia o
cance cells di e om hose o heal hy cells in memb ane pola iza ion,
displaying −220 and −160 mV, espec i ely [8]. This di e ence allows
o he as and selec i e up ake o posi i ely cha ged molecules. This
p ope y led o he de elopmen o di e se mi ochond ia- a ge ing
ec o s [9]. Vec o izing an icance compounds wi h selec ed ca ionic
de i a i es ep esen s an ex ensi ely explo ed s a egy wi h p omising
esul s (Fig. 1A).
Indubi ably, he mos s udied o such ca ions is he iphenylphos-
phonium (TPP) ion. TPP has been widely used o ob ain mi ocans gi en
i s accessibili y h ough chemical syn hesis [10]. TPP possesses bo h a
posi i e cha ge (delocalized o e he h ee phenyl g oups and s abilized
by esonance) and lipophilici y o acili a e i s in e naliza ion in o he
mi ochond ia [11]. These hyd ophobic ca ions can di use h ough he
lipid bilaye s because hei ne cha ge is sca e ed along i s su ace.
Mo eo e , TPP is easy o link and clinical ials demons a ed compa -
ibili y wi h physiological condi ions and a lack o ele an oxici y, e en
a ela i ely high doses [12]. Chemo he apeu ic d ugs such as doxo u-
bicin, amoxi en, chlo ambucil, doce axel and gami inib, and o he
subs ances as honokiol, cu cumin, be ulinic acid (BA) and u solic acid
(UA) a e jus some examples o an ip oli e a i e molecules bea ing TPP
ion o enhance hei po ency and selec i i y [8]. Ea lie , we epo ed he
mi ochond io opic ac i i y o masked phenolic sca olds ollowing his
s a egy [13]. These no el mi ochond io opic agen s showed p omising
an ip oli e a i e p o iles, displaying ema kable po ency and selec i i y,
including mul id ug- esis an cells, upon co-adminis a ion wi h a
pump-e lux inhibi o .
TPP ca ions o pen acyclic i e penoids showed po en ial an icance
ac i i y. In pa icula , de i a i es o glycy he inic acid (GA, enoxolone)
[14], UA [15] and BA [16–18] displayed ac i i y agains cance cell
lines (Fig. 1B). Encou aged by hese esul s, we u ned ou a en ion o
oleanolic acid (OA) wi h he idea o apply he same s a egy o ob ain
new mi ocans. OA is ano he pen acyclic i e penoid p esen in mo e
han 1620 plan s (bo h, edible and medicinal). I is ob ained in i s pu e
o m o wi h s uc u al modi ica ions. These pen acyclic i e penoids
–including OA– bea wo unc ional g oups ha migh be used o
linking he TPP mo i , a hyd oxy g oup a posi ion C-3, and a ca boxylic
acid on C-30 o GA, and C-28 o UA, BA and OA. The esul s o he
biological e alua ion e ealed a be e bioac i i y p o ile o he ca -
boxylic acid de i a i es.
Ou main a ge pu sued he ein has been he de elopmen o a sho
se ies o OA-TPP hyb ids as an example o o ganelle-di ec ed an icance
he apy, and o s udy hei mechanism in i o, and hei e iciency in a
xenog a mice model. Du ing he cou se o ou in es iga ions, Li and
cowo ke s [19] epo ed a se ies o OA de i a i es linked o TPP. In hei
s udy, hey claimed ha po en ial PI3K inhibi ion could be a mode o
ac ion (MoA) ha induce he an ip oli e a i e e ec s o he small mol-
ecules desc ibed. The lead compound was one o he compounds we
we e s udying. In his wo k, we in o m on he an ip oli e a i e ac i i y
obse ed in i o and he s udies ca ied ou o iden i y he MoA o a
small amily o OA-TPP hyb ids. The lead compound unde wen u he
in i o s udies in mice and i s e ec on umo g ow h educ ion will be
shown.
2. Ma e ials and me hods
2.1. Reagen s and chemicals
OA (TCI Eu ope, Belgium), N,N-dime hyl o mamide (DMF, VWR,
USA),
α
,
ω
-dib omoalkanes (Me ck, Ge many), po assium ca bona e
(K
2
CO
3
, Pan eac, Spain), e hyl ace a e (E OAc, VWR, USA), hyd o-
chlo ic acid (HCl, Pan eac, Spain), sodium bica bona e (NaHCO
3
, Pan-
eac, Spain), sodium chlo ide (Pan eac, Spain), sodium sul a e (Na
2
SO
4
,
Pan eac, Spain), cyclohexane (VWR, USA), ace oni ile (MeCN, VWR,
USA), iphenylphosphine (Me ck, Ge many), RPMI 1640 (VWR, USA),
e al bo ine se um FBS, (Biowes , F ance), L-glu amine (Lonza,
Swi ze land), penicillin G/s ep omycin (Lonza, Swi ze land), ypsin/
e hylenediamine e aace ic acid (EDTA) (Lonza, Swi ze land), sul o-
hodamine B (SRB, Me ck, Ge many), DMSO (VWR, USA), cy a abine
(A a-C, Me ck, Ge many), e apamil (VP, Me ck, Ge many), pacli axel
(PTX, Me ck, Ge many), inblas ine (VBL, Me ck, Ge many), phospha e
bu e saline (PBS, Lonza, Swi ze land), me hanol (VWR, USA), c ys al
iole (Me ck, Ge many), sodium dodecyl sul a e (SDS, Me ck, Ge -
many), 3-chlo ophenylhyd azone (CCCP, Me ck, Ge many), hyd ogen
pe oxide (H
2
O
2
, Me ck, Ge many), e ame hyl hodamine e hyl es e
(TMRE, Me ck, Ge many), glyce ol (Me ck, Ge many), 2
′
,7
′
-dichlo o-
luo escein diace a e (DCFDA, Me ck, Ge many), pa a o maldehyde
(PFA, Me ck, Ge many), ammonium chlo ide (NH
4
Cl, Me ck, Ge many),
i on (Me ck, Ge many), 4
′
,6-diamidine-2
′
-phenylindole dihyd o-
chlo ide (DAPI, Me ck, Ge many), bis-benzimide H33342 ihyd o-
chlo ide (Hoechs 33342, Me ck, Ge many), 3-me hyladenine (3-MA,
Me ck, Ge many), chlo oquine (CQ, Me ck, Ge many), cycloheximide
(CHX, Me ck, Ge many), Z-DEVD-FMK (Me ck, Ge many), luci e in (D-
Luci e in, po assium sal , DELTACLON S.L., Spain).
2.2. Chemis y
2.2.1. Gene al p ocedu e o he p epa a ion o b omoalkyl de i a i es I–IV
To a solu ion o OA (1) (300 mg, 0.66 mmol) in DMF (5 mL), was
added he co esponding
α
,
ω
-dib omoalkane (5.0 equi .) and K
2
CO
3
(1.0 equi .). The mix u e was s i ed a o 12 h. Then, i was dilu ed
wi h H
2
O (30 mL) and ex ac ed wi h E OAc (2x15 mL). The combined
o ganic ac ions we e washed wi h 1 M HCl (20 mL), sa . aq. NaHCO
3
(20 mL) and b ine (20 mL), d ied o e Na
2
SO
4
and il e ed. The il a e
was concen a ed o d yness and pu i ied by column ch oma og aphy
(98:2 → 95:5 cyclohexane–E OAc).
