Chiral amide derivatives of ricinoleic acid and 3-hydroxynonanoic acid synthesis and cytotoxic activity

Article abstract of DOI:10.1007/s00044-019-02348-y

A series of chiral ricinoleic and 3-hydroxynonanoic acid derivatives were synthesized in this study using various chemical and biochemical procedures. An effective method for preparation of methyl esters of 3-hydroxynonanoic acid from castor oil or methyl ricinoleate by ozonolysis and oxidation was developed. Simple, fast, and efficient procedures were applied to obtain different primary and secondary, cyclic and acyclic amides, including hydroxamic acids. Among 24 synthesized derivatives of ricinoleic and 3-hydroxynonanoic acids, i.e., methyl esters, amides, and hydroxamic acids, 16 compounds were obtained and described for the first time. The synthesized compounds showed activity against the tested cancer cells, but the best cytotoxic effect was observed for hydroxamic acid derived from 3-hydroxynonanoic acid (11) against HeLa cells. In general, most of the tested compounds were more toxic against HT29 than HeLa cancer cells. The results also showed that there was no significant difference between activities of (R)- and (S)-enantiomer of particular derivatives.

Full text of DOI:10.1007/s00044-019-02348-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02348-y
ORIGINAL RESEARCH
Chiral amide derivatives of ricinoleic acid and 3-hydroxynonanoic
acid synthesis and cytotoxic activity
1 ●
Józef Kula Alina Błaszczyk2
1 ●
Sylwia Matysiak
Received: 24 December 2018 / Accepted: 16 April 2019
The Author(s) 2019
©
Abstract
A series of chiral ricinoleic and 3-hydroxynonanoic acid derivatives were synthesized in this study using various chemical
and biochemical procedures. An effective method for preparation of methyl esters of 3-hydroxynonanoic acid from castor oil
or methyl ricinoleate by ozonolysis and oxidation was developed. Simple, fast, and efficient procedures were applied to
obtain different primary and secondary, cyclic and acyclic amides, including hydroxamic acids. Among 24 synthesized
derivatives of ricinoleic and 3-hydroxynonanoic acids, i.e., methyl esters, amides, and hydroxamic acids, 16 compounds
were obtained and described for the first time. The synthesized compounds showed activity against the tested cancer cells,
but the best cytotoxic effect was observed for hydroxamic acid derived from 3-hydroxynonanoic acid (11) against HeLa
cells. In general, most of the tested compounds were more toxic against HT29 than HeLa cancer cells. The results also
showed that there was no significant difference between activities of (R)- and (S)-enantiomer of particular derivatives.
●
●
●
●
●
Keywords Cytotoxicity 3-Hydroxynonanoic acid Fatty acid amides MTT assay Ozonolysis Ricinoleic acid
Introduction
material for chemical and biochemical syntheses. There is a
lot of information about RA and its derivatives and in many
publications, their great biological activity is described
(Pabiś and Kula 2016). For instance, amides, esters, or
glycosides exert (in vitro) a potent antiproliferative effect on
tumor cell lines and also have antimicrobial activity (Nar-
asimhan et al. 2007; D’Oca et al. 2010; Dos Santos et al.
2015; Kuppala et al. 2016).
It is worth noting that most of the published results relate
to the naturally available R configured RA, while there is
little information about its S enantiomer (Fig. 1). Limited
data on this compound result from the fact that it have not
been identified yet in any natural source. However, in 2014
an efficient method for the preparation of (S)-RA was
developed (Kula et al. 2014), and then, its derivatives were
obtained and described for the first time (Matysiak et al.
Hydroxy fatty acids occur naturally in small amounts and
the only representative of this group available in sufficient
quantities is (R)-ricinoleic acid (RA) [(R,Z)-12-hydro-
xyoctadec-9-enoic acid] (Salywon et al. 2005). This
hydroxy fatty acid is an important feedstock commonly
used in different fields of industry. The most important
source of RA is castor oil, in which it represents about
8
0–90% of all fatty acids. It is characterized by the presence
of a hydroxy group in the homoallilic position and one
double bond of the cis configuration making it a great raw
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02348-y) contains supplementary
material, which is available to authorized users.
2017, 2018).
It is well known that both the direction of biological
*
Sylwia Matysiak
activity and the strength of the action of chiral compounds
are largely determined by the stereochemistry of the mole-
cule. Therefore, it can be hypothesized that the configura-
tion of the acid moiety of hydroxy fatty acids can affect the
bioactivity of their derivatives. It was confirmed by our
previous study, in which different (R)- and (S)-RA amides
and their acetates were synthesized and examined in terms
of anticancer and antimicrobial activity. The tested
sylwia.matysiak@edu.p.lodz.pl
1
Institute of General Food Chemistry, Faculty of Biotechnology
and Food Sciences, Lodz University of Technology, 90-924
Lodz, Poland
2
Department of General Genetics, Molecular Biology and Plant
Biotechnology, Laboratory of Cytogenetics, Faculty of Biology
and Environmental Protection, University of Lodz, 90-237
Lodz, Poland

Medicinal Chemistry Research
Fig. 1 Chemical structures of enantiomeric forms of ricinoleic acid (RA)
compounds exhibited significant pharmacological potential,
and what is more, differences in the activity of the opposite
enantiomers were observed in some cases (Matysiak et al.
a DSQII mass spectrometer (Thermo Scientific, Waltham,
Massachusetts, USA) equipped with an Rxi-1 ms capillary
column (60 m long, 0.25 mm inside diameter, and 0.25 μm
film thickness), temperature program from 220 (2 min) to
2
017, 2018).
−
1
−1
Based on this knowledge and our experience in ozono-
330 °C at 6 °C min , split 150 mL min (280 °C), carrier
1
lysis (Kula and Masarweh 1998; Kula 1999; Kula et al.
000) we decided to synthesize chiral 9-carbon skeleton
gas—helium (300 kPa), Fid 300 °C, injection 0.5 μL. H
1
3
2
NMR (250 MHz), and C NMR (62.90 MHz) were recor-
ded using deuterochloroform solution (99.8% CDCl3;
Sigma-Aldrich), unless otherwise specified, with TMS (δ =
0 ppm) as internal standard (Bruker DPX-250 Avance;
derivatives of RA. Having at disposal both forms of RA
methyl esters, we obtained analogous esters of (R)- and (S)-
3
-hydroxynonanoic acid by a multistage process involving
1
3
ozonolysis and oxidation of castor oil and methyl (S)-rici-
noleate, respectively.
Bruker, Hanau, Germany). C NMR multiplicity was
determined using distortionless enhancement by polariza-
tion transfer experiments. The purity of the products was
Then, all of the obtained compounds were transformed
into corresponding derivatives, i.e., amides and hydroxamic
acids, and used for evaluation of their cytotoxic activity.
