Synthesis of 9-hydroxystearic acid derivatives and their antiproliferative activity on HT 29 cancer cells

Article abstract of DOI:10.3390/molecules24203714

9-Hydroxystearic acid (9-HSA) is an endogenous cellular lipid that possesses antiproliferative and selective effects against cancer cells. A series of derivatives were synthesized in order to investigate the effect of the substituent in position 9 and on the methyl ester functionality on the biological activity. The two separate enantiomers of methyl 9-hydroxystearate and of methyl 9-aminostearate showed antiproliferative activity against the HT29 cell line. This indicates the importance of position 9 groups being able to make hydrogen bonding with the molecular target. Further, this effect must be preserved when the carboxy group of 9-HSA is esterified. The biological tests showed that the amines, contrarily to methyl esters, resulted in cytotoxicity. A deep investigation on the effect of methyl (R)-9-hydroxystearate on HT29 cells showed an antiproliferative effect acting through the CDKN1A and MYCBP gene expression.

Full text of DOI:10.3390/molecules24203714

molecules
Article
Synthesis of 9-Hydroxystearic Acid Derivatives and
Their Antiproliferative Activity on HT 29 Cancer Cells
Natalia Calonghi 1,*, Carla Boga 2,
* ,
, Dario Telese 2, Silvia Bordoni 2, Giorgio Sartor 1
Chiara Torsello 1 and Gabriele Micheletti 2
1
Department of Pharmacy and Biotechnology, University of Bologna, 40127 Bologna, Italy;
giorgio.sartor@unibo.it (G.S.); chiara.torsello@virgilio.it (C.T.)
2
Department of Industrial Chemistry ‘Toso Montanari’, Alma Mater Studiorum Università di Bologna Viale
Del Risorgimento 4, 402136 Bologna, Italy; dario.telese2@unibo.it (D.T.); silvia.bordoni@unibo.it (S.B.);
gabriele.micheletti3@unibo.it (G.M.)
*
Correspondence: natalia.calonghi@unibo.it (N.C.); carla.boga@unibo.it (C.B.); Tel.: +39-051-2091231 (N.C.);
+39-051-2093616 (C.B.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Luigi A. Agrofoglio
Received: 27 September 2019; Accepted: 12 October 2019; Published: 15 October 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: 9-Hydroxystearic acid (9-HSA) is an endogenous cellular lipid that possesses antiproliferative
and selective effects against cancer cells. A series of derivatives were synthesized in order to investigate the
effect of the substituent in position 9 and on the methyl ester functionality on the biological activity. The two
separate enantiomers of methyl 9-hydroxystearate and of methyl 9-aminostearate showed antiproliferative
activity against the HT29 cell line. This indicates the importance of position 9 groups being able to make
hydrogen bonding with the molecular target. Further, this effect must be preserved when the carboxy
group of 9-HSA is esterified. The biological tests showed that the amines, contrarily to methyl esters,
resulted in cytotoxicity. A deep investigation on the effect of methyl (R)-9-hydroxystearate on HT29 cells
showed an antiproliferative effect acting through the CDKN1A and MYCBP gene expression.
Keywords: 9-hydroxystearic acid; methyl 9-hydroxystearate; methyl 9-aminostearate; cancer
1. Introduction
9-Hydroxystearic acid (9-HSA, Figure 1) is an endogenous cellular lipid that possesses a natural
negative regulatory activity of tumor cell proliferation. [
diminished in cancer cells with respect to the corresponding normal line [
administration to adenocarcinoma (HT29), [ ] osteosarcoma (U2OS, [ ] or SaOS [
lines results in a significant inhibition of the proliferation rate, as well as a significant increase of
p53-independent p21 expression. [ ] The expression of the cell cycle kinase inhibitor CDKN1A (P21)
is induced in neoplastic cells by inhibitors of histone deacetylase 1 (HDAC1). Actually, the authors
found that 9-HSA acts as a competitive inhibitor of the HDAC1 (histone deacetylase 1) isoform [ ].
The studies of molecular docking [ ] showed an interaction between the 9-HSA carboxy group and
1
–
3
] Its content has been found greatly
] and its exogenous
]) cancer cell
1
4
5
6
4
7,8
7
the zinc ion in the active site of HDAC1, by a mechanism similar to that already known for HDAC
inhibitors such as valproate, butyrate, trichostatin A (TSA), and suberoylanilidehydroxamic acid
(SAHA, Vorinostat) [9]. The molecular docking also predicted a more favorable binding energy for
the HDAC1-(R)-9-HSA complex, with respect to the complex formed with the opposite enantiomer
(S)-9-HSA. Enantiopure (R)-9-HSA is more easily accessible within the natural chiral pool since its
precursor, (S)-dimorphecholic acid, is one of the main components of seeds of the genus Dimorphotheca
plants [10]. Thus, both the (R)-9-HSA and its enantiomer were synthesized, and the docking prediction
experimentally confirmed [11]. Based on the above, the importance of both the carboxy- and the
hydroxyl- group in inducing the antiproliferative activity of 9-HSA is apparent.
Molecules 2019, 24, 3714; doi:10.3390/molecules24203714
www.mdpi.com/journal/molecules

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Figure 1. The two enantiomeric forms of 9-hydroxystearic acid, an HDAC inhibitor.
Hence, this study made some modifications on these key positions to better enlighten the role
played by these functionalities and to design, if possible, some structure-activity relationships. Herein,
the results obtained are reported.
2. Results and Discussion
2.1. Synthesis of 9-HSA Derivatives
The 9-HSA derivatives synthesized and investigated are in Figure 2.
Figure 2. 9-HSA derivatives synthesized and biologically tested.
Compounds (R)
difference might give information on the free hydroxyl functionality about the biological action. Further,
this study planned to prepare the relative amine (S) isosteric structure, to gain information on the
biological behavior of the methyl 9-hydroxystearate and methyl 9-aminostearate. This study decided to
test also the biological activity of the azide (S) species, as precursor of the mentioned amine species.
-2 and 3 bear an ester and an ether group bound to the C-9, respectively, and this
-
5
-
4
All compounds shown in Figure 2 were obtained starting from methyl (9R)-9-hydroxystearate
(R)-1], which in turn was obtained from Dimorphotheca sinuata seeds, which is oil, as well as the
[
seeds of other plants of the genus Dimorphotheca. These contain high amounts of (9S,10E,12E)-
9-hydroxyoctadeca-10,12-dienoic acid [(S)-dimorphecholic acid] [11], a precursor of (R) and of
-
1
(R)-9-HSA. The natural availability of (S)-dimorphecholic acid is a green and efficient way to obtain
9-HSA derivatives with the C-9 chiral carbon atom in enantiopure form, permitting all the difficulties
exhibited by alternative synthetic methods [12] to be overcome. Scheme 1 shows the synthetic strategy
adopted to prepare compounds (R)-1, (R)-2, 3, (S)-4 and (S)-5.

