Antimicrobial Potential of Chiral Amide Derivatives of Ricinoleic and 3-Hydroxynonanoic Acid

Article abstract of DOI:10.1002/aocs.12292

The growing role of fatty acid amides in medicinal chemistry has recently been observed. Therefore, using simple and fast methods, a series of chiral amide derivatives (24 compounds) of ricinoleic and 3-hydroxynonanoic acid was obtained with 31–95% yields. Then, the evaluation of their antimicrobial activity against 13 microorganisms representing Gram-positive and Gram-negative bacteria, yeast, and molds was performed. The obtained compounds showed antimold potential; however, the tested species of molds were more susceptible to derivatives of 3-hydroxynonanoic acid than to amides obtained from ricinoleic acid (RA). Interestingly, hydroxamic acids derived from RA exhibited the best activity against Candida albicans and Candida tropicalis. On the other hand, hydroxamic acids derived from 3-hydroxynonanoic acid showed the best antimicrobial potential against the remaining tested microorganisms, especially against Pseudomonas cedrina. The obtained derivatives can be considered compounds of potential pharmacological significance, which is important due to the increasing problem of microbial resistance.

Full text of DOI:10.1002/aocs.12292

J Am Oil Chem Soc (2019)
DOI 10.1002/aocs.12292
ORIGINAL ARTICLE
Antimicrobial Potential of Chiral Amide Derivatives of
Ricinoleic and 3-Hydroxynonanoic Acid
2
1
2
Sylwia Matysiak1 · Julia Zabielska · Józef Kula · Alina Kunicka-Styczynska
ꢀ
Received: 2 May 2019 / Revised: 4 September 2019 / Accepted: 9 September 2019
© 2019 AOCS
Abstract The growing role of fatty acid amides in medic-
inal chemistry has recently been observed. Therefore, using
simple and fast methods, a series of chiral amide deriva-
tives (24 compounds) of ricinoleic and 3-hydroxynonanoic
acid was obtained with 31–95% yields. Then, the evalua-
tion of their antimicrobial activity against 13 microorgan-
isms representing Gram-positive and Gram-negative
bacteria, yeast, and molds was performed. The obtained
compounds showed antimold potential; however, the tested
species of molds were more susceptible to derivatives of
3-hydroxynonanoic acid than to amides obtained from
ricinoleic acid (RA). Interestingly, hydroxamic acids derived
from RA exhibited the best activity against Candida albicans
and Candida tropicalis. On the other hand, hydroxamic acids
derived from 3-hydroxynonanoic acid showed the best anti-
microbial potential against the remaining tested microorgan-
isms, especially against Pseudomonas cedrina. The obtained
derivatives can be considered compounds of potential phar-
macological significance, which is important due to the
increasing problem of microbial resistance.
Introduction
Ricinoleic acid (RA) [(12R,9Z)-hydroxyoctadecenoic acid;
18:1-OH] is the main component of castor oil obtained
from the seeds of Ricinus communis L. Data from literature
concerning this hydroxy fatty acid are extensive because of
a significant interest in this compound and its huge utiliza-
tion in various branches of industry. It is used for the pro-
duction of aroma substances, polymers and many other
important compounds, also biologically active (Achaya,
ꢀ
1971; Ogunniyi, 2006; Pabis and Kula, 2016).
Naturally occurring RA has one double bond in the cis
configuration and one R-configured, asymmetrically sub-
stituted carbon atom. The main source of this compound is
castor oil, in which it represents about 80–90% of all fatty
acids (Achaya et al., 1964; Borch-Jensen et al., 1997). (S)-
RA has not yet been identified in any natural source, and
therefore, there is very limited information about it. In 2014,
an efficient methyl ester of the (S)-RA production method
was published (Kula et al., 2014), and since then, some stud-
ies on this enantiomer have been carried out. A series of
amides derived from both forms of RA was obtained and
tested in terms of cytotoxicity and antimicrobial activity. The
promising results showed the significant biological potential
of these derivatives. Interestingly, in the case of some tested
compounds, differences in the activity of the opposite enan-
tiomers could also be observed (Matysiak et al., 2017, 2018).
Motivated by the above-mentioned interesting results, we
focused on the search for other amide derivatives. A number
of primary amides, hydroxamic acids, and N-methyl amides
were obtained by amidation of both enantiomers of RA
methyl esters. The second group of chiral compounds synthe-
sized in this study included 9-carbon skeleton derivatives.
For this purpose, we used our experience of the ozonolysis
process (Kula, 1999; Kula et al., 2000; Kula and Masarweh,
Keywords Amides ꢀ Antibiotics ꢀ Antifungal agents ꢀ
Ricinoleic acid ꢀ 3-hydroxynonanoic acid ꢀ Ozonolysis
J Am Oil Chem Soc (2019).
* Sylwia Matysiak
sylwia.matysiak@edu.p.lodz.pl
1
Institute of General Food Chemistry, Faculty of Biotechnology
and Food Sciences, Lodz University of Technology,
Stefanowskiego 4/10, 90-924, Łódź, Poland
2
Institute of Fermentation Technology and Microbiology, Faculty
of Biotechnology and Food Sciences, Lodz University of
ꢀ
Technology, Wólczanska 171/173, 90-924, Łódź, Poland
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
1998) in RA degradation, resulting in the formation of chiral
methyl 3-hydroxynonanoates. By performing ozonolysis of
castor oil or (S)-RA, we obtained dimethyl acetals of
3-hydroxynonanal, which were then oxidized to the
corresponding methyl esters. Chiral methyl 3-hydroxy-
nonanoates prepared in this way were used as starting mate-
rials for the synthesis of various cyclic and acyclic amides
and hydroxamic acids.
All the produced derivatives were then examined as
potentially bioactive compounds against 13 microorgan-
isms, including Gram-positive and Gram-negative bacteria,
yeast, and molds.
positive bacteria (Bacillus subtilis ATCC 6633, Micrococ-
cus flavus LOCK 0949, and Staphylococcus aureus ATCC
6538) and five Gram-negative bacteria (Escherichia coli
ATCC 8739, Pseudomonas aeruginosa ATCC 15442,
Pseudomonas fluorescens PCM 2123, Pseudomonas
cedrina DH1, and Serratia liquefaciens—the last two spe-
cies isolated from cosmetics). The selected yeast and molds
were as follows: Candida albicans ATCC 10231, Candida
krusei fo/MP/02, and Candida tropicalis fo/BM/01 (the last
two isolated from food) and Aspergillus brasiliensis ATCC
16404 and Penicillium expansum LOCK 0536. The micro-
organisms named ATCC originated from American Type
Culture Collection, those termed PCM were derived from
the Polish Collection of Microorganisms, and strains ter-
med LOCK were obtained from the Culture Collection of
Microorganisms of Institute of Fermentation Technology
and Microbiology (Lodz University of Technology). Bacte-
rial inocula were obtained from activated culture on a
Trypticase Soy Agar (TSA, Merck, Germany) medium,
Materials and Methods
Chemicals
2-Amino-2-methyl-1-propanol, ethanolamine, pyrrolidine,
morpholine, hydroxylammonium chloride, Dowex 50WX8,
Novozyme 435 (Candida antarctica lipase), and N,O-Bis
(trimethylsililyl)trifluoroacetamide + chlorotrimethylsilane
(BSTFA + TMCS) were purchased from Sigma-Aldrich
ꢁ
which were incubated for 24 hours at 30/37 C depending
on the strain. Yeast and molds inocula were prepared from
biomass previously incubated for 72 hours at 30 ꢁC on
Sabouraud Dextrose Agar (Merck, Germany).
ꢀ
(Poznan, Poland).
Preparation of (R)- and (S)-Ricinoleic Acid Derivatives
Silica gel for flash chromatography (catalog no. 7024-2;
J.T. Baker Co.) was purchased from Avantor Performance
Materials B.V. (Deventer, Netherlands). Gas chromatography
- mass spectrometry (GC–MS) was performed using a Trace
GC Ultra chromatograph coupled with a DSQII mass spec-
trometer (Thermo Scientific, Waltham, MA, USA) equipped
with a Rxi-1 ms capillary column (60 m long, 0.25 mm inside
diameter, and 0.25 μm film thickness), temperature program
RA derivatives were obtained from methyl esters of (R)-
and (S)-RA, which were prepared according to the previ-
ously published method (Kula et al., 2014). These substrates
were used for synthesis of chiral primary amides (2),
hydroxamic acids (3), and N-methyl amides (4).