Our attention was drawn to fatty acid amides and hydro-
xoamic acids, because these two groups of compounds have
recently gained more and more importance in medicinal
chemistry (Farrell and Merkler 2008; Pinto and Silva 2016).
Amides play a crucial role in all living organisms and are
commonly used in medicine as it is confirmed by the
Comprehensive Medicinal Chemistry database which shows
that the carboxamide group is present in more than 25% of
known drugs (Montalbetti and Falque 2005).
confirmed by thin-layer chromatography (TLC) and 13
C
NMR. Optical rotations were measured with an Autopol IV
polarimeter (Rudolph Research, Flanders, New Jersey,
USA) and IR spectra were obtained with the FT-IR spec-
trometer Nicolet 6700 (Thermo Scientific).
Cell lines and culture conditions
The experiments were performed with HT29 and HeLa
cancer cells (human colorectal adenocarcinoma cell line and
human cervical adenocarcinoma cell line, respectively). The
cancer cells were obtained from the American Type Culture
Collection (ATCC, Rockville, USA). The HT29 cells were
cultured in RPMI 1640 medium supplemented with FBS
Materials and methods
(10%), antibiotics: penicillin and streptomycin (1%) and
Chemicals
MEM non-essential amino acids solution (1%). The HeLa
cells were cultured in IMDM medium containing FBS
(10%) and antibiotics: penicillin and streptomycin (1%),
and 5 × 10− M β-mercaptoethanol (1%). All the cells were
cultured at 37 °C in a humidified atmosphere of 95% air and
5% CO2.
2
-Amino-2-methyl-1-propanol, ethanolamine, pyrrolidine,
hydroxylammonium chloride, Dowex 50WX8, N,O
Bis(trimethylsililyl)trifluoroacetamide+chlorotrimethylsilane
BSTFA + TMCS), and Novozyme 435 (Candida antarctica
5
-
(
lipase) were purchased from Sigma-Aldrich. MTT [3(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], dime-
thyl sulfoxide (DMSO), penicillin–streptomycin solution sta-
bilized and buffered saline (PBS) were purchased from Sigma
Chemical Co. Foetal bovine serum (FBS), phytohemagglutinin,
RPMI 1640 medium with Glutamax, Iscove’s modified Dul-
becco’s medium, and tripsin-EDTA were supplied by Cytogen
GmbH.
Silica gel for flash chromatography (catalog no. 7024-2;
J.T. Baker Co.) was purchased from Avantor Performance
Materials B.V. (Deventer, Netherlands). GC-MS was per-
formed with a Trace GC Ultra chromatograph coupled with
Cytotoxicity analysis
The effects of the tested compounds on cancer cell growth
and proliferation were determined by a colorimetric MTT
assay. In the living cells the water-soluble tetrazolium salt
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide; thiazolyl blue) is converted to an insoluble purple
formazan by cleavage of the tetrazolium ring by mitochon-
drial dehydrogenase enzymes. The formazan product is
solubilized with organic solvent and the absorbance is mea-
sured spectrophotometrically at 595 nm. The cytotoxicity of

Medicinal Chemistry Research
the tested compounds is expressed as the IC50 value (con-
centration of the tested compound that reduces the absorbance
by 50% when compared to the negative control). The assay
was optimized for the cell lines and chemical compounds
used in the experiments.
(S,Z)-12-hydroxyoctadec-9-enamide (S-2)
The method for its preparation was analogously as for its
enantiomer R-2. Process ran with 52% yield providing pure
2
3
(>98%, GC) amide S-2. ½αŠ −2.95 (c 5.05, CHCl ), mp
D
3
The cancer cells were grown for 24 h on 96-well plates
64.1–66 °C (acetone). Its IR, MS, and NMR spectra mat-
ched those determined for compound R-2.
3
at density of 6–8 × 10 cells/well. Then the cells were
treated with the tested compounds for 72 h (2–200 µM).
At the end of the incubation MTT dissolved in PBS was
added to each plate well (20 µL, 5 mg/mL) for 4 h. The
medium was removed and purple crystals formed in
cancer cells after reduction of MTT were dissolved in
DMSO (100 mL/well). Absorbance was measured with a
spectrophotometer PowerWave XS (BioTek Instruments,
Inc.). IC50 values were calculated by the GraphPad Prism
(R,Z)-N,12-dihydroxyoctadec-9-enamide (R-3)
A solution of potassium hydroxide (1.1 g, 0.019 mol) in
methanol (5 mL) and a solution of hydroxylamine hydro-
chloride (0.9 g, 0.013 mol) in methanol (10 mL) were
obtained by heating at the boiling point of the solvent.
Then, both were cooled to 30 °C and the solution of KOH
6
.0 (GraphPad Prism Software Inc., USA). Three inde-
was added to the NH OH·HCl solution. The mixture was
2
pendent experiments were done. All the results were
presented as the mean ± SEM.
stirred in an ice bath for 15 min and then the precipitated
potassium chloride was filtered off. The filtrate was added
to methyl ester of (R)-RA (R-1; 2 g, 0.006 mol) and after
thorough mixing kept at room temperature for 24 h. Then,
Dowex 50W-X8 resins were added to obtain a pH of 6 and
the solution was filtered. Evaporation of methanol delivered
an orange solid, therefore, crystallization in acetone was
performed giving pure (>96%, GC after derivatization of
the sample) hydroxamic acid R-3 (0.104 g) with 52% yield.
Preparation of (R)- and (S)-RA derivatives
Methyl esters of (R)- and (S)-RA (R-1, S-1)
Methyl (R)-ricinoleate (R-1) and methyl (S)-ricinoleate (S-
1
) were obtained according to the previously described
2
3
method (Kula et al. 2014).
½αŠ +1.26 (c 5.0, CHCl ), mp 64.9–66.3 °C (acetone).
D
3
Derivatization with BSTFA + TMCS reagent was
necessary to perform GC analysis of this compound.
Therefore, 100 µL of silylating reagent was added to ca.
10 mg of sample and heated to 60 °C for 20 min. Then,
silylated compound was dissolved in methanol in 10%
concentration and subjected to GC-MS analysis.
(
R,Z)-12-hydroxyoctadec-9-enamide (R-2)
Methyl ester of (R)-RA (1 g, 0.003 mol) was added to a 2%
solution of ammonia in dioxane (25 ml) and Novozyme 435
(
9
380 mg). The mixture was shaken at 30 °C and 250 rpm for
6 h monitoring the progress of the reaction by TLC. The
−
1
IR (cm , neat): 3403.3, 3278.5, 3015.5, 2918.6, 2847.6,
enzyme was filtered off and washed by dichloromethane.