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Scheme 1. Synthetic routes to 9-HSA derivatives.
The procedure appears to be the first reaction of transmethylation of the triglycerides, obtained from
the extraction of the seeds and subsequent hydrogenation over the Adam’s catalyst. After purification of the
crude product by silica gel chromatography, the (9R)-methyl-9-hydroxystearate [(R)
optical purity was ascertained by 1H NMR upon derivatization with (R)-O-acetylmandelic acid [10].
Compound (R) was then transformed by treatment with tosyl chloride into (R) . The reaction
of (R) with refluxing methanol produced the methyl ether . For the latter, the absolute configuration
of the C-9 chiral center is not defined due to the possibility of occurrence, together with an inversion
of the configuration, and also of a SN1 mechanism producing racemization. The addition to (R)
of sodium azide in N,N-dimethylformamide (DMF) at 80–90 ◦C produced the azide (S)
. However,
-1] was isolated and its
-
1
-2
-2
3
-
2
-4

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due to difficulties of the work-up and purification steps, an alternative method was adopted by reacting
(R) with diphenylphosphoryl azide (DPPA) under Mitsunobu conditions yielding (S) in satisfactory
yield (77%).
-2
-4
The last step was devoted to the reduction of the azide. The adoption of Bayley’s method
(1,3-propandithiol and Et3N) [13] suffered a particularly hard work-up: After the separation of the
amine from by-products through immobilization on the Dowex resin and subsequent release with
triethylamine, a further treatment by liquid-liquid extraction was required and the desired amine was
recovered in 13% yield. This encouraged the study to try the alternative synthetic route shown in
Scheme 2 that gave, after purification, the amine (S)-5 in 36% overall yield from (R)-1.
Scheme 2. Synthetic pathway to obtain aminoderivative (S)-5 from (R)-1.
The enantiopurity of the new amine (S)
of the (S)-O-acetylmandelic acid (
) (Scheme 3) and by calculating, from the 1H-NMR spectrum of
the crude, the ratio between the two diastereomeric amides (S,S)-8 and (R,S) . The signals were
-5 was ascertained by reacting it with the acyl chloride
7
-
8
falling at 3.669 and 3.665 ppm, belonging to the (S,S) and (R,S) diastereomer, respectively, from which
a 9/1 dr was calculated, which was an indication of an almost complete inversion of the configuration
during synthesis (see Figures S1–S17). This was confirmed by adding a little amount of the (R,S)-8
diastereomer (obtained as described below) to the above mixture: A sensitive increase of the area of
the peak at 3.665 ppm was observed.
Scheme 3. Derivatization of the amine
5 with (S)-2-chloro-2-oxo-1-phenylethyl methyl carbonate (7) to
determine the (S,S)-8/(R,S)-8 diastereomeric ratio.
As reported in Scheme 1, several steps of the synthetic pathway imply the inversion of configuration
on C-9 producing the compounds and with the (S) configuration. Since the two enantiomers showed
4
5
distinct antiproliferative activity by the preliminary tests in the case of 9-HSA, this study planned to
prepare the opposite enantiomers of those compounds that, in preliminary tests, were biologically
active. Thus, (S)-1 and (R)-5 were prepared in order to compare the biological activity of the two
enantiomers (Figure 2), and to evaluate the trend in biological activity ascribed to both the isosters
with same configuration.
The procedure utilized is shown in Scheme 4: (S)
center of (R) through the Mitsunobu reaction followed by the treatment of the intermediate
methanolic KOH and esterification with BF3/CH3OH. Then, (R) was obtained from (S) by the same
procedure depicted in Scheme 2 to obtain the opposite enantiomer.
-
1
was obtained by the inversion of the chiral
-1
9
with
-
5
-1

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Scheme 4. Synthesis of (S)-1 and (R)-5.
The enantiopurity degree of (S) was measured [11 14] by reacting it with (R)-(
-
1
,
−
)-O-acetylmandelic
acid: A diastereomeric ratio > 5/95 between the (R,R) and (S,R) diastereomers was calculated from the
1H-NMR of the crude reaction mixture. Analogously, the (R)
-5 enantiopurity degree was evaluated as
a 10/90 diastereomeric ratio, by analyzing 1H-NMR spectrum of the reaction with the acyl chloride of
(S)-O-acetylmandelic acid.
2.2. Biological Activity
2.2.1. Preliminary Biological Activity of (R)-2, 3, (S)-4, and (S)-5
In order to gain information on the effect of the change of the group bound to C-9, preliminary
biological tests on IC50 value for compounds (R)
-
1,
(R)-2
,
3,
(S)-4
, and (S)
-5 were carried out. From the
MTT tests, the only active compounds were the methyl ester (R)
-1
and the amino derivative (S)-5
(see below). The absence of significant activity in the case of compounds functionalized on C-9 with
groups not able to make hydrogen bonds might be considered as a strong indication of the importance
of the presence of a group bearing hydrogen atoms bound to a heteroatom on this position. The results
obtained with (R)-1 and (S)-5 encouraged this study to also synthesize the corresponding enantiomer
in order to compare their biological effects.
2.2.2. Effect of Methyl (9R)-9-hydroxystearate [(R)-1], Methyl (9S)-9-hydroxystearate [(S)-1], Methyl
(9R)-9-aminostearate [(R)-5] and Methyl (9S)-9-aminostearate [(S)-5] on Proliferation of HT29
The in vitro IC50 growth inhibitory concentration was determined for (R)-1, (S)-1, (R)-5 and (S)-5,
incubating the HT29 with increasing concentrations of the compounds for 24 h. The data obtained
from the MTT analyses were examined to assess the concentration of compounds required for 50%
inhibition of cell viability (IC50): The values corresponded to 49
(S)-1, 57 ± 2.1 µM for (R)-5 and 43 ± 4.5 µM for (S)-5 (Figure 3).
The effects on HT29 cell proliferation after treatment with (R)
±
1.3
µ
M for (R)
-
1, 51
±
1.1 µM for
-1
and (S)
M (S)-1 are reported. The results
and (S)-1 treatment show a significantly greater effect for the (R)-enantiomer, recalling
the already reported behavior of the two enantiomers of 9-hydroxystearic acid. In panel B, the effects
-1 and with (R)-5 and (S)-5
are reported in Figure 4. In panel A, the effects of 49
for the (R)
µM (R)-1 and 51 µ
-
1

