Dal on
T ansac ions
PAPER
Ci e his: DOI: 10.1039/d4d 01490a
Recei ed 21s May 2024,
Accep ed 20 h Augus 2024
DOI: 10.1039/d4d 01490a
sc.li/dal on
Effec o mono- and dinuclea hiosemica bazone
pla inacycles in he p oli e a ion o a colo ec al
ca cinoma cell line†
F ancisco Reigosa-Chamo o,‡
a
Sand a Co dei o,‡
b,c
M. Te esa Pe ei a,
a
Bea iz Filipe,
b,c
Ped o V. Bap is a,
b,c
Alexand a R. Fe nandes *
b,c
and
José M. Vila *
a
He ein, we desc ibe he syn hesis and cha ac e iza ion o a se ies o hiosemica bazone pla inacycles.
Thei ac i i y owa ds HCT116 and A2780 cance cell lines as well as no mal fib oblas s was explo ed and
conclusions abou he influence o hei s uc u es we e d awn based on he esul s. Ligands L1–3, e a-
nuclea compounds [P (L1–3)]
4
,[P (L1–3)(PPh
3
)], and [P (L1–L3)
2
{Ph
2
P(CH
2
)
4
PPh
2
}], and phosphine
de i a i es, we e deemed unp omising owing o hei lack o ac i i y. Howe e , mono-coo dina ed dipho-
sphine complexes [P (L1–L3)(Ph
2
PCH
2
PPh
2
-P)] showed high selec i i y and low IC
50
alues, and hei
an ip oli e a i e ac i i y was u he s udied. The h ee s udied de i a i es 3a,3b and 3c showed a as
in e naliza ion o HCT116 colo ec al cance cells wi h simila IC
50
alues, which induced a depola iza ion
o mi ochond ial memb ane po en ial, wi h he subsequen igge ing o apop osis and au ophagy in he
case o 3c. In he case o compounds 3a and 3b, cell dea h mechanisms (ex insic and in insic apop osis,
espec i ely) we e igge ed ia he induc ion o eac i e oxygen species (ROS). The h ee compounds
we e no oxic o a chicken emb yo in i o (a e 48 h), and, impo an ly, showed an an i-angiogenic
po en ial a e exposu e o he IC
50
o compounds 3a,3b and 3c.
In oduc ion
Since he seminal pape by Cope and Siekman
1
desc ibing
cyclome alla ed compounds, i.e., me allacycles, achie ing he
ac i a ion o a oma ic C–H bonds using ansi ion me als was
published, he chemis y o such species has a ac ed much
a en ion. Al hough many ansi ion me als can po en ially be
applied o achie e co esponding me allacycles, he p ima y
ones include palladium and pla inum. This is a esul o he
nume ous applica ions o hese wo ansi ion me als in
eac i i y,
2–4
me allomesogens,
5,6
and syn he ic chemis y,
7,8
whe e hey a e used in he unc ionaliza ion o a oma ic
ca bons h ough inse ion eac ions.
9,10
Palladium and
pla inum ha e also ound applica ion in ca alysis a e he
disco e y o phosphine palladacycles by He mann e al.
11–13
Fu he mo e, being o pa icula no e a e he Suzuki
14–19
and
Mizo oki–Heck c oss-coupling eac ions.
19,20
One applica ion
ha has expanded he mos o ob ious easons is hei usage
as an ineoplas ic subs ances, whe e he unc ionali y o me al-
lacycles is mainly ma ked by palladium o pla inum.
21–26
The
choice o he ligand in he syn hesis o new compounds is o
g ea impo ance, especially when bioac i e applica ions a e o
be conside ed, since hei p ope ies will depend on he inal
s uc u e o he compound. Thiosemica bazone ligands a e
ypical ligands in cyclome alla ion chemis y owing o he ease
o hei p epa a ion ia a simple condensa ion eac ion, which
is also compa ible wi h a numbe o unc ional g oups. Thei
selec ion is enhanced because hey a e bioac i e on hei
own
27
as well as when hey a e combined wi h he me als o
coo dina ion compounds and cyclome alla ed species.
28
Thei
effec s as an iplasmodic agen s we e s udied o yea s, bu
esea ch in e es s and new indings ha e shi ed hei appli-
ca ion o o he diseases such as cance .
29
We ha e epo ed
ha in hiosemica bazone me allacycles, he o ganic ligand is
able o bind o a me al cen e as iden a e [C,N,S] in a e a-
nuclea
30
s uc u e h ough M–S
chela ing
and M–S
b idging
bonds.
†Elec onic supplemen a y in o ma ion (ESI) a ailable. CCDC 2309646 (1b) and
2309120 (2c). Fo ESI and c ys allog aphic da a in CIF o o he elec onic o ma
see DOI: h ps://doi.o g/10.1039/d4d 01490a
‡Bo h au ho s con ibu ed equally.
a
Depa amen o de Química Ino gánica, Uni e sidade de San iago de Compos ela,
A enida das Ciencias s/n, 15782 San iago de Compos ela, Spain.
E-mail: [email p o ec ed]
b
Associa e Labo a o y i4HB –Ins i u e o Heal h and Bioeconomy, NOVA School o
Science and Technology, NOVA Uni e si y Lisbon, 2819-516 Capa ica, Po ugal.
E-mail: [email p o ec ed]
c
UCIBIO, Depa amen o de Ciências da Vida, Faculdade de Ciências e Tecnologia,
Uni e sidade No a de Lisboa, Po ugal
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This a ou s he addi ion o an app op ia e lea ing g oup on
he me al in he esul ing mononuclea compound, holding
as he hiosemica bazone/me al moie y. This sa egua ds
me allacycle s uc u al in eg i y du ing he anspo in bio-
logical luids un il i eaches cance cells, secu ing i s biologi-
cal/d ug ac i i y. This also allows o modi y he cha ac e is ics
o he compounds wi h di e se ancilla y ligands, which has
p o ed o be i al in he p o iciency o he pa en compound in
ca aly ic eac ions, spec oscopic and emissi e chemilumines-
cence and an ip oli e a i e effec . In his sense, phosphine
ligands can be a g ea way o add a iabili y o he esul ing
s uc u es by using diffe en ligands and changing he eac ion
condi ions. Ou p e ious expe ience wi h cyclome alla ed com-
pounds es ed he po en ial o mononuclea and dinuclea
species as an icance agen s. The esul s allowed us o con-
clude ha he inclusion o a second me al ia palladacycle
me allo-ligands bea ing a monocoo dina ed diphosphine did
no p oduce a no iceable imp o emen , and ha he combi-
na ion o he bo onic acid unc ion and dppm showed g ea
po en ial. Ne e heless, he enhanced effec could no be
a ibu ed o one ac o alone. Fu he mo e, i was unclea i
all ypes o bo onic acid de i a i es, ega dless o he ancilla y
ligands, could be as effec i e.
26
The esul s p esen ed he e
con i m he effec i eness o he pla inum analogues, and
poin o he dppm ligand as a key s uc u al moie y in he bio-
logical effec o hese species. In he p esen wo k, we aimed
o expand on he knowledge o he amily o hiosemica ba-
zone pla inacycles by adjus men o he imine g oups and he
ancilla y ligands in a se ies o compounds de i ed om ace yl-
phenylbo onic acid o de e mine he effec o his uning in
hei bioac i i y as an icance d ugs.
Expe imen al sec ion
Gene al p ocedu es
All sol en s we e used wi hou any p e ious pu i ica ion. All
chemicals we e o eagen g ade. The phosphines PPh
3
( i-
phenylphosphine), PPh
2
(CH
2
)PPh
2
[bis(diphenylphosphino)
me hane, dppm] and PPh
2
(CH
2
)
4
PPh
2
[bis(diphenylpho-
sphino)-bu ane, dppb] we e pu chased om Sigma-Ald ich.
