Synthesis of short retinoidal amides related to fenretinide: antioxidant activities and differentiation-inducing ability

Article abstract of DOI:10.1007/s00280-017-3265-1

Purpose: By a scaffold shortening strategy, a small series of retinoidal amides fenretinide (4-HPR) analogs have been synthesized from α, β-ionones and tested for their antiproliferative and differentiating activities, and antioxidant effect. Methods: The

Full text of DOI:10.1007/s00280-017-3265-1

Cancer Chemother Pharmacol
DOI 10.1007/s00280-017-3265-1
ORIGINAL ARTICLE
Synthesis of short retinoidal amides related to fenretinide:
antioxidant activities and differentiation-inducing ability
Maria Anzaldi1 · Maurizio Viale2 · Chiara Macciò1 · Patrizio Castagnola2 ·
Valentina Oliveri3 · Camillo Rosano4 · Alessandro Balbi1
Received: 20 January 2017 / Accepted: 20 February 2017
© Springer-Verlag Berlin Heidelberg 2017
Abstract
TEAC). Docking analysis was performed to study the bind-
Purpose By a scaffold shortening strategy, a small series
of retinoidal amides fenretinide (4-HPR) analogs have been
synthesized from α, β-ionones and tested for their anti-
proliferative and differentiating activities, and antioxidant
effect.
ing features to the Retinoic Acid Receptor alpha (RARα).
Results While no pharmacologically relevant antiprolif-
erative activity was evidenced, some of our short retinoids
showed a differentiating and antioxidant activity similar
to that of 4-HPR. In particular, compound 2b6 displayed
a scavenging activity two times more efficient than 4-HPR
itself. Finally, the docking analysis showed that these short
retinoids, like 4-HPR, bind to the RARα protein with good
fitness scores.
Methods The antiproliferative activity and trigger-
ing of apoptosis of our short retinoids were evaluated by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay and 4′-6-diamidino-2-phenylindole stain-
ing and microscope evaluation after 3- or 6-day exposure,
while their differentiating activity was established by the
analysis of the expression of the CD11b marker of differ-
entiation in treated HL60 target cells and by the superox-
ide production assayed colorimetrically by the nitro blue
tetrazolium-reducing activity assay. Finally, the antioxidant
activity was determined by the 2,2′-azino-bis-(3-ethylbenz-
thiazoline-6-sulphonic acid) diammonium salt radical cat-
ion decolourisation assay utilizing the antioxidant Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
as reference (Trolox equivalent antioxidant capacity, or
Conclusion Our data could pave the way for the design
of new potent and less toxic antioxidant and differentiating
compounds related to 4-HPR.
Keywords Retinoidal amides · 4-HPR · Antioxidant ·
Differentiation
Introduction
Retinoids are receiving continuous considerable attention
as agents that may be useful for both cancer prevention and
treatment due to their cell differentiation, proliferation, and
antioxidant effects, and have been shown to inhibit micro-
somal lipid peroxidation. The applicability of retinoid rem-
edies is limited due to the toxic effects of natural retinoids
[1]. Therefore, many efforts have been made to synthesize
new retinoidal compounds with lower toxic and/or side
effects.
* Alessandro Balbi
balbi@unige.it
1
Dipartimento di Farmacia, Università di Genova, Viale
Benedetto XV, 3, 16132 Genoa, Italy
2
IRCCS Azienda Ospedaliera Universitaria San Martino,
IST Istituto Nazionale per la Ricerca sul Cancro, U.O.C.
Bioterapie, L.go R. Benzi, 10, 16132 Genoa, Italy
We have already reported that the so-called “short reti-
noids” formed by a cyclohexenyl group linked to a suitable
residue by a short ethylenic chain present activities which
could be assigned to classical retinoids [2–4]. Moreover,
in the papers previously mentioned, the heterocyclic nuclei
linked to an ethenylic chain deeply affected the biological
3
Dipartimento di Scienze Chimiche, University of Catania,
Viale A. Doria, 6, 95125 Catania, Italy
4
IRCCS Azienda Ospedaliera Universitaria San Martino,
IST Istituto Nazionale per la Ricerca sul Cancro, U.O.S.
Biopolimeri e Proteomica, L.go R. Benzi, 10, 16132 Genoa,
Italy
Vol.:(0123456789)
1 3

Cancer Chemother Pharmacol
activities. We would like to show that some of the biologi-
cal activities of retinoids are kept and sometimes improved
only by shortening the polyene chain of all-trans retinoic
acid (ATRA) or its derivatives.
different malignancies [10–14]. It also exhibits antioxidant
activities [15] and could cause the induction of cell differ-
entiation [16–19] in a number of human cancer cell lines.
These abilities may also contribute to its chemopreventive/
chemoprotective properties.
We focused our attention on N-(4-hydroxyphenyl)reti-
namide (fenretinide, 4-HPR) (Fig. 1), a chemopreventive
and antiproliferative agent with pharmacological activity
in preclinical setting [5–8], less toxic and substantially less
teratogenic than ATRA [9], and thus involved in various
clinical trials [10–14], where it showed favourable results
when used for the chemoprevention in patients treated for
We thus set out to study a new series of “short retinoids”
structurally related to 4-HPR. We maintained the cyclohex-
enyl ring and the amide functional group, linking the two
with an ethylenic chain. Therefore, seven short retinoidal
N-phenilamides analogs and one adamantyl derivative have
been synthesized (Fig. 2). The anilide derivatives showed
both antioxidant and differentiating-induced activities
when –OH and/or –COOH groups are present. Compound
2b6 exhibited similar differentiating activity of 4-HPR and
was much more efficient than 4-HPR towards the antioxi-
dant activity (Fig. 1).
