Synthesis and biological screening of a library of macamides as TNF-α inhibitors

Article abstract of DOI:10.1039/d0md00208a

Thirty-five macamide analogues were synthesised by modifying the initial molecular structure. The resulting structures were confirmed using NMR and MS. Cytotoxicity and the anti-inflammatory activity of these synthetic macamides were evaluated in the THP-1 cell line. Preliminary biological evaluation indicated that most of these synthetic macamides did not present cytotoxicity (MTT assay) in the tested cell line with respect to the control (actinomycin D). Regarding the anti-inflammatory activity, several analogues had a greater potential for inhibition of TNF-α than natural macamides. Synthetic macamide 4a was the most active (IC50 = 0.009 ± 0.001 μM) compared to the C87 (control). Through looking at the link between the chemical structure and the activity, our study proves that changes made to natural macamides at the level of the alkyl chain, the benzyl position, the amide bond, and the addition of two methyl groups to the aromatic ring (meta position) lead us to obtaining new macamides with greater anti-inflammatory activity. This journal is

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RESEARCH ARTICLE
Synthesis and biological screening of a library of
macamides as TNF-α inhibitors†
Cite this: DOI: 10.1039/
d0md00208a
Víctor Tena Pérez,a Luis Apaza Ticona,
Andreea Madalina Serban,c
ab
*
Javier Acero Gómeza and Ángel Rumbero Sáncheza
Thirty-five macamide analogues were synthesised by modifying the initial molecular structure. The resulting
structures were confirmed using NMR and MS. Cytotoxicity and the anti-inflammatory activity of these
synthetic macamides were evaluated in the THP-1 cell line. Preliminary biological evaluation indicated that
most of these synthetic macamides did not present cytotoxicity (MTT assay) in the tested cell line with
respect to the control (actinomycin D). Regarding the anti-inflammatory activity, several analogues had a
greater potential for inhibition of TNF-α than natural macamides. Synthetic macamide 4a was the most
active (IC50 = 0.009 0.001 μM) compared to the C87 (control). Through looking at the link between the
chemical structure and the activity, our study proves that changes made to natural macamides at the level
of the alkyl chain, the benzyl position, the amide bond, and the addition of two methyl groups to the
aromatic ring (meta position) lead us to obtaining new macamides with greater anti-inflammatory activity.
Received 16th June 2020,
Accepted 27th July 2020
DOI: 10.1039/d0md00208a
rsc.li/medchem
disorders related to diseases with various etymologies. These
diseases can come from an infectious origin (meningitis,
1. Introduction
Our immune system is an incredible network of cells that
protects us from all kinds of diseases. However, sometimes
the system can be faulty and misinterpret the signals, causing
our immune system to not recognise its own body and start
attacking itself.1 The functioning of the immune system is
regulated by the activity of pro and anti-inflammatory
mediators. The imbalance of any of these mediators can lead
to the development of increased susceptibility to infections,
autoimmune diseases, and chronic inflammatory conditions.2
Currently, therapeutic investigations have focused on
suppressing the production of pro-inflammatory mediators
and on inhibiting the activation of the immune response,
using modulation of TNF-α, NF-kB, STAT3 and IFN-γ
signalling pathways.3
tuberculosis, acquired immunodeficiency syndrome),
a
tumour (prostate cancer, lung cancer, breast cancer) or have
an autoimmune origin (inflammatory bowel disease,
rheumatoid
arthritis,
lupus).5
Several
drugs
(e.g.