Elemen al analyses we e pe o med in a THERMO FINNIGAN,
model FLASH 1112. IR spec a we e acqui ed wi h a JASCO FT/
IR-4600 spec ome e equipped wi h an ATR, model ATR-PRO
ONE. The NMR spec a we e acqui ed on Va ian INOVA 400 o
B uke DPX-250 spec ome e s, using he sol en signal
(CDCl
3
,δ
1
H = 7.26, DMSO-d
6
,δ
1
H = 2.50; ace one-d
6
,δ
1
H=
2.05), o ex e nal H
3
PO
4
(85%), as app op ia e. Coupling con-
s an s a e epo ed in Hz.
Syn hesis o he ligands
L1 (a) 4-Ace ylphenylbo onic acid (500 mg, 3.05 mmol) was
added o 3- hiosemica bazide (277.92 mg, 3.05 mmol), hyd o-
chlo ic acid (35%, 0.65 cm
3
) and wa e (40 cm
3
), esul ing in a
clea solu ion ha was s i ed a oom empe a u e (RT) o
3 h. The o med whi e solid powde was il e ed off, washed
wi h cold wa e , and d ied in acuo. Whi e solid. Yield:
650.7 mg, 90%. Anal. Found: 45.8; H, 5.2; N, 17.7; S, 13.5%;
C
9
H
12
BN
3
O
2
S (237.08 g mol
−1
) equi es C, 45.6; H, 5.1; N, 17.7;
S, 13.5%. IR cm
−1
ν(O–H) 3458; ν(N–H) 3272, 3330; ν(C N)
1596; ν(B–O) 1352; ν(C S) 813.
1
H NMR (400 MHz, DMSO-d
6
,
δ): 10.24 (s, 1H, NNH), 8.31 (s, 1H, NH
2
), 8.14 (s, 2H, B(OH)
2
),
7.96 (s, 1H, NH
2
), 7.89 ( d, N= 7.8 Hz, 2H, H2/H6), 7.78 ( , N=
7.8 Hz, 2H, H3/H5), 2.29 (s, 3H, MeC N).
L2 (b) was p epa ed simila ly om 3.05 mmol o ace ylphe-
nylbo onic acid and me hyl hiosemica bazone. Whi e solid.
Yield: 719.8 mg, 94%. Anal. Found: C, 47.6; H, 5.5; N, 16.8; S,
12.8%; C
10
H
14
BN
3
O
2
S (251.11 g mol
−1
) equi es C, 47.8; H, 5.6;
N, 16.7; S, 12.8%. IR cm
−1
ν(O–H) 3500; ν(N–H) 3314, 3341;
ν(C N) 1595; ν(B–O) 1363; ν(C S) 831.
1
H NMR (400 MHz,
DMSO-d
6
,δ): 10.26 (s, 1H, NNH), 8.49 (b , 1H, NHMe), 8.17 (s,
2H, B(OH)
2
), 7.90 (d, N= 7.6 Hz, 2H, H2/H6), 7.80 ( d, N= 7.8
Hz, 2H, H3/H5), 3.03 (d,
3
J= 4.0 Hz, 3H, NHMe), 2.29 (s, 3H,
MeC N).
L3 (c) was made om 3.05 mmol o ace ylphenylbo onic
acid and me hyl hiosemica bazone ollowing he same p o-
cedu e. Whi e solid. Yield: 768.1 mg, 95%. Anal. Found: C,
50.0; H, 6.1; N, 16.0; S, 12.0%; C
11
H
16
BN
3
O
2
S (265.14 g mol
−1
)
equi es C, 49.8; H, 6.1; N, 15.9; S, 12.1%. IR cm
−1
ν(O–H)
3450; ν(N–H) 3317, 3321; ν(C N) 1592; ν(B–O) 1357; ν(C S)
819.
1
H NMR (400 MHz, DMSO-d
6
,δ): 10.16 (s, 1H, NNH), 8.55
(b , 1H, NHE ), 8.13 (s, 2H, B(OH)
2
), 7.88 ( d, N= 7.8 Hz, 2H,
H2/H6), 7.80 ( d, N= 7.8 Hz, 2H, H3/H5), 3.62 (dq,
3
J= 7.0 Hz,
2H, CH
2
), 2.28 (s, 3H, MeC N), 1.14 ( ,
3
J= 7.0 Hz, 3H, Me).
[P L1]
4
(1a).L1 (50 mg, 0.21 mmol) was added o a suspen-
sion o po assium e achlo opla ina e (0.18 mmol, 1 eq.) in a
mix u e o e hanol (20 mL) and wa e (0.5 mL). The mix u e
was s i ed a 55 °C o 48 h. E hanol was emo ed unde
educed p essu e and he esul ing o ange solid was washed
wi h wa e , cen i uged, and d ied in acuo. Yield: 84.9 mg,
92%. Anal. Found: C, 24.8; H, 2.2; N, 9.6; S, 7.6%;
(C
9
H
10
BN
3
O
2
P S)
4
(1720.6 g mol
−1
) equi es C, 25.1; H, 2.3; N,
9.8; S, 7.5%. IR cm
−1
ν(O–H) 3448; ν(N–H) 3289, 3162; ν(C N)
1577; ν(B–O) 1317.
1
H NMR (400 MHz, DMSO-d
6
,δ): 7.83 (s,
1H, H5), 7.16 (d,
3
J= 7.6 Hz, 1H, H3), 6.60 (s, 2H, NH
2
), 6.41
(d,
3
J= 7.6 Hz, 1H, H2), 1.88 (s, 3H, MeC N).
[P L2]
4
(1b) was p epa ed om L2 (50 mg, 0.20 mmol) and
po assium e achlo opla ina e (75 mg, 0.18 mmol) ollowing
he same p ocedu e. Yield: 72.5 mg, 82%. Anal. Found: C,
27.0; H, 2.5; N, 9.3; S, 7.5%; (C
10
H
12
BN
3
O
2
P S)
4
(1776.72 g
mol
−1
) equi es C, 27.1; H, 2.7; N, 9.5; S, 7.2%. IR cm
−1
ν(O–H)
3472; ν(N–H) 3305, 3210; ν(C N) 1582; ν(B–O) 1351.
1
H NMR
(400 MHz, DMSO-d
6
,δ): 7.93 (s, 1H, H5), 7.58 (s, 2H, B(OH)
2
),
7.36 (d,
3
J= 7.6 Hz, 1H, H3), 6.70 (d,
3
J= 7.6 Hz, 1H, H2), 6.49
(s, 1H, NHMe), 2.93 (d,
3
J= 4.7 Hz, 3H, NHMe), 1.51 (s, 3H,
MeC N).
[P L3]
4
(1c) was p epa ed employing L3 (53 mg, 0.20 mmol)
and po assium e achlo opla ina e (75 mg, 0.18 mmol) as
s a ing ma e ials. Yield: 69.1 mg, 80%. Anal. Found: C, 28.9;
H, 3.1; N, 9.4; S, 7.2%; (C
11
H
14
BN
3
O
2
P S)
4
(1832.84 g mol
−1
)
equi es C, 28.8; H, 3.1; N, 9.2; S, 7.0%. IR cm
−1
ν(O–H) 3419;
ν(N–H) 3244, 2972; ν(C N) 1575; ν(B–O) 1320.
1
H NMR
Pape Dal on T ansac ions
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(400 MHz, DMSO-d
6
,δ): 7.92 (s, 1H, H5), 7.35 (d,
3
J= 7.6 Hz,
1H, H3), 6.68 (d,
3
J= 7.6 Hz, 1H, H2), 6.29 (s, 1H, NHE ), 1.54
(s, 3H, MeC N), 1.11 ( ,
3
J= 7.2 Hz, 3H, Me).