OH
O
N
H
Materials and methods
N-(4-Hydroxyphenyl)retinamide
Synthesis and characterization
O
The syntheses of short retinoidal amides 4-HPR analogs
are outlined in Fig. 2.
X
N
H
Compounds 1a and 1b, which were obtained from a and
β-ionones by haloform reaction, dissolved in thionyl chlo-
ride, gave the acyl chloride intermediates (not isolated)
which, in turn, treated with the appropriate amines, pro-
vided the 4-HPR analogs 2a and 2b.
Short retinoidal amides
Fig. 1 Structure of fenretinide and short retinoidal amides
Fig. 2 Scheme for the synthesis
O
O
O
of the short retinoidal amides
SOCl2
1) Br2/NaOH
OH
Cl
2) H+
α;β-ionone
1a, 1b
O
NH2R
Py
NHR
2a, 2b
2a1, 2b1 R=
2b2 R=
2b5 R=
(CH2)3COOH
CH2COOH
OH
2b3 R=
SO2NH2
COOH
OH
OCH3
HO
2b4 R=
2b7 R=
R=
2b6
OH
1 3

Cancer Chemother Pharmacol
Melting points were determined with Fisher–Johns appa-
ratus and are uncorrected. The IR spectra were recorded
in film or in potassium bromide disks on a Perkin-Elmer
buffer (10 mM, pH 7.4), such that the absorbance of the
solution at 734 nm was 0.70 0.02. This radical solution
was combined with sample antioxidants, in varying con-
centrations, and allowed to react for 6 min. All samples
were diluted to provide 20–80% inhibition of the blank
absorbance. The decrease in absorbance caused by each
compound (solution absorbance), measured at 1, 3, and
6 min, reflected the ABTS⋅+ radical cation scavenging
capacity and was plotted against the concentration of the
tested compound.
1
398 spectrometer. The H-NMR spectra were recorded on
Varian Gemini 200 (200 MHz) spectrometers in CDCl3
or DMSO-d6 with tetramethylsilane as the internal stand-
ard (δ=0). The purity of all compounds was checked by
thin-layer chromatography on silica gel 60-F-254 precoated
plates and the spots were located in UV light or by vanillin
in sulfuric acid. Elemental analyses were performed on a
Carlo Erba 1106 Elemental Analyser in the Microanalysis
Laboratory in our Department.
The TEAC value represents the ratio between the slope
of this linear plot for scavenging of ABTS⋅+ radical cation
by the compound compared to the slope of this plot for
ABTS⋅+ radical cation scavenging by Trolox. Each result-
ant slope was normalized with respect to that obtained for
Trolox to give the TEAC value for each time point (1, 3,
6 min).
Evaluation of antiproliferative activity, by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay, and apoptosis, by DAPI (4′-6-diamid-
ino-2-phenylindole) staining and microscope evaluation
Clearly, the TEAC value of Trolox is 1.00. As a conse-
quence, a compound with TEAC value higher than 1.00 has
a superior antioxidant ability compared to Trolox.
All determinations were carried out at least three times
and in triplicate for each separate concentration of the sam-
ples to obtain consistent data with statistical error.
Human cell lines A2780 (ovary, adenocarcinoma), DOHH2
(follicular lymphoma), A549 (lung, carcinoma), and HL60
(lymphoma) were seeded at 104/ml, 105/ml, 0.89×104/
ml, and 1.9×104/ml, respectively in 180-μl RPMI 1640
complete medium into flat- or round-bottomed microtiter
plates. After 6–8 h, 20 μl containing five (four for HL60)
1:10 (1:3 for HL60) fold compound concentrations diluted
in FCS containing 1, 2, or 8% DMSO were added to the
culture medium (final DMSO concentrations: 0.1, 0.2, and
0.8%). Seventy-two hours later (and also 6 days for HL60),
cells were processed as described elsewhere [20]. The
IC50s values were calculated on the base of single concen-
tration–response curves. Each experiment was repeated 3–8
times.
Analysis of cell differentiation
Human promyelocytic leukaemia HL-60 cells (0.35×104/
ml) were exposed to 30 mM of 4-HPR-like compounds and
1-mM 4-HPR for 3 or 6 days. After 6-day exposure, cells
were assayed for the induction of differentiation by the flow
cytometry analysis of the CD11b differentiation marker.
Cells were stained using the phycoerythrin-conjugated
mouse anti-human CD11b (eBioscience Inc., San Diego,
CA, USA). Untreated controls as well as mock staining of
the cells with a phycoerythrin-conjugated antibody, of the
same CD11b IgG isotype, were always made. Analysis of
fluorescence was performed on a FACScan flow cytometer
(Becton Dickinson Italia, Buccinasco, Italy).
The triggering of apoptosis was evaluated on A2780 and
A549 cells treated for 72 h with 30-mM short retinoids, and
with 4-HPR at its IC50s, as evaluated by the MTT assay on
target cells. Floating and adherent cells were harvested and
were washed twice with phosphate buffered saline (PBS),
fixed with 100–120 μl of 70% ethanol in PBS, and main-
tained at 4°C until analysis. The percentage of apoptotic
cells was evaluated at the fluorescence microscope after
adding 6 μl of a DAPI solution at 10 mg/ml in water.