dexamethasone) commonly used as immune-suppressants
have been used to modulate the TNF-α activity, although
their effects are associated with considerable toxicity.6
In the search for new compounds that involve the inhibition
of TNF-α and its activity in cells, while presenting less toxicity,
with greater potency and, thus, offering better clinical
alternatives, our research suggests
a series of chemical
modifications to natural macamides which have been reported
as having anti-inflammatory potential (TNF-α inhibitors).7
Macamides are secondary metabolites of the species
Lepidium meyenii “Maca”8 and Tropaeolum tuberosum
“Mashua”9 that grow exposed to extreme climatic conditions
(poor soils with a slightly acidic pH between 5–6 and exposure
to ultraviolet radiation at altitudes of 3.800 meters above sea
level), producing this type of compounds considered as
biomarkers of these species. Chemically, macamides consist
of a residue of a benzylamine (product of the hydrolysis of
glucosinolates by the enzyme myrosinase) and a fatty acid
residue (product of the hydrolysis of membrane lipids).10
Recent pharmacological studies have shown that
macamides have also other activities such as antioxidants,
analgesics and antidepressants;11 anti-tuberculosis;12 anti-
In particular, the use of inhibitors against the production
and biological activity of the tumour necrosis factor alpha
(TNF-α) has been widely described and discussed.4 This is
because this factor plays an important role in inflammatory
a Department of Organic Chemistry, Faculty of Sciences, University Autónoma of
Madrid, Cantoblanco, 28049 Madrid, Spain. E-mail: luis.apaza@uam.es,
lnapaza@ucm.es
b Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy,
University Complutense of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
c Maria Sklodowska Curie University Hospital for Children, Constantin
Brancoveanu Boulevard, 077120 Bucharest, Romania
† Electronic supplementary information (ESI) available: 1H- and 13C- NMR
spectra of the synthetic macamides analysed in this study are provided as
supporting information (Fig. 1S–70S). See DOI: 10.1039/d0md00208a
cholesterolemics;13
antifibrogenics;14
anticonvulsants;15
neuroprotectives16 and anticancer,17 being interesting
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molecules for chemical studies due to their wide
pharmacological spectrum use.
The objective of the present work is to conclude on how
the anti-inflammatory potential of macamides can be
improved through the use of synthesised analogues. For this
study, we first determined the cytotoxicity of the synthetic
macamides, as their effect may hide anti-inflammatory
activity. Subsequently, we evaluated their inhibitory activity
on TNF-α in the THP-1 cell line.
Scheme
amidation reaction.
1
Synthesis of macamide derivatives by means of the
double bond. The oleic and linoleic fatty acids were used as
precursors in order to obtain natural macamides23 through
an amidation reaction.
2. Results and discussion
The results reveal that the presence of double bonds
improves TNF-α inhibition, due to the increased lipophilic
capacity of derivatives 1e and 1f with respect to derivative
1d.24 The difference between derivatives 1e and 1f was not
statistically significant (nsp > 0.999) (Table 1). Consequently,
it was decided to use derivative 1f for subsequent
modifications, since this macamide is found in a greater
proportion in nature25 and because it showed considerable
biological activity (14.2 1.47 μM).
After the promising results, it was decided to continue the
pharmacomodulation study with the synthesis of homologues
(through the addition or subtraction of methylene groups) on
the benzylic position. We started from (9Z,12Z)-octadeca-9,12-
dienoyl chloride and used different amines, from aniline (n =
In order to improve the pharmacological profile of
macamides as potential anti-inflammatory agents (TNF-α
inhibition), we proceeded to carry out a chemical synthesis of
structural analogues, by means of various modifications in
the positions considered of greatest importance (Fig. 1).
The synthetic pathway was performed using an amidation
reaction (modification of the Steglich reaction18,19) between
the corresponding carboxylic acid and the benzylamine with
N,N′-dicyclohexylcarbodiimide (DCC), as shown in Scheme 1.
Concerning the alkyl chain, it was observed that its
elongation (derivatives 1a → 1d) does not generate a change in
cytotoxicity (MTT) in the THP-1 cell line. However, synthetic
macamide 1a (four-carbon alkyl chain) did not show a relevant
inhibition on TNF-α, it exhibited the same value as the free
carboxylic acid (oleic acid). This effect is due to the increase in
the number of carbon atoms present in the chain, leading to an
increase in the biological activity.20 In this sense, derivative 1d
0) to 3-phenyl-1-propylamine (n
=
3), obtaining the
corresponding synthetic macamides (1g → 1i) (Scheme 2).
Analysing the results, it was observed that as the
number of methylenes increased, their lipophilic capacity
increased and, in turn, their biological activity was higher.
In the case of derivative 1h, the variation with respect to
derivative 1f was not statistically significant (nsp > 0.999),
but, after the introduction of a second methylene group
showed TNF-α inhibition with an IC50 of 16.6
1.59 μM.
Analysing the n-octanol/water partition coefficients (clog P) of
derivatives 1a → 1d, a relationship between lipophilicity and
biological activity was observed, and this is justified by the fact
that increases in lipophilicity lead to an improvement in
biological activity (Table 1). Free oleic acid was tested as negative
control to determine if it was responsible for the inhibitory
activity. Yet, we saw that free carboxylic acid did not produce
significant activity on the target due to its low liposolubility
(clog P = 3.47) compared to derivative 1d (clog P = 7.68).