2.2.2. Gene al p ocedu e o he p epa a ion o iphenylphosphonium sal s
2–5
To a solu ion o b omoalkyl de i a i es I–IV (1.0 equi .) in MeCN
was added PPh
3
(5.0 equi .). Reac ion was s i ed a 115 ◦C (Fishe -
Po e ube) o 24 h. Then, i was concen a ed o d yness and he
esidue was pu i ied by column ch oma og aphy.
All compounds we e cha ac e ized by spec oscopic (
1
H and
13
C
NMR) and spec ome ic (HRMS) me hods.
1
′
’-{4
′
-[(3
′
’β-Hyd oxyolean-12
′
’–3n-oyl)oxy]bu yl} iphenylphos-
phonium b omide (2). B omide I (100 mg, 0.17 mmol), PPh
3
(222 mg,
0.85 mmol) and MeCN (7.5 mL) we e used. Column ch oma og aphy
(80:1 → 10:1 CH
2
Cl
2
–MeOH a o ded 2 as a whi e solid. Yield: 110 mg
(78 %). [
α
]25
D +32 (c 0.85, CH
2
Cl
2
);
1
H NMR (300 MHz, CDCl
3
) δ
7.92–7.67 (m, 15H, A -H), 5.14 ( , 1H, J
11
′
’
α
,12
′
’
=J
11
′
’
α
,12
′
’
=3.4 Hz, H-
12
′
’), 4.07 (m, 4H, CO
2
CH
2
, CH
2
P), 3.21 (m, 1H, H-3
′
’), 2.75 (b dd, 1H,
J
18
′
’,19
′
’
α
=14.0 Hz, J
18
′
’,19
′
’β
=3.6 Hz, H-18
′
’), 2.12 (quin , 2H, J
H,H
=
7.4 Hz, CH
2
), 1.89–1.77 (m, 4H, H-11
′
’
α
,β, H-16
′
’
α
,β), 1.71–1.06 (m,
20H, H-1
′
’β, H-2
′
’
α
,β, H-6
′
’
α
,β, H-7
′
’
α
,β, H-9
′
’, H-15
′
’
α
,β, H-19
′
’
α
,β, H-
22
′
’
α
,β, H-21
′
’
α
,β, 2CH
2
), 1.09 (s, 3H, Me-27
′
’), 0.99 (s, 3H, Me-23
′
’),
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
3
0.91–0.87 (m, 1H, H-1
′
’
α
), 0.89 (s, 3H Me-30
′
’) 0.87, 0.85 (2 s, 3H each,
Me-25
′
’, Me-29
′
’), 0.78, (s, 3H, Me-24
′
’), 0.70 (d, 1H, J
5
′
’,6
′
’β
=10.2 Hz,
H-5
′
’), 0.63 (s, 3H, Me-26
′
’) ppm;
13
C NMR (75.5 MHz, CDCl
3
) δ 177.7
(C-28), 143.7 (C-13
′
’), 135.1 (d,
4
J
C,P
=3.0 Hz, A -Cp), 133.8 (d,
2
J
C,P
=
10.0 Hz, A -Co), 130.6 (d,
3
J
C,P
=12.6 Hz, A -Cm), 122.4 (C-12
′
’), 118.4
(d,
1
J
C,P
=85.9 Hz, A -Cipso), 79.0 (C-3
′
’), 63.1 (CO
2
CH
2
), 55.3 (C-5
′
’),
47.6 (C-9
′
’), 46.7 (C-17
′
’), 45.8 (C-19
′
’), 41.7 (C-14
′
’), 41.4 (C-18
′
’), 39.3
(C-8
′
’), 38.8 (C-4
′
’), 38.5 (C-1
′
’), 37.1 (C-10
′
’), 33.9 (C-21
′
’), 33.2 (Me-
29
′
’), 32.8 (C-7
′
’), 32.5 (C-22
′
’), 30.7 (C-20
′
’), 29.5 (d,
2
J
C,P
=17.1 Hz,
CH
2
-CH
2
P), 28.2 (Me-23
′
’), 27.7 (C-15
′
’), 27.3 (C-2
′
’), 25.9 (Me-27
′
’),
23.8 (Me-30
′
’), 23.5 (C-11
′
’), 23.1 (C-16
′
’), 22.5 (d,
1
J
C,P
=50.5 Hz,
CH
2
P), 19.6 (d,
2
J
C,P
=3.3 Hz, CH
2
-CH
2
-CH
2
P), 18.4 (C-6
′
’), 17.1 (Me-
26
′
’), 15.7 (Me-24
′
’), 15.4 (Me-25
′
’) ppm; HRESI-MS m/z calcd. o
C
52
H
70
O
3
P ([M–B
-
]
+
): 773.5057, ound: 773.5051.
1
′
’-{5
′
-[(3
′
’β-Hyd oxyolean-12
′
’–3n-oyl)oxy]pen yl} iphenylphos-
phonium b omide (3). B omide II (150 mg, 0.25 mmol), PPh
3
(350 mg,
1.48 mmol) and MeCN (7.5 mL) we e used. Column ch oma og aphy
(80:1 → 5:1 CH
2
Cl
2
–MeOH) a o ded 3 as a whi e solid. Yield: 186 mg
(86 %). [
α
]
25
+33 (c 0.87, CH
2
Cl
2
);
1
H NMR (300 MHz, CDCl
3
) δ
7.88–7.66 (m, 15H, A -H), 5.23 ( , 1H, J
11
′
’a,12
=J
11
′
’b,12
=3.2 Hz, H-
12
′
’), 3.93 ( , 2H, J
H,H
=6.4 Hz, CO
2
CH
2
o CH
2
P), 3.86 (m, 2H, CO
2
CH
2
o CH
2
P), 3.20 (dd, 1H, J
2
′
’
α
,3
′
’
=4.9 Hz, J
2
′
’β,3
′
’
=10.6 Hz, H-3
′
’), 2.79
(dd, 1H, J
18
′
’,19
′
’
α
=13.9 Hz, J
18
′
’,19
′
’β
=4.1 Hz, H-18
′
’), 1.95–1.09 (m,
22H, H-1
′
’β, H-2
′
’
α
,β, H-6
′
’
α
,β, H-7
′
’
α
,β, H-9
′
’, H-15
′
’
α
,β, H-19
′
’
α
,β, H-
22
′
’
α
,β, H-21
′
’
α
,β, 3CH
2
), 1.09 (s, 3H, Me-27
′
’), 1.03–0.92 (m, 1H, H-
1
′
’
α
), 0.97 (s, 3H, Me-23
′
’), 0.88, 0.87 (2 s, 3H each, Me-25
′
’, Me-29
′
’),
0.84 (s, 3H, CH
3
, Me-30
′
’), 0.76 (s, 3H, Me-24
′
’), 0.70 (d, 1H, J
5
′
’,6
′
’β
=
10.4 Hz, H-5
′
’), 0.64 (s, 3H, Me-26
′
’) ppm;
13
C NMR (75.5 MHz, CDCl
3
) δ
177.8 (C-28
′
’), 143.9 (C-13
′
’), 135.1 (d,
4
J
C,P
=2.9 Hz, A -Cp), 133.9 (d,
2
J
C,P
=10.0 Hz, A -Co), 130.6 (d,
3
J
C,P
=12.6 Hz, A -Cm), 122.4 (C-
12
′
’), 118.5 (d,
1
J
C,P
=85.8 Hz, A -Cipso), 79.1 (C-3
′
’), 63.6 (CO
2
CH
2
),
55.3 (C-5
′
’), 47.7 (C-9
′
’), 46.7 (C-17
′
’), 46.0 (C-19
′
’), 41.8 (C-14
′
’), 41.4
(C-18
′
’), 39.4 (C-8
′
’), 38.8 (C-4
′
’), 38.5 (C-1
′
’), 37.1 (C-10
′
’), 34.0 (C-
21
′
’), 33.2 (Me-29
′
’), 32.8, 32.6 (C-7
′
’, C-22
′
’), 30.8 (C-20
′
’), 28.2 (Me-
23
′
’), 28.1 (CH
2
), 27.8 (C-15
′
’), 27.3 (C-2
′
’), 26.7 (d,
2
J
C,P
=16.4 Hz,
CH
2
-CH
2
P), 25.9 (Me-27
′
’), 23.8 (Me-30
′
’), 23.5 (C-11
′
’), 23.1 (C-16
′
’),
22.9 (d,
1
J
C,P
=49.3 Hz, CH
2
-P), 22.3 (d,
3
J
C,P
=4.2 Hz, CH
2
-CH
2
-CH
2
P),
18.4 (C-6
′
’), 17.2 (Me-26
′
’), 15.7 (Me-24
′
’), 15.5 (Me-25
′
’) ppm; HRESI-
MS m/z calcd. o C
53
H
72
O
3
P ([M−B
-
]
+
): 787.5214, ound: 787.5210.