In parallel, after 6-day exposure, superoxide production,
indicative of cell differentiation, was assayed colorimetri-
cally by the nitro blue tetrazolium (NBT)-reducing activ-
ity assay [22]. To this end, 105 alive cells/well were seeded
after the 6-day treatment in round-bottomed microtiter
plates; after centrifugation, the medium was discarded and
cells stained for 60 min with 100-ml nitro blue tetrazolium
(2 mg/ml, Sigma) in PBS containing 200-ng/ml phorbol-
12-myristate-13-acetate (Sigma). The plates were then cen-
trifuged, the medium discarded and the formazan deposits
solubilized by 100-ml ethanol. Plates were measured on a
microculture plate reader 400 ATC (SLT Labinstruments,
Austria) at 560 nm. To verify a time dependence of the
induction of cell differentiation, this procedure was also
applied after 3 day exposure to our short retinoids.
Trolox equivalent antioxidant capacity (TEAC) assay
The antioxidant capacity of the tested compounds was
determined by the 2,2′-azino-bis-(3-ethylbenzthiazoline-
6-sulphonic acid) (ABTS) diammonium salt radical cation
decolourisation assay [21] using 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) as a standard.
Briefly, the radical cation ABTS⋅+ was generated by a reac-
tion between ABTS (7 mM) and persulfate (2.45 mM) in
water. After 16-h incubation (dark, room temperature), the
resulting solution was diluted in a cuvette with phosphate
1 3

Cancer Chemother Pharmacol
Docking analysis
The molecular structures of the ligands we screened “in
silico” (2a1, 2b1–7) were built and energy minimized.
The three-dimensional atomic coordinates of the Retinoic
Acid Receptor alpha (RARα) [23, PDB code 5k13] were
retrieved by the Protein Data Bank. The obtained atomic
coordinates were then energy minimized and stereochem-
istry was further optimized by the program REFMAC5
[24]. To perform docking simulations between the recep-
tor and our ligands, we used the program GOLD v.5.2 (the
Cambridge Crystallographic Data Center, UK). GOLD is a
program using a genetic algorithm allowing us to investi-
gate the full range of ligand conformational flexibility and
a partial protein side chain flexibility. In the case of RAR
we chose, as the binding site, the volume included within
20 Å from the O atom of Leu 269. The default GOLD set-
tings were used running the simulations. Residues Phe228,
Ile273, Met284, Phe286, Met297, and Phe302 were defined
with flexible side chains, allowing their free rotation. Fig-
ure 3 was drawn with the program Chimera [25].
Results
Synthesis
General procedure for the preparation of compounds 1a,
1b
3.0 ml of Br2 was added to a cooled solution (0°C) of
NaOH (9.4 g, 0.23 mol) in water (39 ml) and the reac-
tion mixture was allowed to stir 1 h at room temperature.
A solution of a or b-ionone (2.5 g, 0.01 mol) in dioxane
(5.5 ml) was added dropwise and the solution was further
stirred for 4 h. The hypobromite in excess was destroyed
with 10% aqueous sodium bisulphite and the solution was
extracted with diethyl ether to remove any impurities. Acid-
ification of the alkaline solution with concentrated hydro-
chloric acid gave 1b as a white solid or 1a as oil.
3‑(2,6,6‑trimethyl‑2‑cyclohexen‑1‑yl)‑2‑propenoic acid
(1a)
The oil was extracted with ethyl acetate. The organic
phase was washed with water, dried (MgSO4) and con-
centrated in vacuum to yield 1a, thick yellow oil already
pure. Yield 70%; IR (film) ν 2918, 2671, 1694, 1645, 1417,
1256 cm−1; 1H NMR (200 MHz, CDCl3): δ 0.84 (3H, s,
CH3), 0.90 (3H, s, CH3), 1.18 (1H, m, H-5′), 1.30 (1H, m,
H-5′), 1.55 (3H, s, CH3), 2.01 (2H, m, H-3′), 2.30 (1H, d,
H-1′ J=9.80 Hz), 5.45 (1H, m, H-3′), 5.79 (1H, d, ethene,
J=15.8 Hz), 6.90 (1H, dd, ethene, J=9.80 Hz, 15.80 Hz),
Fig. 3 Concentration–response curves after the evaluation of antipro-
liferative activity of short retinoidal amides 4-HPR on A2780, A549
and DOHH2 cells
10.55 (1H, bs, COOH, deuterium oxide exchangeable);
Calcd. for C12H18O2: C, 74.19; H, 9.34. Found: C, 74.21;
H, 9.32.
1 3

Cancer Chemother Pharmacol
3‑(2,6,6‑trimethyl‑1‑cyclohexen‑1‑yl)‑2‑propenoic acid
ethene, J=15.8 Hz), 6.67 (2H, d, arH), 7.20 (1H, d, ethene,
J=15.8 Hz), 7.43 (2H, d, arH), 9.17 (1H, s, NH, deuterium
oxide exchangeable), 9.75 (1H, s, OH, deuterium oxide
exchangeable); Calcd. for C18H23NO2: C, 75.76; H, 8.12;
N, 4.91. Found: C, 75.79; H, 8.43; N, 4.82.
(1b)
The white solid was recrystallized from AcOEt/n-hex-
ane to give 1b pure. Yield 90%; mp 108–109°C (Lit., mp
108–109°C) [26]; IR (KBr) ν 2930, 2674, 1684, 1629,
1417, 1265 cm−1; 1H NMR (200 MHz, CDCl3): δ 1.11 (6H,
s, CH3), 1.53 (2H, m, H-5′), 1.63 (2H, m, H-4′), 1.81 (3H, s,
CH3), 2.11 (2H, m, H-3′), 5.91 (1H, d, ethene, J=15.8 Hz),
7.60 (1H, d, ethene, J=15.8 Hz),; Calcd. for C12H18O2: C,
74.19; H, 9.34. Found: C, 74.22; H, 9.36.