Primary amides (2-R and 2-S) of RA were synthesized
using the enzymatic method catalyzed by Novozyme
435 (lipase from Candida antarctica) (García et al., 1994).
Hydroxamic acids (3-R and 3-S) and N-methyl amides
(4-R and 4-S) were synthesized based on previously published
methods (Devlin et al., 1975; Hauser and Renfrow, 1939).
Purity of the obtained derivatives was checked by GC
analysis. In some cases, GC analysis required derivatization
with a BSTFA + TMCS reagent. Therefore, 100 μL of
silylating reagent was added to ca. 10 mg of sample and
−1
−1
ꢁ
ꢁ
220 (2 min) to 330 C at 6 C min , split 150 mL min
ꢁ
ꢁ
(280 C), carrier gas—helium (300 kPa), Fid 300 C, and
injection 0.5 μL. H nuclear magnetic (1H NMR; 250 MHz)
1
and 13C NMR (62.90 MHz) were recorded using deutero-
chloroform solution (99.8% CDCl3, Sigma-Aldrich), unless
otherwise specified, with tetramethylsilane; (δ = 0 ppm) as the
internal standard (Bruker DPX-250 Avance; Bruker, Hanau,
Germany). 13C NMR multiplicity was determined using Dis-
tortionless Enhancement by Polarization Transfer (DEPT)
experiments. The purity of the products was confirmed by thin-
layer chromatography (TLC) and 13C NMR. Optical rotations
were measured with an Autopol IV polarimeter (Rudolph
Research, Flanders, NJ, USA), and Infrared (IR) spectra were
obtained with the Fourier-transform infrared spectroscopy
(FT-IR) spectrometer Nicolet 6700 (Thermo Scientific).
ꢁ
heated to 60 C for 20 min. Then, the silylated compound
was dissolved in 10% methanol concentration and sub-
jected to GC–MS analysis. All compounds were analyzed
1
by H NMR, 13C NMR, and IR, which confirmed their
structures. Moreover, specific optical rotations and, in the
case of solid compounds, melting points were determined.
(R,Z)-12-Hydroxyoctadec-9-Enamide (2-R)
Microorganism
Methyl esters of (R)-RA (1 g, 0.003 mol) were mixed with
Thirteen strains, either reference or environmental, were
used in this research. Among them, there were three Gram-
Novozyme 435 (380 mg) in 2% dioxane solution of NH3
(25 mL) at 30 C and centrifuged at 250 rpm for 96 hours.
ꢁ
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
The progress of the reaction was monitored by TLC. The
enzyme was filtered off and washed with dichloromethane.
Evaporation of the solvent provided a crude solid product,
which was purified by crystallization in acetone. Pure
amide 2-R (0.44 g) was a white solid of >98% purity (GC);
1H NMR (δ ppm): 5.54 (m, 3H; NH2; CH ), 5.39 (m,
1H; CH ), 3.60 (qn J = 6.3 Hz, 1H; CHOH), 2.21 (m,
4H; CHCH2CH ; CH2CO ), 2.04 (m, 2H), 1.62
(qn J = 7.3 Hz, 2H), 1.45 (m, 3H), 1.30 (m, 15H), 0.87
(t J = 6.5 Hz; CH3); 13C NMR: 176.08 (s; CO ),
855.2; and GC–MS (EI, 70 eV) m/z (%): 529 (0) [M+],
282 (2), 188 (14), 187 (97), 103 (31), 97 (27), 75 (20),
73 (100), 55 (32), 41 (13).
(S,Z)-N,12-Dihydroxyoctadec-9-Enamide (3-S)
Hydroxamic acid 3-S was synthesized in the same way as
compound 3-R, and its purity was >98% (GC after derivati-
zation of the sample). Its IR, MS, and NMR spectra mat-
ched those determined for enantiomer 3-R.
133.10 (d;
CH ), 125.24 (d;
CH ), 71.39 (d;
CHOH ), 36.74 (t), 35.82 (t), 35.26 (t), 31.75 (t), 29.43
(t), 29.27 (t), 29.02 (t, 2xCH2), 28.94 (t), 27.23 (t), 25.63
(t), 25.38 (t), 22.53 (t), 14.01 (q; CH3); IR (cm−1, neat):
3353.6, 3184.0, 2922.4, 2850.8, 1658.4, 1631.8, 1469.4,
1072.9, 813.1; and GC MS (EI, 70 eV) m/z (%):
297 (0) [M+], 279 (1) [M+–18], 183 (5), 97 (10), 72 (54),
69 (15), 59 (100), 55 (70), 44 (22), 43 (35), 41 (36).
(R,Z)-12-Hydroxy-N-Methyloctadec-9-Enamide (4-R)
A solution of potassium hydroxide (1.1 g, 0.019 mol) in
methanol (5 mL) was added to a solution of methylamine
hyꢁdrochloride (0.9 g, 0.013 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 esters of RA (1-R;
2 g, 0.006 mol) and, after thorough mixing, was maintained
at room temperature for 24 hours. Then, Dowex 50WX8
resin was added to obtain a pH of 6, and the solution was
filtered. The solvent was evaporated, and the crude product
was purified on a silica gel column using ethyl acet-
ate/hexane (40:60, v/v) as an eluent, providing compound
(S,Z)-12-Hydroxyoctadec-9-Enamide (2-S)
It was prepared analogously as compound 2-R. Amide 2-S
was a solid of >98% purity (GC). Its IR, MS, and NMR
spectra matched those determined for compound 2-R.
1
(R,Z)-N,12-Dihydroxyoctadec-9-Enamide (3-R)
4-R of >99% purity (GC); H NMR (δ ppm): 5.54 (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; NHCH3), 2.18
(m, 4H), 2.04 (m, 2H), 1.35 (br.m, 18H), 0.87 (t J = 6.5 Hz,
3H; CH3); 13C NMR: 173.92 (s; CO ), 132.78 (d;
CH ), 125.24 (d; CH ), 71.26 (d; CHOH ), 36.66
(t), 36.36 (t), 35.19 (t), 31.67 (t), 29.36 (t), 29.19 (t), 29.06
(t), 29.01 (t), 28.89 (t), 27.15 (t), 26.01 (q; NHCH3),
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 to the boiling point of the solvent.
ꢁ
Then, both were cooled to 30 C, and a solution of KOH
was added to the NH2OHꢀHCl solution. 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 RA (1-R; 2 g, 0.006 mol) and, after thor-
ough mixing, was maintained at room temperature for
24 hours. Then, Dowex 50WX8 resin was added to obtain
a pH of 6, and the solution was filtered. Evaporation of
methanol delivered a crude product, which was subjected
to crystallization in acetone giving pure (>96%, GC after
25.58 (t), 25.55 (t), 22.44 (t), 13.90 (q; CH3); IR (cm−1
,
neat): 3297.6, 2925.2, 2854.4, 1648.1, 1560.9, 1046.9,
725.7; and GC–MS (EI, 70 eV) m/z (%): 311 (0) [M+],
293 (1) [M+–18], 97 (8), 86 (53), 73 (100), 69 (12),
58 (36), 55 (56), 43 (21), 41 (23).
(S,Z)-12-Hydroxy-N-Methyloctadec-9-Enamide (4-S)
1
derivatization of the sample) hydroxamic acid; H NMR
The method for the preparation of compound 4-S was anal-
ogous to the one used for its enantiomer 4-R (purity >99%,
GC). Its IR, MS, and NMR spectra were the same as those
of derivative 4-R.