This result can be justified by the lipophilicity effect, a
physicochemical parameter that determines the ability of a
substance to cross the biological membrane.21,22 Therefore,
molecules with a longer chain length can more easily cross
the cell barrier by diffusion.
(1i), the effect was more pronounced (IC50 = 8.3
μM). However, derivative 1i showed cytotoxic effect
0.49
a
(CC50 = 80.3 1.31 μM) on THP-1 cells and the difference
was statistically significant with respect to derivative 1f
(***p < 0.001) (Table 1).
These data can be justified based on electronic
distribution, in which nitrogen, being a strong activator,
modifies the electronic distribution of the aromatic ring and,
consequently, its interaction26 as observed when comparing
derivatives 1f and 1i.
The next phase of the investigation of the structure–
activity relationship was to study the influence of
substituents on the benzylic position. Due to the decrease in
yield when the alkyl chain length is increased using an
amidation reaction, (9Z,12Z)-octadeca-9,12-dienoyl chloride
was used as a synthesis precursor instead of oleic acid,
following Scheme 3.
After confirming that the aliphatic chain of C18 is the
most suitable, the next step was to study the influence of the
To understand how this position affects activity, the
following substitutions in the benzyl position have been
tested: hydrazide residue (2a), methyl residue (2b), ethyl
residue (2c), hydroxyethyl residue (2d) and gem-dimethyl
residue (2e). The data revealed that the substitution at the
benzylic position affected biological activity. Synthetic
Fig. 1 General structure of macamide and positions explored on this
study.
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Table 1 Influence of the length of the alkyl chain, degree of unsaturation in the alkyl chain and structural homology of the synthetic macamides on
TNF-α
No.
Derivatives
Viability (CC50 μM) THP-1 cells
TNF-α inhibition (IC50 μM) THP-1 cells
clog Pa
Oleic acid
100 1.99
79.0 1.78
3.47
1a
1b
1c
1d
1e
1f
100 1.71
100 1.66
100 1.98
100 1.83
97.3 1.18
98.4 1.43
78.4 1.19
58.7 1.61
43.1 1.77
16.6 1.59
15.2 1.51
14.2 1.47
1.70
4.01
6.30
7.68
8.31
7.96
1g
1h
1i
97.8 1.22
88.15 1.20
80.3 1.31
21.0 1.45
15.7 1.69
8.3 0.49
7.82
8.28
8.60
a
Values of clog P were calculated by DataWarrior.27
Scheme 2 Synthesis of 1g → 1i macamide derivatives.
Scheme 3 Synthesis of 2a → 2e macamide derivatives.
macamide 2a, which presents a hydrazide bond instead of
amide, improved biological activity, exhibiting an IC50 of 8.9
0.98 μM. Also, substitution in the benzylic position by a
methyl group (2b), significantly improved the activity (IC50 of
5.2 0.81 μM). This result is due to the greater lipophilic
capacity of derivative 2b (clog P = 8.20) compared to derivative
2a (clog P = 7.40) (Table 2).
In the case of derivatives 2c and 2e, although they have a
greater lipophilic capacity than derivative 2b, they showed
less biological activity. This effect can be justified by the fact
that its substituents have a higher volume that prevents
correct interaction with the target. Regarding the low
biological activity of the derivative 2d, this result is due to its
lower lipophilic capacity with respect to derivative 2a.
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Table 2 Influence of the substitution in the benzylic position and modified analogues at the amide bond of the synthetic macamides on TNF-α
No.
Derivatives
Viability (CC50 μM) THP-1 cells
TNF-α inhibition (IC50 μM) THP-1 cells
clog Pa
2a
98.1 1.84
8.9 0.98
7.40
2b
2c
95.8 1.97
96.5 1.79
5.2 0.81
8.20
8.29
19.7 1.94
2d
98.0 1.46
56.8 1.75
7.29
2e
2f
97.3 1.94
89.1 1.23
95.4 1.88
84.3 1.12
13.1 1.74
23.8 1.44
6.4 0.91
5.3 0.75
8.33
8.11
8.10
8.17
2g
2h
2i
2j
98.7 1.97
95.0 1.82
1.6 0.07
8.30
8.31
0.97 0.02
a
Values of clog P were calculated by DataWarrior.27
Once the structure–activity study on the benzylic position
was completed, we proceeded to analyse how the amide bond
influences biological activity. At this point, changes were
made to the amide bond. For this, the nitrogen-methylated
derivative (2f), the benzyl ester (2g) and the benzyl thioester
(2h) were synthesised (Scheme 4).