Evaporation of solvent provided crude solid product which
was purified by crystallization in acetone. Pure amide R-2
1662.6, 1621.2, 1077.4, 855.2. GC-MS (EI, 70 eV), m/z:
+
529 (M , 0), 282 (2), 188 (14), 187 (97), 103 (31), 97 (27),
1
75 (20), 73 (100), 55 (32), 41 (13). H NMR (500 MHz, δ
(
0.44 g) was white solid of 98% purity (GC) and the reac-
ppm): 9.39 (br.s, 1H; -NH-), 5.52 (m, 1H; -CH=), 5.38 (m,
1H; -CH=), 3.62 (qn J = 6 Hz, 1H; -CHOH), 2.20 (m, 2H;
-CHCH CH=), 2.11 (t J = 7.3 Hz, 2H; -CH CO-), 2.03 (m,
23
tion yield was 46%. ½αŠ +2.92 (c 5.2, CHCl ), mp
D
3
6
1
1
4.8–67.7 °C (acetone), lit. mp 64–65.5 °C (Galstukhova
2
2
−
1
960). IR (cm , neat): 3353.6, 3184.0, 2922.4, 2850.8,
658.4, 1631.8, 1469.4, 1072.9, 813.1. GC-MS (EI, 70 eV),
2H; = CHCH CH -), 1.59 (m, 2H), 1.46 (m, 3H), 1.29 (m,
2 2
1
3
16H), 0.87 (t J = 6.8 Hz, 3H; -CH3). C NMR (500 MHz):
171.69 (s; -CO-), 133.24 (d; -CH=), 125.22 (d; -CH=),
71.61 (d; -CHOH-), 36.68 (t), 35.23 (t), 32.84 (t), 31.81 (t),
29.36 (t), 29.33 (t), 28.86 (t), 28.84 (t), 28.82 (t), 27.18 (t),
25.69 (t), 25.28 (t), 22.60 (t), 14.07 (q; -CH3).
+
+
m/z: 297 (M , 0), 279 (M -18, 1), 183 (5), 97 (10), 72 (54),
6
NMR (δ ppm): 5.57 (m, 3H; -NH ; -CH=), 5.40 (m, 1H;
1
9 (15), 59 (100), 55 (70), 44 (22), 43 (35), 41 (36). H
2
-
-
CH=), 3.60 (qn J = 6.3 Hz, 1H; -CHOH), 2.21 (m, 4H;
CHCH CH=, -CH CO-), 2.04 (m, 2H), 1.62 (qn J =
2
2
7
6
.3 Hz, 2H), 1.45 (m, 3H), 1.30 (m, 15H), 0.87 (t J =
.5 Hz; -CH3). C NMR: 176.08 (s; -CO-), 133.10 (d;
(S,Z)-N,12-dihydroxyoctadec-9-enamide (S-3)
1
3
-
CH=), 125.24 (d; -CH=), 71.39 (d; -CHOH-), 36.74 (t),
5.82 (t), 35.26 (t), 31.75 (t), 29.43 (t), 29.27 (t), 29.02 (t,
The reaction was carried out in the same way as for com-
pound R-3. Pure (>98%, GC after derivatization of the
sample) hydroxamic acid (S-3) was obtained with 40%
3
2
1
xCH ), 28.94 (t), 27.23 (t), 25.63 (t), 25.38 (t), 22.53 (t),
2
2
3
4.01 (q; -CH3).
yield. ½αŠ −1.38 (c 5.25, CHCl ), mp 64.6–65.7 °C
D
3

Medicinal Chemistry Research
2
D
1
−1
(
acetone). Conducting the GC analysis required a derivati-
½αŠ −13.7 (c 0.9, MeOH) (Kula et al. 2000). IR (cm ,
sation of the sample which was done analogously as for
compound R-3. Its IR, MS, and NMR spectra matched
those reported for enantiomer R-3.
neat): 3432.7, 2927.2, 2856.1, 1457.5, 1192.4, 1120.4,
1054.4, 820.7. GC-MS (EI, 70 eV), m/z: 204 (M , 0), 173
(2), 119 (5), 97 (3), 87 (33), 75 (100), 59 (36), 58 (13), 55
+
(7), 43 (4). The enantiomeric purity of the product was
(
R,Z)-12-hydroxy-N-methyloctadec-9-enamide (R-4)
>99% ee, which was determined by chiral GC analysis
described elsewhere (Kula et al. 2014).
A solution of potassium hydroxide (1.1 g, 0.019 mol) in
methanol (5 mL) was added to a solution of methylamine
hydrochloride (0.9 g, 0.013 mol) in methanol (10 mL) at
Dimethyl acetal of (S)-3-hydroxynonanal (S-5)
0
°C. The mixture was stirred in an ice bath for 15 min and
Methyl (S)-ricinoleate (S-1) (71.5 g, 0.23 mol) was dis-
then the precipitated potassium chloride was filtered off.
The filtrate was added to methyl ester of (R)-RA (R-1; 2 g,
solved in methanol (250 mL) and the mixture was cooled to
−10 °C. Oxygen containing 160 g of ozone in Nm at the
3
0
.006 mol) and after thorough mixing kept at room tem-
gas flow 0.4 g/min was passed through the solution until a
positive test with potassium iodide (ozonation time: 2.5 h).
When the reaction was completed, the peroxides were
reduced by addition of dimethyl sulphide (DMS, 50 g,
0.8 mol). DMS was added with stirring at temperature
below 0 °C for 1.5 h. Then, the reaction mixture was left
overnight at room temperature until the total decomposition
of peroxides, which was controlled by the potassium iodide
test. Next, the solution containing (S)-3-hydroxynonanal as
intermediate was cooled to −5 °C and 35% HCl (4.5 mL) in
methanol (75 mL) was dropped carefully during intensive
stirring, maintaining the temperature below 0 °C. Stirring
was continued for 2 h at room temperature and left over-
night. In the next step, KOH (40 g, 0.07 mol) in water
(50 mL) was added at 0–5 °C. Excess of DMS with a little
of methanol (entirely 100 mL) were distilled off and the
residue was refluxed for 3 h. In this way, present in the
mixture glycerides were converted into water-soluble
potassium salts easy to remove during extraction. After
vacuum evaporation of methanol and addition of water
(100 mL), the extraction with diethyl ether (3 × 100 mL)
was carried out. The ether solution was washed neutral with
perature for 24 h. Then, Dowex 50W-X8 resins were added
to obtain a pH of 6 and the solution was filtered. The sol-
vent was evaporated and the crude product was purified on
a silica gel column by using the mixture of ethyl acetate/
hexane (40:60, v/v) as eluent. Pure (>99%, GC) compound
23
R-4 (1.24 g) was obtained with 62% yield. ½αŠ +2.35 (c
D
−
1
5
1
3
.05, CHCl ). IR (cm , neat): 3297.6, 2925.2, 2854.4,
3
648.1, 1560.9, 1046.9, 725.7. GC-MS (EI, 70 eV), m/z:
+
+
11 (M , 0), 293 (M –18, 1), 97 (8), 86 (53), 73 (100), 69
1
(
5
12), 58 (36), 55 (56), 43 (21), 41 (23). H NMR (δ ppm):
.54 (m, 2H; -CH=, -NH-), 5.38 (m, 1H; -CH=), 3.60 (qn
J = 5.9 Hz, 1H; -CHOH), 2.79 (d J = 4.8 Hz, 3H;
-
NHCH ), 2.18 (m, 4H), 2.04 (m, 2H), 1.35 (br.m, 18H),
3
13
0
1
3
2
2
.87 (t J = 6.5 Hz, 3H; -CH3). C NMR: 173.92 (s; -CO-),
32.78 (d; -CH=), 125.24 (d; -CH=), 71.26 (d; -CHOH-),
6.66 (t), 36.36 (t), 35.19 (t), 31.67 (t), 29.36 (t), 29.19 (t),
9.06 (t), 29.01 (t), 28.89 (t), 27.15 (t), 26.01 (q; -NHCH3),
5.58 (t), 25.55 (t), 22.44 (t), 13.90 (q; -CH3).