(500 MHz, δ 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; CHCH2CH ), 2.11 (t J = 7.3 Hz,
2H; CH2CO ), 2.03 (m, 2H; CHCH2CH2 ), 1.59 (m,
2H), 1.46 (m, 3H), 1.29 (m, 16H), 0.87 (t J = 6.8 Hz, 3H;
CH3); 13C 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); IR (cm−1, neat): 3403.3,
3278.5, 3015.5, 2918.6, 2847.6, 1662.6, 1621.2, 1077.4,
Preparation of (R)- and (S)-3-Hydoxynonanoic Acid
Derivatives
The first step was preparation of dimethyl acetals of
3-hydroxynonanal, which were then oxidized to corresponding
methyl esters (5-R and 5-S) in the way described by Takeda
et al. (Takeda et al., 1997).
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
ꢁ
Methyl esters of (R)- and (S)-3-hydroxynonanoic acid
(5-R and 5-S) were used as substrates for the production of
corresponding derivatives. A series of chiral cyclic and acy-
clic amides was synthesized using different methods.
Primary amides (6-R and 6-S) of 3-hydroxynonanoic
acids were obtained using a simple reaction of methyl
esters (R)- and (S)-3-hydroxynonanoic acid with water
solution of ammonia.
Chiral hydroxamic acids (7-R and 7-S) derived from
3-hydroxynonanoic acid were obtained from the reaction of
methyl esters with hydroxylamine in the presence of KOH
in methanol based on previously described methods
(Devlin et al., 1975; Hauser and Renfrow, 1939).
methanol (200 mL) and cooled to 0 C. After that, 35%
HCl (15.5 g, 0.15 mol) and then 30% H2O2 (17 g,
0.15 mol) were added carefully during intensive ꢁstirring
while the temperature was maintained at below 5 C. The
stirring was continued for ꢁ30 min in an ice bath. Then, the
solution was heated to 40 C and stirred while maintaining
this temperature for 3 hours. About 100 mL of methanol
was evaporated, and after addition of water (80 mL),
extraction with diethyl ether was performed (3 × 100 mL).
The organic layer was washed with aqueous sodium sulfide
(80 mL) and then with water (80 mL) and was dried over
anhydrous MgSO4. The solvent was evaporated, and the
crude product was purified on column chromatography
using hexane/ethyl acetate (80:20 v/v) as an eluent to
obtain pure (>97%, GC) methyl ester of (R)-
3-hydroxynonanoic acid (5-R) (9.1 g, 49% yield from ace-
tal and 37% considering castor oil); 1H NMR (δ ppm): 3.95
N-methyl amides of 3-hydroxynonanoic acid (8-R and
8-S) were obtained in the same manner as analogues com-
pounds derived from RA, i.e., 4-R and 4-S. Methyl esters
of 3-hydoxynonanoic acids were subjected to a reaction
with methanolic alkaline solution of methylamine.
(m, 1H;
CHOH), 3.66 (s, 3H;
OCH3), 2.47
Amides of 3-hydroxynonanoic acid with 2-amino-
2-methyl-1-propanol (9), ethanolamine (10), morpholine
(11), and pyrrolidine (12) were synthesized from methyl
esters of (R)- and (S)-3-hydroxynonanoic acid and the
corresponding amine, i.e., 2-amino-2-methyl-1-propanol,
ethanolamine, morpholine, and pyrrolidine (amines used in
sixfold molar excess). The reagents were mixed together
and heated at a suitable temperature, maintaining gentle
reflux for 2–12 hours. The progress of the reaction was
monitored using TLC analysis. Then, the crude amides
were purified by flash chromatography with silica gel using
ethyl acetate/hexane or ethyl acetate/methanol as an eluent.
Analogues methods for preparation were used in our previ-
ous studies in which the corresponding amides of RA were
produced (Matysiak et al., 2018).
(dd J = 16.3 Hz, J = 3.8 Hz, 1H; CHCH2CO ), 2.36
(dd J = 16.3 Hz, J = 8.8 Hz, 1H; CHCH2CO ), 1.31
(br. m, 11H), 0.83 (t J = 6.6, 3H; CH3); 13C NMR:
173.34 (s;
CO ), 67.90 (d;
CHOH), 51.57 (q;
OCH3), 41.12 (t), 38.48 (t), 31.65 (t), 29.07 (t), 25.33 (t),
22.47 (t), 13.92 (q; CH3); IR (cm−1, neat): 3473.3,
2953.9, 2927.9, 1731.1, 1162, 1055.6, 864.6; and GC MS
(EI, 70 eV) m/z (%): 188 (0) [M+], 170 (1) [M+–18],
138 (4), 103 (100), 96 (9), 74 (36), 71 (38), 61 (16),
55 (21), 43 (43), 41 (22).
Methyl Ester of (S)-3-Hydroxynonanoic Acid (5-S)
Methyl (S)-ricinoleate (1-S) (71.5 g, 0.23 mol) was dis-
solved in methanol (250 mL), and the mixture was cooled
3
ꢁ
Purity of the obtained derivatives was confirmed by GC
analysis. In some cases, derivatization with BSTFA +
TMCS reagent (described vide supra) was necessary to per-
form GC analysis. Moreover, all compounds were analyzed
to −10 C. Oxygen containing 160 g of ozone in Nm at a
gas flow of 0.4 g min−1 was passed through the solution
until a positive test with potassium iodide was obtained
(ozonation time: 2.5 hours). Then, dimethyl sulfide (DMS,
50 g, 0.8 mol) was added, and the mixture was stirred at
1
by H NMR, 13C NMR, and IR, which made it possible to
ꢁ
confirm their structures. For all the obtained derivatives,
specific optical rotations were verified. What is more, in the
case of solid compounds, melting points were determined.
temperatures below 0 C for 1.5 hours. The reaction mix-
ture was left overnight at room temperature until the total
decomposition of peroxides occurred, which was controlled
by the potassium iodide test. Next, the solution was cooled
to −5 ꢁC, and 35% HCl (4.5 mL) in methanol (75 mL) was
added carefully during intensive stirring at a temperature
Methyl Ester of (R)-3-Hydroxynonanoic Acid (5-R)
ꢁ
Dimethyl acetal of (R)-3-hydroxynonanal was obtained
from castor oil by ozonolysis, which was performed
according to a previously described method (Kula et al.,
2000). Pure acetal (>96%, GC) was isꢁolated 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).
In the next step, oxidation of (R)-3-hydroxynonanal
dimethyl acetal to a corresponding methyl ester (5-R) was
carried out. Acetal (20 g, 0.098 mol) was dissolved in
below 0 C. Stirring was continued for 2 hours at room
temperature and left overnight. The next step was the addi-
tion of KOH (40 g, 0.07 mol) solution in water (50 mL) at
ꢁ
0–5 C. Excess of DMS with a bit of methanol (entirely
100 mL) was distilled, and the residue was refluxed for
3 hours. After vacuum evaporation of methanol and addi-
tion of water (100 mL), extraction with diethyl ether
(3 × 100 mL) was carried out. The ether solution was neu-
trally washed with water and dried over anhydrous MgSO4.
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
Then, the solvent was evaporated, and the crude product
was isolated by vacuum distillation giving pure (>97%,
GC) acetal of (S)-hydroxynonanal (32 g, 68% yield); bp:
temperature for 24 hours. White crystals appearing in the
mixture were dissolved by heating the methanol solution;
then, Dowex 50WX8 resin was added to obtain a pH of
6. The mixture was filtered and evaporated by vacuum dis-
tillation. Pure (>98%, GC after derivatization of sample)
hydroxamic acid 7-R (1.91 g) was obtained by crystalliza-
89–88 ꢁC/0.2 Torr; ½αꢂD23 –11.95 (c = 5.08, CHCl3).
Next, the obtained acetal was oxidized to corresponding
methyl ester in the same way as compound 5-R. Pure (97%,
GC) methyl ester of (S)-3-hydroxynonanoic acid (5-S;
15.1 g) was obtained with 51% yield considering dimethyl
acetal of 3-hydroxynonanal. The yield of the whole process
was 35%. Its IR, MS, and NMR spectra matched those
reported for enantiomer 5-R.
