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h ps://doi.o g/10.1021/j 400796p ."
Selec i e cy o oxic ac i i y o new lipophilic 1
hyd oxy y osol alkyl e he de i a i es 2
3
4
José Manuel Calde ón-Mon año
†
, And és Mad ona
‡
, Es e anía Bu gos-Mo ón
†
, Manuel Luis 5
O a
§
, San iago Ma eos
§
, José Luis Espa e o
‡
and Miguel López-Láza o
†*
6
7
†
Depa men o Pha macology, Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain 8
‡
Depa men o O ganic and Pha maceu ical Chemis y, Facul y o Pha macy, Uni e si y o 9
Se ille, Spain
10
§
Depa men o Cell Biology, Facul y o Biology, Uni e si y o Se ille, Spain
11
12
13
14
*Co esponding Au ho (Tel: + 34 954 55 63 48; Fax: + 34 954 55 60 74; E-mail: 15
[email p o ec ed]) 16
2
Abs ac 17
18
Recen da a sugges ha hyd oxy y osol, a phenolic compound o i gin oli e oils, has 19
an icance ac i i y. This communica ion epo s he syn hesis o decyl and hexadecyl 20
hyd oxy y osyl e he s, as well as he cy o oxic ac i i y o hyd oxy y osol and a se ies o 21
se en hyd oxy y osol alkyl e he de i a i es agains A549 lung cance cells and MRC5 non-22
malignan lung ib oblas s. Hyd oxy y osyl dodecyl e he (HTDE) showed he highes 23
selec i e cy o oxici y and possible mechanisms o ac ion we e in es iga ed; esul s sugges 24
ha HTDE can mode a ely inhibi glycolysis, induce oxida i e s ess and cause DNA damage 25
in A549 cells. The combina ion o HTDE wi h he an icance d ug 5- lu ou acil induced a 26
syne gis ic cy o oxici y in A549 cance cells bu no in non-malignan MRC5 cells. HTDE 27
also displayed selec i e cy o oxici y agains MCF7 b eas cance cells s MCF10 no mal 28
b eas epi helial cells in he 1-30 mic omola ange. These esul s sugges ha he cy o oxici y 29
o HTDE is mo e po en and selec i e han ha o pa en compound hyd oxy y osol. 30
31
Keywo ds: oli e oil, lipophilic de i a i es, phenolic compounds, cance , an icance , 32
cy o oxic 33
3
INTRODUCTION 34
Epidemiological s udies sugges ha a Medi e anean die may educe he isk o 35
de eloping degene a i e pa hologies such as ca dio ascula diseases and cance .
1-4
Since 36
oli e oil is he main sou ce o a in he Medi e anean die , he numbe o epo s desc ibing 37
he bene icial p ope ies o oli e oil has no ably inc eased in ecen yea s. This oli e oil 38
popula i y has mainly been a ibu ed o i s high con en on monounsa u a ed a y acids and 39
o i s ichness in phenolic compounds.
3-9
40
The p esence o phenolic compounds in oli e oil has a ac ed much a en ion due o 41
hei known biological ac i i ies and heal h e ec s. Pa icula a en ion has been placed on 3,4 42
dihyd oxyphenyle hanol (hyd oxy y osol), an o-diphenolic compound ha is p esen in i gin 43
oli e oil as secoi idoid de i a i es o ace a e es e s and has shown a a ie y o 44
pha macological ac i i ies.
10-15
Accumula ing p eclinical e idence sugges s ha 45
hyd oxy y osol has cance chemop e en i e and chemo he apeu ic po en ial. In i o 46
expe imen s ha e shown ha hyd oxy y osol inhibi s p oli e a ion and induces apop osis in a 47
a ie y o cance cell lines om di e en o igin, including b eas , colon and leukemia cell 48
lines.