4‑[[1‑oxo‑3‑(2,6,6‑trimethyl‑1cyclohexen1yl)2propen1yl]
amino]benzenebutanoic acid (2b2)
From 4-amino-benzenbutanoic acid and 1b for 5 h. After
evaporation to dryness, the residue was chromatographed
on SiO2 eluting first with cyclohexane and then with
cyclohexane/AcOEt 1:1. The second eluate gave 2b2 as
white solid; yield 52%; mp 108–109°C. IR (KBr) ν 2931,
2866, 1712, 1600, 1539, 1515 cm−1; 1H NMR (200 MHz,
CDCl3): δ 1.05 (6H, s, CH3), 1.46 (2H, m, H-5′), 1.58 (2H,
m, H-4′), 1.74 (3H, s, CH3), 1.92 (2H, m, CH2), 2.04 (2H,
m, H-4′), 2.34 (2H, t, CH2), 2.62 (2H, t, CH2), 5.91 (1H,
d, ethene, J=15.8 Hz), 7.12 (2 H, d, arom-H), 7.38–7.53
(3H, m, arH and ethene). Calcd. for C22H29NO3: C, 74.33;
H, 8.22; N, 3.94. Found: C, 74.34; H, 8.12; N, 3.94.
General procedure for the preparation of compounds 2a,
2b
The solution of 1a or 1b (3.1 mmol) in SOCl2 (10 ml) was
stirred for 1.30 h at 50°C. After evaporation of SOCl2 in
excess, the residue was dissolved in pyridine (15 ml) and
added with 3.1 mmol of the appropriate amine. The result-
ing solution was then stirred at 60°C and, after cooling,
was poured into water. The suspension was extracted with
AcOEt and the organic phase was washed with HCl 2 N,
NaHCO3, water and dried over MgSO4.
N‑[4‑(aminosulfonyl)phenyl]‑3‑(2,6,6‑trimethyl‑1‑cy‑
clohexen‑1‑yl)‑2‑propenamide (2b3)
N‑(4‑hydroxyphenyl)‑3‑(2,6,6‑trimethyl‑2‑cy‑
clohexen‑1‑yl)‑2‑propenamide (2a1)
From 4-amino-benzensulfonamide and 1b for 24 h. After
evaporation to dryness, the resulting white solid was crys-
tallized from AcOEt; yield 35%; mp 257–258°C (dec). IR
(KBr) ν 3351, 2924, 1670, 1595, 1531, 1317, 1154 cm−1;
1H NMR (200 MHz, DMSO-d6): δ 1.06 (6H, s, CH3),
1.44 (2H, m, H-5′), 1.55 (2H, m, H-4′), 1.75 (3H, s, CH3),
2.05 (2H, m, H-4′), 6.19 (1H, d, ethene, J=15.8 Hz), 7.24
(2H, s, NH2, deuterium oxide exchangeable), 7.32 (1H, d,
ethene, J=15.8 Hz), 7.76 (4H, m, arH), 10.35 (1H, s, NH,
deuterium oxide exchangeable); Calcd. for C18H24N2O3S:
C, 62.04; H, 6.94; N, 8.04; S, 9.20. Found: C, 61.81; H,
6.87; N, 8.12; S, 8.65.
From 4-amino-phenol and 1a for 5 h. After evaporation to
dryness, the residue was chromatographed on SiO2 eluting
first with cyclohexane and then with cyclohexane/AcOEt
1:1. The second eluate gave 2a1 as yellow solid; yield 30%;
mp: 205–208°C; IR (KBr) ν 3435, 2946, 1645, 1577, 1470,
1256 cm−1; 1H NMR (200 MHz, DMSO-d6): δ 0.78 (3H, s,
CH3), 0.86 (3H, s, CH3), 1.18 (1H, m, H-5′), 1.30 (1H, m,
H-5′), 1.53 (3H, s, CH3), 1.88 (2H, m, H-3′), 2.30 (1H, d,
H-1′, J=9.80 Hz), 5.42 (1H, m, H-3′), 6.03 (1H, d, ethene,
J=15.8 Hz), 6.50 (1H, dd, ethene, J=9.80, 15.80 Hz), 6.65
(2H, d, arH), 7.41 (2H, d, arH), 9.10 (1H, s, NH, deuterium
oxide exchangeable), 9.75 (1H, s, OH, deuterium oxide
exchangeable); Calcd. for C18H23NO2: C, 75.76; H, 8.12;
N, 4.91. Found: C, 75.71; H, 8.30; N, 4.78.
N‑(3‑hydroxy‑4‑methoxyphenyl)‑3‑(2,6,6‑trimethyl‑1‑cy‑
clohexen‑1‑yl)‑2‑propenamide (2b4)
From 5-amino-2-methoxyphenol and 1b for 20 h. After
evaporation to dryness, the residue was chromatographed
on SiO2 eluting first with cyclohexane and then with
cyclohexane/AcOEt 1:1. The second eluate gave 2b4 as
brown oil; yield 60%; IR (film) ν 3263, 2934, 1657, 1606,
1510 cm−1; 1H NMR (200 MHz, CDCl3): δ 1.05 (6H, s,
CH3), 1.44 (2H, m, H-5′), 1.59 (2H, m, H-4′), 1.75 (3H,
s, CH3), 2.04 (2H, m, H-4′), 3.86 (3H, s, OCH3), 5.96
(1H, d, ethene, J=15.8 Hz), 6.78 (1H, d, arH), 7.20–7.13
(2H, m, arH), 7.42 (1H, d, ethene, J=15.8 Hz); Calcd. for
C19H25NO3: C, 74.33; H, 8.22; N, 3.94. Found: C, 74.38;
H, 8.12; N, 3.90.