(
S,Z)-12-hydroxy-N-methyloctadec-9-enamide (S-4)
Reaction was carried out analogously as for compound (R-
) starting from methyl (S)-ricinoleate (S-1) as a substrate.
water and dried over anhydrous MgSO . Then, the solvent
was evaporated and crude product was isolated by vacuum
4
4
Purification on column chromatography delivered com-
pound S-4 with >99% purity (GC) and 55% yield.
distillation giving pure (>97%, GC) acetal S-5 (32 g, 68%
2
3
yield). Bp 89–88 °C/0.2 torr; ½αŠ −11.95 (c 5.08, CHCl ).
D
3
23
½
አ−2.31 (c 5.1, CHCl ). IR, MS, and NMR spectra were
IR, MS, and NMR spectra were in accordance with com-
D
3
in accordance to compound R-4.
pound R-5. The enantiomeric purity of the product was
94.7% ee, which was determined by chiral GC analysis
Preparation of dimethyl acetals of (R)- and (S)-3-
described elsewhere (Kula et al. 2014).
hydroxynonanal
Preparation of (R)- and (S)-3-hydroxynonanoic acid
Dimethyl acetal of (R)-3-hydroxynonanal (R-5)
derivatives
Reaction was carried out according to the previously
Methyl ester of (R)-3-hydroxynonanoic acid (R-6)
described method (Kula et al. 2000). Ozonation conditions:
3
1
60 g of ozone in Nm , gas flow 0.4 g/min, time 5 h. Pure
Oxidation of (R)-3-hydroxynonanal dimethyl acetal (R-5) to
corresponding methyl ester (R-6) was carried out similarly
as published by Takeda et al. (Takeda et al. 1997). Com-
pound R-5 (20 g, 0.098 mol) was dissolved in methanol
acetal R-5 (>96%, GC) was isolated by vacuum distillation
with 74% yield. Bp 96–104 °C/0.8 torr, lit. bp 90–92 °C/0.7
torr (Kula et al. 2000); ½αŠ +12.2 (c 5.16, CHCl ); lit.
23
D
3

Medicinal Chemistry Research
(
(
200 mL) and cooled to 0 °C. In the next step, 35% HCl
15.5 g, 0.15 mol) and then 30% H O (17 g, 0.15 mol) were
1H; -CHCH CO-), 1.41 (br.m, 10H), 0.88 (t J = 6.6 Hz, 3H;
2
1
3
-CH3). C NMR: 175.05 (s; -CO-), 68.48 (d; -CHOH),
42.01 (t), 36.87 (t), 31.73 (t), 29.15 (t), 25.42 (t), 22.55 (t),
14.01 (q; -CH3).
2
2
dropped carefully during intensive stirring maintaining
temperature below 5 °C. The stirring was continued for an
additional 30 min in an ice bath. Then, the solution was
heated to 40 °C and stirred in this temperature for 3 h.
About 100 mL of methanol was evaporated and after
addition of water (80 mL) the extraction with diethyl ether
was performed (3 × 100 mL). The organic layer was washed
with aqueous sodium sulfide (80 mL), then with water
(S)-3-hydroxynonandamide (S-7)
The reaction was carried out in the same manner as for
compound R-7. Amide S-7 of >99% purity (GC) was
2
D
3
obtained with 58% yield. ½αŠ +26.24 (c 0.32, CHCl ), mp
3
(
80 mL) and dried over anhydrous MgSO . The solvent was
101.4–102.4 °C (acetone). Its IR, MS, and NMR spectra
matched those reported for enantiomer R-7.
4
evaporated and crude product was purified on column
chromatography using hexane/ethyl acetate (80:20 v/v) as
eluent to obtain pure (>97%, GC) methyl ester of (R)-3-
hydroxynonanoic acid (R-6) (9.1 g, 49% yield). ½αŠ −19.3
N-(2-hydroxyethyl)-(3R)-hydroxynonanamide (R-8)
2
3
D
2
D
5
(
1
(
8
1
(
-
3
8
6
c 5.10, CHCl ); lit. ½αŠ −20.0 (Odham and Samuelsen
Methyl ester of (R)-3-hydroxynonanoic acid (R-6; 0.5 g,
0.0027 mol) was mixed with ethanolamine (1 g, 0.016 mol)
and heated at 130 °C (gentle reflux) for 2 h by monitoring
the progress of the reaction by TLC. The crude amides were
purified by flash chromatography with silica gel using ethyl
acetate/methanol (97:3, v/v) as eluent to deliver pure amide
R-8 (0.43 g; 99% purity confirmed by GC) with 74% yield.
Product was white solid, mp 74.3–75.9 °C (ethyl acetate/
3
2
D
5
970), ½αŠ −16.0 (c 1.48, CHCl ) (Jiang et al. 2010). IR
3
−1
cm , neat): 3473.3, 2953.9, 2927.9, 1731.1, 1162, 1055.6,
+
+
64.6. GC-MS (EI, 70 eV), m/z: 188 (M , 0), 170 (M –18,
), 138 (4), 103 (100), 96 (9), 74 (36), 71 (38), 61 (16), 55
1
21), 43 (43), 41 (22). H NMR (δ ppm): 3.95 (m, 1H;
CHOH), 3.66 (s, 3H; -OCH ), 2.47 (dd J = 16.3 Hz, J =
3
.8 Hz, 1H; -CHCH CO-), 2.36 (dd J = 16.3 Hz, J =
2
2
3
.8 Hz, 1H; -CHCH CO-), 1.31 (br. m, 11H), 0.83 (t J =
methanol), 79.6–80.6 °C (acetone), ½αŠ −9.7 (c 5.1,
2
D
1
3
−1
.6, 3H; -CH3). C NMR: 173.34 (s; -CO-), 67.90 (d;
CHCl ). IR (cm , neat): 3298.6, 2920.9, 2848.5, 1643.6,
3
-
2
CHOH), 51.57 (q; -OCH ), 41.12 (t), 38.48 (t), 31.65 (t),
9.07 (t), 25.33 (t), 22.47 (t), 13.92 (q; -CH3).