16-20
Se e al mechanisms o ac ion ha e been shown o pa icipa e in i s cy o oxic 49
ac i i y, such as cell cycle a es , cy och ome c elease, ac i a ion o caspase 3, c-jun o bcl-2, 50
and inhibi ion o CDK6, HER2, a y acid syn hase, o PI3K/Ak /NF-kappa B pa hway 51
( e iewed in e e ence 11). Recen animal expe imen s ha e e ealed ha hyd oxy y osol can 52
also inhibi cance cell g ow h in i o.
21
53
Ou p e ious wo k epo ed he syn heses o lipophilic hyd oxy y osol de i a i es in 54
he o m o es e s
22
and e he s
23
, and he e alua ion o hei an ioxidan ac i i y in ela ion o 55
ha o hyd oxy y osol.
24-26
These s udies e ealed ha he in oduc ion o an acyl o alkyl 56
side chain in he hyd oxy y osol s uc u e did no educe, o e en imp o ed, he an ioxidan 57
4
capaci y o hyd oxy y osol. We ha e s udied he abso p ion, me abolism and diges i e 58
s abili y o hese compounds
27-29
and ha e also obse ed ha some o hese hyd oxy y osol 59
de i a i es exhibi an ipla ele , an i-in lamma o y and neu op o ec i e e ec s.
30,31
In his 60
a icle we epo he syn hesis o wo new alkyl de i a i es o hyd ox y osol, as well as he 61
e alua ion o he cy o oxic ac i i y on cance and non-malignan cells o hyd oxy y osol and a 62
se ies o i s e he de i a i es (e hyl, bu yl, hexyl, oc yl, decyl, dodecyl and hexadecyl e he s). 63
In e es ingly, hyd oxy y osol isola ed om oli e oil was e wa e s (OOWW) has been used
o 64
he p epa a ion o hese compounds o gi e an added alue o his ype o esidue. 65
66
MATERIAL AND METHODS 67
68
Chemicals 69
Hyd oxy y osol was ob ained om oli e oil was e wa e s as desc ibed elsewhe e.
23
70
Hyd oxy y osol alkyl e he de i a i es we e p epa ed om hyd oxy y osol as desc ibed 71
below. Mn(III) e akis(1-me hyl-4-py idyl)po phy in pen achlo ide (MnTMPyP) was 72
pu chased om Biomol In e na ional. All o he compounds used in his wo k we e ob ained 73
om Sigma. All compounds we e dissol ed in DMSO and added o he cells a e app op ia e 74
dilu ions in cell cul u e medium. Final DMSO concen a ions we e ne e highe han 0.3%. 75
76
Cell Lines 77
The human A549 lung cance cell line, he human emb yo lung ib oblas ic MRC5 cell 78
line and he human MCF7 b eas adenoca cinoma cell line we e main ained in DMEM 79

5
supplemen ed wi h 2 mM glu amine, 50 µg/mL penicillin, 50 µg/mL s ep omycin and 10% 80
e al bo ine se um. The human MCF10 b eas epi helial cell line (kindly p o ided by D . D. 81
Ruano and D . P. Daza) was main ained in a 1:1 mix u e o Dulbecco's modi ied Eagle's 82
medium and Ham's F12 medium supplemen ed wi h 20 ng/mL epide mal g ow h ac o , 100 83
ng/mL chole a oxin, 10 µg/mL insulin and 500 ng/mL o hyd oco isone (95%) and ho se 84
se um (5%). To s udy he possible DNA damage esponse induced by he es ed compounds, 85
he ollowing pa en al and DNA epai -de icien cell lines we e used: AA8: pa en al Chinese 86
hams e o a y cells; V3-3: AA8 cells mu a ed in XRCC7 (DNA-PK), non-homologous end 87
joining (NHEJ) de icien ; KO40: AA8 cells mu a ed in FANCG, Fanconi anemia (FA) 88
de icien ; VC8: V79 Chinese hams e lung cells mu a ed in BRCA2, homologous 89
ecombina ion (HR) de icien ; VC8B2: VC8 cells complemen ed wi h human BRCA2 (HR 90
p o icien ); EM9-V: AA8 cells mu a ed in XRCC1 (DNA ligase III), base excision epai 91
(BER) de icien ; EM9-XH: EM9 cells complemen ed wi h XRCC1 (BER p o icien ), HCT-92
116: human colon cance cells mu a ed in MLH1, misma ch epai (MMR) de icien ; and 93
HCT-116 + c3: HCT-116 cells complemen ed wi h ch omosome 3 (wi h MLH1 gene; MMR 94
p o icien ).