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for (R)-5 and (S)-5 at the respective concentrations of 57
µM and 43
µM are reported: In this case,
after 24 h of treatment, the cell proliferation strongly reduced, and after 48 h and 72 h of treatment,
the cell numbers still diminished, indicating a continuous cytotoxic effect.
Figure 3. Dose response curves of HT29 cell viability upon treatment with different concentrations of
(A) (R)-1, of (B) (S)-1, of (C) (R)-5 and (D) (S)-5.
These findings prompted this study to gain more information on the biological effect of the methyl
(9R)-9-hydroxystearate (R)-1.
Figure 4. Effects on cell proliferation. (
and 72 h of treatment as compared with the control. (
A
) Antiproliferative effect of (R)
-
1
and (S)
-
1
on HT29 at 24, 48,
or (S) in the
B) Antiproliferative effect of (R)
-
5
-5
same experimental conditions. The analysis was carried out by two-way analysis of variance (ANOVA)
followed by the Bonferroni multiple comparisons. The differences of at least p < 0.05 were considered
significant. Statistical analysis was carried out using Prism GraphPad software.

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2.2.3. Cell Cycle Analysis
In order to assess whether the antiproliferative effect of (R)-1 was associated with the interference
of the cell cycle progression, DNA profiles of cultured cells were examined by flow cytometry. The cells
were exposed to the compound for 48 h prior to processing and analysis. As shown in Figure 5,
the exposure to (R)
73.91 ± 0.9%) phase, while reducing their number in the S (13.18
G2/M (6.64 0.2%versus 8.12 0.1%) phases, thus indicating that the treatment leads to a cell cycle
arrest. The flow cytometric data correlates with cell counting over time. Indeed, the administration of
(R) reduced the number of cells by 23% at 24 h, by 35% at 48 h and by 49% at 72 from the treatment,
indicating that the cells did not duplicate anymore.
-
1
resulted in an increase in the number of the G0/G1 (80.18
±
0.7% versus
±
0.5% versus 17.97 ± 0.2%) and
±
±
-1
Figure 5. Effect of (R)
-1 on cell cycle. The cells were incubated for 48 h with the vehicle (A) or with
49 M of (R)-1 ), afterward the cell cycle distribution was determined by flow cytometry. The two
µ
(B
panels report the cytofluorimeter outputs obtained in one typical experiment repeated twice with
similar results.
2.2.4. Effect of (R)-1 on Histone Acetylation
Since the above findings parallel those found for the parent (R)-9-HSA [11], an investigation of
whether the molecular target might be histone deacetylase, as in the case of 9-HSA, was undertaken.
To identify the state of histones acetylation, these proteins were extracted from the control HT29
and treated for 6 h with 49
lysine monoclonal antibody. The histone acetylation signals were quantified by densitometry and
normalized on histone H3. As shown in Figure 6, the treatment with (R) for 6 h increased histone H4
acetylation by 50%, while the acetylation status of histones H2/H3 did not change.
µM of (R)-1. Acetylation was detected by a western blot using an anti-acetyl
-1

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Figure 6. Effect of (R)
(control) or presence of 49
a western blot analysis for acetylated (ac) histone H2/H3, histone H4 and histone H3 as loading controls.
) A representative experiment, repeated three times with similar results and densitometric analysis
-
1
on histone H4 acetylation. (
A) The cells were cultured for 6 h in the absence
µ
M (R) The cell nuclear extracts were prepared and subjected to
-1.
(
B
of the bands (mean ± SEM; n = 3), are shown. * p < 0.05.
2.2.5. (R)-1 Induces Gene and Relative Protein Expression Modulations
The induction of p21 is one of the common phenomena observed after treatment with HDAC
inhibitors such as TSA, NaBu, SAHA or 9-HSA [
is causally involved in cancers, principally through its capacity to drive tumor cell proliferation and
promote angiogenesis, invasion, and metastasis [18 21]. The downregulation of Myc is an essential
part of the anti-proliferative response to differentiation signals. Thus, a western blot analysis was
performed to assess the expression of p21 and Myc in HT29 cells after (R) treatment. As expected,
p21 was induced by the treatment with (R) compared to its expression level in the untreated control
cells, while the Myc expression decreased significantly by (R)-1 treatment (Figure 7A,B).
The observed cytostatic effects of (R) transcription levels of P21 and MYCBP (MYC) were analysed
by quantitative Real Time-Polymerase Chain Reaction (RT-PCR. The genes were analysed after 6 h of
treatment. The qRT-PCR was performed on cDNA of the control and (R) -treated cells, and the ∆∆CT
7,15–17]. Myc is a transcription factor whose activity
–
-
1
-1
-1
-1
method was used with the GAPDH gene as the housekeeping gene. The relative transcription levels,
expressed as a means of fold changes, are reported in Figure 7C. (R)-1 administration transcription
significantly increases P21 gene at 6 h of treatment, while MYC is significantly reduced.