1
′
’-{6
′
-[(3
′
’β-Hyd oxyolean-12
′
’–3n-oyl)oxy]hexyl} iphenylphos-
phonium b omide (4). B omide III (100 mg, 0.16 mmol), PPh
3
(212 mg,
0.81 mmol) and MeCN (7.5 mL) we e used. Column ch oma og aphy
(80:1 → 10:1 CH
2
Cl
2
–MeOH) a o ded 4 as a whi e solid. Yield: 120 mg
(85 %). [
α
]
25
+28 (c 0.81, CH
2
Cl
2
);
1
H NMR (300 MHz, CDCl
3
) δ
7.91–7.66 (m, 15H, A -H), 5.23 ( , 1H, J
11a,12
=J
11b,12
=3.5 Hz, H-12),
3.93 ( , 2H, J
H,H
=6.7 Hz, CO
2
CH
2
´
o CH
2
P), 4.02–3.89 (m, 2H, CO
2
CH
2
´
o CH
2
P), 3.20 (dd, 1H, J
2
α
,3
=4.8 Hz, J
2β,3
=10.4 Hz, H-3), 2.82 (dd, 1H,
J
18,19
α
=14.1 Hz, J
18,19β
=4.5 Hz, H-18), 1.93–1.21 (m, 26H),
1.17–0.99 (m, 24H, H-1
′
’β, H-2
′
’
α
,β, H-6
′
’
α
,β, H-7
′
’
α
,β, H-9
′
’, H-15
′
’
α
,β,
H-19
′
’
α
,β, H-22
′
’
α
,β, H-21
′
’
α
,β, 4CH
2
), 1.11 (s, 3H, Me-27
′
’), 0.98 (s, 3H,
Me-23
′
’), 0.97–0.93 (m, 1H, H-1
′
’
α
), 0.89, 0.88 (2 s, 3H each, Me-25
′
’,
Me-29
′
’), 0.85 (s, 3H, Me-30
′
’), 0.77 (s, 3H, Me-24
′
’), 0.71 (d, 1H, J
5
′
’,6
′
’β
=11.6 Hz, H-5
′
’), 0.67 (s, 3H, Me-26
′
’) ppm;
13
C NMR (75.5 MHz,
CDCl
3
) δ 177.8 (C-28
′
’), 143.9 (C-13
′
’), 135.1 (d,
4
J
C,P
=2.9 Hz, A -Cp),
133.8 (d,
2
J
C,P
=10.0 Hz, A -Co), 130.6 (d,
3
J
C,P
=12.5 Hz, A -Cm),
122.3 (C-12
′
’), 118.5 (d,
1
J
C,P
=85.8 Hz, A -Cipso), 79.0 (C-3
′
’), 64.1
(CO
2
CH
2
), 55.3 (C-5
′
’), 47.6 (C-9
′
’), 46.7 (C-17
′
’), 45.9 (C-19
′
’), 41.8 (C-
14
′
’), 41.4 (C-18
′
’), 39.4 (C-8
′
’), 38.8, 38.5 (C-1
′
’, C-4
′
’), 37.1 (C-10
′
’),
33.9 (C-21
′
’), 33.2 (Me-29
′
’), 32.8 (C-7
′
’), 32.5 (C-22
′
’), 30.8 (C-20
′
’),
30.1 (d,
2
J
C,P
=16.0 Hz, CH
2
-CH
2
-P), 28.3 (CH
2
), 28.2 (Me-23
′
’), 27.7
(C-15
′
’), 27.2 (C-2
′
’), 26.0, 25.9 (Me-27
′
’, CH
2
), 23.8 (Me-30
′
’), 23.5 (C-
11
′
’), 23.0 (C-16
′
’), 22.8 (d,
1
J
C,P
=49.7 Hz, CH
2
-P), 22.8 (d,
3
J
C,P
=
49.7 Hz, CH
2
-CH
2
-CH
2
-P), 18.4 (C-6
′
’), 17.1 (Me-26
′
’), 15.7 (Me-24
′
’),
15.4 (Me-25
′
’) ppm; HRESI-MS m/z calcd. o C
54
H
74
O
3
P ([M−B
-
]
+
):
801.5370, ound: 801.5357.