The synthetic macamide 2i was obtained reducing
derivative 2b with LiAlH4 in a 90% yield (Scheme 5).
The synthetic macamide 2j was quantitatively synthesised
from the reaction of the derivative 2b and Lawesson's reagent
(Scheme 6).
The data revealed that the methylated nitrogen derivative
2f showed a lower biological activity (IC50 of 23.8 1.44 μM)
compared to derivative 1f (IC50 of 14.2 1.47 μM). This effect
is due to the absence of hydrogen in the amide bond that
allows the macamide to form a hydrogen bond with the
target. Similarly, it was observed that derivative 2f did not
show cytotoxicity (≥90%).
Through the substitution of N–H in the amide bond by
other heteroatoms such as oxygen and sulphur, derivatives 2g
and 2h had a favourable effect on the inhibition of TNF-α,
exhibiting IC50 values of 6.4 0.91 and 5.3 0.75 μM similar
Scheme 4 Synthesis of 2f → 2h macamide derivatives.
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Scheme 5 Synthesis of 2i macamide derivative.
Scheme 6 Synthesis of 2j macamide derivative.
to those of derivative 2b (IC50 = 5.2
0.81 μM), yet the
Subsequently, it was decided to introduce electron donating
groups (EDG) and electron withdrawing groups (EWG) in the
aromatic ring to observe its effect on the biological activity.
Although all substitutions improved activity with respect to
derivative 2b, the trend shows that substitutions with strong
EDG and EWG28 (3d and 3j) generate worse results with respect
to derivatives that have weak EDG and EWG29,30 (3f, 3h and 3i).
This effect is due to the greater lipophilic capacity of these
derivatives (3f = 8.14; 3h = 8.04 and 3i = 8.37) (Table 3).
Regarding the cytotoxicity of this series of derivatives, the
synthetic macamides 3g, 3h and 3i showed a CC50 of 82.3
1.73; 82.5 1.86 and 81.4 1.80 μM, being cytotoxic (≥90%)
on THP-1 cells. In the case of derivative 3j, its CC50 of 89.8
1.30 μM was not statistically significant (nsp > 0.999) with
respect to the negative control (untreated cells).
difference was not statistically significant between them (nsp
> 0.999). Likewise, the similarity between these derivatives
with respect to their activity is due to their lipophilic capacity
in the range of 8.10–8.20 (Table 2).
To observe the influence of the amide bond on the
biological activity, the carbonyl group of derivative 2b was
modified obtaining the synthetic macamides (2j) and (2i). In
both cases, there was an increase in TNF-α inhibition, with
the value of derivative 2j standing out (IC50 = 0.97 0.02 μM).
These results denote that alteration in the amide bond is key
to biological activity. Likewise, it was observed that the
increase in the biological activity of derivatives 2j and 2i was
due to their greater lipophilic capacity with respect to
derivative 2b (Table 2).
To end the first step of our study on the structure–activity
relationship, we investigated the influence of the aromatic ring
on the inhibition of TNF-α. Changes were made to the position
of the aromatic ring. For this, it was investigated if its presence
was influential and how and if the introduction of substituents
was relevant. In this sense, two types of derivatives were
prepared: those in which the aromatic ring was replaced by
alkyl groups (3a → 3c), and those in which the aromatic ring
had different types of substituents (3d → 3j). In all cases, the
synthetic methodology followed Scheme 7.
As it can be seen in Table 3, the changes made to the
aromatic ring have a very favourable effect on the activity.
When the benzene ring is replaced by a cyclohexyl group (3a),
a tert-butyl group (3b), or an adamantyl group (3c), an
improvement in biological activity occurs, showing IC50
Moreover, analysing previous works, we observed that the
natural macamide N-oleoyldopamine (IC50 = 3.54 0.02 μM)
showed a lower activity with respect to the natural macamide
N-(2-hydroxyethyl)-7Z,10Z,13Z,16Z-docosatetraenamide (IC50
=
1.77 0.07 μM).9 This effect is due to the fact that the second
macamide showed a greater length of the alkyl chain with a
greater number of double bonds. In our work, synthetic
macamide 3d is an improved derivative of natural macamide
N-oleoyldopamine. From a chemical point of view, the increase
in the activity of the 3d derivative with respect to
N-oleoyldopamine is due to the presence of an additional
double bond, decrease of methylene groups in the benzyl
position and addition of a methyl residue in the benzyl position.