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Figure 7. The effects of (R)
colon cancer cell line. ) Representative western blots for p21 and MYC in HT29 cells following
6 h treatment with (R) -actin was used as the control. ) Densitometry from western blots for
HT29 following 6 h treatment with (R)-1. Protein quantification was normalized to the -actin band.
The mean SEM, n = 3, and * p < 0.05. ) Changes in mRNA expression of P21 and MYC. GAPDH
were used as a control gene. CT values were obtained, the data was normalized against GAPDH and
-1 on the expression of cell growth regulatory genes and proteins in HT29
A
-
1.
β
B
β
±
C
the fold change was calculated by
experiments. * p < 0.05.
∆
CT method. The data are the mean
±
SEM of three independent
3. Materials and Methods
3.1. Chemical Syntheses
Dimorphotheca sinuata L. seeds were bought from Galassi Sementi Srl (Gambettola, FC, Italy).
The reagents used, unless stated otherwise, were purchased from Sigma-Aldrich (Milan, Italy). For flash
chromatography (FC), silica gel 0.037–0.063 mm (Merck KGaA, Darmstadt, Germany) was used as the
stationary phase. Thin layer chromatography (TLC) was carried out on silica gel 60 (Fluka Analytical,
Buchs, Switzerland) and the spots were revealed, depending on the compound to be analyzed, using
UV light, and an aqueous solution of (NH4)6MoO24 (2.5%) and (NH4)4Ce(SO4)4 (4%) in 10% H2SO4,
a solution of ninhydrin 0.013 M in n-butanol/acetic acid, or a basic solution of potassium permanganate.
Anhydrous THF was freshy distilled over sodium benzophenone ketyl. Pyridine, trimethylamine and
methanol were dried by standard methods. The hydrogenation was carried out on a Parr hydrogenator
model 3911EKX at a hydrogen gas pressure of approximately 40 psi. The nuclear magnetic resonance
spectra (1H-NMR 13C-NMR,) were recorded at 25 ◦C on Varian spectrometers Gemini 300, Mercury
400, or Inova 600 (Varian, Palo Alto, CA, USA). The signal multiplicities were established by DEPT-135
experiments. The chemical shifts were referenced to the solvent (CDCl3, δ = 7.27 and 77.0 ppm
for 1H and 13C-NMR, respectively). The GC-MS analyses were carried out by gas chromatograph
directly interfaced with a mass selective detector (injection temperature: 250 ◦C; oven temperature was
programmed as follows: 60 ◦C for 2 min, increased up to 260 ◦C at the rate of 20◦/min, followed by
260 ◦C for 20 min; the carrier gas was helium, used at a flow rate of 1 mL; the transfer line temperature

Molecules 2019, 24, 3714
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was 280 ◦C; the ionization was obtained by electron impact (EI); the acquisition range was 50–500 m/z).
The ESI-MS spectra were recorded using a Waters 2Q 4000 instrument (Waters Corporation, Milford,
MA, USA). The melting points were measured on a Büchi apparatus (Stone, Staffs, UK) and were not
corrected. The Fourier transform infrared (FT-IR) spectra of the organic compounds were recorded
using a Perkin-Elmer FT-IR MOD 1600 spectrophotometer (Norwalk, CT, USA).
3.1.1. Synthesis of Methyl (9R)-9-hydroxyoctadecanoate [(R)-1]
Dimorphotheca sinuata L. seeds (12.0 g) were ground and suspended in a CHCl3/MeOH mixture
(2:1 v/v, 200 mL) and the mixture was kept under nitrogen atmosphere, in the dark, and stirred for
24 h. After filtration and washing of the solid with CHCl3 (3
NaCl aqueous solution was added to the green solution. After extraction with CHCl3 (3
the combined o◦rganic layers were dried over anhydrous MgSO4. After filtration and solvent removal
in vacuo at 30 C, 4.0 g of residue was recovered. The crude yellow oil was dissolved in CH3OH
(40 mL) under nitrogen atmosphere and the solution was kept at 0 ◦C by immersion in an external
water/ice bath. Then, CH3ONa (2.16 g) was added and the mixture was magnetically stirred at 0 ◦C
×
20 mL), 20 mL of 0.1 M HCl and 0.1 M
15 mL),
×
for 2 h. The mixture was acidified to pH 4.0 and extracted with n-hexane (3
×
20 mL). The combined
organic layers were dried over anhydrous MgSO4. After filtration, the solvent was removed under
reduced pressure and the residue (2.2 g) dissolved in 10 mL of ethyl acetate and transferred into the
hydrogenation vessel. A catalytic amount of PtO2 (Adam’s catalyst) was added and the mixture was
subjected to hydrogenation (40 psi H2 pressure). The reaction course was monitored by 1H-NMR
spectroscopy. Usually, the hydrogenation is complete after approximately 40 min. The mixture was
then filtered over celite and the solution concentrated under vacuum. Flash chromatography (eluent:
petroleum ether/diethyl ether 7/3) of the residue gave methyl (9R)-9-hydroxyoctadecanoate
(C19H38O3) as a white solid (0.74 g), m.p.: 50–51 ◦C (Lit. [22]: 50–51.5 ◦C). 1H-NMR (600 MHz, CDCl3):
(ppm) = 3.67 (s, 3 H, OCH3), 3.61–3.55 (m, 1 H, CHOH), 2.30 (t, 2 H, J = 7.6 Hz, CH2COO), 1.62
(quint, 2 H, J = 7.4 Hz, CH2CH2COO), 1.48–1.21 (m, 27 H, incl. OH), 0.88 (t, 3 H, J = 7.0 Hz, CH3).
13C-NMR (100.6 MHz, CDCl3)
(ppm) = 174.1, 71.7, 51.3, 37.4, 37.3, 33.9, 31.8, 29.6, 29.56, 29.5, 29.4,
29.2, 29.1, 28.9, 25.5, 25.4, 24.8, 22.6, 14.05. IR (CHCl3): 3427, 1728 cm−1. MS (EI) m/z (%): 283 (M+
[(R)-1]
δ
δ
−
OCH3, 2), 264 (4), 187(45), 159 (11), 158 (53), 155 (100), 129 (6), 115 (18), 109 (14), 87 (50), 74 (33), 69 (18),
55 (31). The enantiomeric excess was determined by derivatization with (R)-(–)-O-acetyl mandelic acid
as previously reported [12,14] and was found to be 80%.
3.1.2. Synthesis of Methyl (R)-9-(tosyloxy)octadecanoate [(R)-2]
Methyl (9R)-9-Hydroxyoctadecanoate [(R)-1, 0.190 g (0.60 mmol], 0.607 g (3.18 mmol) of TsCl
and 10 mL of anhydrous pyridine were kept at 0 ◦C for 1 h then at 4 ◦C. The reaction was monitored
by TLC (n-hexane/AcOEt 1:1, RF = 0.54) and, when it was complete (after approximately 5 days), it
was quenched with water (40 mL). The mixture was extracted with Et2O and the combined organic
layers were acidified with HCl 1 M. The organic layer was dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. Compound (R)
-
2
(C26H44O5S) was obtained as colorless oil
1
in 70% yield. H-NMR (300 MHz, CDCl3):
δ (ppm) = 0.87 (t, J = 6.9 Hz, 3 H, CH3), 1.13–1.30 (m, 27
H, (CH2)13), 1.55 (m, 2 H, CH2CH2COOCH3), 2.27 (t, J = 7.56 Hz, 2 H, CH2COOCH3), 2.42 (s, 3 H,
PhCH3), 3.65 (s, 3 H, COOCH3), 4.52 (q, J = 6.1 Hz, 1 H CHOSO2), 7.31(d, 2 H, J = 4 Hz, phenyl), 7.77
(d, J = 4.4 Hz, 2 H, phenyl). 13C-NMR (75.44 MHz, CDCl3):
δ (ppm) = 174.3, 144.3, 134.8, 129.6, 127.7,
84.5, 51.4, 34.1 (two signals overlapped), 34.0, 31.9, 29.43, 29.38, 29.3(two signals overlapped), 29.1, 29.0,
28.9, 24.8, 24.7, 24.6, 22.6, 21.6, 14.1. ESI-MS (m/z): 481 [M + Na]+, 469 [M + H]+
3.1.3. Synthesis of Methyl 9-MethoxyOctadecanoate (3)
Compound (R)
refluxed for 7 h. After removal of the solvent under reduced pressure, brine (20 mL) was added and the
mixture was extracted with Et2O (4 20 mL). The combined organic layers were dried over anhydrous
-2 (0.167 g, 0.357 mmol) was dissolved in CH3OH (5 mL) and the solution was
×