[P L1(PPh
3
)] (2a). Compound 1a (50 mg, 0.029 mmol) and
iphenylphosphine (30.5 mg, 0.11 mmol, 1 : 4) we e added in
a ca ousel eac ion lask i ed wi h a s i ing od, and
acuum/a gon cycles we e pe o med; hen, deoxygena ed
ace one was added h ough a sy inge. This mix u e was s i ed
o 6 h o yield a clea solu ion. A e sol en emo al, he
esul ing solid was i u a ed wi h dichlo ome hane/n-hexane
and cen i uged. The co al solid was hen d ied unde acuum.
Yield: 63.6 mg, 79%. Anal. Found: 46.6; H, 3.6; N, 6.2; S, 4.8%;
C
27
H
25
BN
3
O
2
PP S (692.44 g mol
−1
) equi es C, 46.8; H, 3.7; N,
6.1; S, 4.6%. IR cm
−1
ν(O–H) 3452; ν(N–H) 3049; ν(C N) 1574;
ν(B–O) 1328.
1
H NMR (400 MHz, ace one-d
6
,δ): δ7.71–7.64 (m,
6H, o-PPh
3
), 7.49–7.39 (m, 11H, PPh
3
), 7.31 (dd, J= 7.5, 1.2 Hz,
1H, H3), 6.98 (d, J= 7.6 Hz, 1H, H2), 6.96 (q, J= 1.8 Hz, 1H,
H5), 6.05 (s, 2H, NH
2
), 2.43 (s, 3H, MeC N).
31
P–{
1
H} NMR
(400 MHz, ace one-d
6
)δ23.91,
1
J(P P) = 3892.86 Hz.
The emaining phosphine and/o diphosphine de i a i es
we e p epa ed simila ly.
[P L2(PPh
3
)] (2b) was p epa ed om 0.1 mmol o iphenyl-
phosphine and 0.025 mmol o 1b. Yield: 56.5 mg, 71%. Anal.
Found: C, 47.4; H, 3.8; N, 6.0; S, 4.4%; C
28
H
27
BN
3
O
2
PP S
(706.47 g mol
−1
) equi es C, 47.6; H, 3.8; N, 5.9; S, 4.5%. IR
cm
−1
ν(O–H) 3412; ν(N–H) 3324; ν(C N) 1574; ν(B–O) 1335.
1
H NMR (400 MHz, ace one-d
6
,δ): 7.75–7.63 (m, 6H, PPh
3
),
7.47–7.35 (m, 9H, PPh
3
), 7.30 (d,
3
J= 7.5 Hz, 1H, H3), 6.98 (d,
3
J= 8.0 Hz, 1H, H2), 6.94 (d,
4
J= 1.6 Hz, 1H, H5), 6.04 (s, 1H,
NHMe), 2.96 (d,
3
J= 4.5 Hz, 3H, NHMe), 2.42 (s, 3H, Me).
31
P–
{
1
H} NMR (400 MHz, ace one-d
6
)δ23.91
1
J(P P) = 3888 Hz.
[P L3(PPh
3
)] (2c) was p epa ed om 0.1 mmol o iphenyl-
phosphine and 0.025 mmol o 1c. Yield: 62.9 mg, 80%. Anal.
Found: C, 48.1; H, 4.0; N, 5.9, S, 4.6%; C
29
H
29
BN
3
O
2
PP S
(720.50 g mol
−1
) equi es C, 48.3; H, 4.1; N, 5.8, S, 4.5%. IR
cm
−1
ν(O–H) 3434; ν(N–H) 3052; ν(C N) 1574; ν(B–O) 1330.
1
H NMR (400 MHz, ace one-d
6
,δ): 7.67 (dd,
3
J(PH) = 11.3 Hz,
3
J= 7.7 Hz, 6H, o-PPh
3
), 7.45 (dd, J= 8.2, 5.2 Hz, 9H), 7.31 (d,
3
J= 7.6 Hz, 1H, H3), 6.99 (d,
3
J= 8.3 Hz, 1H, H2), 6.96 (s, 1H,
H5), 6.11 (s, 1H, NHE ), 3.40 (p,
3
J= 6.9 Hz, 2H, CH
2
), 2.44 (s,
3H, Me), 1.17 ( d,
3
J= 7.1, 2.3 Hz, 3H, Me).
31
P–{
1
H} NMR
(400 MHz, ace one-d
6
)δ23.83
1
J(P P) = 3912.3 Hz.
[P L1(dppm-P)] (3a) was p epa ed om 0.2 mmol o dppm
and 0.05 mmol o 1a. Yield: 101.3 mg, 57% Anal. Found: C,
49.8; H, 3.7; N, 5.1; S, 3.8%; C
34
H
32
BN
3
O
2
P
2
P S (814.55 g
mol
−1
) equi es C, 50.1; H, 3.9; N, 5.2; S, 3.9. IR cm
−1
ν(O–H)
3444; ν(N–H) 3051; ν(N–H) 1575; ν(B–O) 1330.
1
H NMR
(400 MHz, ace one-d
6
,δ): 7.90–7.82 (m, 4H, PPh
2
), 7.42–7.33
(m, 6H, PPh
2
), 7.33–7.27 (m, 4H, PPh
2
), 7.21–7.13 (m, 5H,
PPh
2
), 7.03 (d, J= 1.6 Hz, 1H, H5), 6.89 (d,
3
J= 7.6 Hz, 1H, H2),
6.28 (s, 2H, B(OH)
2
), 6.08 (s, 2H, NH
2
), 3.72–3.51 (m, 2H,
PCH
2
P), 2.37 (s, 3H, MeC N).
31
P–{
1
H} NMR (400 MHz,
ace one-d
6
)δ12.54 (d,
2
J(PP) = 78.9 Hz,
3
J(P P) = 3863.70 Hz),
−23.51 (d,
2
J(PP) = 79.0 Hz,
3
J(P P) = 77.76 Hz).
[P L2(dppm-P)] (3b) was p epa ed om 0.2 mmol o dppm
and 0.05 mmol o 1b. Yield: 88.8 mg, 62%. Anal. Found: C,
50.6; H, 4.1; N, 5.0; S, 3.8%; C
35
H
34
BN
3
O
2
P
2
P S (828.58 g
mol
−1
) equi es C, 50.7; H, 4.1; N, 5.1; S, 3.9%. IR cm
−1
ν(O–H)
3391; ν(N–H) 3051; ν(C N) 1574; ν(B–O) 1331.
1
H NMR
(400 MHz, ace one-d
6
,δ): 7.86 ( , J= 9.7 Hz, 4H, PPh
2
), 7.33 (q,
J= 7.3 Hz, 10H, PPh
2
), 7.18 (p, J= 7.7 Hz, 6H, PPh
2
), 7.03 (s,
1H, H5), 6.90 (d,
3
J= 7.6 Hz, 1H, H2), 6.32 (s, 2H, B(OH)
2
), 6.12
(s, 1H, NHMe), 3.72–3.55 (m, 2H, PCH
2
P), 2.99 ( ,
3
J= 3.5 Hz,
3H, NHMe), 2.37 (s, 3H, MeC N).
31
P–{
1
H} NMR (400 MHz,
ace one-d
6
)δ12.55 (d,
2
J(P–P) = 79.5 Hz,
1
J(P –P) = 3860.46
Hz), −23.59 (d,
2
J(PP) = 79.0 Hz,
3
J(P P) = 38.6 Hz).
[P L3(dppm-P)] (3c) was p epa ed om 0.2 mmol o dppm
and 0.05 mmol o 1c. Yield: 89.4 mg, 63%. Anal. Found: C,
51.1; H, 4.2; N, 5.1; S, 3.9%; C
36
H
36
BN
3
O
2
P
2
P S (842.60 g
mol
−1
) equi es C, 51.3; H, 4.3; N, 5.0; S, 3.8%. IR cm
−1
ν(O–H)
3391; ν(N–H) 3054; ν(C N) 1574; ν(B–O) 1334.