1
tion in acetone; H NMR (CD3OH, δ ppm): 5.05 (br.s, 3H;
NH ; 2xOH), 3.95 (m, 1H; CHOH), 2.20 (m, 2H;
CHCH2CO ), 1.45 (m, 3H), 1.30 (m, 7H), 0.89
(t J = 6.5 Hz, 3H; CH3); 13C NMR (CD3OH): 170.96 (s;
CO ), 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); IR (cm−1
,
neat): 3361.1, 3294.3, 2956.2, 2922.6, 2850.9, 1647.6,
1606.4, 1081.2, 977.2; and GC–MS (EI, 70 eV) m/z (%):
405 (0) [M+], 187 (63), 103 (19), 100 (19), 97 (8), 75 (15),
73 (100), 69 (26), 55 (39), 45 (12), 43 (11).
(R)-3-Hydroxynonanamide (6-R)
Methyl ester of (R)-3-hydroxynonanoic acid (5-R; 2 g,
0.011 mol) was mixed with 25% water solution of ammo-
ꢁ
nia (8 mL) at 30 C for 24 hours. The progress of the reac-
(S)-N,3-Dihydroxynonanamide (7-S)
tion was monitored by TLC. The precipitated crystals were
filtered off and purified by crystallization in acetone. Amide
6-R (0.91 g) was a white solid of >99% purity (GC); H
Hydroxamic acid 7-S (purity >97%, GC after derivatization
of sample) was prepared in the same way as its enantiomer
7-R. Its IR, MS, and NMR spectra matched those deter-
mined for compound 7-R.
1
NMR (δ ppm): 5.94 (s, 1H; NH2), 5.77 (s, 1H; NH2),
4.00 (m, 1H; CHOH), 3.40 (br.s, 1H; OH), 2.41
(dd J = 15.5 Hz, J = 3 Hz, 1H; CHCH2CO ), 2.30
(dd J = 15.5 Hz, J = 8.8 Hz, 1H; CHCH2CO ), 1.41 (br.
m, 10H), 0.88 (t J = 6.6 Hz, 3H; CH3); 13C 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); IR (cm−1, neat): 3349, 3176.1, 2951.8, 2921.5,
2844.4, 1631.4, 1599.4, 1095.4, 862.3; and GC–MS (EI,
70 eV) m/z (%): 173 (0) [M+], 155 (1) [M+–18], 88 (100),
71 (10), 59 (49), 55 (17), 45 (16), 44 (24), 43 (32),
41 (21), 39 (16).
(R)-3-Hydroxy-N-Methylnonanamide (8-R)
Pure (>99%, GC) amide 8-R was obtained in the same
1
manner as compounds 4-R and 4-S; H NMR (δ ppm):
6.18 (br.s, 1H; NH ), 3.95 (m, 1H; CHOH), 3.74 (br.s,
1H; OH), 2.79 (d J = 4.8 Hz, 3H; NHCH3), 2.35
(dd J = 15.3 Hz, J = 3 Hz, 1H; CHCH2CO ), 2.22
(dd J = 15.3 Hz, J = 8.8 Hz, 1H; CHCH2CO ), 1.40 (br.
m, 10H), 0.85 (t J = 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; NHCH3), 25.40 (t), 22.52 (t), 14.01
(q; CH3); IR (cm−1, neat): 3289.7, 3092, 2925.2, 2846.1,
1640.5, 1559.8, 1402.1, 1071.7, 861.4; and GC–MS (EI,
70 eV) m/z (%): 186 (1) [M+–1], 102 (100), 73 (56), 69 (8),
58 (63), 55 (23), 45 (32), 43 (33), 41 (23), 39 (6).
(S)-3-Hydroxynonanamide (6-S)
The reaction was carried out analogously as performed for
compound 6-R, producing amide 6-S of >99% purity (GC).
Its IR, MS, and NMR spectra matched those reported for
enantiomer 6-R.
(S)-3-Hydroxy-N-Methylnonanamide (8-S)
(R)-N,3-Dihydroxynonanamide (7-R)
Compound 8-S was obtained according to the method
described for amides 4-R and 4-S. Crystallization in ace-
tone produced pure (>99%, GC) amide 8-S. Its IR, MS,
and NMR spectra determined for this compound matched
those obtained for its enantiomer 8-R.
By heating to the boiling point of the solvent, a solution of
potassium hydroxide (3.6 g, 0.064 mol) in methanol
(10 mL) and a solution of hydroxylamine hydrochloride
(3 g, 0.043 mol) in methanol (20 mL) were obtained. In the
ꢁ
next step, both were cooled to 30 C, and the solution of
KOH was added to the NH2OHꢀHCl solution. The mixture
was stirred in an ice bath for 15 min, and then, the precipi-
tated potassium chloride was filtered off. The filtrate was
mixed with methyl ester of (R)-3-hydroxynonanoic acid
(5-R; 4 g, 0.021 mol) and then maintained at room
N-(1-Hydroxy-2-Methylpropan-2-Yl)-(3R)-
hydroxynonanamide (9-R)
ꢁ
Reaction conditions: 130 C, 2.5 hours. Flash chromatogra-
phy: silica gel, ethyl acetate/hexane (50:50, v/v). Amide 9-R
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
was a light yellow dense liquid of 98% purity (GC after
derivatization of the sample); H NMR (δ ppm): 6.65 (s,
Morpholinyl-(3R)-hydroxynonanamide (11-R)
1
ꢁ
1H; NH ), 5.15 (br.s, 1H; CHOH), 4.24 (br.s, 1H;
CH2OH), 3.83 (m, 1H; CHOH), 3.51 (d J = 4 Hz, 1H;
CH2OH), 3.40 (d J = 4 Hz, 1H; CH2OH), 2.25
(dd J = 5.4 Hz, J = 1.1 Hz, 1H; CHCH2CO ), 2.14
(dd J = 5.3 Hz, J = 3.3 Hz, 1H; CHCH2CO ), 1.40 (m,
1H), 1.32 (m, 2H), 1.19 (m, 13H), 0.88 (t J = 6.8 Hz, 3H;
CH2CH3); 13C NMR: 173.20 (s; CO ), 69.51 (t;
CH2OH), 68.59 (d; CHOH), 55.55 (s; C(CH3)2 ),
43.29 (t; CH2CO ), 36.89 (t), 31.55 (t), 29.00 (t), 25.26
(t), 24.28 (q; C(CH3)2 ), 23.94 (q; C(CH3)2 ), 22.35
(t), 13.82 (q; CH2CH3); IR (cm−1, neat): 3243.2, 3074.5,
2926.6, 2856.4, 1638.9, 1553.2, 1056.8, 784.9; and GC–MS
(EI, 70 eV) m/z (%): 461 (0) [M+], 374 (4), 286 (22),
246 (6), 187 (7), 145 (13), 144 (33), 75 (23), 73 (73),
58 (100), 55 (12).
Reaction conditions: 120 C, 12 hours. Flash chromatogra-
phy: silica gel, hexane/ethyl acetate (50:50, v/v). The reac-
tion provided amide 11-R with 96% purity, confirmed by
1
GC; H NMR (500 Hz; δ ppm): 4.01 (m, 2H; CHOH;
OH), 3.62 (m, 5H; CH2OCH2CH2 ), 3.53 (m, 1H;
OCH2CH2 ), 3.40 (m, 2H;
OCH2CH2 ), 2.40
(d J = 16 Hz, 1H; CHCH2CO ), 2.27 (dd J = 16.5 Hz,
J = 9.5 Hz, 1H; CHCH2CO ), 1.49 (m, 1H), 1.38 (m,
2H), 1.23 (br. m, 7H), 0.82 (t J = 6.3 Hz, 3H; CH3); 13C
NMR: 171.14 (s; CO ), 67.78 (d; CHOH), 66.61 (t;
CH2O ), 66.32 (t; CH2O ), 45.56 (t; CH2N ),
41.57 (t; CH2N ), 39.11 (t), 36.32 (t), 31.63 (t), 29.11
(t), 25.38 (t), 22.44 (t), 13.93 (q; CH3); IR (cm−1, neat):
3429.2, 2924.2, 2856.4, 1624.4, 1441, 1114.4, 1035.5,
852.8; and GC–MS (EI, 70 eV) m/z (%): 243 (3) [M+],
225 (16) [M+–18], 158 (99), 129 (48), 114 (92), 86 (61),
70 (59), 57 (100), 55 (65), 43 (75).