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MgSO4. After filtration and concentration, the residue was purified by FC (eluent: n-hexane/CH2Cl2
from 6:4 to 1:1 to 3:7 to 2:8 until CH2Cl2). Pure methyl 9-methoxyoctadecanoate ( , (C20H40O3) was
obtained in 55% (0.064 g, 0.195 mmol). 1H-NMR (300 MHz, CDCl3):
(ppm) = 0.86 (t, J = 6.9 Hz,
3
δ
3 H, CH3), 1.24–1.32 (m, 27 H, (CH2)13), 1.60 (m, 2 H, CH2CH2COOCH3), 2.29 (t, J = 7.49 Hz, 2 H,
CH2COOCH3), 3.10 (m, 1 H, CHOCH3), 3.30 (s, 3 H, CHOCH3), 3.65 (s, 3 H, COOCH3). 13C-NMR
(150.80 MHz, CDCl3):
δ
(ppm) = 174.3, 80.9, 56.3, 51.4, 34.1, 33.42, 33.41, 31.9, 29.9, 29.66, 29.65, 29.58,
1, 0.5) 297 (3), 264 (14), 222 (6),
29.3, 29.2, 29.1, 25.3, 25.26, 24.9, 22.7, 14.1. MS (EI) m/z (%): 327 (M+
−
201 (100), 171 (50), 137 (29), 123 (8), 109 (19), 97 (43), 83 (72), 69 (57), 55 (83).
3.1.4. Synthesis of Methyl (9S)-9-azidooctadecanoate [(S)-4]
Method 1:
Compounds (R)-2 (0.147 g, 0.314 mmol), NaN3 (0.102 g, 1.57 mmol), and DMF (10 mL) were heated
at reflux for 3 h. The mixture was treated once with brine (20 mL) then extracted with Et2O (3
then two folds with water (30 mL and 50 mL) and each time extracted with Et2O (3 15 mL), finally
with brine (20 mL) then extracted with Et2O (3 15 mL). Each time the organic layer was dried over
×
15 mL),
×
×
anhydrous MgSO4 and filtered: The absence of DMF was checked. The combined organic layers were
concentrated and the residue chromatographed on silica gel (eluent: 7:3 n-hexane/AcOEt). Compound
1
(S)-4 (C19H37N3O2, 0.064 g, 58%) was recovered as colorless oil. H-NMR (300 MHz, CDCl3):
δ (ppm)
= 0.88 (t, J = 6.6 Hz, 3 H, CH3), 1.24–1.33 (m, 26 H, (CH2)13), 1.61 (m, 2 H, CH2CH2COOCH3), 2.30
(t, J = 7.9 Hz, 2 H, CH2COOCH3), 3.21(q, J = 6.1 Hz, 1 H, CHN3), 3.65 (s, 3 H, COOCH3). 13C-NMR
(150.80 MHz, CDCl3):
δ (ppm) =174.3, 63.1, 51.4, 34.4, 34.3, 34.0, 31.9, 30.3, 29.5, 29.4, 29.3, 29.2, 29.1,
29.0, 26.1, 26.0, 24.9, 22.6, 14.1; IR-Neat (cm−1): 2928.0, 2856.0, 2361.2, 2098.7, 1742.6, 1465.4, 1257.5,
1171.1; ESI-MS (m/z): 352 [M + Na]+, 340 [M + H]+
Method 2:
Compound (R)-1 (0.230 g, 0.732 mmol) was dissolved in anhydrous THF (7.5 mL) and under
nitrogen atmosphere. PPh3 (0.398 g, 1.52 mmol) and DIAD 94% (300
µL, 1.52 mmol) were added
and the yellow mixture was stirred. After a few minutes, DPPA 97% (330
µL, 1.52 mmol) was added.
The solution became cloudy and after 18 h, had come clear. The crude was subjected to FC (eluent:
n-hexane/AcOEt 9:1) and pure (S)-4 (0.190 g, 77%) was recovered.
3.1.5. Synthesis of Methyl (9S)-9-aminooctadecanoate [(S)-5]
Compound (R)-1 (0.222 g, 0.70 mmol) was dissolved in anhydrous THF (8.0 mL) in a flame
dried apparatus immersed in an ice-bath and kept under nitrogen atmosphere. PPh3 (0.103 g
(0,70 mmol) and phthalimide (0.103 g (0,70 mmol) were added. Through a funnel, DIAD (0.2 mL,
0.70 mmol) in 2.0 mL of THF was added dropwise. After 12 h, the reaction appeared complete (TLC:
petroleum light/diethyl ether 6:4). The reaction mixture was concentrated. FC on silica gel (light
petroleum/diethyl ether: 20:1) of the residue gave 0.280 g (0.63 mmol, 90% yield) of the intermediate
(9S)-9-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) octadecanoate [(S)-6] (C27H41NO4) as a colorless oil:
1H-NMR (400 MHz, CDCl3),
3.63 (s, 3 H, OCH3), 2.25 (t, J = 7.43 Hz, 2 H), 2.11–1.97 (m, 2 H), 1.74–1.61 (m, 2 H), 1.61–1.49 (m, 2
H), 1.32–1.14 (m, 22 H), 0.84 (t, J = 6.97 Hz, 3 H, CH3). 13C-NMR (100.56 MHz, CDCl3),
(ppm) =
δ (ppm) = 7.83–7.79 (m, 2 H), 7.71–7.68 (m, 2 H), 4.26–4.06 (m, 1 H),
δ
174.2, 168.8, 133.8, 131.8, 123.0, 52.2, 51.4, 34.0, 32.5, 32.4, 31.8, 29.45, 29.43, 29.23, 29.21, 29.04 (2 signals
overlapped), 29.0, 26.6, 26.5, 24.8, 22.6, 14.1. ESI-MS (m/z): 444 [M + H]+.
Sodium borohydride (0.06 g, 1.6 mmol) in 2-propanol (3.0 mL) was added to a solution of the
compound (S)-6 (0.131 g, 0.29 mmol) and the mixture was magnetically stirred at room temperature
◦
for 12 h. Glacial acetic acid (0.3 mL) was added and the mixture was heated at 80 C for 2 h.
After concentration, water (5 mL), and then saturated aqueous solution of NaHCO3 (5 mL) were added.
After extraction with ethyl acetate (3
magnesium sulfate. After filtration and removal of the solvent under reduced pressure, the crude
×
10 mL), the combined organic layer was dried over anhydrous
was subjected to FC (eluent: CH2Cl2/CH3OH 10/1), the spots being evidenced with ninhydrin stain.