1
H NMR
(400 MHz, ace one-d
6
,δ): 7.86 ( , J= 9.8 Hz, 4H), 7.49 (s, 1H),
7.33 (q, J= 7.5 Hz, 10H), 7.18 ( , J= 8,4 Hz, 6H), 7.03 (s, 1H,
H5), 6.89 (dd,
3
J= 7.6, 2.5 Hz, 1H, H
2
), 6.31 (d, J= 2.5 Hz, 2H,
B(OH)
2
), 6.12 (s, 1H, NHE ), 3.62 (dd, J= 17.9, 9.8 Hz, 2H,
PCH
2
P), 3.43 (p,
3
J= 7.2 Hz, 2H, PCH
2
P), 2.36 (d, J= 2.5 Hz,
3H, MeC N), 1.20 ( d,
3
J= 7.2, 2.3 Hz, 3H, Me).
31
P–{
1
H} NMR
(400 MHz, ace one-d
6
)δ12.55 (d,
2
J(P–P) = 78.9 Hz,
1
J(P –P) =
3859.6 Hz), −23.53 (d,
2
J(P–P) = 78.4 Hz,
3
J(P –P) = 38.7 Hz).
[(P L1)
2
(μ-dppb)] (4a) was p epa ed om 0.05 mmol o
dppb and 0.025 mmol o 1a. Yield: 53.8 mg, 72%. Anal. Found:
C, 42.6; H, 3.7; N, 6.7; S, 4.7%; C
46
H
48
B
2
N
6
O
4
P
2
P
2
S
2
(1286.78 g mol
−1
) equi es C, 43.0; H, 3.8; N, 6.5; S, 5.0%. IR
cm
−1
ν(O–H) 3389; ν(N–H) 3050; ν(C N) 1574; ν(B–O) 1331.
1
H NMR (400 MHz, ace one-d
6
)δ7.84–7.71 (m, 8H, PPh
2
), 7.50
(q, J= 9.4, 8.1 Hz, 6H, PPh
2
), 7.40 ( d, J= 5.2, 2.7 Hz, 6H,
PPh
2
), 7.37–7.30 (m, 3H, PPh
2
), 7.05 (d,
3
J= 7.5 Hz, 2H, H2),
6.94 ( , J= 6.6 Hz, 2H, H5), 6.43 (s, 4H, B(OH)
2
), 6.17 (s, 4H,
NH
2
), 2.35 (d, J= 5.9 Hz, 6H, MeC N), 2.30–2.16 (m, 4H,
PCH
2
), 1.92–1.85 (m, 4H, CH
2
).
31
P–{
1
H} NMR (400 MHz,
ace one-d
6
)δ16.04, 15.06,
1
J(P P) = 3818.23 Hz.
[(P L2)
2
(μ-dppb)] (4b) was p epa ed om 0.05 mmol o
dppb and 0.025 mmol o 1b. Yield: 50.3 mg, 68%. Anal.
Found: C, 44.3; H, 4.1; N, 6.2; S, 4.7%; C
48
H
52
B
2
N
6
O
4
P
2
P
2
S
2
(1314.84 g mol
−1
) equi es C, 43.9; H, 4.0; N, 6.4; S, 4.9%. IR
cm
−1
ν(O–H) 3394; ν(N–H) 3054; ν(C N) 1576; ν(B–O) 1333.
1
H NMR (400 MHz, ace one-d
6
)δ7.82–7.75 (m, 6H, PPh
2
),
7.55–7.46 (m, 6H, PPh
2
), 7.39 (dq, J= 6.1, 2.5 Hz, 4H, PPh
2
),
7.36–7.32 (m, 4H, PPh
2
), 7.07 ( , J= 3.8 Hz, 2H, H5), 6.96 (d,
3
J
= 7.5 Hz, 2H, H2), 6.33 (s, 4H, B(OH)
2
), 6.10 (s, 2H, NHMe),
2.98 (dd, J= 8.6, 4.6 Hz, 6H, NHMe), 2.40 (d, J= 7.8 Hz, 6H,
MeC N), 2.31–2.17 (m, 4H, PCH
2
), 1.92–1.76 (m, 4H, CH
2
).
31
P–{
1
H} NMR (400 MHz, ace one-d
6
)δ16.04, 15.06,
1
J(P P) =
3818.23 Hz.
[(P L3)
2
(μ-dppb)] (4c) was p epa ed om 0.05 mmol o
dppb and 0.025 mmol o 1c. Yield: 57.1 mg, 78%. Anal. Found:
C, 44.9; H, 4.3; N, 6.1; S, 4.6%; C
50
H
56
B
2
N
6
O
4
P
2
P
2
S
2
(1342.89 g mol
−1
) equi es C, 44.7; H, 4.2; N, 6.3; S, 4.8%. IR
cm
−1
ν(O–H) 3399; ν(N–H) 3046; ν(C N) 1572; ν(B–O) 1330.
1
H NMR (400 MHz, ace one-d
6
)δ7.82–7.75 (m, 6H, PPh
2
),
7.55–7.46 (m, 6H, PPh
2
), 7.39 (d , J= 6.1, 2.1 Hz, 4H, PPh
2
),
7.36–7.32 (m, 4H, PPh
2
), 7.09–7.05 (m, 2H, H5), 6.96 (d, J= 7.5
Dal on T ansac ions Pape
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Hz, 2H, H2), 6.31 (s, 4H, B(OH)
2
), 6.17 (s, 2H, NHE ), 3.43
(ddd, J= 9.2, 7.1, 5.1 Hz, 4H, CH
2
), 2.40 (d, J= 7.1 Hz, 6H,
MeC N), 2.34–2.17 (m, 4H, PCH
2
), 1.94–1.73 (m, 4H, CH
2
),
1.19 (q,
3
J= 7.1 Hz, 6H, Me).
31
P–{
1
H} NMR (400 MHz, ace one-
d
6
)δ16.06, 15.01,
1
J(P P) = 3821.58 Hz.
The compound syn hesis eac ions a e shown in Scheme 1.
Cell cul u e
The HCT116 colo ec al cance cell line and he p ima y human
de mal ib oblas s we e ob ained om Ame ican Type Cul u e
Collec ion (ATCC®, Manassas, VA, USA) and cul u ed in
Dulbecco’smodi iedEagle’s medium (DMEM), while he A2780
ca cinoma cell line was acqui ed om Sigma-Ald ich (Mad id,
Spain) and cul u ed in Roswell Pa k Memo ial Ins i u e (RPMI)
medium. Media we e supplemen ed wi h 10% ( / ) e al bo ine
se um (FBS) and 1% ( / ) o a penicillin/s ep omycin solu ion.
The media and supplemen s we e ob ained om The mo Fische
Scien i ic (Wal ham, Massachuse s, USA).
Cells we e cul u ed in 25 cm
2
and/o 75 cm
2
T- lasks in a
CO
2
incuba o wi h a humidi ied a mosphe e a 37 °C and 5%
( / ) CO
2
(SANYO CO
2
Incuba o , Elec ic Biomedical Co.,
Osaka, Japan).
Cell iabili y assays
Cells we e ini ially seeded in 96-well pla es a a cell densi y o
0.75 × 10
5
cells pe mL and incuba ed (37 °C, 5% ( / ) CO
2
) o
24 h. A e his pe iod, he media we e eplaced wi h esh
media con aining he desi ed compounds (concen a ions
anging om 0.1 µM o 50 µM), 0.1% ( / ) o DMSO o 0.4 µM
doxo ubicin (Dox). A e 48 h o incuba ion unde he same
condi ions, he CellTi e 96® aqueous one solu ion cell p o-
li e a ion assay ki (P omega, Madison, USA) was used o
e alua e he cell iabili y by measu ing he abso bance a
490 nm in a Tecan In ini e M200 mic opla e eade (Tecan,
Männedo , Swi ze land). Da a we e analysed wi h he
G aphPad P ism 8 so wa e, whe e iabili y-concen a ion
esponse cu es acili a ed he calcula ion o he concen a ion
o compounds ha induced a 50% educ ion in cell iabili y
(IC
50
).