32,33
These DNA epai -de icien cell lines, kindly p o ided by D . Thomas 95
Helleday, we e cul u ed in DMEM. All cell lines we e cul u ed a 37 °C in a humidi ied 96
a mosphe e con aining 5% CO
2
. Cell cul u e eagen s we e ob ained om Li e Technologies. 97
98
P epa a ion o e he de i a i es o hyd oxy y osol 99
Hyd oxy y osyl alkyl e he s (10–16) we e p epa ed om hyd oxy y osol (1) using a 100
me hod p e iously desc ibed by us.
23
The ollowing compounds ha e been syn hesized o he 101
i s ime: 102
6
1,2-bis(benzyloxy)-4-(2’-decyloxye hyl)benzene (7): whi e solid (86% yield).
1
H-NMR 103
(500 MHz, DMSO-d
6
) δ ppm 7.37 (m, 10H, 2xPh), 6.96 (d, J=2.0 Hz, 1H, H
4
), 6.92 (d, J=8.2 104
Hz, 1H, H
7
), 6.71 (dd, 1H, H
8
), 5.07 (s, 2H, CH
2
Ph in pos. 5), 5.06 (s, 2H, CH
2
Ph in pos. 6), 105
3.48 ( , J=6.9 Hz, 2H, H
1
), 3.32 ( , J=6.5 Hz, 2H, H
1’
), 2.68 ( , 2H, H
2
), 1.43 (m, 2H, H
2’
), 106
1.23 (m, 14H, H
3’
−H
9’
), 0.83 ( , 3H, H
10’
).
13
C-NMR (125 MHz, DMSO-d
6
) δ ppm 148.2 (C
5
), 107
146.6 (C
6
), 137.4 y 137.3 (C
ipso
, Bn g oups), 132.2 (C
3
), 128.2−127.4 (C
3’’
, C
4’’
y C
5’’
, g oups 108
Bn), 121.2 (C
8
), 115.4 (C
4
), 114.6 (C
7
), 70.9 (C
1
), 70.2 (CH
2
Ph in pos. 6), 70.1 (CH
2
Ph in pos. 109
5), 69.8 (C
1’
), 35.0 (C
2
), 29.1 (C
2’
), 29.0−28.6 (C
4’
−C
7’
), 25.6 (C
3’
), 31.2 (C
8’
), 22.0 (C
9’
), 13.8 110
(C
10’
). Elem. Anal. Calcd. o C
32
H
42
O
3
: C, 80.97; H, 8.92. Found: C, 88.85; H, 8.78. 111
1,2-bis(benzyloxy)-4-(2’-hexadecyloxye hyl)benzene (9): whi e solid (86% yield).
1
H-112
NMR (500 MHz, DMSO-d
6
) δ ppm 7.37 (m, 10H, 2xPh), 6.96 (d, J=2.0 Hz, 1H, H
4
), 6.93 (d, 113
J=8.2 Hz, 1H, H
7
), 6.72 (dd, 1H, H
8
), 5.08 (s, 2H, CH
2
Ph in pos. 5), 5.07 (s, 2H, CH
2
Ph in 114
pos. 6), 3.49 ( , J=7.0 Hz, 2H, H
1
), 3.33 ( , J=6.5 Hz, 2H, H
1’
), 2.69 ( , 2H, H
2
), 1.45 (m, 2H, 115
H
2’
), 1.23 (m, 10H, H
3’
−H
15’
), 0.84 ( , 3H, H
16’
).