Molecules 2019, 24, 3714
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Compound (S)-5 (0.027 g, 0.09 mmol) was obtained in a 30% yield. An alternative method was as
follows: Hydrazine hydrate (0.16 mL, 3.2 mmol) was added to a solution of compound (S)-6 (0.280 g,
0.63 mmol) in ethanol (5 mL) and the mixture was heated and refluxed for 3 h. The formation of a white
solid was observed. After cooling, 1 M HCl was added until pH = 6. Then, the solution was filtered on
a Buchner funnel. The mixture was treated with saturated solution of NaHCO3 until reaching pH ~ 9
and then it was extracted with CH2Cl2 (4
and removal of the solvent under reduced pressure, the light yellow oil was subjected to FC (eluent:
CH2Cl2/CH3OH 10/1), obtaining the product in 28% yield. Compound (S)-5 (C19H39NO2): H-NMR
×
10 mL) and dried over anhydrous MgSO4. After filtration
1
(400 MHz, CDCl3):
2 H, CH2CH2COOCH3), 2.29 (t, J = 7.7 Hz, 2 H, CH2COOCH3), 3.13 (m, 1 H, CHNH2), 3.66 (s, 3 H,
COOCH3), 8.30 (br.s, 2 H, NH2). 13C-NMR (100.56 MHz CDCl3):
(ppm) = 174.2, 51.6, 51.4, 35.92,
δ (ppm) = 0.88 (t, J = 6.9 Hz, 3 H, CH3), 1.24–1.32 (m, 26 H, (CH2)13), 1.58–1.65 (m,
δ
35.86, 34.0, 31.9, 29.63, 29.55 (two signals overlapped), 29.4, 29.3, 29.12, 29.06, 25.8, 25.7, 24.9, 22.6, 14.1.
MS (m/z): 282 (5), 267(1), 250(1), 207(3), 186(100), 156(99), 109(4), 97(2), 83(4), 70(5), 56(11). ESI-MS
(m/z): 336 [M + Na]+, 314 [M + H]+; IR (cm−1): 3423.5, 2950.0, 2870.0, 1710, 1635.8.
The synthesis of (R)-5 was carried out with the same procedure starting from (S)-1.
3.1.6. Synthesis of (S)-2-Chloro-2-oxo-1-phenylethyl Methyl Carbonate (7)
In a flame-dried apparatus and under nitrogen atmosphere, (S)-O-acetylmandelic acid (1.046 g,
5,4 mmol) and anhydrous CH2Cl2 (10 mL) were added. To the solution, oxalyl chloride (0.47 mL,
5.4 mmol) dissolved in anhydrous CH2Cl2 (2 mL) were added dropwise. After 2 h at room temperature
and under magnetic stirring, the solvent was distilled at room pressure. The residue was distilled at
0.01 mmHg and the colorless oil with b.p.= 102–103 ◦C found to be compound 7 (C10H9ClO4).
δ
1 H-NMR (300 MHz, CDCl3), (ppm): 7.59–7.37 (m, 5 H), 6.09 (s, 1 H), 2.22 (s, 3 H). 13 C-NMR
(75.44 MHz, CDCl3), δ (ppm): 170.7, 169.8, 130.8, 130.2, 129.2, 128.4, 80.8, 20.4.
3.1.7. Methyl (9S)-9-{[(2S)-2-(Acetyloxy)-2-Phenylacetyl]amino}octadecanoate [(S,S)-8] and Methyl
(9R)-9-{[(2S)-2-(Acetyloxy)-2-Phenylacetyl]amino}octadecanoate [(R,S)-8]
1
The optical purity of (S)-5 was determined by H-NMR analysis after derivatization with
7,
the acyl chloride of the (S)-(–)-O-acetylmandelic acid. Compound
7 (2.8 mg) in 0.5 mL of CD2Cl2
was put directly into the NMR spectroscopy tube and methyl 9(S)-9-aminostearate [(S)-5] (4 mg,
0.013 mmol) dissolved in CD2Cl2 (0.5 mL) was added. Then, triethylamine (2.7
µ
L) was added
1
and the H-NMR spectrum of the crude was recorded. A diastereomeric ratio of ~9:1 between
methyl (9S)-9-{[(2S)-2-(acetyloxy)-2-phenylacetyl]amino}octadecanoate and methyl (9R)-9-{[(2S)-
2-(acetyloxy)-2-phenylacetyl]amino}octadecanoate was calculated from the integration of the signals
belonging to the methoxy group of the two diastereomers (see spectrum SI-17). This relative ratio was
calculated from the spectrum of the crude reaction mixture. The product (S,S)-8 was then purified by
preparative TLC on silica gel eluting with CH2Cl2 and scraping the spot with RF ~ 0.21. The pure
compound (S,S)-8 was extracted with CHCl3 and analyzed by NMR.
Methyl (9S)-9-{[(2S)-2-(acetyloxy)-2-phenylacetyl]amino}octadecanoate [(S,S)-8] (C29H47NO6):
1H-NMR (600 MHz, CDCl3),
δ (ppm): 7.45–7.41 (m, 2 H), 7.39–7.32 (m. 3H), 6.03 (s, 1 H), 5.66 (br. d., J
= 9.44 Hz, 1 H, NH), 3.94–3.85 (m, 1 H. H-9), 3.669 (s, 3 H, OCH3), 2.29 (t, J = 7.61 Hz, 2 H), 2.19 (s, 3 H,
CH3CO), 1.64–1.14 (m, 28 H), 0.88 (t, J = 7.04 Hz, 3 H, CH3).
The same procedure was used to determine the optical purity of methyl (9R)-9-{[(2S)-
1
2-(acetyloxy)-2-phenylacetyl]amino}octadecanoate [(R,S)
-8
] (C29H47NO6): H-NMR (600 MHz, CDCl3),
δ
(ppm): 7.45–7.42 (m, 2 H), 7.39–7.33 (m. 3H), 6.03 (s, 1 H), 5.6 (br. d., J = 9.04 Hz, 1 H, NH), 3.95–3.85
(m, 1 H. H-9), 3.665 (s, 3 H, OCH3), 2.29 (t, J = 7.61 Hz, 2 H), 2.17 (s, 3 H, CH3CO), 1.64–1.14 (m, 28 H),
0.88 (t, J = 7.04 Hz, 3 H, CH3).
In order to verify if some signals of the two diastereisomers could be separated in the 1H-NMR
spectrum, a mixture of (S,S)-8 and (R,S)-8 was prepared by mixing different amounts of the two species,