Scheme 1 Reac ion sequence o he syn hesis o he epo ed compounds.
Pape Dal on T ansac ions
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Complex s abili y in biological media
The s abili y o he h ee pla inum compounds (3a,3b and 3c)
was analysed ia UV-Visible spec oscopy (Shimadzu Scien i ic
Ins umen s) wi h a qua z cu e e (1 cm pa h leng h) o e a
wa eleng h ange o 220–700 nm o h ee diffe en incuba ion
imes, namely 0, 3, 24 and 48 h. Compounds we e dilu ed in
RPMI medium wi hou phenol ed and FBS a e being i s
dissol ed in 100% ( / ) DMSO. Compounds 3a and 3c we e
analysed a a inal concen a ion o 50 μM, while 3b was ana-
lysed a a inal concen a ion o 150 μM.
ICP-AES (induc i ely coupled plasma-a omic emission
spec ome y)
To e alua e he compound in e naliza ion in HCT116 cells,
induc i ely coupled plasma-a omic emission spec oscopy
(ICP-AES) was pe o med. HCT116 cells we e seeded in 25 cm
2
T- lasks a a densi y o 5 × 10
5
cells/T- lask and incuba ed o
24 h. The cul u e medium was hen eplaced wi h esh medium
wi h 10× IC
50
concen a ions o he s udied compounds o 0.1%
( / ) DMSO, and cells we e incuba ed o 3 h. A e his pe iod,
he cell cul u e medium was eco e ed in a 15 mL Falcon ube
and he cells we e washed wi h PBS. A e his washing s ep, he
PBS washing solu ion was also eco e ed in he 15 mL Falcon
ube. Cells we e de ached om he T- lask wi h 2 mL o T ypLE
Exp ess and cen i uged a 750g(Sigma 3-16K Sa o ius,
Ge many) o 5 min. The supe na an was emo ed and added o
he 15 mL Falcon ube, and he cell pelle was washed 2 mo e
imeswi hPBS.Aqua egia(3:1HCl/HNO
3
) was p epa ed, and
added o he 15 mL Falcon ubes con aining he p e ious
washing solu ions and he supe na an (non-cellula ac ion) o
o he cell pelle s (cellula ac ion). Samples we e incuba ed a
oom empe a u e (RT) o 24 h in a hood ume, and hen deli-
e ed o Labo a ó io de Análises/LAQV o quan i y he pla inum
le els by ICP-AES.
Analysis o apop osis induc ion by low cy ome y
Apop osis was e alua ed using he Alexa Fluo ® 488 Annexin V/
dead cell apop osis ki (In i ogen, The mo Fishe Scien i ic, MA,
USA). HCT116 cells we e seeded in 6-well pla es (a a densi y o 2
×10
5
cellspe well) o 24h,andla e incuba ed o 48hpe iod
wi h he IC
50
concen a ions o compounds 3a,3b and 3c. Cells
ea ed wi h 0.1% ( / ) DMSO we e used as he ehicle con ol,
while cells ea ed wi h 0.4 μM Dox we e used as posi i e con-
ols. A e 48 h o incuba ion, cells we e washed wi h PBS and
de ached om he wells wi h T ypLE Exp ess (In i ogen), and
hen washed again wi h PBS. Las ly, cells we e incuba ed wi h
he Alexa® Fluo 488-Annexin V solu ion and 100 μgmL
−1
o p o-
pidium iodide (PI) a RT o 15 min.
Samples we e e alua ed by he A une® acous ic ocusing
low cy ome e (Li e Technologies, Ca lsbad, USA), and he
esul s we e analysed wi h he A une® Cy ome ic so wa e.
Quan i ica ion o BAX and BCL-2 p o ein le els by wes e n blo
HCT116 cells we e cul i a ed, incuba ed and collec ed, as
desc ibed p e iously.
32
A e collec ion, cells we e submi ed o
5 ul asound pulses on ice (2 min on ul asound, ollowed by
30 s on ice; Elma D-78224; Singen/H w, Ge many) and cen i-
uged a 1000g o 5 min. The o al amoun o p o ein in he
supe na an ex ac s was quan i ied wi h Pie ce 660 nm
P o ein Assay Reagen (The mo Fishe Scien i ic, Wal ham,
MA, USA). Fo SDS-PAGE, 20 μg o p o ein was loaded on 10%
polyac ylamide gel and ans e ed o a 0.45 μm PVDF mem-
b ane (GE Heal hca e Li e Sciences, Ge many) (BAX and BCL-2
p o eins). The memb ane was blocked o 2 h wi h 5% (w/ )
non- a milk in TBST (50 mM T is-HCl, pH 7.5, 150 mM NaCl,
0.1% ( / ) Tween-20), and incuba ed a oom empe a u e o
1 h unde cons an agi a ion wi h he espec i e p ima y an i-
body solu ion (an i-BAX, 1 : 5000, Abcam, Uni ed Kingdom;
and an i BCL-2, 1 : 1000, Sigma, S Louis, MO, USA). A e incu-
ba ion, memb anes we e washed wi h TBST buffe h ee imes,
o a du a ion o 5 min each (p ocedu e also epea ed a e he
seconda y an ibody incuba ion) and exposed o he seconda y
an ibody solu ion (1 : 3000, an i-mouse IgG, ho se adish pe -
oxidase HPR-linked an ibody o 1 : 2000, an i- abbi IgG, HPR-
linked an ibody; Cell Signalling Technology, USA). Memb anes
we e ea ed wi h he Wes e nB igh ECL subs a e (Ad ans a,
USA) o 5 min and la e exposed o a ilm in a da k oom.
A e ha , o no malize he esul s, memb anes we e incu-
ba ed wo imes wi h s ipping buffe (0.1 M glycine, 20 mM
magnesium ace a e, 50 mM KCl, pH 2.0) o a du a ion o 10
and 20 min, espec i ely, and hen incuba ed wi h an i-βac in
(1 : 5000; Sigma, S Louis, USA). BAX and BCL-2 p o ein quan i-
ica ion (densi ome y) was pe o med using he Image J so -
wa e. BAX and BCL-2 p o ein le els we e no malized o βac in
le els.
Mi ochond ial memb ane po en ial (ΔΨ
m
) analysis by low
cy ome y
ΔΨ
m
was analysed using he JC-1 mi ochond ial memb ane
po en ial assay ki (Abno a Co po a ion, Walnu , CA, USA).
HCT116 cells we e seeded in 6-well pla es wi h a densi y o 2 ×
10
5
cells pe well, incuba ed o 24 h, and la e incuba ed o
ano he 48 h pe iod wi h he IC
50
concen a ions o com-
pounds 3a,3b and 3c o wi h 0.1% ( / ) DMSO. As posi i e
con ols, 0.4 μM Dox and 5 μM Cis we e used. A e ha , cells
we e washed wi h PBS, de ached wi h T ypLE Exp ess, and
washed wi h DMEM medium. Cells we e hen esuspended in
DMEM medium wi hou phenol ed + 5% ( / ) FBS, and
s ained wi h he JC-1 s aining solu ion o 20 min a 37 °C.
Las ly, cells we e esuspended in DMEM medium wi hou
phenol ed + 5% ( / ) FBS, and analysed in he A une®
Acous ic Focusing Flow Cy ome e (Li e Technologies,
Ca lsbad, CA, USA).
Caspase-8 ac i i y
HCT116 cells we e seeded in 25 cm
2
T- lasks a a densi y o 2 ×
10
6
cells/T- lasks. A e 24 h incuba ion, unde he same con-
di ions as desc ibed p e iously, he medium was eplaced wi h
esh medium con aining DMSO 0.1% ( / ), 0.4 µM Dox, 5 µM
Cis o IC
50
concen a ions o he compounds 3a,3b and 3c,
and incuba ed o 48 h. A e ha , cells we e de ached using
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cold PBS and a cell sc ape , and cen i uged a 500g o 5 min.