N‑(4‑hydroxyphenyl)‑3‑(2,6,6‑trimethyl‑1‑cy‑
clohexen‑1‑yl)‑2‑propenamide (2b1)
From 4-amino-phenol and 1b for 5 h. After evaporation to
dryness, the residue was chromatographed on SiO2 elut-
ing first with cyclohexane and then with cyclohexane/
AcOEt 1:1. The second eluate gave 2b1 [27] as white
solid; yield 50%; mp 220–223°C. IR (KBr) ν 3301, 2951,
1657, 1598, 1513, 1242 cm−1; 1H NMR (200 MHz, DMSO-
d6): δ 1.04 (6H, s, CH3), 1.46 (2H, m, H-5′), 1.55 (2H, m,
H-4′), 1.73 (3H, s, CH3), 2.04 (2H, m, H-3′), 6.12 (1H, d,
1 3

Cancer Chemother Pharmacol
Table 1 Antiproliferative activity of fenretinide-like compounds
4‑[[1‑oxo‑3‑(2,6,6‑trimethyl‑1‑cyclohexen‑1‑yl)‑2‑pro‑
pen‑1‑yl]amino]‑benzenacetic acid (2b5)
Compounds
Cell lines
A2780
A549
DOHH2
From 4-amino-benzenacetic acid and 1b for 20 h. After
evaporation to dryness, the residue was chromatographed
on SiO2 eluting first with cyclohexane and then with
cyclohexane/AcOEt 1:1. The second eluate gave 2b5
as yellow solid; yield 47%; mp 158–160°C. IR (KBr) ν
3300, 2931, 1713, 1660, 1602, 1538, 1516 cm−1; 1H NMR
(200 MHz, CDCl3): δ 1.04 (6H, s, CH3), 1.44 (2H, m,
H-5′), 1.59 (2H, m, H-4′), 1.74 (3H, s, CH3), 2.04 (2H, m,
H-4′), 3.58 (3H, s, CH2), 5.91 (1H, d, ethene, J=16.1 Hz),
7.19 (1H, d, arH), 7.39–7.60 (3H, m, arH and ethene);
Calcd. for C20H25NO3: C, 73.37; H, 7.70; N, 4.28. Found:
C, 73.34; H, 7.68; N, 4.24.
2b1
2b2
2b3
2b4
2b5
2b6
2b7
2a1
34.1 8.9a
>100
>100
41.6 2.6
>100
>100
>100
60.5 6.6
4.2 1.2
50.6 6.8
>100
>100
48.8 3.8
>100
>100
68.1 7.5
>100
10.3 3.0
56.9 5.6
>100
>100
49.2 6.8
>100
>100
57.4 5.1
64.9 8.5
15.4 3.1
4-HPR
The values (μM) express the mean SD of 4–8 experiments
3‑Hydroxy‑4‑[[1‑oxo‑3‑(2,6,6‑trimethyl‑1‑cy‑
but, in any case, higher than 30 μM, which is the arbitrary
limit, we considered to define a substance with a pharma-
cologically significant antiproliferative activity (Table 1;
Fig. 3). It is of note that 4-HPR showed always IC50s lower
than this arbitrary limit (Table 1), thus being the more
cytotoxic molecule among those analyzed. In particular, in
Fig. 3, the mean curves show the absence of antiprolifera-
tive activity of our compounds and graphically confirm the
higher activity of 4-HPR.
clohexen‑1‑yl)‑2‑propen‑1‑yl]amino]‑ benzoic acid (2b6)
From 4-amino-3-hydroxybenzoic acid and 1b for 20 h.
After evaporation to dryness, the resulting white solid
was crystallized from AcOEt; yield 62%; mp 243–246°C.
IR (KBr) ν 3405, 2936, 2918, 1673, 1602, 1518 cm−1; 1
H
NMR (200 MHz, DMSO-d6): δ 1.06 (6H, s, CH3), 1.45
(2H, m, H-5′), 1.54 (2H, m, H-4′), 1.75 (3H, s, CH3), 2.05
(2H, m, H-4′), 6.56 (1H, d, ethene, J=15.8 Hz), 7.27–7.42
(3H, m, arH and ethene), 9.55 (1H, s, OH, deuterium
oxide exchangeable), 10.32 (1H, s, NH, deuterium oxide
exchangeable), 12.61 (1H, s, COOH, deuterium oxide
exchangeable); Calcd. for C19H23NO4: C, 69.28; H, 7.04;
N, 4.25. Found: C, 69.06; H, 7.40; N, 4.30.
To confirm the lack of cytotoxic effects for our short reti-
noids, we also evaluate their apoptosis triggering on A2780
and A549 cells. In all cases, they did not display signifi-
cant apoptosis (% range apoptotic cells in all combinations
of A549 cells: 1.3–3.0%, control 2.0 1.7%; % range apop-
totic cells in all combinations of A2780 cells: 1.0–2.7%,
control 1.3 1.5%). On the contrary, 4-HPR administered
at its IC50, caused 6.4 2.1 and 58.7 4.0% apoptotic cells
in A549 and A2780 cells (control 2.2 1.7 and 1.0 0.9%),
respectively.