1560.7, 1081.1, 1058.4, 860.9. GC-MS (EI, 70 eV), m/z:
3
+
+
217 (M , 1), 199 (M –18, 1), 174 (16), 139 (25), 132
100), 114 (29), 88 (30), 62 (35), 60 (40), 55 (61), 43 (71).
(
1
Methyl ester of (S)-3-hydroxynonanoic acid (S-6)
H NMR (δ ppm): 7.04 (t J = 5.1 Hz, 1H; -NH-), 4.41 (br.s,
H), 3.95 (m, 1H; -CHOH), 3.63 (m, 2H; -CH OH), 3.42
2
2
The reaction was carried out in the same way as for com-
pound R-6. Pure (97%, GC) methyl ester of (S)-3-hydro-
xynonanoic acid (S-6) was obtained with 51% yield.
(m, 1H; -NHCH -), 3.29 (m, 1H; -NHCH -), 2.38 (dd J =
2
2
14.6 Hz, J = 2.4 Hz, 1H; -CHCH CO-), 2.24 (dd J =
2
14.6 Hz, J = 9.4 Hz, 1H; -CHCH CO-), 1.39 (br.m, 10H),
2
23
13
½
አ+18.1 (c 5.01, CHCl ). Its IR, MS, and NMR spectra
0.85 (t J = 6.8 Hz, 3H, -CH3). C NMR: 173.32 (s; -CO-),
D
3
matched those reported for enantiomer R-6.
R)-3-hydroxynonandamide (R-7)
68.70 (d, -CHOH), 61.25 (t, -CH OH), 43.27 (t,
2
-
CHCH CO-), 42.03 (t, -NHCH -), 37.19 (t), 31.73 (t),
2
2
(
29.18 (t), 25.51 (t), 22.54 (t), 14.00 (q, -CH3).
2
5% water solution of ammonia (8 mL) was added to
methyl ester of (R)-3-hydroxynonanoic acid (R-6; 2 g,
.011 mol). The mixture was shaken at 30 °C for 24 h and
N-(2-hydroxyethyl)-(3S)-hydroxynonanamide (S-8)
0
The reaction was carried out analogously to the compound
R-8, however, in order to obtain pure (>99%, GC) product it
was necessary to perform additional purification process,
the progress of the reaction was monitored by TLC. The
precipitated crystals were filtered off and purified by crys-
tallization in acetone. Amide R-7 (0.913 g) was white solid
i.e., crystallization in acetone. Pure amide S-8 was obtained
23
23
D
of >99% purity (GC) obtained with 50% yield. ½αŠ −26.77
with 39% yield, and it was a white solid. ½αŠ +10.48 (c 5.1,
D
−
1
(
c 0.31, CHCl ), mp 101.8–103.7 °C (acetone). IR (cm ,
CHCl ); mp 79.8–80.5 °C (acetone).
3
3
neat): 3349, 3176.1, 2951.8, 2921.5, 2844.4, 1631.4,
+
1
0
(
599.4, 1095.4, 862.3. GC-MS (EI, 70 eV), m/z: 173 (M ,
N-(1-hydroxy-2-methylpropan-2-yl)-(3R)-
hydroxynonanamide (R-9)
+
), 155 (M –18, 1), 88 (100), 71 (10), 59 (49), 55 (17), 45
16), 44 (24), 43 (32), 41 (21), 39 (16). H NMR (δ ppm):
1
5
.94 (s, 1H; -NH ), 5.77 (s, 1H; -NH ), 4.00 (m, 1H;
2-amino-2-methyl-1-propanol (6.4 g, 0.072 mol) was added
to methyl ester of (R)-3-hydroxynonanoic acid (R-6; 2.3 g,
0.012 mol) and the mixture was heated at 130 °C for 2.5 h.
2
2
-
CHOH), 3.40 (br.s, 1H; -OH), 2.41 (dd J = 15.5 Hz, J =
Hz, 1H; -CHCH CO-), 2.30 (dd J = 15.5 Hz, J = 8.8 Hz,
3
2

Medicinal Chemistry Research
The progress of the reaction was monitored by TLC ana-
lysis. The crude product was purified by passing through a
silica gel column using ethyl acetate/hexane (50:50, v/v) as
eluent. The obtained with 95% yield product (R-9, 2.85 g)
was a light yellow dense liquid of 98% purity (GC after
3H, -CH3). 13C NMR: 171.27 (s; -CO-), 67.83 (d; -CHOH),
46.44 (t), 45.31 (t), 40.46 (t), 36.37 (t), 31.65 (t), 29.13 (t),
25.76 (t), 25.36 (t), 24.17 (t), 22.44 (t), 13.92 (q; -CH3).
Pyrrolidinyl-(3S)-hydroxynonanamide (S-10)
23
derivatization of the sample). ½αŠ = −14.76 (c 5.45,
D
CHCl ). GC analysis required a previous derivatization of
Amide S-10 was obtained in the same way as compound R-
10 with 86% yield. Its purity was high (>99%, GC) and the
obtained IR, MS, and NMR spectra were in accordance with
3
the sample which was done analogously as for compound
−
1
R-3. IR (cm , neat): 3243.2, 3074.5, 2926.6, 2856.4,
2
D
3
1
4
1
638.9, 1553.2, 1056.8, 784.9. GC-MS (EI, 70 eV), m/z:
those for enantiomer R-10. ½αŠ +42.30 (c 5.30, CHCl ).
3
+
61 (M , 0), 374 (4), 286 (22), 246 (6), 187 (7), 145 (13),
1
44 (33), 75 (23), 73 (73), 58 (100), 55 (12). H NMR (δ
(R)-N,3-dihydroxynonanamide (R-11)
ppm): 6.65 (s, 1H; -NH-), 5.15 (br.s, 1H; -CHOH), 4.24 (br.
s, 1H; -CH OH), 3.83 (m, 1H; -CHOH), 3.51 (d J = 4 Hz,
Hydroxamic acids were obtained according to methods
described in literature (Hauser and Renfrow 1939; Devlin
et al. 1975). A solution of potassium hydroxide (3.6 g,
0.064 mol) in methanol (10 mL) and a solution of hydro-
xylamine hydrochloride (3 g, 0.043 mol) in methanol
(20 mL) were obtained by heating at the boiling point of the
solvent. Then, both were cooled to 30 °C and the solution of
2
1
H; -CH OH), 3.40 (d J = 4 Hz, 1H; -CH OH), 2.25 (dd J
2
2
=
5.4 Hz, J = 1.1 Hz, 1H; -CHCH CO-), 2.14 (dd J =
2
5
.3 Hz, J = 3.3 Hz, 1H; -CHCH CO-), 1.40 (m, 1H), 1.32
2
(
m, 2H), 1.19 (m, 13H), 0.88 (t J = 6.8 Hz, 3H, -CH CH ).