Then, he ins uc ions p o ided in he caspase-8 assay ki
(Abcam) p ocedu e we e ollowed. Caspase 8 ac i i y was
ob ained by measu ing he abso bance a 400 nm o each
sample. Caspase 8 ac i i ies we e no malized o he co es-
ponding alue in DMSO samples.
Analysis o au ophagy by low cy ome y
The au ophagic po en ial o HCT116 cells was e alua ed using
he au ophagy assay ki (ab139484) (Abcam, Camb idge,
Uni ed Kingdom). HCT116 cells we e seeded in 6-well pla es
wi h a densi y o 2 × 10
5
cells pe well and incuba ed o 24 h.
A e he eplacemen o he cul u e medium wi h esh
medium con aining he IC
50
concen a ions o compounds 3a,
3b and 3c, cells we e incuba ed o ano he 48 h. 0.1% ( / )
DMSO, 0.4 μM Dox and 5 μM Cis we e used as con ols. In
addi ion, 15 h be o e inishing he 48 h incuba ion ime,
0.5 μM Rapamycin was added o he espec i e wells. A e
ha , cells we e washed wi h PBS, de ached wi h T ypLE
Exp ess, washed wi h DMEM medium wi hou phenol ed +
5% ( / ) FBS, and hen incuba ed wi h he g een s ain solu ion
in DMEM medium wi hou phenol ed + 5% ( / ) FBS o
30 min a RT. A e his pe iod, cells we e washed, esus-
pended in he assay buffe , and hen analysed wi h he
A une® Acous ic Focusing Flow Cy ome e (Li e Technologies,
Ca lsbad, CA, USA). The esul s we e analysed wi h he
A une® Cy ome ic so wa e.
Analysis o eac i e oxygen species (ROS) p oduc ion by low
cy ome y
HCT116 cells we e seeded in 6-well pla es a a densi y o 2 ×
10
5
cells pe well and incuba ed o 24 h. The cul u e medium
was eplaced wi h esh medium con aining he IC
50
concen-
a ions o compounds 3a,3b and 3c, 0.1% ( / ) DMSO, 0.4 μM
Dox, 5 μM cispla in o 30 μM TBHP (posi i e con ol as indi-
ca ed by he manu ac u e ), and cells we e incuba ed o
ano he 48 h pe iod. Cells we e hen washed wi h PBS,
de ached wi h T ypLE Exp ess, washed wi h PBS, and la e
incuba ed wi h 10 μMo 2′,7′-dichlo odihyd o luo escein dia-
ce a e (H2DCF-DA) (The mo Fishe Scien i ic, Wal ham, MA,
USA) in PBS o 20 min. Cells we e la e analysed wi h he
A une® Acous ic Focusing Flow Cy ome e (Li e Technologies,
Ca lsbad, USA), and he esul s we e ea ed wi h he A une®
Cy ome ic so wa e.
Ex o o CAM assay
The p o-/an i-angiogenic po en ial o he compounds was e al-
ua ed wi h he ex o o cho ioallan oic memb ane (CAM) assay,
as p e iously desc ibed in (Reigosa-Chamo o e al.,
2021
26
).
30,31
Chicken emb yos we e i s incuba ed o 24 h
and la e exposed o he IC
50
concen a ions o compounds 3a,
3b and 3c dissol ed in PBS (in he cen e o O- ings) o 0.1%
( / ) DMSO. The compounds’and DMSO’s dis ibu ion in he
emb yos we e always pe o med in a diffe en o de . Then,
emb yos we e incuba ed o ano he 48 h a 37 °C and i s
images we e cap u ed a e 0, 24 and 48 h wi h a digi al USB
Mic oscope Came a (Op i-Tekscope OT-V1) o manually coun
he newly o med blood essels ia ImageJ so wa e. The ex o o
CAM assay ul ils he Di ec i e 2010/63/EU o he Eu opean
Pa liamen o p o ec he animal models o scien i ic
pu poses.
S a is ical analysis
The p esen ed da a a e e e ed o as mean ± SEM om a
leas h ee biological independen expe imen s, unless o he -
wise s a ed. One-way ANOVA o S uden ’s - es we e pe o med
o de e mine he s a is ical signi icance (p< 0.05) wi h he
G aphPad P ism 8 so wa e (G aphPad So wa e, San Diego,
CA, USA).
Resul s and discussion
The compounds and espec i e eac ions a e shown in
Scheme 1 o simpli ica ion. The hiosemica bazone ligands
L1–L3,a,band cwe e p epa ed by eac ion o 4-ace ylphenyl-
bo onic acid wi h he co esponding hiosemica bazide as
pu e ai -s able solids, as app op ia e (see Expe imen al
sec ion). The NNHp o on esona ed ca. 10.2 ppm, whils he
NH
2
p o ons, a, ga e ise o wo cha ac e is ic esonances in
he
1
H NMR spec um which we e a ibu ed o he es ic ed
o a ion o he NH
2
g oup abou he C(S)NH
2
bond axis. The
NHR p o ons, b,c, showed a b oad esonance a ca. δ8.5 ppm.
The cha ac e is ics o he ligand spec a included wo i ual
double s s emming om he a oma ic AA′XX′spin sys em wi h
an N alue a ca. 8.0–7.8 ppm. F om hem, he new cyclome al-
la ed compounds we e ob ained as desc ibed in Scheme 1.
The p epa a i e de ails and cha ac e is ic mic oanaly ical and
spec oscopic da a a e gi en in he Expe imen al sec ion.
Reac ion o a,bo c, as app op ia e, wi h po assium e achlo -
opla ina e in wa e /e hanol ga e clea solu ions. These solu-
ions we e used o isola e he e anuclea compounds 1a–1c
as pu e ai -s able solids, wi h he ligand in he E,Zcon igu -
a ion. Absence o he NH esonance ag eed wi h dep o ona ion
a he hyd azine g oup. The
1
H NMR spec a showed he
absence o he AA′XX′spin sys em upon me ala ion o he
pa a-subs i u ed phenyl ing, and dis inc esonances we e
acco dingly assigned o he H2, H3 and H5 nuclei (see
Expe imen al).
Reac ion o [P (L1–3)]
4
1a–1c wi h e ia y phosphines PPh
3
,
Ph
2
PCH
2
PPh
2
(dppm), and Ph
2
P(CH
2
)
4
PPh
2
(dppb), in 1 : 4 o
1 : 2 mola a ios, as app op ia e, ga e he compounds
[P (L1–3)(PPh
3
)] 2a–2c, [P (L1–L3)(Ph
2
PCH
2
PPh
2
-P)] 3a–3c, and
[P (L1–L3)
2
{μ-Ph
2
P(CH
2
)
4
PPh
2
}] 4a–4c as pu e ai -s able solids
(Scheme 1). The mic oanaly ical and spec oscopic da a a e
p esen ed in he Expe imen al sec ion. The use o excess
diphosphine only ga e clea age o he P –S
b idging
. The e o e,
in compounds wi h diphosphines, coo dina ion o he me al
was only h ough one phospho us a om. Usage o excess phos-
phine did no clea e he P –S
chela e
bond. The
1
H NMR spec a
showed he high- ield shi o he H5 esonance a ca. 1 ppm
wi h espec o he co esponding e anuclea compound,
Pape Dal on T ansac ions
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owing o he shielding effec o he phosphine phenyl ings.
The
31
P NMR spec a o he 3a–3c compounds showed wo
double s assigned o he wo non-equi alen phospho us
nuclei. The lowe ield esonance, a ca. 12.5 ppm, was
assigned o he
31
P nucleus bonded o pla inum, in ag eemen
wi h he J(P –P) alues. Meanwhile, o 4a–4c, he
31
P eso-
nance was a single signal in acco dance wi h he exis ence o
equi alen phospho us nuclei; he chemical shi alues we e
consis en wi h a phospho us o ni ogen ans geome y.