Molecules 2019, 24, 3714
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previously purified by preparative TLC on silica gel (20
showed separate methoxyl hydrogen signals (see Figures S1–S18).
×
20 cm glass plates). The spectrum (600 MHz)
Finally, to confirm the assignment of the signals at 3.669 and 3.665 ppm to the relative diastereomers,
a small portion of purified (R,S)-8 was added to a solution of a 9:1 diastereomeric mixture of 8, and the
peak at 3.665 ppm increased, as expected.
3.2. Cell Culture and Treatments
The human colon cancer cell line HT29 was obtained from American Type Culture Collection
(Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Labtek Eurobio, Milan, Italy),
supplemented with 10% FCS (Euroclone, Milan, Italy) and 2 mM l-glutamine (Sigma-Aldrich, Milan,
Italy), at 37 ◦C and 5% CO2 atmosphere. These conditions were used in all cell incubation steps for the
experiments described below. (R)
stock solution then diluted in culture medium to obtain the required concentrations. The cells were
plated at a density of 1.0
104 cells/well in 6-well culture plates (Orange Scientific, Braine-l’Alleud,
Belgium). Twenty-four hours after plating, (R) or (S) were added to a final concentration of 49
or 51 M for each drug, and incubated for 24, 48, and 72 h. Moreover, HT29 cells were seeded onto
6-well plates, and after 24 h, they were treated with (R) at the concentration of 57 M or with (S) at
the concentration of 43 M for 24, 48 and 72 h. The Trypan Blue exclusion dye method was used to
-1, (S)-1, (R)-5 and (S)-5 were dissolved in ethanol to obtain a 33 mM
×
-
1
-
1
µM
µ
-5
µ
-5
µ
determine the number of viable cells.
3.2.1. MTT Assay
To evaluate (R)
or with concentrations between 1 nM–5 mM (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1
M, 10 M, 50 M, 100 M, 500 M, 1 mM and 5mM) of the test samples. The culture medium was
-
1, (S)
-
1, (R)
-
5
and (S)
-
5
activity, the cells were treated for 24 h without (control)
µM,
5
µ
µ
µ
µ
µ
removed and the cells were further incubated for 2 h with 0.2 mg/mL MTT in PBS. After removal of the
medium, the cells were lysed with 0.1 mL of iso-propanol. The absorbance of the solubilized formazan
pellet at 540 nm was determined by Victor2, Multilabel plate reader (Perkin Elmer, MI, Italy). The IC50
was determined from three different experiments of the dose-response curve by using GraphPadPrism
5 (GraphPad Software, Inc., La Jolla, CA, USA) fitting a symmetrical sigmoidal shaped curve.
3.2.2. Cell Cycle Analysis by Flow Cytometry
The HT29 cells were seeded in 25 cm2 flasks at a density of 2
cell cycle were studied 48 h after treatment with 49 M (R)- . To determine the cell cycle distribution
at the end of incubation, HT29 cells were stained according to Busi et al. [23]. Briefly, 1
106 cells were
pelleted and resuspended in trisodium citrate 0.1%, RNAse 0.1 mg/L, Igepal 0.01%, Propidium Iodide
(PI) 50
g/L. After 30 min at 37 ◦C in the dark, the cells were analyzed on a Coulter Epics Elite flow
×
104 cells/cm2. The effects on the
µ
1
×
µ
cytometer (Beckman Coulter) equipped with an argon ion laser tuned at 488 nm. PI fluorescence was
collected on a linear scale at 600 nm and the DNA distribution was analyzed by the Modfit 5.0 software
(ModFit Verity Software House http://www.vsh.com/).
3.2.3. Histone Extraction and Western Blot
HT29 cells were cultured with compounds (R)-1 for 6 h, and the histone fraction was extracted.
The cells were harvested using 0.11% trypsin and 0.02% EDTA, washed twice with PBS, and nuclei
were isolated according to Amellem et al. [24]. The nuclear histones were extracted, and proteins were
quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA). The histones were examined by 15%
SDS-PAGE and a western blot analysis against acetylated lysines using anti-acetylated lysine (Cell
Signaling Technology, Danvers, MA #9441, USA). The detection of the immunoreactive bands was
performed with a secondary antibody conjugated with horseradish peroxidase (Amersham, Uppsala,
SE NA931V, Sweden) and developed with the enhanced chemiluminescence system Clarity Western