The substituent that exhibited the best biological activity
was derivative 3f, which has a weak EDG (methyl residue) at
the para position of the aromatic ring, with an IC50 of 0.62
0.03 μM. Based on this result, it was verified how the
introduction of an ethyl group in the para position (3j) affects
the activity, and it was observed that the introduction of one
more carbon atom generates a decrease in biological activity
(IC50 of 1.21 0.04 μM).
values of 0.71
respectively, with respect to derivative 2b (5.2
0.03, 1.00
0.03 and 0.78
0.02 μM,
0.81 μM).
Likewise, derivatives 3a and 3c showed a greater lipophilic
capacity with respect to derivative 2b, determining their
greater biological activity. These data demonstrate that
aromaticity is not a key effect for activity.
Scheme 7 Synthesis of 3a → 3j macamide derivatives.
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Table 3 Influence of the aromatic ring and the position of methyl on the aromatic ring of the synthetic macamides on TNF-α
No.
Derivatives
Viability (CC50 μM) THP-1 cells
TNF-α inhibition (IC50 μM) THP-1 cells
clog Pa
3a
93.2 1.76
0.71 0.03
8.64
3b
3c
94.7 1.25
90.9 1.22
1.00 0.03
0.78 0.02
8.07
8.45
3d
3e
93.5 1.58
93.6 1.53
1.84 0.03
1.71 0.03
8.00
8.14
3f
95.0 1.98
82.3 1.73
0.62 0.03
1.52 0.04
8.31
7.99
3g
3h
3i
82.5 1.86
81.4 1.80
89.8 1.30
0.78 0.03
0.74 0.03
1.21 0.04
8.04
8.37
8.52
3j
3k
3l
92.5 1.64
91.7 1.19
0.54 0.03
0.33 0.02
8.20
8.31
3m
93.2 1.53
0.08 0.001
8.50
a
Values of clog P were calculated by DataWarrior.27
In view of the data, it was decided to analyse how the
position of methyl on the aromatic ring influences biological
activity. The variation of the position within the ring causes a
considerable improvement in the biological activity; this
effect is especially relevant when the substitution is made in
the meta position (3l), where the corresponding amide had
an IC50 of 0.33 0.02 μM. Based on this value, it was decided
to study the di-substitution at the meta position, obtaining
the best derivative substituted in the aromatic ring (3m),
presenting an IC50 of 0.08
0.001 μM. These data
demonstrate the strong dependence between the geometry of
the synthetic macamide and the biological activity, resulting
in the modification on the aromatic ring, showing that this is
the position of the most influential pharmacophore (Table 3).
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Accounting for these results, it was decided to analyse
how the position of the methyl in the aromatic ring
influences biological activity. The variation of the position
within the ring caused a considerable improvement in the
biological activity. This effect was especially relevant when
the substitution was made in the meta position, where the
derivative 3l presented an IC50 of 0.33 0.02 μM. Based on
this observation, we decided to study the di-substitution in
the meta position, obtaining the derivative 3m, with an IC50
of 0.08 0.001 μM. Likewise, derivative 3m showed a greater
lipophilic capacity with respect to derivative 2b. These data
demonstrate the strong dependence between the geometry of
the synthetic macamide and the biological activity,
confirming once more that this is the position of the most
influential pharmacophore (Table 3).
with LiAlH4. Finally, to obtain synthetic macamide 4c,
derivative 3m was reacted with Lawesson's reagent.
The introduction of the thiocarbonyl group (4c) achieved a
slight improvement in activity with an IC50 of 0.06 0.001 μM.
In the case of synthetic macamide 4b, as expected, in view of
the previous results, the effect was more pronounced with an
IC50 of 0.05 0.001 μM. Finally, synthetic macamide 4a showed
the highest inhibitory effect with an IC50 of 0.009 0.001 μM.
This effect could be justified on the basis of the difference
between the amide bond and the ester bond, since the latter
has less rigidity than the amide bond and could be adapted to
the active centre of the target. Similarly, it was observed that, in
this series of derivatives, synthetic macamide 4c showed a
higher cytotoxicity (71.5 1.13 μM) and synthetic macamide 3b
showed a moderate cytotoxicity (80.9 1.96 μM) with respect to
untreated cells (negative control) (Table 4).