1
′
’-{12
′
-[(3
′
’β-Hyd oxyolean-12
′
’–3n-oyl)oxy]dodecyl} iphenyl-
phosphonium b omide (5). B omide IV (160 mg, 0.23 mmol), PPh
3
(298
mg, 1.15 mmol) and MeCN (8 mL) we e used. Column ch oma og aphy
(80:1 → 5:1 CH
2
Cl
2
–MeOH) a o ded 5 as a whi e solid. Yield: 190 mg
(85 %). [
α
]25
D +42 (c 0.82, CH
2
Cl
2
);
1
H NMR (300 MHz, CDCl
3
) δ
7.85–7.66 (m, 15H, A -H), 5.24 ( , 1H, J
11
′
’
α
,12
′
’
=J
11
′
’β,12
′
’
=3.3 Hz, H-
12
′
’), 3.96 (m, 2H, CO
2
CH
2
), 3.71 (m, 2H, CH
2
P), 3.18 (dd, 1H, J
2
′
’
α
,3
′
’
=
5.1 Hz, J
2
′
’β,3
′
’
=10.3 Hz, H-3
′
’), 2.83 (dd, 1H, J
18
′
’,19
′
’
α
=13.7 Hz,
J
18
′
’,19
′
’β
=4.4 Hz, H-18
′
’), 1.98–1.81 (m, 6H, H-11
′
’
α
, H-11
′
’β, H-16
′
’
α
,
H-16
′
’β, CH
2
), 1.66–1.34 (m, 19H, H-1
′
’β, H-2
′
’
α
, H-2
′
’β, H-6
′
’
α
, H-7
′
’
α
,
H-9
′
’, H-15
′
’β, H-19
α
, H-21
′
’
α
, H-22
′
’
α
, H-22
′
’β, 4CH
2
), 1.30–1.00 (m,
15H, H-6
′
’β, H-7
′
’β, H-15
′
’
α
, H-19
′
’β, H-21
′
’β, 5CH
2
), 1.11 (s, 3H, Me-
27
′
’), 0.95 (s, 3H, Me-23
′
’), 0.89 (s, 3H, Me-30
′
’), 0.92–0.84 (m, 1H, H-
1
α
), 0.86 (s, 6H, Me-25
′
’, Me-29
′
’), 0.74 (s, 3H, Me-24
′
’), 0.68 (s, 3H, Me-
26
′
’), 0.68 (d, J
5
′
’,6
′
’β
=11.2 Hz, H-5
′
’) ppm;
13
C NMR (75.5 MHz, CDCl
3
)
δ 177.9 (C=O), 143.9 (C-13
′
’), 135.1 (d,
4
J
C,P
=2.9 Hz, A -Cp), 133.8 (d,
2
J
C,P
=9.9 Hz, A -Co), 130.6 (d,
3
J
C,P
=12.5 Hz, A -Cm), 122.4 (C-12
′
’),
118.5 (d,
1
J
C,P
=85.8 Hz, A -Cipso), 79.0 (C-3
′
’), 64.4 (CO
2
CH
2
), 55.3
(C-5
′
’), 47.7 (C-9
′
’), 46.7 (C-17
′
’), 46.0 (C-19
′
’), 41.8 (C-14
′
’), 41.4 (C-
18
′
’), 39.4, 38.9 (C-4
′
’, C-8
′
’), 37.1 (C-10
′
’), 34.0 (C-21
′
’), 33.2 (Me-29
′
’),
32.8 (C-7
′
’), 32.6 (C-22
′
’), 30.8 (C-20
′
’), 30.6 (d,
2
J
C,P
=15.7 Hz, CH
2
-
CH
2
P), 29.7 (x2) (2CH
2
), 29.4(x2), 29.3 (3CH
2
), 28.7 (CH
2
), 28.2 (Me-
23
′
’), 27.7 (C-15
′
’), 27.3 (C-2
′
’), 26.2 (CH
2
), 26.0 (Me-27
′
’), 23.7 (Me-
30
′
’), 23.5 (C-11
′
’), 23.1 (C-16
′
’), 22.9 (d,
1
J
C,P
=49.5 Hz, CH
2
P), 22.8
(d,
3
J
C,P
=4.6 Hz, CH
2
-CH
2
-CH
2
P), 18.4 (C-6
′
’), 17.1 (Me-26
′
’), 15.7
(Me-24
′
’), 15.4 (Me-25
′
’) ppm; HRESI–MS: calcd. o C
60
H
86
O
3
P
([M–B
-
]
+
): 885.6309, ound: 885.6293.
2.3. Cell lines, g ow h and main enance
The human solid umo cell lines om he di e en issues used in
his s udy we e ob ained om he Ame ican Type Cul u e Collec ion
(ATCC, Manassas, VA). The cell lines we e es ed o hei au hen ica ion
by PCR p o iling using sho andem epea s, which was pe o med by
BaseClea (Leiden, The Ne he lands).
All cells we e g own in RPMI 1640 medium supplemen ed wi h hea
inac i a ed 5 % FBS, 2 mM L-glu amine, 100 U/mL o penicillin G and
0.1 mg/mL o s ep omycin. Cell cul u es we e main ained in 60 mm o
100 mm issue cul u e- ea ed dishes (depending on he desi ed numbe
o cells) placed in a 37 ◦C, 5 % CO
2
and 95 % humidi ied ai incuba o .
Fo passaging, cells we e de ached om he pla e, washed wice wi h
PBS wi hou calcium and magnesium and hen incuba ed o 4 min a
37 ◦C wi h a solu ion o ypsin/EDTA (170000 U ypsin/L, 200 mg/L
EDTA). F esh medium was added o neu alize he ac ion o ypsin and
he cells we e coun ed and seeded in a new dish a he desi ed con-
cen a ion o main ain hei g ow h o pe o m an expe imen .
2.4. An ip oli e a i e assays in 96-well pla es
An in-house e sion o he SRB assay o he Na ional Cance Ins i u e
(NCI, USA) was he p e e ed me hod o assess he e ec s o he small
molecules unde s udy on cell popula ion g ow h [20]. Cell seeding
densi y was 2,500–10,000 cells/well depending on he doubling ime o
he cell line. A e 24 h o incuba ion, se ial en- old dilu ions o com-
pounds in he cell cul u e medium we e added. The maximum es
concen a ion was 100 µM. As a nega i e con ol, he cells we e ea ed
wi h he maximal pe cen age o DMSO in he es wells (0.25 % / ).
A e 48 h o exposu e o he compounds, he SRB p o ocol [21] was
applied and he op ical densi y (OD) o each well was measu ed a 530
nm using a mic opla e eade (Powe Wa e XS, BioTek, USA). In-
e e ences om he pla e ma e ials we e educed by measu ing each
well in he dual mode a 620 nm. This dual wa eleng h me hod was
p e e ed in o de o a oid olume di e ences ha would a ec he pa h
leng h o he beam o ligh . Fo backg ound co ec ion, wells con aining
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
4
cell cul u e medium (blank) we e used.
2.4.1. An ip oli e a i e ac i i y
The pe cen age g ow h (PG) was calcula ed wi h espec o un ea ed
con ol cells (C) a each d ug concen a ion based on he di e ence in
OD a he beginning (T
0
) and he end o d ug exposu e (T), acco ding o
NCI o mulas. Thus, 50 % g ow h inhibi ion (GI
50
) ep esen s he d ug
concen a ion a which PG is +50.
2.4.2. E lux chemo esis ance
The cell line-based assay o p edic whe he a ce ain chemo he a-
peu ic agen could de elop chemo esis ance when beha ing as a P-gp
subs a e equi es a wild ype cell line (SW1573), and i s a ian o e -
exp essing P-gp (SW1573/Pgp). The me hod in ol es he calcula ion o
he GI
50
alues (as desc ibed abo e) o each compound unde s udy,
and in he absence o p esence o VP, which is known o inhibi P-gp.
The cell cul u e medium con aining VP was p epa ed by adding VP a
inal concen a ion o 10 µM. The s anda d P-gp subs a es PTX and VBL
we e used as he posi i e con ols. In his assay, he esis ance ac o (R )
o a ce ain compound is he a io o he GI
50
alue in he cell line
o e exp essing P-gp (mu an , m ) and in he wild ype (w ). R is
calcula ed in he absence and p esence o VP.
2.5. Cell colony o ma ion assay
A ange om 450 o 800 cells we e seeded on o a six-well pla e,
depending on he p oli e a i e p o ile he cell line. A e 24 h, he me-
dium was e eshed and compounds we e added a he desi ed con-
cen a ion. Cells we e incuba ed o a leas se en days un il a high
numbe o colonies could be obse ed mic oscopically in he con ol
wells, de ining a colony as a g oup o a leas 50 cells. The medium was
emo ed and he cells we e insed wi h PBS and ixed wi h me hanol o
2 min. Excess o me hanol was insed wi h ano he wash o PBS and he
cells we e s ained using c ys al iole (0.5 % w/ , deionized wa e ).
Colonies we e coun ed using Au oCellSeg, a MATLAB implemen a-
ion o au oma ic segmen a ion. The OD o each well was de e mined
dissol ing he s ained colonies in 1 % SDS in PBS and subsequen ly
measu ing he abso bance a 590 nm using a BioTek Powe Wa e XS
Abso bance Mic opla e Reade .