N-(1-Hydroxy-2-Methylpropan-2-Yl)-(3S)-
hydroxynonanamide (S-9)
Morpholinyl-(3S)-hydroxynonanamide (11-S)
Pure (97%, GC after derivatization of the sample) amide 9-
S was obtained analogously as compound 9-R. Its IR, MS,
and NMR spectra matched those reported for enantiomer
9-R.
Compound 11-S was obtained in the same way as amide
11-R (>99% purity, GC). The IR, MS, and NMR spectra
determined for this compound matched those obtained for
its enantiomer 11-R.
N-(2-Hydroxyethyl)-(3R)-hydroxynonanamide (10-R)
Pyrrolidinyl-(3R)-hydroxynonanamide (12-R)
ꢁ
Reaction conditions: 130 C, 2 hours. Flash chromatogra-
ꢁ
phy: silica gel, ethyl acetate/methanol (97:3, v/v). Amide
10-R was of 99% purity, confirmed by GC analysis; H
Reaction conditions: 75–80 C, 3 hours. Flash chromatog-
1
raphy: silica gel, ethyl acetate/hexane (95:5, v/v). The prod-
uct was obtained with >99% purity (GC); 1H NMR
(500 Hz, δ ppm): 3.96 (m, 2H; CHOH, OH), 3.41
NMR (δ ppm): 7.04 (t J = 5.1 Hz, 1H; NH ), 4.41 (br.s,
2H), 3.95 (m, 1H; CHOH), 3.63 (m, 2H; CH2OH), 3.42
(m, 1H; NHCH2 ), 3.29 (m, 1H; NHCH2 ), 2.38
(dd J = 14.6 Hz, J = 2.4 Hz, 1H; CHCH2CO ), 2.24
(dd J = 14.6 Hz, J = 9.4 Hz, 1H; CHCH2CO ), 1.39 (br.
m, 10H), 0.85 (t J = 6.8 Hz, 3H; CH3); 13C NMR:
(t J
=
6.8 Hz, 2H;
NCH2CH2 ), 3.34 (m, 2H;
NCH2CH2 ), 2.38 (dd J = 16.3 Hz, J = 2.3 Hz, 1H;
CHCH2CO ), 2.22 (dd J = 16.5 Hz, J = 9.5 Hz, 1H;
CHCH2CO ), 1.91 (qn J = 6.6 Hz, 2H; NCH2CH2 ),
1.82 (m, 2H; NCH2CH2 ), 1.50 (m, 1H), 1.38 (m, 2H),
1.24 (br.m, 7H), 0.82 (t J = 6.8 Hz, 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); IR (cm−1, neat):
3410.1, 2926.9, 2857.4, 1618.2, 1449.7, 1044.6, 857.9; and
GC–MS (EI, 70 eV) m/z (%): 227 (2) [M+], 209 (10),
156 (7), 142 (100), 113 (60), 98 (94), 85 (15), 70 (50),
55 (50), 43 (43).
173.32 (s;
CO ), 68.70 (d;
CHOH), 61.25 (t;
CH2OH), 43.27 (t;
CHCH2CO ), 42.03 (t;
NHCH2 ), 37.19 (t), 31.73 (t), 29.18 (t), 25.51 (t), 22.54
(t), 14.00 (q; CH3); IR (cm−1, neat): 3298.6, 2920.9,
2848.5, 1643.6, 1560.7, 1081.1, 1058.4, 860.9; and GC–
MS (EI, 70 eV) m/z (%): 217 (1) [M+], 199 (1) [M+–18],
174 (16), 139 (25), 132 (100), 114 (29), 88 (30), 62 (35),
60 (40), 55 (61), 43 (71).
N-(2-Hydroxyethyl)-(3S)-hydroxynonanamide (10-S)
Pyrrolidinyl-(3S)-hydroxynonanamide (12-S)
The reaction was carried out analogously to the compound
10-R, which gave a white solid amide 10-S of >99% purity
(GC). Its IR, MS, and NMR spectra matched those reported
for enantiomer 10-R.
Amide 12-S of >99% purity (GC) was obtained in the same
way as compound 12-R. The obtained IR, MS, and NMR
spectra were in accordance with those for enantiomer 12-R.
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
Evaluation of Antimicrobial Activity
Results and Discussion
The antimicrobial activity of the obtained derivatives of
ricinoleic and 3-hydroxynonanoic acids was evaluated by
the microdilution method described earlier (Khalifa et al.,
2013). The results were presented as the minimal inhibitory
concentrations (MIC).
A total of 24 compounds were synthesized and character-
ized in this study. They represented derivatives of ricinoleic
and 3-hydroxynonanoic acid, all synthesized as pairs of
enantiomers. The methyl esters of both mentioned acids
were used as starting materials for the preparation of the
corresponding amides. Eight compounds derived from (R)-
and (S)-RA were synthesized, including methyl esters (1),
primary amides (2), hydroxamic acids (3), or N-methyl
amides (4) (Table 1; Scheme 1). Methyl ricinoleates have
previously been synthesized and characterized spectrally,
which is why we used the method described for their prepa-
ration (Kula et al., 2014). Moreover, they were tested in
terms of antimicrobial activity in our previous study
(Matysiak et al., 2018).
First, depending on the solubility of the tested deriva-
tives, 20% or 10% ethanol solutions (w/w) were prepared.
Then, 96-well microtiter plates were filled with 0.1 mL of
medium (Tripticase Soy Broth for bacteria and Sabouraud
broth for yeast and molds) with the tested substance in an
appropriate concentration. Each well was inoculated with
activated bacterial, yeast, or mold cell suspension in saline
(~106 CFU mL−1). Bacteria were incubated for 24 hours at
ꢁ
30/37 C (depending on the strain), ꢁand yeast and molds
were grown for up to 3 days at 30 C. After incubation,
0.01 mL of 0.01% resazurin solution was added to the
plates inoculated with bacteria, and they were incubated for
2 hours in 30 ꢁC. The MIC values were noted based on the
change of color of the solution in the well from blue to
pink, which indicates bacteria growth. For yeast and molds,
MIC values were estimated by relying on macroscopic
observation of the growth in the well. The experiments
were conducted in triplicate for each microorganism. Gen-
tamicin and nystatin were used as reference drugs for bacte-
ria and fungi, respectively. Moreover, the noninoculated
growth media with 20% or 10% ethanol solutions of the
tested compounds and the growth media inoculated by
microbial suspension were used as negative and positive
controls, respectively.
Primary amides of ricinoleic (2-R and 2-S) acid were
synthesized using a method catalyzed by Novozyme
435 and described by Garcia et al. (1994). The reaction was
ꢁ
performed in 2% solution of NH3 in dioxane at 30 C for
96 hours followed by crystallization in acetone, which gave
pure products with good yield of 46–52%. Information
about derivative 2-R could be found in the literature
(Galstukhova, 1960), while its S-enantiomer was a new
compound synthesized and described for the first time.
Chiral methyl ricinoleates were also used for the produc-
tion of hydroxamic acids (3-R and 3-S) and N-methyl
amides (4-R and 4-S). Simple procedures based on previ-
ously published methods (Devlin et al., 1975; Hauser and
Renfrow, 1939) were used and developed to obtain these
derivatives with a yield of 40–62%. Literature data provide
Table 1 (R)- and (S)-ricinoleic acid derivatives
NR1R2
Yielda [%]
Melting point [ꢁC]
23
Symbol
Esters
½αꢂD
1-R
OMe
62
54
+2.8 (c = 2.23, CHCl3)
—
—
1-S
−2.9 (c = 2.21, CHCl3)
Amides
2-R
2-S
3-R
3-S
4-R
4-S
NH2
46
52
52
40
62
55
+2.92 (c = 5.2, CHCl3)
−2.95 (c 5.05, CHCl3)
+1.26 (c = 5.0, CHCl3)
−1.38 (c = 5.25, CHCl3)
+2.35 (c = 5.05, CHCl3)
−2.31 (c = 5.1, CHCl3)
64.8–67.7 (acetone)b
64.1–66.0 (acetone)
64.9–66.3 (acetone)
64.6–65.7 (acetone)
NHOH
NHMe
c
—
—
a Isolated yields.
b Lit. mp: 64–65.5 ꢁC (Galstukhova, 1960).
c Lit. mp: 30.5–31.5 ꢁC (Applewhite et al., 1963), semisolid consistency of this derivative hindered determination of this parameter during our
research.