13
C-NMR (125 MHz, DMSO-d
6
) δ ppm 116
148.1 (C
5
), 146.6 (C
6
), 137.4 y 137.3 (C
ipso
, Bn g oups), 132.2 (C
3
), 128.2−127.4 (C
3’’
, C
4’’
y 117
C
5’’
, Bn g oups), 121.3 (C
8
), 115.5 (C
4
), 114.7 (C
7
), 70.9 (C
1
), 70.2 (CH
2
Ph in pos. 6), 70.1 118
(CH
2
Ph in pos. 5), 69.8 (C
1’
), 35.0 (C
2
), 31.2 (C
14’
), 29.1 (C
2’
), 29.0−28.6 (C
4’
−C
13’
), 25.6 119
(C
3’
), 22.0 (C
15’
), 13.8 (C
16’
). Elem. Anal. Calcd. o C
38
H
54
O
3
: C, 81.67; H, 9.74. Found: C, 120
81.27; H, 9.35. 121
4-(2’-decyloxye hyl)benzene-1,2-diol (14): colou less oil (83% yield).
1
H-NMR (500 122
MHz, DMSO-d
6
) δ ppm 8.63 (s, 1H, OH in pos. 6), 8.56 (s, 1H, OH in pos. 5), 6.59 (d, J=8.0 123
Hz, 1H, H
7
), 6.57 (d, J=2.1 Hz, 1H, H
4
), 6.43 (dd, 1H, H
8
), 3.44 ( , J=7.2 Hz, 2H, H
1
), 3.33 ( , 124
J=6.6 Hz, 2H, H
1’
), 2.58 ( , 2H, H
2
), 1.41 (m, 2H, H
2’
), 1.24 (m, 10H, H
3’
−H
9’
), 0.84 ( , 3H, 125
H
10’
).
13
C-NMR (125 MHz, DMSO-d
6
) δ ppm 144.8 (C
5
), 143.3 (C
6
), 129.7 (C
3
), 119.3 (C
8
), 126
116.1 (C
4
), 115.3 (C
7
), 71.4 (C
1
), 69.9 (C
1’
), 34.9 (C
2
), 29.1 (C
2’
), 29.0−28.6 (C
4’
−C
7’
), 25.6 127
7
(C
3’
), 31.2 (C
8’
), 22.0 (C
9’
), 13.8 (C
10’
). Elem. Anal. Calcd. o C
18
H
30
O
3
: C, 73.43; H, 10.27. 128
Found: C, 73.41; H, 10.39. 129
4-(2’-hexadecyloxye hyl)benzene-1,2-diol (16): whi e solid (83% yield).
1
H-NMR (500 130
MHz, DMSO-d
6
) δ ppm 8.59 (s, 2H, OH in pos. 5 and 6), 6.60 (d, J=8.0 Hz, 1H, H
7
), 6.58 (d, 131
J=2.1 Hz, 1H, H
4
), 6.43 (dd, 1H, H
8
), 3.44 ( , J=7.2 Hz, 2H, H
1
), 3.33 ( , J=6.6 Hz, 2H, H
1’
), 132
2.59 ( , 2H, H
2
), 1.45 (m, 2H, H
2’
), 1.23 (m, 10H, H
3’
−H
15’
), 0.85 ( , 3H, H
16’
).