2.6. Mi ochond ial memb ane po en ial dis up ion
To e alua e changes in he mi ochond ial memb ane po en ial
(MMP), TMRE was used as a luo escen p obe in bo h luo escence
mic oscopy and mic opla e eade measu emen s.
Fo luo escence mic oscopy, cells we e seeded on o co e slips,
ea ed, and incuba ed o he indica ed imes. Then, 30 min be o e he
ending poin o he expe imen , he medium was eplaced wi h esh
medium con aining TMRE (0.5 µM) and incuba ed un il eaching he
endpoin a 37 ◦C unde da k condi ions. Then, cells we e insed wice
wi h PBS o a oid cell dislodgemen . Gi en ha he use o ixa i e agen s
is no compa ible wi h TMRE because o he educ ion o he TMRE
signal, co e slips we e moun ed di ec ly on o mic oscopy slides o e a
d op o glyce ol solu ion (10 % in PBS). A leas 6 andom ield luo-
escence images we e aken using a Leica DM 4000B® wi h a Qwin
so wa e (Leica Mic osys ems) equipped wi h N2.1 exci a ion il e
(bandpass [BP] il e =515–560 nm) a 40X magni ica ion.
Fo mic opla e eade measu emen s, cells we e seeded in 96 well
da k-wall clea -bo om pla es a a densi y o 10,000 cells/well. T ea -
men and incuba ion wi h TMRE dye ollowed as p e iously desc ibed
o luo escence mic oscopy assay. Then, cells we e insed e y ca e ully
wi h PBS, 100 µL o PBS was added, and he luo escence was measu ed
using a Va ioskan Lux Mul imode pla e eade (The mo Fishe Scien i ic,
USA) using 549/575 nm exci a ion/emission wa eleng hs.
Fluo escence uni s we e no malized o he cell numbe in e e y well
using c ys al iole s aining. B ie ly, he cells we e ixa ed wi h
me hanol o 1 min, insed wi h PBS and s ained wi h c ys al iole (0.5
% w/ , deionized wa e ). Dye was esuspended using SDS (1 % in PBS)
and measu ed a 590 nm using BioTek’s Powe Wa e XS Abso bance
Mic opla e Reade .
The TMRE s anda d cu e was ob ained by measu ing di e en
concen a ions o he luo escen dye in RPMI medium wi hou phenol
ed. E e y concen a ion was added in iplica es o S e ilin™ Mic o i-
e ™ 96 well black bo om mic opla es in a o al olume o 100 µL/well.
The luo escence in ensi y was measu ed by eading om he op, as
p e iously desc ibed.
2.7. Reac i e oxygen species de ec ion
Changes in he o ma ion o eac i e oxygen species (ROS) we e
iden i ied using DCFDA. Cells we e seeded on o 96 well da k-wall clea -
bo om pla es as desc ibed p e iously. P io o he exposu e o he
selec ed compounds, he medium was emo ed and he cells we e
incuba ed wi h 40 µM o DCFDA in RPMI wi hou phenol ed o 30 min.
Then, he compounds we e added o he medium con aining DCFDA
conside ing he inal concen a ion desi ed. Fluo escence in ensi y was
measu ed using a Va ioskan Lux Mul imode pla e eade a 492/520 nm
exci a ion/emission wa eleng hs.
2.8. DNA s aining
P io o cell seeding, s e ile co e slips we e placed on o a 6-well
pla es. Then, cells we e pla ed on op o he co e slip a a densi y o
100,000 cells/well. A e o e nigh incuba ion, he compounds we e
added o he app op ia e concen a ion. A he desi ed expe imen al
ime s op, cells we e ixed using PFA solu ion (1 mL, 4 % in PBS) o 10
min a oom empe a u e. PFA solu ion was emo ed and cells we e
washed wi h NH
4
Cl solu ion (50 mM in PBS) o 15 min ( o educe PFA
backg ound) and insed wi h PBS a e wa ds. A T i on solu ion (0.1 % in
PBS) was used o pe meabilize he cells. A e 10 min o incuba ion, he
cells we e insed 2–3 imes wi h PBS o 5 min each. S aining was pe -
o med using a DAPI solu ion (750 µL, 1 µg/mL in me hanol) o 10 min
in he absence o ligh and mode a e oscilla ion. The DAPI solu ion was
eco e ed and cells we e insed again 2–3 imes main aining da kness
condi ions. Co e slips we e moun ed on o a mic oscopy slide o e a
d op o Mowiol moun ing medium and sealed using nail polish o a oid
e apo a ion. A leas 6 andom ields we e imaged using 20X and 40X
magni ie s in Leica DM 4000 B® mic oscope (Leica Mic osys ems) wi h
he A exci a ion il e (BP =340–380).
Quan i ica ion o he mean luo escence o DAPI-s ained cells was
pe o med using FIJI/ImageJ so wa e (NIH, USA).
2.9. Con inuous li e-cell imaging
D ug-induced pheno ypic changes ela ed o he di e se MoA o
compounds subjec ed o s udy we e obse ed in a con inuous manne
using a li e cell mic oscopy CX-A label- ee cell imaging sys em,
(Nanoli e S.A, Swi ze land). Cells we e seeded on 35 mm high glass-
bo om µ-dish (IBIDI, G ¨
a el ing, Munich, Ge many) a a concen a ion
o 80,000–100,000 cells pe mL, depending on he cell ype. A e 24 h,
he medium was eplaced wi h RPMI 1640 wi hou phenol ed since he
pH indica o would in e e e wi h he e ac i e index measu emen s.
Compounds we e added o cells a he desi ed concen a ions and
expe imen al se ings we e con igu ed depending on he ype o expe -
imen (i.e. numbe o cycles and hei du a ion). A eas whe e he
numbe o cells was ep esen a i e we e selec ed o image acquisi ion.
Da a we e ans e ed o FIJI (NIH, USA) o isual inspec ion.
A e he images we e aken o he desi ed pe iod, EVE so wa e
(Nanoli e S.A Swi ze land) was used o cell segmen a ion and analysis
o he e ac i e index (RI) based pa ame e s. These 11 pheno ypic pa-
ame e s a e di ided in o wo g oups: a) Mo phology ela ed pa ame-
e s: cell a ea (%), cell a ea (
μ
m
2
), cell eccen ici y, cell pe ime e , cell
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
5
ex en , cell compac ness, cell o m ac o and cell g anula i y; and b)
Con en ela ed pa ame e s: mean e ac i e index, d y mass (pg) and
a e age d y mass densi y (pg/
μ
m
3
). Li e Cell Dea h Assay (LCDA)
so wa e (Nanoli e S.A., Swi ze land) was used o in eg a e e ac i e
index pa ame e s o ob ain apop osis and nec osis kine ics.
2.10. Fluo escence labelling in li e cell imaging
Hoechs 33,342 was used o s ain he nuclea compa men s o e
ime. B ie ly, cells we e seeded as desc ibed o he e ac i e index li e
cell imaging. Assay concen a ion o Hoechs was p epa ed om a 10
mg/mL s ock solu ion (1:2000 dilu ion) and incuba ed unde da k
condi ions o 10 min. The medium was emo ed and he cells we e
washed once wi h PBS. The compounds unde s udy we e added o RPMI
wi hou phenol ed a he desi ed concen a ion. Re ac i e index
acquisi ion was pe o med as p e iously desc ibed. Fo luo escence
imaging, he exposu e ime and signal ampli ica ion we e selec ed
empi ically o each expe imen and main ained a he same ime o all
he samples measu ed. Fluo escence images we e ob ained using a
DAPI-UV il e e e y 10, 20 o 30 cycles, depending on he expe imen .