32,33
The esonance o he ABXY spin sys em o he PCH
2
P p o ons
appea ed a ca. a δ3.52. A emp s o p oduce mononuclea
species analogous o 3a–3c wi h diphosphines o he han
Ph
2
PCH
2
PPh
2
we e unsuccess ul.
Sui able c ys als o 1b we e g own by slowly e apo a ing an
ace one solu ion o he complex. The ORTEP illus a ion o
compound 1b is shown in Fig. 1. The e anuclea compound
c ys allizes in he Pcan space g oup wi h wo ace one mole-
cules. Wi hin each molecule, he me ala ed moie ies a e dis-
played as wo se s o almos coplana an ipa allel pai s sepa -
a ed a ca. 3.5 Å. Each pla inum a om o he in e nal P
4
S
4
nucleus is bonded o a iden a e C,N,S
chela ing
ligand and o
he sul u a om, S
b idging
, o ano he me ala ed species. The
longe P –S
chela ing
bond leng hs, as opposed o P –S
chela ing
,
e lec he diffe ing ans in luence o he phenyl ca bon and
ni ogen a oms o he ligands.
Sui able c ys als o 2c we e g own by slowly e apo a ing a
chlo o o m solu ion o he complex. The c ys als we e iclinic
wi h P1
ˉspace g oup. The ORTEP illus a ion is shown in
Fig. 2. The s uc u e o compound 2c comp ises a molecule
wi h he pla inum(II) a om bonded in a sligh ly dis o ed
squa e plana coo dina ion o ou diffe en dono a oms, a i-
den a e hiosemica bazone h ough he a yl C(6) ca bon, he
imine N(1) ni ogen, and he hioamide S(1) sul u a om, and
o a phospho us a om P(1) o he iphenylphosphine. The
bond dis ances and angles a e wi hin he expec ed alues o
analogous s uc u es, as was p o ided by Mogul 2020.3 om
he CCDC p og am package. The angles a pla inum a e close
o 90° wi h allowance o he somewha smalle C(6)–P (1)–N
(1) and S(1)–P (1)–N(1) angles, 79.81° and 83.4°, espec i ely,
and la ge C(6)–P (1)–P(1) and S(1)–P (1)–P(1) angles, 99.80°
and 96.97°, also espec i ely, consequen upon chela ion. The
c ys al packing shows he molecules a e a anged in shee s
held oge he by means o hyd ogen bonding in e ac ions
be ween he bo onic acid unc ion, he chlo ine a om o he
sol en molecule and he amide ni ogen, as well as by π–π
s acking.
An ip oli e a i e ac i i y
The in i o an ip oli e a i e po en ial o compounds 1a–c,2a–
c,3a–c,4a–cand he espec i e ligands a–cwas assessed using
he CellTi e 96®Aqueous non- adioac i e cell p oli e a ion
assay (MTS assay), as desc ibed in he Expe imen al sec ion.
34
This an ip oli e a i e ac i i y was analysed by exposu e o he
o a ian ca cinoma (A2780) and colo ec al ca cinoma (HCT116)
cell lines and no mal human p ima y de mal ib oblas s o
0.1–50 µM o all compounds o 48 h (ESI Fig. S1†). As shown
in Fig. S1,†a educ ion o he cell iabili y is obse ed upon
inc ease o he compounds concen a ions. Cell iabili y-con-
cen a ion cu es (G aphPad so wa e) allowed us o calcula e
Fig. 1 ORTEP d awing o he pla inacycle 1b wi h he mal ellipsoid plo
shown a he 50% p obabili y le el. Hyd ogen a oms and sol en mole-
cules a e omi ed o cla i y. Selec ed bond dis ances (Å) and angles (°)
o 10: P (1)–C(11) 2.013(8), P (1)–N(10) 1.988(7), P (1)–S(3) 2.287(2), P
(1)–S(4) 2.347(2), S(4)–P (1)–S(3) 100.76(8), N(10)–P (1)–S(3) 174.5(2),
N(10)–P (1)–S(4) 84.1(2), C(11)–P (1)–S(3) 94.6(3), C(11)–P (1)–S(4) 163.9
(3), and C(11)–P (1)–N(10) 80.8(3).
Fig. 2 ORTEP d awing o he pla inacycle 2c wi h he mal ellipsoid plo
shown a he 50% p obabili y le el. Hyd ogen a oms and sol en mole-
cules ha e been omi ed o cla i y. Selec ed bond dis ances (Å) and
angles (°) o 10: P (1)–C(6) 2.038(6), P (1)–N(1) 2.032(5), P (1)–S(1)
2.3558(17), P (1)–P(1) 2.2234(18), S(1)–P (1)–P(1) 96.97(6), N(1)–P (1)–S
(1) 83.44(16), N(1)–P (1)–P(1) 179.24(18), C(6)–P (1)–S(1) 162.95(19),
C(6)–P (1)–P(1) 99.79(19), and C(6)–P (1)–N(1) 79.8(2).
Dal on T ansac ions Pape
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he ela i e IC
50
alues o each compound o each cell line
(Table 1). Mo eo e , as posi i e con ols, he an ip oli e a i e
ac i i y o he Dox and Cis was e alua ed h ough he exposu e
o A2780, HCT116 and Fib oblas cells o 0.1–50 µM o hese
an i umo d ugs o 48 h (ESI Fig. S2†). The selec i i y index
(SI) o each compound – he a io IC
50
in ib oblas s/IC
50
in
he umou cell line –was de e mined, and i is epo ed in
Table 1 o e alua e he selec i i y o he compound owa ds
umou cell lines. Highe SI alues we e co ela ed wi h highe
selec i i y o he compound o a pa icula umou cell line
compa ed o he no mal cells.
35
Compa ing he IC
50
alues ob ained o he HCT116 cance
cell line o all o he es ed compounds (Table 1), compounds
3a–cp esen ed he lowes IC
50
alues (2.1, 1.9 and 8.6 µM,
espec i ely) wi h he ollowing cy o oxici y o de : 3b >3a >3c.
Mo eo e , A2780 cells compounds 3a–calso p esen ed he
lowes IC
50
alues (5.0, 1.9, 8.0, espec i ely), main aining he
same cy o oxici y o de o 3b >3a >3c (Table 1). In e es ingly,
in he HCT116 cells, hese 3 compounds p esen ed a highe
cy o oxic po en ial when compa ed o he chemo he apeu ic
agen , cispla in (IC
50
alue o 15.6 µM). All he o he com-
pounds do no demons a e cy o oxici y in he HCT116 colo -
ec al ca cinoma cell line (IC
50
> 50 µM). This clea ly indica es
ha coo dina ion wi h dppm –Ph
2
PCH
2
PPh
2
-Pp o ides a
highe cy o oxici y compa ed o he o he phosphines (PPh
3
o
dppb) (Table 1).
In A2780 o a ian ca cinoma cells, compounds 1a–c,2b, and
4a–cshow mode a e cy o oxici y alues (10 µM < IC
50
< 50 µM)
(Table 1). Among all compounds, i seems ha compounds
wi h subs i u ion b(R Me) show mo e cy o oxic effec s,
al hough ligands 1,2and 3did no p esen any cy o oxici y
when es ed sepa a ely (Table 1). We ha e ye o ind a plaus-
ible explana ion o he diffe ing alues o compounds 3a,3b
as compa ed o 3c. The modi ica ion o he –NHR g oup was
no hough o ha e a signi ican effec on he modes o ac ion
o he compounds, bu mo e in he ine- uning o in e naliz-
a ion and hei p ope ies. I is possible ha R g oups wi h
longe ca bon chains affec in e ac ions wi h he biological
memb anes and/o a ge s. This pu s o wa d he need o
examine dis inc op ions when designing a me allod ug.