13
C-NMR (125 133
MHz, DMSO-d
6
) δ ppm 144.8 (C
5
), 143.3 (C
6
), 129.7 (C
3
), 119.3 (C
8
), 116.1 (C
4
), 115.3 (C
7
), 134
71.4 (C
1
), 69.9 (C
1’
), 35.0 (C
2
), 31.2 (C
14’
), 29.1 (C
2’
), 29.0−28.6 (C
4’
−C
13’
), 25.6 (C
3’
), 22.0 135
(C
15’
), 13.8 (C
16’
). Elem. Anal. Calcd. o C
24
H
42
O
3
: C, 76.14; H, 11.18. Found: C, 76.41; H, 136
10.71. 137
138
Cell p oli e a ion assay 139
The MTT assay is a colo ime ic echnique o he quan i a i e de e mina ion o cell 140
iabili y. I is based on he capabili y o iable cells o ans o m he MTT sal (3-(4,5-141
dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) in o a pu ple o mazan dye. 142
Exponen ially g owing cells we e seeded in o 96-well pla es and d ugs we e added 24 h la e . 143
Cells we e exposed o he d ugs o 48 h (excep in he expe imen s wi h he pa en al and 144
DNA epai -de icien cell lines, in which cells we e exposu e o 24 h and we e hen allowed 145
o g ow o addi ional 24 h in d ug- ee medium o le hem epai he possible DNA damage 146
induced by he d ugs). A e hese 48 h, medium was emo ed and 125 µL MTT (1 mg/mL in 147
medium) was added o each well o 5 h. Then, 80 µL 20% SDS in 20 mM HCl was added o 148
dissol e he insoluble pu ple o mazan p oduc , pla es we e incuba ed o 10 hou s a 37 ºC, 149
and op ical densi ies we e measu ed a 540 nm on a mul iwell pla e spec opho ome e eade . 150
Cell iabili y was exp essed as pe cen age in ela ion o con ols. All da a we e a e aged om 151
8
a leas h ee independen expe imen s and we e exp essed as means ± s anda d e o o he 152
means (SEM). 153
154
Inhibi ion o glycolysis 155
Glycolysis inhibi ion was assessed by measu ing concen a ions o glucose (ini ial 156
p oduc o glycolysis) and lac a e ( inal p oduc o glycolysis) in con ol and ea ed cells. 157
B ie ly, 10
6
cells we e exposed o he es ed compounds o 8 h, and glucose and lac a e 158
concen a ions we e de e mined in cell supe na an s by using he Accu end
®
Plus analyze 159
oge he wi h Accu end glucose s ips and BM-Lac a e S ips (Roche Diagnos ics). A e 160
calib a ing he ins umen wi h glucose and lac a e calib a ion s ips, es s ips we e used o 161
de e mine glucose and lac a e le els ia colo ime ic-oxidase media o eac ions acco ding o 162
he manu ac u e 's ins uc ions.
34
Resul s a e exp essed as pe cen age o lac a e p oduc ion 163
and pe cen age o glucose consump ion in ela ion o un ea ed cells, and a e shown as he 164
means ± s anda d e o o he means (SEM) o h ee independen expe imen s. 165
166
Come assay 167
The single-cell gel elec opho esis assay (come assay) is a well es ablished echnique 168
o DNA damage de ec ion. The images ob ained wi h his assay esemble a "come " wi h a 169
dis inc head and ail; he head is composed o in ac DNA, while he ail consis s o damaged 170
(single-s and o double-s and b eaks) DNA. This assay has been desc ibed in de ail by Singh 171
e al.
35
We ollowed his p o ocol wi h mino modi ica ions desc ibed p e iously.
36
B ie ly, 172
s anda d slides we e imme sed in 1% no mal mel ing aga ose a 55 °C, le o allow he 173
aga ose o solidi y, and kep a 4 °C un il use. A e cell ea men s, app oxima ely 10000 174
15
Figu e 5A shows ha HTDE 3 µM can inhibi he g ow h o MCF7 b eas cance cells 321
wi hou a ec ing he g ow h o MCF10 no mal b eas cells. I is unknown whe he he o al 322
adminis a ion o HTDE can lead o such concen a ions in plasma and issues. P e ious 323
expe imen s sugges ha hyd oxy y osol alkyl e he de i a i es a e s able when diges ed in 324
i o, a e apidly abso bed, and a e me abolized only pa ially.