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
Scheme 1 Synthesis of (R)- and (S)-ricinoleic acid derivatives (presented in Table 1)
some information about R-configured N-methyl amide of
RA 4-R, including the method for its preparation and its
basic characterization (Applewhite et al., 1963).
research (Table 2; Scheme 2). The work began with the
preparation of chiral substrates, which were
3-hydroxynonanoate methyl esters (5-R and 5-S). The liter-
ature review presents some methods for the production of
5-R by ozonolysis of castor oil (Ishmuratov et al., 2006,
A
series of 16 derivatives of (R)- and (S)-
3-hydroxynonanoic acid (5–12) was synthesized during our
Table 2 (R)- and (S)-3-hydroxynonanoic acid derivatives
23
Symbol
Esters
5-R
NR3R4
Yielda [%]
Melting point [ꢁC]
½αꢂD
OMe
37
35
−19.3 (c = 5.10, CHCl3)b
+18.1 (c = 5.01, CHCl3)
-
-
5-S
Amides
6-R
NH2
50
58
47
41
31
31
95
87
−26.77 (c = 0.31, CHCl3)
+26.24 (c = 0.32, CHCl3)
−4.36 (c = 2.75, MeOH)
+4.11 (c = 2.65, MeOH)
−25.03 (c = 5, CHCl3)
+24.83 (c = 5, CHCl3)
−14.76 (c = 5.45, CHCl3)
+16.01 (c = 5.65, CHCl3)
101.8–103.7 (acetone)
101.4–102.4 (acetone)
104.8–107.8 (acetone)
104.8–108.6 (acetone)
78.5–80.4 (acetone)
81.1–82.9 (acetone)
-
6-S
7-R
7-S
8-R
8-S
9-R
9-S
NHOH
NHMe
-
10-R
10-S
11-R
11-S
74
66
49
52
−9.70 (c = 5.1, CHCl3)
+9,98 (c = 5, CHCl3)
75,8–76.6 (ethyl acetate)
75.5–77.4 (ethyl acetate)
−41.05 (c = 5.25, CHCl3)
+36.10 (c = 5.15, CHCl3)
-
-
12-R
12-S
67
86
−42.05 (c = 5.20, CHCl3)
+42.30 (c = 5.30, CHCl3)
-
-
a Isolated yields.
b Lit. ½αꢂ2D5–20.0 (Odham and Samuelsen, 1970), ½αꢂD25–16.0 (c 1.48, CHCl3) (Jiang et al., 2010).
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
Scheme 2 Synthesis of (R)- and (S)-3-hydroxynonanoic acid derivatives (see Table 2)
2007, 2009). However, none of them led to a pure product
because mixtures of compounds were obtained in all publi-
shed studies. The multistep method of obtaining
3-hydroxynonanoate methyl esters from castor oil and
methyl (S)-ricinoleate described in this article using
ozonolysis, and the subsequent oxidation, is a new proce-
dure, although it is based on known reactions. The total
yield of the developed method was 35–37%, which is a sat-
isfactory result considering the complexity of the reaction.
In the next steps of our research, simple and efficient
methods for preparation of different primary and second-
ary, cyclic and acyclic amides, including hydroxamic acids,
were developed. Primary amides of (R)- and (S)-
3-hydroxynonanoic acid were obtained by mixing starting
methyl esters (5-R and 5-S) with 25% NH3 solution in
pyrrolidine (12-R and 12-S) were synthesized using a free-
of-solvent procedure. Reactions were conducted by heating
(under gentle reflux) of starting methyl esters (5-R and 5-S)
with the corresponding amines. Pure amides (9–12) were
obtained by purification using column chromatography
with a 39–95% yield. Great performances were observed in
the case of amides with 2-amino-2-methyl-1-propanol (9-R
and 9-S). The obtained results (87–95% yield) were quite
surprising considering our previous studies in which ana-
logues amides were synthesized from methyl esters of
RA. The reactions with methyl ricinoleates were conducted
in a similar manner as in the present studies, but even after
9 hours of heating, they resulted in significantly lower
yields (43–48%) (Matysiak et al., 2018). In general, the
proposed method for the preparation of amides 9–12 was
simple, fast, and efficient, and due to the lack of solvent
during the reaction, it can be considered environment
friendly.
In these studies, 24 chiral compounds (12 pairs of enan-
tiomers), including methyl esters, hydroxamic acids, and
amides derived from ricinoleic and 3-hydroxynonanoic
acids, were obtained, and 19 of them were synthesized and
spectrally and polarimetrically characterized for the first
time. All derivatives were of high purity, more than 96%,
which was confirmed by GC analysis, in some cases pre-
ceded by derivatization of the samples. The structures of
the synthesized compounds were confirmed using 1H
NMR, 13C NMR, GC–MS, and IR. Moreover, optical rota-
tions and, in the case of solid products, melting points were
also determined.
ꢁ
water at 30 C, which led to compounds 6-R and 6-S with
more than 50% yield.
Based on articles describing the preparation of
hydroxamic acids (Devlin et al., 1975; Hauser and
Renfrow, 1939), simple and efficient methods for the pro-
duction of hydroxamic acids (7-R and 7-S) and N-methyl
amides (8-R and 8-S) derived from 3-hydroxynonanoic
acid have been developed. These compounds were pre-
pared
using
reactions
of
starting
methyl
3-hydroxynonanoates (5-R and 5-S) with the corresponding
amines salts in methanol, conducted at room temperature
for 24 hours. Hydroxamic acids (7-R and 7-S) and N-
methyl amides (8-R and 8-S) were obtained with 50–58%
and 31% yields, respectively.
Then, amides of (R)- and (S)-3-hydroxynonanoic acids
with 2-amino-2-methyl-1-propanol (9-R and 9-S), ethanol-
amine (10-R and 10-S), morpholine (11-R and 11-S), and
In the next step, the microdilution method was used to
evaluate the antimicrobial activity of the obtained amide
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
Reference druga
Table 3 Antimicrobial activity of (R)- and (S)-ricinoleic acid derivatives in MIC (%)
Microorganism
2-R
2-S
3-R
3-S
MIC (%)
4-R
4-S
Gram-positive bacteria
Bacillus subtilis
1.25
10
1.25
10
5
2.5
2.5
2.5
0.0049
0.0006
0.0098
Micrococcus flavus
Staphylococcus aureus
Gram-negative bacteria
Escherichia coli
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.5
>10
1.25
1.25
1.25
>10
>10
1.25
1.25
2.5
>10
1.25
1.25
2.5
>10
2.5
>10
5
0.0098
0.0049
0.0012
0.0195
0.0390
Pseudomonas aeruginosa
Pseudomonas cedrina
Pseudomonas fluorescens
Serratia liquefaciens
Yeast
1.25
1.25
>10
1.25
2.5
1.25
2.5
10
>10
>10
>10
Candida albicans
Candida krusei
5
1.25
5
5
1.25
5
<0.078
0.625
<0.078
0.625
10
10
10
>10
>10
10
0.0006
<0.0003
<0.0003
Candida tropicalis
Molds
<0.078
<0.078
Aspergillus brasiliensis
Penicillium expansum
1.25
1.25
1.25
1.25
0.625
0.156
0.312
0.156
1.25
2.5
0.0006
0.625
0.625
<0.0003
a Gentamicin for bacteria resistance evaluation; nystatin for yeast and mold resistance evaluation.
derivatives of ricinoleic and 3-hydroxynonanoic acids
against the selected microorganisms, including bacteria,
yeast, and molds. In this investigation, the MIC values of
all synthesized compounds were determined, and the results
are presented in Tables 3 and 4.
It can be observed that (R)- and (S)-RA derivatives differed
in antimicrobial activity depending on the microorganism
tested. Their antibacterial activity against both Gram-positive
and Gram-negative bacteria varied substantially. However,
the MIC at a level of 1.25% against bacteria from the genus
Pseudomonas, which are usually resistant to many
antibacterial substances, indicates their promising antimicro-
bial potential (Gross and Loper, 2009; Poole, 2011). The
weakest inhibitory activity was observed against the follow-
ing bacteria: M. flavus, S. aureus, E. coli, and S. liquefaciens
with MIC values equal or greater than 10%.