N‑(5‑hydroxytricyclo[3.3.1.13,7]dec‑2‑yl)‑3‑(2,6,6‑trime‑
thyl‑1‑cyclohexen‑1‑yl)‑2‑propenamide (2b7)
From 4-aminoadamantan-1-ol and 1b for 20 h. After evap-
oration to dryness, the resulting white solid was crystal-
lized from cyclohexane; yield 33%; mp 184–186°C. IR
All these data confirm the lower antiproliferative and
cytotoxic activities of our short retinoids as compared to
the parent compound 4-HPR.
1
(KBr) ν 3309, 2922, 2853, 1660, 16211 1544, 1353; H
NMR (200 MHz, CDCl3) δ ppm: 1.03 (6H, s, CH3), 1.46
(2H, m, H-5′), 1.57 (2H, m, H-4′), 1.62 (2H, m, adaman-
tyl), 1.73 (3H, s, CH3), 1.96–2.04 (8H, m, adamantyl), 2.28
(2H, m, H-4′), 5.21 (1H, s, OH, deuterium oxide exchange-
able), 6.56 (1H, d, ethene, J=15.8 Hz), 7.25 (1H, d, ethene,
J=15.8 Hz); Calcd. for C22H33NO2: C, 76.92; H, 9.68; N,
4.08. Found: C, 76.97; H, 9.83; N, 3.91.
TEAC antioxidant assay
To assess the antioxidant activity of these derivatives, the
TEAC assay was performed. This method examines the
quenching of 2,2-azino-bis(3-ethylbenzthiazoline-6-sul-
fonic acid) radical cation (ABTS⋅+) in the presence of anti-
oxidant compounds by means of UV–Vis spectroscopy.
The results, expressed as the TEAC, indicated the ability of
these derivatives to scavenge free radical ABTS⋅+ after 1, 3,
and 6 min. TEAC values are plotted in Fig. 4.
Antiproliferative activity and apoptosis
When we analyzed the antiproliferative activity against
A2780, DOHH2, and A549 cells, our 4-HPR derivatives
showed very high IC50s, from 34.1 8.9 to >100 μM, and
only 2b1, 2a1, and 2b4 displayed IC50s lower than 100 μM,
2a1, 2b1, and 2b4 showed similar TEAC values com-
pared to that of 4-HPR, whereas 2b2, 2b5, and 2b7 dis-
played a low scavenging activity. 2b3 was found to be inac-
tive in the range of investigated concentrations (≤95 μM).
1 3

Cancer Chemother Pharmacol
a strong expression of the CD11b marker of differentiation
after 6 days of treatment (Table 2; Fig. 5).
In particular, while 2a1 stimulated a weak expression of
CD11b in one-third of the cells, all the other compounds
induced more than 70% HL60 cells to express on their sur-
face a high amount of this differentiation marker (Table 2).
4-HPR showed a similar differentiating activity without
a significantly different impact on the expression of the
CD11b marker, as shown in Table 2.
The choice of a 6-day time exposure was linked to our
effort to show a long-lasting differentiating effect of our
short retinoids. In this experimental conditions, the antipro-
liferative activity of our selected 4-HPR-like compounds
on HL60 cells demonstrated a discrete variability (Table 3)
that was irrelevant after 3-day exposure (IC50 >30 μM,
vs. 4.94 1.24 and 3.88 1.14 μM of 4-HPR and ATRA,
respectively).
Fig. 4 TEAC values after treatment with 4-HPR and 2a1 and 2b
compounds. TEAC values at 1, 3, and 6 min for the derivatives.
Means are the average of three independent trials, and error bars
show standard deviations. Due to the 2b3 precipitation occurring in
the TEAC assay conditions (see the paragraph: “TEAC antioxidant
activity” of Results), the data about this compound were not entered
in the graph
At higher concentrations, the compound precipitation
occurred in the assay conditions and thereof TEAC value
cannot be determined. Finally, 2b6 presented the high-
est capacity to scavenge free radicals, which is generally
associated with a more protective function against oxida-
tive stress. In particular, the most active compound 2b6
toward ABTS⋅+ was two times more efficient than 4-HPR.
TEAC values can be related to the structure of the 4-HPR
derivatives and the compounds considerably active toward
ABTS⋅+ possess at least one hydroxyl group in the aro-
matic ring. Free radical scavenging capacity is primarily
attributed to the high reactivity of hydroxyl substituents on
aromatic rings, and the characteristics of substituents (elec-
tron-withdrawing, electron donor, and inductive effects)
have an influence on the H-donating ability of hydroxyl
group.
The data about the expression of the CD11b marker of
differentiation were largely confirmed by the simultane-
ous analysis of the NBT reducing activity. In fact, also in
this case, while 2a1 showed a quite low reducing activity
(+9.4 7.0% compared to untreated controls), the other
compounds tested, 2b1, 2b4, and 2b6, showed reducing
activities 72.2 1.65, 80.3 20.0, and 74.6 1.7% higher
than those of untreated controls, respectively (Table 2). In
this case, 4-HPR showed a slightly higher differentiating
activity compared to that observed for 2b1, 2b4, and 2b6
(72.2 1.6, 80.3 20.0, and 74.6 1.7 vs. 117.2 10.7).
It is noteworthy that even better results for the NBT
assay were obtained after 3-day exposure with the selected
compounds (Table 2). In this case, also 2a1 displayed a rel-
evant differentiating activity that was nearly absent after
6-day exposure.