One o he main easons and in e es o de elop new pla i-
num-based compounds is o ci cum en he high cy o oxici y
o app o ed pla inum d ugs, and o educe he side effec s in
heal hy issues. The e o e, i is impo an ha compounds 3a–
cha e much highe IC
50
alues in a no mal human cell line
when compa ed o he alues ob ained o he s udied umo
cell lines. Fib oblas s we e used as heal hy cells due o hei
impo ance in he umo mic oen i onmen ,
36
and he IC
50
alues ob ained a e epo ed in Table 1.
In e es ingly, when analysing he IC
50
alues ob ained o
ib oblas s (Table 1), all o he compounds showed low cy o-
oxic po en ial (IC
50
alues >50 µM), which is e lec ed in he
high SI alues. A he limi , i we conside he IC
50
o com-
pounds 3a–cin ib oblas s as equal o 50 µM, compounds 3a
and 3b showed he highes SI alues (23.8 and 26.3, espec -
i ely) in he HCT116 cance cell line, meaning ha hose com-
Table 1 Rela i e IC
50
alues and SI ob ained o each o he pla inum
compounds and espec i e ligands in HCT116, A2780 and fib oblas cell
lines. IC
50
alues a e exp essed as he mean ± SEM o a leas h ee bio-
logical independen assays
Complex Cell line IC
50
(µM) SI
1a HCT116 >50 —
A2780 10 < IC
50
<50 —
Fib oblas s >50 —
1b HCT116 >50 —
A2780 35.7 >1.4
Fib oblas s >50 —
1c HCT116 >50 —
A2780 10 < IC
50
<50 —
Fib oblas s >50 —
2a HCT116 >50 —
A2780 >50 —
Fib oblas s >50 —
2b HCT116 >50 —
A2780 31.9 >1.6
Fib oblas s >50 —
2c HCT116 >50 —
A2780 >50 —
Fib oblas s >50 —
3a HCT116 2.1 >23.8
A2780 5.0 >10
Fib oblas s >50 —
3b HCT116 1.9 >26.3
A2780 1.9 >26.3
Fib oblas s >50 —
3c HCT116 8.6 >5.8
A2780 8.0 >6.3
Fib oblas s >50 —
4a HCT116 >50 —
A2780 10 < IC
50
<50 —
Fib oblas s >50 —
4b HCT116 >50 —
A2780 31.0 >1.6
Fib oblas s >50 —
4c HCT116 >50 —
A2780 10 < IC
50
<50 —
Fib oblas s >50 —
L1 HCT116 >50 —
A2780 >50 —
Fib oblas s >50 —
L2 HCT116 >50 —
A2780 >50 —
Fib oblas s >50 —
L3 HCT116 >50 —
A2780 >50 —
Fib oblas s >50 —
Doxo ubicin (Dox) HCT116 0.5 ± 0.10 24.2
A2780 0.1 ± 0.04 121
Fib oblas s 12.1 ± 0.20 —
Cispla in (Cis) HCT116 15.6 ± 5.30 0.6
A2780 1.9 ± 0.20 4.6
Fib oblas s 8.8 ± 2.90 —
> indica es ha he IC
50
is highe han he alue indica ed; –SI
alues no calcula ed.
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pounds a e a leas 23.8× mo e cy o oxic o HCT116 cance
cells han o ib oblas s. Conside ing he same app oach,
compounds 3a and 3b also show highe SI alues in he A2780
cance cell line (10 and 26.3, espec i ely). Compound 3c p e-
sen ed a SI alue o 5.8 in he HCT116 cell line, and a sligh ly
highe SI alue (6.3) o he A2780 cell line. I is also in e es -
ing o no e ha all o he ligands (a,band c) do no show a
cy o oxic effec in he es ed cell lines (IC
50
> 50 µM in umo
and no mal cells). In e es ingly, ou p e ious da a
37
show high
cy o oxici y o he ee PPh
3
(a ligand ha is p esen in com-
pounds 2a,2b and 2c) and o o he ee diphenylphosphines
in no mal ib oblas s. In he p esen wo k, we show a high
cy o oxici y o compounds, pa icula ly hose ha bou ing
dppm, in umo cell lines and no cy o oxici y in ib oblas s.
This means ha his effec is p obably a ibu ed o he effec s
o he compounds pe se, and no o he ee ligands, e en
hough ee phosphines also ha e high cy o oxici y in hese
umo cell lines.
37
Thus, compounds 3a,3b and 3c show an
o e all e y good he apeu ic window and po en ial in he
HCT116 cell line and om now on, all o he emaining s abi-
li y and biological analysis will be assessed o hese 3 com-
pounds in he HCT116 cell line.
Complex s abili y in biological media
A e selec ion o he bes compounds o pu suing addi ional
biological cha ac e iza ion, he s abili y o he compound (3a,
3b and 3c) in biological medium was analyzed using
UV-Visible spec oscopy o e a wa eleng h ange o 220 o
700 nm o diffe en incuba ion imes (−0, 3 h, as shown in
Fig. 3; 0, 24, 48 h in ESI Fig. S5†).
Analysing he spec a o all compounds, high-ene gy
abso p ion bands co esponding o π→π* and n →π* an-
si ions wi h peaks in he 230–330 and 330–400 nm anges,
espec i ely, ha a e associa ed wi h he a oma ic ings o e -
py idines can be obse ed.
38
Acco ding o Fig. 3, o com-
pound 3a, bands a e obse ed a app oxima ely 235 nm,
265 nm and 360 nm a 0 h. Fo compound 3b, ou cha ac e -
is ic bands can be iden i ied a app oxima ely 293, 325, 352
and 371 nm. Finally, compound 3c p esen s bands a app oxi-
ma ely 235 nm, 265 nm, 327 nm, 365 nm, 380 nm and
530 nm. As obse ed in Fig. 3, om 0 h o 3 h, he e is an
inc ease o abso bance ha is mos p obably due o a be e
solubiliza ion o he complexes in he medium, main aining
hei cha ac e is ic bands. Howe e , analysing he da a om
ESI Fig. S5,†i is possible o obse e a change in he peak a
265 nm o 272 nm om 0 h o 48 h o compound 3c.
Howe e , o 3a a 24 h, he band a 360 nm is no longe
obse ed. Fu he mo e, a 48 h he e is a peak a app oxima ely
325 nm, which may indica e ha he complex is no s able in
solu ion (ESI Fig. S5†). Fu he mo e, he e is no a la ge
dec ease in he abso bance alues h oughou he spec um
be ween he diffe en acquisi ion imes. Rega ding compound
3b, he ou cha ac e is ic bands emained a he same wa e-
leng h h oughou he 48 h. Howe e , be ween 0 h and 24 h,
he e is a sligh dec ease in all abso bances h oughou he
spec um (ESI Fig. S5†).
Conside ing he s abili y o all compounds du ing he i s
h ee hou s, we u he accessed hei in e naliza ion in
HCT116 cells in his pe iod. Ne e heless, o ensu ing he
bes solubili y and s abili y o he compounds in all biological
assays, hey we e always pe o med wi h eshly p epa ed solu-
ions o hese compounds.
Compound cellula in e naliza ion by ICP-AES
The in e naliza ion o he pla inum compounds 3a,3b and 3c
in HCT116 cells was e alua ed by ICP-AES echnique, which
allows a quan i a i e measu emen o he amoun o me al
p esen in each sample. This assay allows us o unde s and i
he compounds unde s udy a e indeed in e nalized by
HCT116 cells.
39
To pe o m his echnique, HCT116 cells we e
Fig. 3 E alua ion o he s abili y o compounds 3a (A), 3b (B) and 3c (C)
ia UV- isible spec oscopy o 3 h. Abso bance spec a o 50 µM o
compounds 3c and 3a and 150 µM 3b in an RPMI medium wi hou
phenol ed and FBS a diffe en incuba ion imes: 0 h (o ange) and 3 h
(blue).
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