Most of the synthesized RA derivatives showed weak anti-
fungal potential to the selected yeast and molds, apart from
the hydroxamic acids 3-R and 3-S. These compounds were
very toxic to the selected yeast, especially against two spe-
cies, i.e., C. albicans and C. tropicalis (MIC values amounted
to less than 0.078%). The susceptibility of C. krusei to these
compounds was about 10 times greater than the other can-
didas tested. Compounds 3-R and 3-S showed the best activ-
ity against molds as well. However, P. expansum was more
susceptible to these derivatives (with MIC 0.156%) than the
second selected mold A. brasiliensis, for which higher MIC
values (0.312% and 0.625%) were determined. Moreover, dif-
ferences in the activity of the opposite enantiomers against
A. brasiliensis were observed, and the S-configured one
showed better activity.
The selected yeast proved to be strongly resistant to
compounds 4-R and 4-S (MIC amounted 10% and >10%),
which were N-methyl amides of RA. On the other hand,
these derivatives were active against the tested molds, and
it was observed that P. expansum was susceptible to them
at MIC values of 0.625%. Compounds 4-R and 4-S
exhibited two and four times weaker activity against
A. brasiliensis, respectively. Inhibitory activity of primary
amides 2-R and 2-S against the tested yeast and molds was
weak, with the MIC values ranging from 1.25% to 5%.
The antimicrobial activity of (R)- and (S)-
3-hydroxynonanoic acid derivatives (Table 4) against the
tested microorganisms was very diverse depending on sub-
stituents implemented in the structure of 3-hydroxynonanoic
acid. Starting methyl esters of 3-hydroxynonanoic acid 5-R
and 5-S showed weak activity against Gram-negative and
Gram-positive bacteria and most of the tested yeast. How-
ever, it was observed that they were active against C. krusei
yeast (MIC values 0.625%) and molds, but P. expansum was
more susceptible to these compounds, demonstrating an MIC
of 0.156%, than A. brasilienis, for which the MIC values
were 0.312%.
Interesting results were observed for primary amides 6-R
and 6-S and hydroxamic acids 7-R and 7-S derived from
3-hydroxynonanoic acid. Compounds 6-R and 6-S showed
inhibitory activity against most of the selected microorgan-
isms, especially against Gram-negative bacteria P. cedrina
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
(MIC of 0.312%) and S. liquefaciens (MIC of 0.312%) and
all tested yeast and molds (MIC values ranging from
0.312% to 0.625%). However, it was observed that the S-
configured derivative 6-S was more toxic to some of the
tested microorganisms, i.e., bacteria B. subtilis, S. aureus,
and E. coli and molds P. expansum. The MIC values deter-
mined for these microorganisms were two to eight times
higher for compound 6-S than its R-enantiomer. The results
obtained for S. aureus deserved special attention because
R-configured primary amides 6-R showed weak toxicity
against these bacteria with an MIC of 2.5%. On the other
hand, S. aureus was very susceptible to its opposite enan-
tiomer 6-S (MIC value of 0.312%).
Hydroxamic acids 7-R and 7-S showed the best antimicro-
bial activity against the tested microorganisms among all the
synthesized compounds, both derivatives of RA and
3-hydroxamic acid. They were active against Gram-positive
and Gram-negative bacteria, yeast, and molds with MIC values
ranging from <0.078% to 0.625%, with the only exception of
E. coli. The best activity was observed against P. cedrina
(MIC <0.078%) and both tested molds A. brasiliensis and
P. expansum (MIC 0.156%). Moreover, they were also very
toxic to B. subtilis and the rest of bacteria from the genus
Pseudomonas, as well as to yeast C. albicans and C. krusei
(MIC 0.312%). However, R-configured hydroxamic acid 7-R
was more active with regard to B. subtilis, showing an MIC of
0.156%, while its opposite enantiomer 7-S’ MIC values were
determined to be two times higher. In general, most of the
tested microorganisms turned out to be very susceptible to
compounds 7-R and 7-S. S. aureus, which is pathogenic and
resistant to many antibiotics (Kurlenda and Grinholc, 2012),
and also showed susceptibility to these derivatives (MIC
values of 0.625%). The weakest activity with MIC, which was
1.25%, was determined for bacteria E. coli. Moreover, differ-
ences in the activity of the opposite enantiomers were
observed. In the case of B. subtilis and S. liquefacies, the R-
configured derivative 7-R was more toxic to these microorgan-
isms than S-enantiomer 7-S.
i.e., amides of 3-hydroxynonanoic acid with morpholine
and pyrrolidine was observed. It was observed that
P. expansum was very susceptible to these compounds, and
the MIC were 0.078% (11-R and 11-S) or less than 0.078%
(12-R and 12-S). P. expansum is a mold expressing a
mycotoxin patulin and is one of the main causes of contam-
ination and rot of fruits (Okull and LaBorde, 2004). Deriva-
tive 11-R was also active against A. brasliensis, commonly
found in all environments, with an MIC value of <0.078%,
while its opposite enantiomer 11-S showed much weaker
activity against these molds (MIC of 0.312%). Compounds
8-R and 8-S also exhibited differences in the antimold
activity depending on the configuration of the asymmetric
carbon atom. The S-enantiomer derivative (8-S) was more
active against both of the tested mold species, and its MIC
values were two times lower than that of 8-R.
Conclusion
Twenty-four chiral compounds, including methyl esters
and amides derived from ricinoleic and 3-hydroxynonanoic
acids, were obtained, and 19 of them were synthesized and
characterized for the first time. All derivatives were evalu-
ated in terms of antimicrobial activity against 13 different
microorganisms representing Gram-negative and Gram-
positive bacteria, yeast, and molds.
The obtained compounds showed diverse antimicrobial activ-
ity and promising antimold potential. It was found that the tested
species of molds were more susceptible to derivatives of
3-hydroxynonanoic acid than to amides obtained from
RA. Nevertheless, hydroxamic acids derived from RA exhibited
the best activity against C. albicans and C. tropicalis. On the
other hand, hydroxamic acids derived from 3-hydroxynonanoic
acid showed the best antimicrobial potential against the
remaining microorganisms, especially against P. cedrina.
Comparing the differences between the antimicrobial activ-
ity of RA derivatives (current and previous (Matysiak et al.,
2018)) and analogous derivatives of 3-hydroxynonanoic acid,
a clear increase in the activity of short-chain compounds can
be seen (with the exception of yeast). Such structural changes
that result from an alteration in the carbon chain length and a
distance of both functional groups also cause an increase in
the polarity and hydrophilicity of the molecule, and these, in
turn, may be reflected in the interactions’ quality.
The remaining (R)- and (S)-3-hydroxynonanoic acid deriv-
atives (8–12) showed weak activity against the selected bacte-
ria and yeast, and their MIC values were 1.25% or 2.5%. The
only exceptions were compounds 8-R, 10-R, 10-S, 11-R, and
11-S, which were slightly more active against P. cedrina and
P. fluorescens, and 11-S derivative against B. subtilis showing
MIC equal to 0.625%. Inhibitory activity against bacteria from
the genus Pseudomonas deserves special attention due to the
resistance of these microorganisms to a wide range of antimi-
crobial compounds (Gross and Loper, 2009; Poole, 2011).
Therefore, the obtained MIC for these compounds, ranging
from 0.612% to 1.25%, can be considered promising results.
On the other hand, compounds 8–12 showed antimold
potential with MIC values ranging from less than 0.078%
to 0.625%. The best activity of derivatives 11 and 12,
The obtained results also indicate that the bacteriostatic
activity of the tested derivatives depends not only on the
length of the fatty acid chain but also on the cyclic or acy-
clic structure of the amide group. One of the intriguing fea-
tures of perceived bioactivity is the well-known dramatic
effect that can be wrought by a “small” change in the struc-
ture of the molecule, such as the stereogenicity that we face
J Am Oil Chem Soc (2019)

J Am Oil Chem Soc
hydroxylamine hydrochloride. Russian Journal of Organic Chemis-
try, 43:1114–1119. https://doi.org/10.1134/S1070428007080039
Ishmuratov, G. Y., Yakovleva, M. P., Shayakhmetova, A. K.,
in our research. Although the differences in the toxicity of
individual enantiomer pairs are sometimes quite pro-
nounced (2–8 times), they are difficult to generalize. On the
other hand, one slight enantiodifferentiation could probably
be observed in the case of 3-hydroxynonamide, of which
only the S-enantiomer for the five microorganisms is more
toxic (and the other not even once). However, for most
derivatives, there is no clear correlation between enantio-
mers of tested compounds and their activities.