Fluo escence and RI images we e moun ed and analysed using
STEVE so wa e (Nanoli e S.A., Swi ze land).
2.11. Animals and housing
All expe imen s ollowed he PI87/20 esea ch p ocedu es app o ed
by he e hics commi ee o animal expe imen s o he Uni e si y o
Za agoza. Animal ca e and use was ca ied ou in acco dance wi h he
Spanish policy o animal p o ec ion RD53/2013 and he Eu opean
Union di ec i e 2010/63 o he p o ec ion o animals used o expe i-
men al and o he scien i ic pu poses. Fo he in i o oxicology s udy, 14
male RjO l:SWISS we e used. Eigh een adul emale Rj:ATHYM-
Foxn1
nu/nu
mice (15 weeks) we e used in he in i o oncological e icacy
s udies. All animals we e pu chased om Jan ie Labo a o ies. Up o a
maximum o 5 animals we e kep in 30 x 20 x 15 cm boxes, wi h access
o osmo ized and au ocla ed wa e and i adia ed ood ad libi um (2914
Teklad Global 14 % p o ein oden main enance die ). The oom em-
pe a u e was main ained a 23 ±1 ◦C wi h a 12 h ligh cycle (s a ing a
8:00 a.m.).
2.12. In i o oxicology assay
Be o e es ing compound 3 in an in i o e icacy s udy, we e alua ed
he in i o oxici y o he mos p omising compound in i o. Fo such a
pu pose, an acu e o al (PO) and in ape i oneal (IP) oxici y es was
pe o med based on he OECD Tes No. 425: Acu e O al Toxici y: Up-
and-Down P ocedu e. The highes dose es ed was 17.5 mg/kg and he
s a ing dose was 0.55 mg/kg, applying a slope o 2. The es was pe -
o med in male RjO l: SWISS mice o 11–14 weeks wi h dosing in e als
o 48 h. Then, oxici y was e alua ed a e a single adminis a ion.
D ugs we e adminis e ed o al (PO) o conscious mice wi h a s anda d
olume o 10 mL kg
−1
body weigh . as a suspension in physiological
saline (5 % DMSO). Animals we e moni o ed indi idually a leas once
du ing he i s 30 min a e injec ion, pe iodically du ing he i s 24 h
(wi h special a en ion o he i s 4 h) and daily he ea e o a o al o
14 days.
Heal h s a us o he animals was moni o ed using a modi ied scale
based on he p oposal by Mo on and G i i hs. Gene al appea ance
(0–3), weigh (0–2), spon aneous beha io (0–3), and p o oked
beha io (0–3) we e sco ed.
2.13. In i o oncological e icacy assay
4T1 mouse mamma y umo cells exp essing luci e ase was
implan ed subcu aneously in mice and isualized using op ical imaging
(IVIS LUMINA 100) o moni o umo g ow h non-in asi ely in a
longi udinal s udy.
The s udy was ca ied ou in 2 expe imen al g oups: con ol and
ea ed (compound 3). Each expe imen al g oup consis ed o 9 emale
mice o he Rj:ATHYM-Foxn1
nu/nu
s ain (Jan ie Labs) wi h an age o
15 weeks and a mean weigh o 38.36 g (±2.46 g).
On day 0, all animals (18) we e implan ed by subcu aneous admin-
is a ion in he lumba egion wi h 1x10
6
umo cells in a olume o 200
µL o phospha e bu e ed saline (PBS). On day 2, a e op ical imaging
ollow-up, he expe imen al g oups we e es ablished. A ha ime, he
mean adiance o each expe imen al g oup was se be ween 2.83 x 10
7
and 3.15 x 10
7
pho on/sec/cm
2
/s . The s a o ea men was on day 2
once he g oups had been es ablished.
The ial had a o al du a ion o 22 days om he day o inocula ion
o he umo cells. Op ical imaging and calipe ollow-up, weigh change
and ea men we e pe o med on days 2, 6, 8, 10, 13, 15, 17 and 20,
wi h a dose o 0.55 mg/Kg (3 imes/week). T ea men was pe o med
o ally wi h a s e ile me al eeding ube 22ga x 25 mm (FTSS-22S-25,
Ins ech) a he end o op ical imaging ollow-up a a concen a ion o 1
mg/kg. D ugs we e adminis e ed o ally (PO) o conscious mice wi h a
s anda d olume o 10 mL kg
−1
body weigh as a suspension in physi-
ological saline (5 % DMSO). Fi e minu es be o e op ical imaging
acquisi ion, a olume o 10 mL kg
−1
body weigh o a luci e in solu ion
(15 mg/mL) was adminis e ed in ape i oneally o each mouse.
Th ough he calipe measu emen s, he mean umo olume was
calcula ed wi h he ollowing o mula (Leng h*Wid h
2
/2).
3. Resul s
3.1. Syn hesis and an ip oli e a i e e ec s o OA-TPP hyb ids
In he design o OA-TPP hyb ids, we en isioned he linkage be ween
OA and he TPP agmen using a lexible and lipophilic hyd oca bon
chain. To connec bo h key s uc u al mo i s we used linea alky chains
con aining 4, 5, 6 o 12 me hylene g oups. The gene al o mula o hese
hyb id molecules is OA-(CH
2
)
n
-TPP. OA-TPP hyb ids we e accessed in a
wo-s ep syn he ic pa hway (Table 1): 1) chemoselec i e alkyla ion o
he ca boxylic moie y o OA wi h an excess o
α
,
ω
-dib omoalkanes wi h
di e en leng hs unde basic condi ions (K
2
CO
3
), 2) ollowed by
nucleophilic displacemen o he e minal b omine a om wi h PPh
3
in a
Fishe -Po e ube. As a i s s ep, he OA-TPP hyb ids 2–5 as well as
pu e OA (1), we e e alua ed as an ip oli e a i e agen s agains a panel
o human solid umo cell lines (Table 1).
Al hough he s uc u al a ia ion o he compounds was e y sub le
–p esen ing only di e ences in he numbe o me hylene uni s ha
con o m o he linke – a p elimina y s uc u e–ac i i y ela ionship was
es ablished. The leng h o he linke signi ican ly a ec ed he po ency o
he compounds. Mino di e ences we e obse ed in he an ip oli e a i e
e ec s o he C
4
, C
5
and C
6
compounds (2, 3 and 4, espec i ely), which
exhibi ed po encies wi hin he nanomola ange. Howe e , mi ocan 5
(bea ing a C
12
linke ) showed a clea loss o po ency compa ed o he
o he hyb ids. Hence, compounds wi h sho chains (n =4–6) showed
GI
50
alues in he nanomola ange, whe eas long alkyl chains (n =12)
p oduced a signi ican dec ease in po ency, wi h GI
50
alues a he
mic omola le el. This is consis en wi h he epo ed p edominan
an ip oli e a i e e ec s o pu e OA on cance cells [22].