27-29
Indeed, HTDE and ela ed 325
hyd oxy y osol alkyl e he de i a i es we e ound o exe a neu op o ec i e e ec in a s 326
when adminis e ed o ally, he e o e sugges ing ha HTDE is bioa ailable a e o al 327
adminis a ion. In any case, he possible low o al bioa ailabili y and high me abolism o 328
HTDE could be o e come wi h a sus ained in a enous in usion. This s a egy could be used 329
o ob ain and main ain cy o oxic concen a ions o HTDE in plasma and issues, which would 330
maximize i s he apeu ic po en ial. Fu u e animal s udies a e needed o e alua e he an icance 331
po en ial o HTDE in i o. 332
The possible exploi a ion o he biological p ope ies o hyd oxy y osol alkyl e he 333
de i a i es desc ibed in his and p e ious epo s
25,26,30,31
would equi e an adequa e supply o 334
hese de i a i es. Se e al million ons pe yea o was es a e es ima ed o be p oduced 335
wo ldwide by he oli e oil indus y and, in e es ingly, hyd oxy y osol alkyl e he de i a i es 336
can be syn hesized easily and in high yield using hyd oxy y osol ob ained om oli e oil 337
was e wa e s.
23,48
338
In summa y, since accumula ing da a sugges ha he oli e oil cons i uen 339
hyd oxy y osol has an icance ac i i y, we ha e e alua ed he selec i e cy o oxic ac i i y o a 340
se ies o hyd oxy y osol alkyl e he de i a i es in ela ion o ha o hyd oxy y osol, and ha e 341
in es iga ed possible mechanisms in ol ed in he cy o oxici y o he mos selec i e 342
compound (HTDE, 15). Ou esul s showed ha he cy o oxic ac i i y o HTDE was mo e 343
po en and selec i e han ha o i s pa en compound hyd oxy y osol. 344

16
345
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347
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496
497
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We hank he Conseje ía de Inno ación, Ciencia y Emp esa o he Jun a de Andalucía (Spain) 498
o inancial suppo (g an no. P09-AGR-5098). 499
24
FIGURE CAPTIONS 500
Figu e 1. Syn he ic p ocedu e o he alkyl hyd oxy y osyl e he de i a i es (10–16). 501
502
Figu e 2. Selec i e cy o oxici y o hyd oxy y osol and se e al alkyl hyd oxy y osyl e he 503
de i a i es (10–16). The pe cen age o cell iabili y (± SEM) in A549 cance cells and MRC5 504
no mal cells exposed o 48 h o hyd oxy y osol (1), i s alkyl e he de i a i es (10-16) and 5-505
luo ou acil (5-FU) was de e mined wi h he MTT assay. 506
507
Figu e 3. Possible mechanisms o ac ion in ol ed in he cy o oxici y o hyd oxy y osyl 508
dodecyl e he (HTDE, 15). A) Pe cen age o lac a e p oduced by cells exposed o 8 h o 509
HTDE (15), dichlo oace a e (DCA) and 5- luo ou acil (5-FU) in ela ion o un ea ed cells 510
(con ol). B) Pe cen age o glucose consumed by cells exposed o 8 h o HTDE, DCA and 5-511
FU in ela ion o un ea ed cells. C) P e en ion o HTDE -induced cell dea h in A549 cells by 512
he supe oxide dismu ase mime ic MnTMPyP (MTT assay); A549 cells we e exposed o 513
HTDE o 48 h in he p esence and absence o MnTMPyP (added 1 h be o e HTDE). D) 514
Rep esen a i e pho og aphs o un ea ed cells, o cells ea ed wi h he DNA damaging agen 515
camp o hecin (cp ), and o cells exposed o 2 h and 24 h o HTDE 32 µM and 100 µM (come 516
assay). E) Quan i ica ion o DNA damage (come assay) exp essed as pe cen o DNA 517
damage in ail and as ail momen ( ail leng h × pe cen age o DNA in he ail). F) Cells 518
mu a ed in XRCC1 (de icien in base excision epai ) a e mo e sensible han pa en al cells o 519
he cy o oxic ac i i y o HTDE. Bo h cell lines we e ea ed wi h se e al concen a ions o 520
HTDE o 24 h and, a e a eco e y pe iod o 24 h, cell iabili y was es ima ed wi h he MTT 521
assay. 522
31
TOC G aphic
H
O
HO
O
Hyd oxy y osyl dodecyl e he (15)