Effect of 2a and 2b compounds on cell differentiation
Docking analysis
Once the antioxidant activity was assayed, the active com-
pounds were selected and analyzed for their effect on cell
differentiation. To this end, we treated the HL60 target cells
with the selected compounds that, except for 2a1, induced
Docking simulations were performed using as a target
the atomic structure of RARα (I1; PDB code 5k13) as
determined by X-ray crystallography, using the program
GOLD (CCDC Ltd., Cambridge, UK). We performed, for
Table 2 Effect of 2a and 2b compounds on the induction of differentiation of lymphoma HL60 human cells as evaluated by the expression of
cd11b and NBT reduction
Differentiation parameters
CTRa Compounds
2a1
2b1
2b4
2b6
4-HPR 1 μM 4-HPR 0.5 μM ATRA 0.5 μM
% CD11b positive cells (day 6)
MFI (day 6)
5
77
2
34 2.0
69
93
72
1
4
2
63
104
80 20
3
3
68
1
74
4
8
ND
ND
ND
ND
ND
ND
7
74
9
1
7
105 1.0 106
75 117 11
% NBT increase positivity (day 6)b 100
2
% NBT increase positivity (day 3)
100
183 120 107 15 660 203 219 76 564 313
126 72
102 28
aThe data represent the mean SD of 5–9 experiments
bAs compared to untreated HL60 control cells
1 3

Cancer Chemother Pharmacol
Fig. 5 Representative histograms of CD11b expression on
HL60 cells treated for 6 days with 2a1 and 2b compounds and
4-HPR. Expression of the CD11b in cultured HL60 cells treated per
6 days with the indicated compounds or mock treated (Control cells).
Concentrations were: 2a1, 2b1, 2b4 and 2b6, 30 mM; 4-HPR, 1 μM.
In each panel the white profiles refer to cells incubated with a mouse
IgG1 isotype control antibody conjugated with phycoerythrin (PE);
the gray profiles refer to cells incubated with the mouse IgG1 mAb
specific for the CD11b antigen conjugated with PE (eBioscience Inc.,
San Diego, CA, USA); the black profiles refer to CD11b positive cells
obtained by subtraction of the white profiles from the gray ones. In
the Control cells panel the gray profile is almost coincident whit the
white one, which is therefore not visible
all the ligand tested, a “blind docking”: i.e., the docking
of small molecules to their targets was done without a
priori knowledge of the binding mode of the ligand in the
active site of the protein, and we compared our result to
the crystallographic structure of RARα in complex with
the known antagonist 4-{5-(3-tert-butylphenyl)-1-[4-
(methylsulfonyl)phenyl]-1H-pyrazol-3-yl}benzoic acid
(6Q7). As expected, the binding mode of 6q7 (fitness
value of 150.59, Fig. 6a) to its target was correctly identi-
fied by our docking simulation, with the docked moiety
pose perfectly superposed to the one determined experi-
mentally through X-ray diffraction (RMSD between the
superposed structures less than 0.18 Å). This validation
procedure allows us to assess that our docking proce-
dure has been carried over correctly. Once validated, we
applied our protocol to calculate the binding modes of
the different ligands. Using RARα atomic coordinates as
the molecular target, 2b5 and 2b6 were determined as the
best ligands (fitness 110.94 and 110.47) followed by 2b3
and 2b7 (fitness 109.87 and 100.17 respectively) (Fig. 6).
Discussion
The aim of the present work was to develop new com-
pounds related to 4-HPR with a shorter alkyl chain and
to explore their antioxidant, antiproliferative, and dif-
ferentiating activities. The shortening of the alkyl chain
was suggested by the paper of Takahashi et al. [28] where
the authors demonstrated that cell growth inhibition and
superoxide scavenging ability were directly or inversely
dependent on the elongation of the alkyl chain. Since
we were looking for compounds with increased anti-
oxidant and differentiating activities, low (cyto)toxicity,
and original molecular structures, this indication seemed
to justify in part the modification of the parent 4-HPR
molecule.
Furthermore, several clinical studies have shown, in
general, that retinoids are not effective as expected on the
basis of preclinical studies in the prevention and treatment
of solid tumors [29–31], while a more significant clinical
effect in the leukaemia field against the cutaneous T-cell
1 3

Cancer Chemother Pharmacol
Fig. 6 Docking simulation of the binding modes of ligands 2b4,
2b5, and 2b7 on the crystallographic structure of the RAR in com-
plex with 4-HPR. Schematic representation of the binding modes
of different ligands to the Retinol Binging Protein (RAR). Protein
is represented as grey ribbons, residues involved in ligand binding
are drawn as sticks. Panel a shows the crystallographic structure of
RAR in complex with the agonist 4-{5-(3-tert-butylphenyl)-1-[4-
(methylsulfonyl)phenyl]-1H-pyrazol-3-yl}benzoic acid (6q7). Panel
b-f show the binding modes of ligands 2b5, 2b6, 2b3, 2b7 and
4-HPR as resulting from docking simulations
lymphoma and acute promyelocytic leukaemia was clearly
shown and clinically applied [32–35].
[35–37]. Furthermore, its side effects, even though rather
low, and in particular its low bioavailability may further
limit its clinical application.
In particular, 4-HPR has shown significant preven-
tion potential in a clinical trial where it furnished a strong
rationale for a further clinical study in young women at
high risk for breast cancer [35], rendering this drug, which
is not considered as a well-established and effective anti-
cancer drug, a promising agent in the prevention setting
On the other hand, due to the mechanism of action
on retinoic acid receptors, it has been proposed that the
treatment with retinoic acids should be preceded by the
evaluation of RARβ and downstream genes, since this
evaluation could be useful in the selection of patients
1 3

Cancer Chemother Pharmacol
Table 3 Antiproliferative effect of 2a and 2b compounds on HL60
that the sequence of differentiating events is quite rapid and
their effects long-lasting, at least for 2b1, 2b4 and 2b6.