The se o OA-TPP hyb ids was also e alua ed in he non- umo
immo alized cell line BJ-hTERT (Table 1). In e es ingly, none o he
compounds exe ed an ip oli e a i e e ec s on his cell line (GI
50
>10
5
nM), showing ema kable selec i i y agains cance cell lines. The
absence o an e ec on cell g ow h in non- umo human ib oblas s
indica ed p omising selec i i y owa ds heal hy cells. This is a ele an
esul because one o he main d awbacks o he use o mi ocans in
he apy is hei in insic oxici y [9]. The absence o an ip oli e a i e
ac i i y in a heal hy cell line ag eed wi h o he epo s in he li e a u e
ega ding he absence o oxic e ec s when using OA o TPP sepa a ely
[10,22]. In he case o he OA-TPP hyb ids p esen ed he e, he selec i i y
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
6
be ween umo and non- umo cell lines was e en la ge .
Consis en wi h he an ip oli e a i e da a, compound 5 was dis-
ca ded a his poin .
3.2. OA-TPP hyb ids a e no P-gp subs a es
Nex , we checked whe he he OA-TPP hyb ids 2–4 could be sub-
s a es o he e lux pump P-gp, a known mechanism o d ug esis ance
in cance cells. The assay comp ises a wild ype cell line (SW1573) and a
mu an a ian ha o e exp esses P-gp (SW1573/Pgp), and PTX unc-
ions as posi i e con ol. Bo h cell lines we e exposed o he compounds
unde e alua ion. The GI
50
alues we e compu ed a e 48 h o exposu e
in he absence o p esence o 10 µM e apamil (VP) in cell cul u e
(Table 2). In his assay, he esis ance ac o (R ) o any gi en com-
pound is de ined as he a io o i s GI
50
in he cell line o e exp essing P-
gp (mu an , m ) and he wild ype (w ). R was calcula ed in he absence
and p esence o VP. F om he esul s ob ained in he absence o VP, we
can in e om he R alues ha he mi ocans 2–4 we e 10–15 imes less
po en agains he mu an cells. To ou su p ise, he co- ea men wi h
VP sensi ized bo h he wild ype and he esis an cance cell lines. This
is an unusual esul o his ype o assay. VP is used in his assay because
i is a P-gp blocke , which a oids d ug e lux and consequen ly enhances
he ac i i y. Howe e , his esul was no consis en wi h he inc eased
po ency obse ed o he OA-TPP hyb ids. I has been epo ed ha TPP,
benzodiazepines, as well as se e al Ca
2+
an agonis s –like VP– inhibi
mi ochond ial Na
+
/Ca
2+
exchange [23]. Thus, we specula ed ha he
e ec s obse ed in Table 2 o he co- ea men wi h VP we e jus a
esul o he syne gis ic in e ac ion be ween he OA-TPP hyb ids and VP.
Fu he mo e, hese esul s ag ee wi h he p emise ha compounds 2–4
a e mi ocans.
3.3. E ec s on ep oduc i e iabili y
Gi en he po en e ec obse ed du ing he an ip oli e a i e
sc eening, we decided o e alua e he abili y o OA-TPP hyb ids 2–4 o
in e e e wi h he su i al and g ow h abili y o cells a e long- e m
exposu e. To achie e his goal, clonogenic assays we e pe o med by
incuba ing HeLa cells wi h he compounds o 8 days. Conside ing his
ex ended exposu e ime, only he GI
50
and GI
50
/3 concen a ions we e
assayed (Table 1). All h ee compounds we e able o p oduce a dec ease
in he numbe o cell colonies o med when compa ed o un ea ed cells,
and in a concen a ion dependen manne . In addi ion, he compounds
we e unable o educe colony o ma ion when he cells we e exposed o
a lowe dose. In e es ingly, he i s di e ences be ween he compounds
we e obse ed in hese assays. HeLa colonies we e s a is ically signi i-
can less in he ea men wi h GI
50
dose o compounds 3 and 4 bu no
o 2. Since he clonogenic assay conside s a colony as a g oup o 50
cells, o uniquely e alua e he numbe o colonies, ele an di e ences
could be concealed. The images aken depic di e ences in he size o he
colonies be ween he con ol and he ea men g oups (Fig. 2). The
di e ences a e c ys al iole s aining measu emen be ween he un-
ea ed and ea ed samples we e mo e e iden . Th ough his p ocess, all
h ee compounds signi ican ly educed he colony s aining a he highe
dose, and de i a i es 3 and 4 we e able o do so a he lowe dose. These
esul s demons a ed ha he OA-TPP hyb ids we e able o hinde cell
g ow h e en when a single adminis a ion was main ained o 8 days.
Simul aneously, exposu e o he compounds was able o educe no only
he numbe o colonies bu also he numbe o he cells con o ming
hem.
Table 1
Syn hesis and an ip oli e a i e ac i i y (GI
50
, nM) o OA and OA-TPP hyb ids agains human solid umo s cell lines and non- umo cell line BJ-hTERT.
Cell line (o igin)
1(H) 2
(n =4)
3
(n =5)
4
(n =6)
5
(n =12)
A a-C
A549 (lung) 7167 ±1136 17 ±1.5 21 ±2.4 34 ±13 6418 ±2759 75 ±17
HBL-100 (b eas ) 23803 ±7173 31 ±13 31 ±7.4 57 ±11 6845 ±2910 642 ±100
HeLa (ce ix) 13827 ±9121 45 ±8.1 36 ±1.7 41 ±8.5 5558 ±1952 59 ±7.8
T-47D (b eas ) 35847 ±7209 32 ±7.6 26 ±1.9 47 ±7.5 >10
5
9437 ±3705
WiD (colon) 31684 ±8087 18 ±8.9 13 ±1.5 38 ±9.3 >10
5
20296 ±6973
BJ-hTERT ( ib oblas s) >10
5
>10
5
>10
5
>10
5
>10
5
Values ep esen he mean ±s anda d de ia ion o wo o i e independen expe imen s. A a-C was used as he posi i e con ol.
Table 2
An ip oli e a i e ac i i y (GI
50
, nM) o OA-TPP hyb ids and PTX agains SW1573
and SW1573/Pgp cell lines being co-incuba ed wi h o wi hou VP.
w/o Ve apamil w Ve apamil
Compound SW1573 SW1573/
Pgp
R SW1573 SW1573/
Pgp
R
224 ±3.6 240 ±27 10 0.12 ±
0.02
14 ±2.4 117
315 ±2.2 230 ±20 15 0.88 ±
0.20
18 ±4.8 21
421 ±4.9 220 ±26 10 0.46 ±
0.04
18 ±1.9 38
PTX 0.053 ±
0.022
300 ±110 564 0.46 ±
0.21
0.31 ±
0.15
0.7
Values ep esen he mean ±s anda d de ia ion o a leas h ee independen
expe imen s.
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
7
3.4. OA-TPP hyb ids p oduce mi ochond ial memb ane depola iza ion
and eac i e oxygen species p oduc ion
The mi ochond ial memb ane po en ial and eac i e oxygen species
(ROS) o HeLa cells a e ea men wi h he OA-TPP de i a i es was
e alua ed h ough di e se commonly used me hods. Fo compa ison
pu poses, ca bonyl cyanide 3-chlo ophenylhyd azone (CCCP) and
hyd ogen pe oxide we e used as posi i e con ols, espec i ely. CCCP
ac s as a p o onopho e ha dissipa es he p o on g adien necessa y o
main aining he mi ochond ial memb ane po en ial (MMP) [24], hus
p oducing a depola iza ion e ec . To moni o changes in he MMP, we
i s used e ame hyl hodamine e hyl es e (TMRE). This lipophilic
ca ionic dye ac s as a luo escen p obe o mi ochond ial pola iza ion,
accumula es in heal hy pola ized mi ochond ia bu eleases wi h
memb ane depola iza ion. The educ ion in he luo escence signal
poin ed ou MMP depola iza ion, wi h TMRE being eleased in o he
cy osol and subsequen ly washed ou (Fig. 3A–B). Pu e OA (1) was
included in he assay o show he ele ance o he TPP ion agmen in
he MoA o he compounds unde s udy. Al hough a e y modes e ec
was obse ed, he e was no signi ican educ ion in ela i e luo escence
Fig. 2. E ec s on ep oduc i e iabili y: (A) Rep esen a i e images o he colony o ma ion assay. HeLa cells we e exposed o de i a i es 2–4 a wo concen a ions
(GI
50
and GI
50
/3) o 8 days and s ained wi h c ys al iole ; (B) Rela i e numbe o colonies compa ed o con ol; (C) Abso bance o c ys al iole s aining esus-
pended in PBS wi h 1 % SDS ela i e o he con ol. *p <0.05; **p <0.005.