Botsman, L. P., Nasibullina, G. V., Muslukhov, R. R.,
&
Tolstikov, G. A. (2006) Ozonolytic transformations of olefinic deriva-
tives of L-menthol and ricinolic acid. Chemistry of Natural Com-
pounds, 42:631–635. https://doi.org/10.1007/s10600-006-0240-1
Jiang, H., Gschwend, B., Albrecht, Ł., & Anker Jørgensen, K. (2010)
Organocatalytic preparation of simple β-hydroxy and β-amino
esters: Low catalyst loadings and gram-scale synthesis. Organic
Letters, 12:5052–5055. https://doi.org/10.1021/ol102164y
Khalifa, R. A., Nasser, M. S., Gomaa, A. A., Osman, N. M., &
Salem, H. M. (2013) Resazurin microtiter assay plate method for
detection of susceptibility of multidrug resistant Mycobacterium
tuberculosis to second-line anti-tuberculous drugs. Egyptian Jour-
nal of Chest Diseases and Tuberculosis, 62:241–247. https://doi.
org/10.1016/j.ejcdt.2013.05.008
Conflict of Interest The authors declare that they have no
conflict of interest.
Kula, J. (1999) Safer ozonolysis reactions: A compilation of labora-
tory experience. Chemical Health and Safety, 6:21–22. https://doi.
org/10.1016/S1074-9098(99)00046-5
Kula, J., Bonikowski, R., Szewczyk, M., & Ciolak, K. (2014) Synthe-
sis of (S)-ricinoleic acid and its methyl ester with the participation
of ionic liquid. Chemistry and Physics of Lipids, 183:137–141.
https://doi.org/10.1016/j.chemphyslip.2014.06.005
References
Achaya, K. T. (1971) Chemical derivatives of castor oil. Journal of
the American Oil Chemists’ Society, 48:758–763. https://doi.org/
10.1007/BF02638537
Achaya, K. T., Craig, B. M., & Youngs, C. G. (1964) The component
fatty acids and glycerides of castor oil. Journal of the American Oil
Kula, J., & Masarweh, A. (1998). 12-nor-Alloisolongifolan-11-one, a
ketone with amber-like odour from longifolene. Flavour and Fra-
grance Journal, 13:277–278. https://doi.org/10.1002/(SICI)
1099-1026(1998070)13:4<277::AID-FFJ746>3.0.CO;2–9
Chemists’
BF03373657
Society,
41:783–784.
https://doi.org/10.1007/
Applewhite, T. H., Nelson, J. S., & Goldblatt, L. A. (1963) Castor-
based derivatives : Synthesis of some amides. Journal of the Ameri-
can Oil Chemists’ Society, 40:101–104. https://doi.org/10.1007/
BF02640699
Borch-Jensen, C., Jensen, B., Mathiasen, K., & Mollerup, J. (1997)
Analysis of seed oil from ricinus communis and dimorphoteca
pluvialis by gas and supercritical fluid chromatography. Journal of
the American Oil Chemists’ Society, 74:277–284. https://doi.org/
10.1007/s11746-997-0136-7
Devlin, J. P., Ollis, W. D., & Thorpe, J. E. (1975). Studies concerning
the antibiotic actinonin. Part V. Synthesis of structural analogues of
actinonin by the anhydride-ester method. Journal of the Chemical
Society, Perkin Transactions 1, 0:846–848. DOI: https://doi.org/10.
1039/P19750000846
Galstukhova, N. (1960) Some reactions of ricinoleic acid and its
derivatives. The Russian Journal of General Chemistry, 30:
1387–1389.
García, M. J., Rebolledo, F., & Gotor, V. (1994) Lipase-catalyzed
aminolysis and ammonolysis of β-ketoesters. Synthesis of optically
active β-ketoamides. Tetrahedron, 50:6935–6940. https://doi.org/
10.1016/S0040-4020(01)81346-6
Gross, H., & Loper, J. E. (2009) Genomics of secondary metabolite
production by Pseudomonas spp. Natural Product Reports, 26:
1408–1446. https://doi.org/10.1039/b817075b
Hauser, C. R., & Renfrow, W. B. J. (1939) Benzohydroxamic acid.
Organic Syntheses, 19:15. https://doi.org/10.15227/orgsyn.019.0015
Ishmuratov, G. Y., Legostaeva, Y. V., Botsman, L. P.,
Yakovleva, M. P., Shakhanova, O. O., Muslukhov, R. R., &
Tolstikov, G. A. (2009) Transformations of peroxide ozonolysis
products of natural olefins by N-containing organic compounds in
methanol. Chemistry of Natural Compounds, 45:318–321. https://
doi.org/10.1007/s10600-009-9354-6
Kula, J., Quang, T. B.,
& Sikora, M. (2000) Synthesis of
enantiomerically pure volatile compounds derived from (R)-
3-hydroxynonanal. Tetrahedron: Asymmetry, 11:943–950. https://
doi.org/10.1016/S0957-4166(00)00019-7
Kurlenda, J., & Grinholc, M. (2012) Alternative therapies in Staphylo-
coccus aureus diseases. Acta Biochimica Polonica, 59:171–184.
Matysiak, S., Chmiel, A., Skolimowski, J., Kula, J., Pasternak, B., &
Blaszczyk, A. (2017) Synthesis and cytotoxicity of (R)- and (S)-
ricinoleic acid amides and their acetates. Chirality, 29:616–622.
https://doi.org/10.1002/chir.22733
ꢀ
Matysiak, S., Zabielska, J., Kula, J., & Kunicka-Styczynska, A.
(2018) Synthesis of (R)- and (S)-ricinoleic acid amides and evalua-
tion of their antimicrobial activity. Journal of the American Oil
Chemists’ Society, 95:69–77. https://doi.org/10.1002/aocs.12013
Odham, G., & Samuelsen, B. (1970) On the methylation and demeth-
ylation of optically active aliphatic hydroxy compounds. Acta
Chemica Scandinavica, 24:468–472. https://doi.org/10.3891/acta.
chem.scand.24-0468
Ogunniyi, D. S. (2006) Castor oil: A vital industrial raw material. Bio-
resource Technology, 97:1086–1091. https://doi.org/10.1016/j.
biortech.2005.03.028
Okull, D. O., & LaBorde, L. F. (2004) Activity of electrolyzed oxidiz-
ing water against Penicilium expansum in suspension and on
wounded apples. Journal of Food Science, 69:23–27. https://doi.
org/10.1111/j.1365-2621.2004.tb17872.x
ꢀ
Pabis, S., & Kula, J. (2016). Synthesis and bioactivity of (R)-ricinoleic
acid derivatives: A review. Current Medicinal Chemistry, 23:
4037–4056.
104453
https://doi.org/10.2174/0929867323666160627
Poole, K. (2011) Pseudomonas aeruginosa: Resistance to the max.
Frontiers in Microbiology, 2:65. https://doi.org/10.3389/fmicb.
2011.00065
Ishmuratov, G. Y., Shayakhmetova, A. K., Yakovleva, M. P.,
Legostaeva, Y. V., Shitikova, O. V., Galkin, E. G.,
&
Tolstikov, G. A. (2007) Ozonolysis of alkenes and study of reac-
tions of polyfunctional compounds: LXVIII. Investigation of trans-
formations of peroxide products of olefins ozonolysis treated with
Takeda, T., Watanabe, H., & Kitahara, T. (1997) Direct oxidation of
acetals and aldehydes to esters. Synlett, 1997:1149–1150. https://
doi.org/10.1055/s-1997-996
J Am Oil Chem Soc (2019)