2 3
1
3
C NMR: 173.20 (s; -CO-), 69.51 (t, -CH OH), 68.59 (d,
2
-
CHOH), 55.55 (s, -C(CH ) -), 43.29 (t, -CH CO-), 36.89
3 2 2
(
t), 31.55 (t), 29.00 (t), 25.26 (t), 24.28 (q; -C(CH ) -),
KOH was added to the NH OH·HCl solution. The mixture
3
2
2
2
3.94 (q; -C(CH ) -), 22.35 (t), 13.82 (q, -CH CH ).
was stirred in an ice bath for 15 min and then the pre-
cipitated potassium chloride was filtered off. The filtrates
were added to methyl ester of (R)-3-hydroxynonanoic acid
(R-6; 4 g, 0.021 mol) and after thorough mixing kept at
room temperature for 24 h. Appeared in the mixture white
crystals of potassium (R)-3-hydroxynonanhydroxamate
were dissolved by heating the methanol solution. Then,
Dowex 50W-X8 resin was added to obtain a pH of 6, next,
the solution was filtered and evaporated. Crystallization in
acetone was performed giving pure (>98%, GC after deri-
vatization of sample) hydroxamic acid R-11 (1.91 g) with
3
2
2
3
N-(1-hydroxy-2-methylpropan-2-yl)-(3S)-
hydroxynonanamide (S-9)
The reaction was carried out in the same way as for com-
pound R-9. Pure (97%, GC after derivatization of the
sample) amide S-9 was obtained with 87% yield.
23
½
አ+16.01 (c 5.65, CHCl ). Its IR, MS, and NMR spectra
D
3
matched those reported for enantiomer R-9.
2
3
Pyrrolidinyl-(3R)-hydroxynonanamide (R-10)
47% yield. ½αŠ −4.36 (c 2.75, MeOH), m.p.:
D
104.8–107.8 °C (acetone). It was necessary to perform
The mixture of methyl ester of (R)-3-hydroxynonanoic acid
derivatization of the sample for GC analysis which was
done the same way as for compound R-3. IR (cm , neat):
−
1
(
R-6; 1.1 g, 0.006 mol) and pyrrolidine (2.5 g, 0.035 mol)
was heated under gentle reflux (75–80 °C). The lack of
visible progress of the reaction on TLC was the reason for
completing the process after 3 h. Pure (>99%, GC) product
was obtained by purification on a silica gel column using
ethyl acetate/hexane (5:95, v/v) as eluent. The reaction yield
3361.1, 3294.3, 2956.2, 2922.6, 2850.9, 1647.6, 1606.4,
1081.2, 977.2. GC-MS (EI, 70 eV), m/z: 405 (M , 0), 187
+
(63), 103 (19), 100 (19), 97 (8), 75 (15), 73 (100), 69 (26),
1
55 (39), 45 (12), 43 (11). H NMR (CD OH, δ ppm): 5.05
3
(br.s, 3H; -NH-, 2xOH), 3.95 (m, 1H; -CHOH), 2.20 (m,
23
was 67% that gave 0.89 g of compound R-10. ½αŠ −42.05
2H; -CHCH CO-), 1.45 (m, 3H), 1.30 (m, 7H), 0.89 (t J =
D
2
−
1
13
(
c 5.20, CHCl ). IR (cm , neat): 3410.1, 2926.9, 2857.4,
6.5 Hz, 3H, -CH3). C NMR (CD OH): 170.96 (s, -CO-),
3
3
1
2
8
618.2, 1449.7, 1044.6, 857.9. GC-MS (EI, 70 eV), m/z:
69.38 (d, -CHOH), 41.70 (t), 38.11 (t), 32.94 (t), 30.33 (t),
26.55 (t), 23.63 (t), 14.42 (q, -CH3).
+
27 (M , 2), 209 (10), 156 (7), 142 (100), 113 (60), 98 (94),
1
5 (15), 70 (50), 55 (50), 43 (43). H NMR (500 Hz, δ
ppm): 3.96 (m, 2H; -CHOH, -OH), 3.41 (t J = 6.8 Hz, 2H;
NCH CH -), 3.34 (m, 2H; -NCH CH -), 2.38 (dd
(S)-N,3-dihydroxynonanamide (S-11)
-
2
2
2
2
J = 16.3 Hz, J = 2.3 Hz, 1H; -CHCH CO-), 2.22 (dd J =
Hydroxamic acid S-11 was prepared in the same manner as its
enantiomer R-11. Pure compound (>97%, GC after derivati-
zation of sample) in form of solid with a slight yellow tint was
2
1
6.5 Hz, J = 9.5 Hz, 1H; -CHCH CO-), 1.91 (qn J =
2
6
.6 Hz, 2H; -NCH CH -), 1.82 (m, 2H, -NCH CH -), 1.50
2
2
2
2
23
(
m, 1H), 1.38 (m, 2H), 1.24 (br.m, 7H), 0.82 (t J = 6.8 Hz,
obtained with 41% yield. ½αŠ +4.11 (c 2.65, MeOH), mp
D

Medicinal Chemistry Research
1
04.8–108.6 °C (acetone). Its IR, MS, and NMR spectra
Results and discussion
matched those determined for compound R-11.
Preparation of corresponding starting materials, i.e., enan-
tiomeric forms of methyl esters of RA and 3-hydroxamic
acid was the first step (Scheme 1). Methyl (R)-ricinoleate
(R-1) was obtained from castor oil by common transester-
ification with methanol. It was then used as a substrate in
the three-step process of inversion leading to its S enan-
tiomer (S-1) (Kula et al. 2014). In order to obtain 9-carbon
skeleton derivatives of RA we performed ozonolysis pro-
cess similarly as reported elsewhere (Kula et al. 2000).
Castor oil and methyl (S)-ricinoleate were used as substrates
for the production of dimethyl acetals of (R)- and (S)-3-
hydroxynonanal (R-5 and S-5), respectively. Then, the
obtained compounds R-5 and S-5 were transformed into
methyl esters of (R)- and (S)-3-hydroxynonanoic acid (R-6,
S-6) by oxidation with hydrogen peroxide and hydrochloric
acid in methanol with about 50% yield. The oxidation was
performed according to the published method (Takeda et al.