Fig. 3. E ec o OA-TPP hyb ids on mi ochond ia: (A) Rep esen a i e luo escence images o un ea ed HeLa cells o ea ed wi h 3 a 5X GI
50
(180 nM) and
incuba ed o 30 min wi h TMRE. Scale ba : 10 µm; (B) Fluo escence in ensi y o HeLa cells s ained wi h TMRE when exposed o 30 min o CCCP and 1–4 a 5X hei
GI
50
(70.000, 225, 180 and 200 nM, espec i ely). E o ba s: SD o a leas h ee independen expe imen s. *p <0.05; **p <0.01; ***p <0.001; (C) E ec o OA-TPP
hyb ids on ROS induc ion. Fluo escence signal o DCF exp essed as RFU ela i e o ha o he con ol. Hyd ogen pe oxide se ed as posi i e con ol. E o ba s
ep esen s anda d de ia ion o a leas h ee independen expe imen s. *p <0.05; **p <0.01.
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
8
uni s (RFU) by 1 when compa ed o he un ea ed samples a e 18 h o
exposu e. Howe e , a signi ican dec ease in he luo escen signal was
obse ed a e ea men wi h he h ee selec ed compounds, and in he
(dec easing) o de 3 >4 >2. In addi ion, hyb id 3 was able o p oduce a
signi ican educ ion in MMP, which was compa able o ha o he
posi i e con ol CCCP. This esul was con i med using luo escence
mic oscopy. As depic ed in Fig. 3A–B, un ea ed cells incuba ed o 30
min wi h TMRE showed b oad luo escence s aining, while cells ea ed
wi h compound 3 leaked he dye in o he ex acellula medium,
esul ing in a loss o signal. These esul s sugges ha he e is an e ec o
OA-TPP hyb ids on mi ochond ial ac i i y, and he TPP ion mo i in he
molecule plays a key ole in p omo ing his e ec . In addi ion, he leng h
o he linke in luences his speci ic ac i i y gi en he di e ences
obse ed be ween he h ee hyb ids, highligh ing he i e-ca bon linke
(3) as he one ha exe ed a highe educ ion in MMP.
The nex s ep was he e alua ion o eac i e oxygen species (ROS)
o ma ion because o exposu e o he compounds. Unbalanced ROS
equilib ium is a clea sign o mi ochond ial dys unc ion in an oxida i e
s ess en i onmen , p eceding apop o ic cell dea h h ough he mi o-
chond ial pa hway [25]. Conside ing ha he compounds p oduced a
educ ion in he MMP, a ia ions in he o ma ion o ROS a sho imes
o exposu e should happen. The e o e, he induc ion o ROS a e 3 h
was measu ed in HeLa cells using he widely known DCFDA assay.
Hyd ogen pe oxide se ed as posi i e con ol. Alike he esul s ob ained
wi h he TMRE assay, he highes dichlo o luo escein (DCF) signal was
obse ed a e exposu e o compound 3, while OA-TPP hyb ids 2 and 4
showed weake luo escence signals (Fig. 3C). Hence, posi i e DCF
luo escence –independen ly o whe he he inc eased signal depends on
o al ROS accumula ion o cy och ome c elease– sugges s pe u ba ion
o he mi ochond ial unc ion by he addi ion o he OA-TPP de i a i es
in he same di ec ion as he educ ion in MMP, as bo h p ocesses a e
indica i e o damage induced o his o ganelle. In e es ingly, co-
adminis a ion wi h ROS inhibi o asco bic acid alongside wi h 3 did
no modi y he an ip oli e a i e e ec s o 3 on HeLa cells.
3.5. Explo ing he mode o cell dea h
One o he hallma ks o cell dea h caused by d ugs is nuclea shape
modi ica ion. DAPI s aining o cell exposed o OA-TPP hyb ids 2–4 o
24 h allowed obse ing educ ion in he numbe o s ained en i ies –as
expec ed by hei an ip oli e a i e e ec s–, nuclea la ening and he
appea ance o apop o ic ea u es, such as nuclea size dec ease (Fig. 4A).
Al hough some ea u es o DNA agmen a ion we e obse ed, especially
in he ea men wi h 2, he e was no signi ican numbe o cells man-
i es ing his e ec .
While DAPI s aining helped isualizing nuclea shape modi ica ions,
li e cell imaging o Hoechs p e-s ained cells allowed o ollow in ime
he ea u es o cell dea h (Fig. 4B, Supplemen a y Videos S1a-c). Cell
sh inkage, memb ane blebbing and o e all cellula compac ion – ypical
apop o ic nuclea pheno ypes– we e obse ed a e eigh hou s o
exposu e o mi ocans 2–4. Kine ics o he s a us o he cell cul u e
showed how he h ee compounds induced cell dea h, mainly h ough
apop osis. Based on image analysis, OA-TPP induced an apop o ic mode
o dea h wi h a la e ansi ion o nec o ic pheno ypes (sligh memb ane
swelling) in some o he cells con o ming he popula ion unde s udy.
Mo eo e , all he compounds induced massi e cy oplasmic acuola ion.
In e es ingly, ime-lapse obse a ion o he cell a e showed how cells
ha we e mo e acuola ed needed mo e ime o show apop o ic phe-
no ypes and die la e han hose ha p esen ed less acuoles in o al.
Cy oplasmic acuoles we e easily obse ed in he space su ounding he
nucleus.
3.6. S udies on acuole o ma ion
As mi ochond ial unc ion should be al e ed by MMP educ ion, i
could be easible ha compensa o y mechanisms would be igge ed o
main ain he me abolic equi emen s o cells [26]. To explain hese e -
ec s p oduced by he OA-TPP hyb ids, mul iple hypo heses we e
o mula ed.
Since all h ee de i a i es (2–4) we e able o induce acuole o -
ma ion ega dless o he size o he linke be ween he wo moie ies, we
specula ed i he inclusion o he TPP was de e minan o his
Fig. 4. Explo ing he mode o cell dea h: (A) Rep esen a i e images o DAPI s aining in HeLa cells a e exposu e o OA-TPP hyb ids o 24 h. Yellow a ows:
Rep esen a i e condensed nuclei. Red a ows: DNA agmen a ion. 40x magni ica ion. Scale ba : 10 µm; (B) Rep esen a i e snapsho s o HeLa cells ea ed wi h 200
nM o OA-TPP hyb ids. Nuclea s aining was pe o med using Hoechs 33,342 and acked o e ime using con inuous li e-cell imaging. Scale ba : 20 µm.
A. Pue a e al.
Biochemical Pha macology 234 (2025) 116807
9
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