Molecules 2019, 24, 3714
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(Bio-Rad, Hercules, CA, USA), and the quantification was done by Fluor-S Max MultiImager (Bio-Rad)
using the histone H3 signal as the control (Cell Signaling Technology, Beverly, MA #3638, USA).
3.2.4. Total Protein Extraction and Western Blot
HT29 cells were treated with 49 µM of (R)-1 for 24 h. Cell were lysed in according to ref. 23. In brief,
the cells were lysed for 20 min in an HEPES buffer and the protein concentration was determined by
using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA). The proteins were resolved on
a 10% density gel and immunoblotted with p21 (Cell Signaling Technology, Danvers, MA#2946, USA)
or Myc (Cell Signaling Technology, Danvers, MA #5605, USA) or β-actin (Cell Signaling Technology,
Danvers, MA #4967, USA) antibodies. The nitrocellulose membrane was incubated with secondary
horseradish peroxidase-conjugated antibodies (GE Healthcare, Chicago, IL #NA931V or #NA9340,
USA), developed as described in the previous section, and the quantification was done by Fluor-S Max
MultiImager (Bio-Rad) using the β-actin signal as the control.
3.2.5. Quantitative Real Time-PCR Analysis
HT29 cells were seeded in 25 cm2 flasks at a density of 2
×
104 cells/cm2 and treated with (R)-1
for 6 h. RT-PCR was assessed as previously described [23]. The gene expression was quantified by
∆∆CT method, by using GAPDH as the housekeeping gene. The following primers were used: 50-
ATTTGGTCGTATTGGGCGCC-30 (forward) and 50-ACGGTGCCATGGAATTTGCC-30 (reverse) for GAPDH
detection, 50-CCTAAGAGTGCTGGGCATTTT-30 (forward) and 50-TGAATTTCATAACCGCCTGTG-30 (reverse)
for P21 detection, 50-TAGCTTCACCAACAGGAACT-30 (forward) and 50-AGCTCGAATTTCTTCCAGAT-30
(reverse) for MYC detection.
3.3. Statistical Analysis
The data for the MTT, western blot, cell cycle and RT-PCR were analysed with a one-way analysis
of variance (ANOVA) followed by Dunnett’s multiple comparison test. The data of the cell growth
were analysed with a two-way analysis of variance (ANOVA) followed by the Bonferroni multiple
comparisons. The differences of at least p < 0.05 were considered significant. A statistical analysis was
carried out using Prism GraphPad software.
4. Conclusions
The novel derivatives of (9R)-9-hydroxystearic acid (9-HSA) have been synthesized and the
influence of the modification at the level of the substituents on C-9 and of the carboxy group have been
studied on HT 29 colon cancer cell line.
The modification on the C-9 by ester (namely methyl (9R)-9-{[(4-methylphenyl)sulphonyl]
oxy}octadecanoate, (R)
did not produce relevant antiproliferative activity on HT 29 cells. On the contrary, methyl
(9S)-9-aminooctadecanoate [(S) ] and its enantiomeric form (R)-5 was active against the above colon
-2), by ether (namely methyl 9-methoxyoctadecanoate, 3), or azide group
-5
tumor cells, but very toxic, as evidenced by the high cytotoxicity after 48 and 72 h of the treatment. This is
an indication of the importance, for a biological effect, to have a group bound to the C-9 with hydrogen
atoms able to interact with the active site of the biological target. When the modification was at the carboxy
group of 9-HSA, as in case of the formation of methyl esters (R)
-1
and (S)-1, an antiproliferative effect was
observed. In particular, (R) showed a more marked cytostatic effect compared to the (S)-1
-
1
enantiomer.
In fact, the number of cells dropped at 24 h and remained unchanged over time, suggesting a halt in cell
growth. The anti-proliferative effect induced by (R)-1 is characterized by an accumulation of cells in the
G0 / G1 phase. The antiproliferative effect of the (R)-1 is due to an increase of the p21WAF/CYP1 and
a decrease of c-myc expression that is reflected on these two protein levels.
Supplementary Materials: The following are available online: Figures S1–S18: 1H and 13C-NMR spectra of
compounds 1–8.

Molecules 2019, 24, 3714
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Author Contributions: Conceptualization, C.B. and N.C.; investigation, C.B., N.C., D.T., C.T., G.M.; resources,
C.B., N.C., G.S., S.B.; writing—original draft preparation, C.B., N.C.; writing—review and editing, S.B., G.S.
All authors have read the final version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Work supported by Alma Mater Studiorum–Università di Bologna (RFO funds). Authors
thank Luca Prati, Isabel Orlando, Arige Othman, and Luca Zuppiroli for running the mass spectra.
Conflicts of Interest: The authors declare no conflicts of interest.
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,
Sample Availability: Samples of the compounds are not available from the authors.
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