1997), however, some modifications of this procedure were
made. Preparation of the methyl esters of (R)-3-hydro-
xynonanoic acid (R-6) by ozonolysis of castor oil was
previously described in literature, however, in all cases the
product of that reaction was a mixture of compounds (Ish-
muratov et al. 2006, 2007, 2009).
(
R)-3-hydroxy-N-methylnonanamide (R-12)
A solution of potassium hydroxide (1.3 g, 0.023 mol) in
methanol (5 mL) was added to a solution of methylamine
hydrochloride (1.1 g, 0.016 mol) in methanol (10 mL) at
0
°C. The mixture was stirred in an ice bath for 15 min and
then the precipitated potassium chloride was filtered off.
The filtrate was added to methyl ester of (R)-3-hydro-
xynonanoic acid (R-6; 1.5 g, 0.008 mol) and after thorough
mixing kept at room temperature for 24 h. Then, Dowex
5
0W-X8 resin was added to obtain a pH of 6 and the
solution was filtered. The solvent was evaporated and the
crude product was purified by crystallization using acetone.
Pure (>99%, GC) amide R-12 (0.463 g) was obtained with
3
(
2
D
3
1% yield. ½αŠ −25.03 (c 5, CHCl ), mp 78.5–80.4 °C
3
−1
acetone). IR (cm , neat): 3289.7, 3092, 2925.2, 2846.1,
640.5, 1559.8, 1402.1, 1071.7, 861.4. GC-MS (EI, 70 eV),
1
+
m/z: 186 (M –1, 1), 102 (100), 73 (56), 69 (8), 58 (63), 55
(
6
-
1
23), 45 (32), 43 (33), 41 (23), 39 (6). H NMR (δ ppm):
.18 (br.s, 1H; -NH-), 3.95 (m, 1H; -CHOH), 3.74 (br.s, 1H;
OH), 2.79 (d J = 4.8 Hz, 3H; -NHCH ), 2.35 (dd J =
3
1
5.3 Hz, J = 3 Hz, 1H; -CHCH CO-), 2.22 (dd J = 15.3 Hz,
2
J = 8.8 Hz, 1H; -CHCH CO-), 1.40 (br.m, 10H), 0.85 (t J
=
Consequently, a series of amides and hydroxamic acids
derived from ricinoleic and 3-hydroxynonanoic acids were
prepared. Starting from the methyl esters of RA (R-1, S-1) 6
compounds (i.e., 3 pairs of enantiomers), among them two
primary amides (R-2, S-2), two hydroxamic acids (R-3, S-
3), and two N-methyl amides (R-4, S-4) were synthesized
2
6.6 Hz; -CH3). 13C NMR: 173.24 (s; -CO-), 68.65 (d;
-
CHOH), 42.29 (t), 36.91 (t), 31.71 (t), 29.15 (t), 26.06 (q;
-
NHCH ), 25.40 (t), 22.52 (t), 14.01 (q; -CH ).
3
3
(
S)-3-hydroxy-N-methylnonanamide (S-12)
(Scheme 2).
Compound S-12 was obtained according to the method
described for amide R-12. Crystallization in acetone gave
pure (>99%, GC) amide S-12 (0.465 g) with 31% yield.
On the other hand, the methyl esters of (R)- and (S)-3-
hydroxynonanoic acid (R-6, S-6) were used for the pre-
paration of 12 compounds (6 pairs of enantiomers). Like in
the case of RA, two primary amides (R-7, S-7), two
hydroxamic acids (R-11, S-11), and two N-methyl amides
(R-12, S-12) were obtained from these 9-carbon skeleton
23
½
አ+24.83 (c 5, CHCl ), mp 81.1–82.9 °C (acetone). Its
D
3
IR, MS, and NMR spectra matched those determined for
compound R-12.
Scheme 1 Synthesis of methyl esters of ricinoleic acid (R-1, S-1), dimethyl acetals of 3-hydroxynonanal (R-5, S-5), and methyl esters of
-hydroxynonanoic acid (R-6, S-6)
3

Medicinal Chemistry Research
Scheme 2 Synthesis of (R)- and (S)-ricinoleic acid derivatives: primary amides R-2 and S-2, hydroxamic acids R-3 and S-3 (R1 = OH), and
N-methyl amides R-4 and S-4 (R1 = CH3)
Scheme 3 Synthesis of (R)- and
(
S)-3-hydroxynonanoic acid
derivatives: primary amides (R-
, S-7), ethanolamine amides
R-8, S-8), 2-amino-2-methyl-1-
7
(
propanol amides (R-9, S-9),
pyrrolidine amides (R-10, S-10),
hydroxamic acids (R-11, S-11;
R4 = OH), and N-methyl amides
(R-12, S-12; R4 = CH3)
fatty acids. Moreover, six other enantiomeric amides of
esters of RA and 3-hydroxynonanoic acid with hydro-
xylamine or methylamine was performed in the presence of
KOH in methanol at room temperature for 24 h. These
reactions proceeded with good yields, ranging from 31
to 62%.
Primary amides derived from RA (R-2, S-2) were syn-
thesized using the enzymatic method catalyzed by Novo-
zyme 435 (lipase from Candida antarctica) and performed
3
2
-hydroxynonanoic acids, i.e., with ethanolamine (R-8, S-8),
-amino-2-methyl-1-propanol (R-9, S-9) and pyrrolidine
(
R-10, S-10) were also synthesized (Scheme 3). We decided
to prepare and investigate these six chiral compounds
because of good anticancer and antimicrobial activity of
analogical amides, especially for derivatives with ethano-
lamine and pyrrolidine derived from (R)- and (S)-RA and
reported earlier (Matysiak et al. 2017, 2018). Moreover,
impact of the stereogenic center on their biological activity
was observed.
Hydroxamic acids and N-methyl amides were synthe-
sized basing on the previously published methods (Hauser
and Renfrow 1939; Devlin et al. 1975). Reaction of methyl
in 2% dioxane solution of NH by mixing all substrates at
3
30 °C (García et al. 1994). The reaction yield was good
ranging from 46 to 52%. In the case of primary amides of
3-hydroxynonanoic acid (R-7 and S-7), another, very sim-
ple and fast method was used. Good yields (50–58%) were
obtained when starting the methyl esters (R-6 and S-6) were

Medicinal Chemistry Research
shaken with 25% water solution of NH at 30 °C without
presence of the enzyme.
able to determine this parameter during our research due to
the semisolid consistency of this derivative. The other
obtained compounds have been neither synthesized nor
described in literature, and all the more tested for cytotoxic
activity. In general, in the group of 24 compounds derived
from ricinoleic and 3-hydroxynonanoic acids, including
methyl esters, hydroxamic acids, and amides, 16 of them
were new compounds. Therefore, we carried out full spec-
tral and polarimetric characterization of all the obtained RA
derivatives. The purity of all compounds was monitored by
3
The amides (8–10) were prepared by a simple reaction of
methyl esters of 3-hydroxynonanoic acid with correspond-
ing amines, i.e., ethanolamine, 2-amino-2-methyl-1-propa-
nol, and pyrrolidine. The reactions were carried out by
heating the substrates at the proper temperature (keeping a
gentle reflux) without a solvent. This kind of method for
preparation of amides was previously used to synthesize
analog derivatives of (R)- and (S)-ricinoleic acids (Matysiak
et al. 2017, 2018). It was not only environment-friendly and
free of the solvent procedure, but it also provided relatively
good performance. The amides (8–10) synthesized in the
present study were obtained in good yields, ranging from
1
3
TLC, C NMR, GC, and measurements of their optical
1
13
rotations. What is more, H NMR, C NMR, GC-MS, and
IR analyses were done for confirmation of their structures.