Finally, to try defining the possible mechanism of action
of our compounds we applied the docking analysis using
as target the RARα, a bio-molecule working as receptor
for ATRA [23], and here considered as a good model for
identifying the similarities between the starting event of the
mechanisms of action of 4-HPR and our short retinoids.
Our results confirm that the short retinoids bind to the same
molecular target that 4-HPR with fitness scores that are in
general similar, although not identical, to that of the par-
ent drug, with a significantly better score obtained by the
compounds 2b6 and 2b5. On the other hand, this result
confirms that the shortening of the polyene chain of 4-HPR
does not reduce in a pharmacological significant way the
binding activity of this compound, and may suggest that the
starting interaction triggering the sequences of event per-
taining to the mechanism of action of these compounds is
the same of the parent compound 4-HPR. Other more tar-
geted experiments need to better define the complete mech-
anism of action of the short retinoidal N-phenylamides here
described.
cells after 6- and 3-day exposure
Compounds
IC50 (μM)
% inhibition of
cell proliferation
at 30 or 1-μM
drug concentra-
tion
6 days
3 days
6 days
3 days
2b1
11.2 1.7
33.9 9.6
2.00 1.00
10.4 2.7
1.44 0.26
<0.13
44.6 6.9
55.5 2.9
97.6 32.1
31.5 1.8
4.94 1.24
3.88 1.14
71
51
77
82
42
90
26
22
29
40
15
36
2b4
2b6
2a1
4-HPR
ATRA
who are likely to respond to the therapy [35]. At the same
time, it is also clear that the synthesis of new, possibly
more active and less toxic, retinoid derivatives might rep-
resent another option to improve the outcome of therapies
based on this kind of pharmaceutical compounds.
It is in this context that, among the seven short reti-
noidal amides analogs synthesized and tested, 2a1, 2b1,
and 2b4 showed an antioxidant activity similar to that
of 4-HPR, whereas 2b2, 2b5, and 2b7 displayed a rather
low scavenging activity. Moreover, while 2b3 was found
to be inactive in the range of investigated concentrations
(≤95 μM), 2b6 was the most active compound, two times
more efficient than 4-HPR itself.
Conclusions
In summary, superoxide scavenging activity of short reti-
noidal N-phenylamides seems to be improved thanks to
the phenolic group, while the carboxylic group increases
this effect if this is directly linked to benzene ring. In fact,
both phenylacetic and phenylbutanoic acid derivatives were
completely inactive in our assays. However, the differen-
tiating activity is always maintained when the phenolic
group is present. All these molecular features always make
the antiproliferative activity of our short retinoids lower
than that of 4-HPR, in practice absent.
As known, free radical scavenging capacity is primar-
ily attributed to the high reactivity of hydroxyl substitu-
ents on aromatic rings. In our conditions, TEAC values
can be related to the structure of the 4-HPR derivatives
and in fact, as supposed, the compounds active toward
ABTS⋅+ possess at least one hydroxyl group in the aro-
matic ring.
With regard to the antiproliferative activity, all our com-
pounds show very high IC50s values, well above those of
4-HPR, and well above the arbitrary limit we considered
to define a substance with a pharmacologically significant
antiproliferative activity (30 μM).
These observations, together with the results of the
docking analysis, confirm the hypothesis that “short reti-
noids” related to 4-HPR can maintain and even improve the
antioxidant activity, preserving a significant differentiating
activity with less cytotoxic effect that could start from the
interaction of this new molecules with the molecular target
RARα.
On the other hand, as regards the differentiating activ-
ity, our results show that after 6-day exposure, compounds
2b1, 2b4, and 2b6 also induced a significant expression
of the CD11b differentiation marker in HL60 human lym-
phoma cells, similar to that of 4-HPR, expressed as percent
of positive cells or MFI. These data were confirmed by the
analysis of superoxide production, another typical marker
of differentiation. In fact, all compounds able to induce
the expression of CD11b in HL60 cells were also able to
increase their NBT reducing activity after 6 days of treat-
ment. Furthermore, this ability was present and even bet-
ter also after a lower time exposure of 3 days, underlying
In conclusion, even due to their relative ease of synthe-
sis, the compounds of this series are further amenable for
structural modifications and will be useful as templates for
the design of new potent and less toxic antioxidant and dif-
ferentiating compounds related to 4-HPR.
Compliance with ethical standards
Conflict of interest Author M. Anzaldi declares that she has no con-
flict of interest; author M. Viale declares that he has no conflict of
1 3

Cancer Chemother Pharmacol
15. Takahashi N (2000) Antioxidant properties of N-(4-hydroxyphe-
nyl)retinamide (fenretinide). Biol Pharm Bull 23:222–225
16. Takahashi N, Sausville EA, Breitman TR (1995) N-(4-hydroxy-
phenyl)retinamide (Fenretinide) in combination with retinoic
acid enhances differentiation and retinoylation of proteins. Clin
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interest; author C. Macciò declares that she has no conflict of inter-
est; author P. Castagnola declares that he has no conflict of interest;
author V. Oliveri declares that she has no conflict of interest; author C.
Rosano declares that he has no conflict of interest; and author A. Balbi
declares that he has no conflict of interest.
17. Janardhanan R, Banik NL, Ray SK (2008) N-(4-Hydroxyphenyl)
retinamide induced differentiation with repression of telomerase
and cell cycle to increase interferon-γ sensitivity for apoptosis in
human glioblastoma cells. Cancer Lett 261:26–36
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.
18. Samuel W, Kutty RK, Sekhar S, Vijayasarathy C, Wiggert B,
Redmond TMJ (2008) Mitogen-activated protein kinase path-
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