Then, the effects of the obtained compounds on cancer
cell growth and proliferation were determined by a colori-
metric MTT assay. Two cancer cell lines—HT29 (human
colorectal adenocarcinoma) and HeLa (human cervical
adenocarcinoma)—were used in the experiments. The cells
were treated with various concentrations of the compounds
for 72 h and the cytotoxicity was expressed as the IC50
value (concentration of the tested compound that reduces
the absorbance by 50% when compared to the negative
control). The results of the cytotoxicity assay are presented
in Table 1.
3
9% to 95%. The best yields (87–95%) were observed for
the derivatives with 2-amino-2-methyl-1-propanol (R-9 and
S-9). It was surprising, because in our previous study in the
case of analogical amides of (R)- and (S)-ricinoleic acids we
observed significantly weaker results ranging from 43 to
4
8% (Matysiak et al. 2018).
In general, we obtained 24 chiral compounds eight of
which are derivatives of RA (1–4), two dimethyl acetals of
-hydroxynonanal (5) and 14 derivatives of 3-
3
hydroxynonanoic acid (6–12). It is worth mentioning that
only the starting methyl esters of RA (R-1, S-1) and the
methyl ester of (R)-3-hydroxynonanoic acid (R-6) have
been fully spectrally and polarimetrically characterized so
far (Odham and Samuelsen 1970; Ishmuratov et al.
Cytotoxic activities of the tested compounds expressed
as IC50 values ranged from 13.22 to 80.00 µM. The results
also showed that there was no significant difference
between the activities of (R)- and (S)-enantiomers of parti-
cular derivatives. For the majority of tested compounds
higher toxicity was observed against HT29 than HeLa
cancer cells. Derivatives 3 and 11, i.e., hydroxamic acids
were exceptions to this rule. Toxicity of the compound 3
was similar both against HT29 and HeLa cells (IC50 from
41.11 to 44.03 µM; Table 1). The most interesting results
were obtained for the hydroxamic acids derived from 3-
hydroxynonanoic acid (11), which were found to be the
most cytotoxic for HeLa cancer cells (IC50 values: 13.22 µM
2
006, 2007, 2009; Jiang et al. 2010; Kula et al. 2014).
Moreover, the compounds R-1 and S-1 were studied with
regard to biological activity. Some information about the
compounds R-2 and R-4 which are amides of (R)-RA can
also be found (Galstukhova 1960; Applewhite et al. 1963).
The methods for their preparation and their melting points
were described, but their specific optical rotations were not
given. Applewhite et al. (1963) described the melting point
of the compound R-4 (30.5–31.5 °C), however, we were not
Table 1 The IC50 values (µM)
Compound
HT29
HeLa
determined after 72-h treatment
of cancer cells with the tested
compounds
(R)-enantiomer
(S)-enantiomer
(R)-enantiomer
(S)-enantiomer
2
3
4
6
7
8
9
41.25 ± 2.72
44.03 ± 3.23
40.42 ± 2.37
43.75 ± 2.51
76.25 ± 3.35
45.42 ± 2.07
43.33 ± 1.49
52.78 ± 2.89
41.42 ± 2.16
50.28 ± 1.73
42.78 ± 4.20
41.11 ± 2.02
42.92 ± 1.72
43.75 ± 2.28
64.50 ± 2.80
47.08 ± 3.33
49.17 ± 2.25
52.50 ± 3.06
42.42 ± 2.15
53.19 ± 2.40
60.56 ± 4.40
41.56 ± 2.83
62.67 ± 2.09
70.56 ± 5.09
79.33 ± 5.70
73.67 ± 4.97
64.00 ± 3.46
54.89 ± 6.42
13.22 ± 1.25
54.00 ± 2.65
57.00 ± 3.12
44.00 ± 2.34
56.89 ± 1.44
77.00 ± 4.96
80.00 ± 3.21
63.89 ± 1.96
66.00 ± 2.27
58.67 ± 3.79
14.33 ± 1.05
57.67 ± 4.64
1
1
1
0
1
2

Medicinal Chemistry Research
and 14.33 for (R)- and (S)-enantiomer, respectively) while
HT29 cells were more resistant to it (IC50 values: 41.42 µM
and 42.42 for (R)- and (S)-enantiomer, respectively).
Comparing these results with our previous findings
considered as compounds of potential pharmacological
significance. The results also showed that there was no
significant difference between activities of (R)- and (S)-
enantiomers of particular derivatives.
(
Matysiak et al. 2017) we can observe that modification of
Acknowledgements The authors want to thank Prof. Krzysztof
Śmigielski for his help in ozonolysis process.
the parent methyl ricinoleate with amines resulted in
increased cytotoxicity. In the case of HT29 cells, methyl
esters of RA showed weak activity (IC50 values:
Compliance with ethical standards
1
64.8–175.3 µM), whereas, the amides (2–4) obtained in this
study were significantly more toxic against the tested cancer
cells with IC50 values ranging from 40.42 to 44.03 µM.
Interestingly, such a relationship was not observed in the
case of 3-hydroxynonanoic acid derivatives (6–12) and their
activities varied depending on cancer cell line. In the case of
HT29 cells, the starting methyl 3-hydroxynonanoates (6;
IC50 value 43.75 µM) showed similar cytotoxicity as their
amide derivatives 8, 9, 11 (IC50 values from 41.42 to
Conflict of interest The authors declare that they have no conflict of
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tivecommons.org/licenses/by/4.0/), which permits use, duplication,
adaptation, distribution, and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
changes were made.
4
7.08 µM), and even better activity than compounds 7, 10,
and 12 (IC50 values: 50.28–76.25 µM). In the case of HeLa
cancer cells, the starting methyl esters 6 and amides 7, 8, 9
exhibited similar activity with IC50 values in the range from
6
4.00 to 80.00 µM. Slightly greater cytotoxicity was
observed for the compounds 10 and 12 (IC50 values
4.00–58.67 µM), and the derivatives 11 were significantly
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