Finally, compiling the information on the influence of the
alkyl chain (aliphatic chain lengthening, 1f), the position of
Our results show that the synthesised macamides and
especially derivative 4a have a pharmacological potency
between 200–400 times better than the natural N-(2-
the benzyl (introduction of
a methyl residue, 2b), the
modification of the amide bond (ester, 2g; amine, 2i and
thioamide, 2j) and the introduction of substituents on the
aromatic ring (two methyl groups at the meta position, 3m),
it was decided to synthesise analogues with the optimal
substituents at each position to analyse their biological
activity. The synthetic methodology used to obtain the
synthetic macamide 4a was the same as that used to
synthesise derivative 2g, reacting the acid chloride with an
alcohol. In the case of the synthetic macamide 4b, it was
obtained starting from the reduction of the derivative 3m
hydroxyethyl)-7Z,10Z,13Z,16Z-docosatetraenamide
and
N-oleoyldopamine macamides that inhibit TNF-α in THP-1
cells with IC50 of 1.77
0.07 and 3.54
0.02 μM,
respectively,7 and 12 times better than the positive control
(C87) with an IC50 of 0.11 0.01 μM (Table 4).
Previous works showed that the anti-inflammatory activity
of natural macamides is due to the inhibitory capacity of NF-
κB activation induced by TNF-α, through the direct inhibition
of the IKKβ subunit and, to a smaller extent, of the IKKα
Table 4 Comparative study of the optimal synthetic macamides against natural macamides and the positive control C87 on TNF-α
No.
Derivatives
Viability (CC50 μM) THP-1 cells
TNF-α inhibition (IC50 μM) THP-1 cells
clog Pa
4a
90.1 1.27
0.009 0.001
9.09
4b
4c
80.9 1.96
71.5 1.13
0.05 0.001
0.06 0.001
9.19
8.75
1
>100
3.54 0.02
8.79
2
>100
1.77 0.07
0.11 0.01
6.58
4.57
C87
10.81 1.23
a
Values of clog P were calculated by DataWarrior.27 1 = N-oleoyldopamine; 2 = N-(2-hydroxyethyl)-7Z,10Z,13Z,16Z-docosatetraenamide.
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subunit of the κB kinase inhibitor complex.7,25 In this sense,
we can indicate that the derivatives synthesised in this work
lead to direct inhibition of IκB, preventing its degradation
and the subsequent inhibition of NF-κB activation.7,31 These
results open the way to the development of new anti-
inflammatory drugs with a better safety profile compared to
the drugs available on the market.
2 × 15 mL. The organic phase was dried with anhydrous
MgSO4, filtered and the solvent was removed under reduced
pressure. (9Z,12Z)-N′-phenyloctadeca-9,12-dienehydrazide (2a)
was purified by flash chromatography with a mixture of Hex–
AcOEt (2 : 1) as eluent; it was obtained as a colourless liquid
with a yield of 70%.
3.4. General procedure for the synthesis of (9Z,12Z)-octadeca-
9,12-dienamide, (9Z,12Z)-octadeca-9,12-dienoate derivatives
and (9Z,12Z)-octadeca-9,12-dienethioate derivatives
3. Material and methods
3.1. General experimental procedures
In a solution of the corresponding amine, alcohol or thiol
(1.0 eq.) in dry CH2Cl2 (2 mL mmol−1), Et3N (1.2 eq.) was
added and stirred at 0 °C. Next, the (9Z,12Z)-octadeca-9,12-
dienoyl chloride was added dropwise. After 10 minutes, the
reaction was observed to have been completed by TLC. HCl
(10%) was added and the aqueous phase 2 × 15 mL of CH2Cl2
was extracted. The organic phase was dried over anhydrous
MgSO4, filtered, and the solvent was removed under reduced
pressure. The crude reaction was purified by flash
chromatography using Hex–AcOEt as eluent.
NMR spectra were performed on a Bruker Avance AV-I
operating at 300 MHz (1H) or 75 MHz (13C). The chemical shifts
(δ) are indicated in ppm relative to the residual signals of
deuterated solvent: δH 7.26 and δC 77.2 for CDCl3. 13C NMR
spectra were acquired in a proton decoupled broadband
system. The following abbreviations were used to describe the
peak patterns: s (singlet), d (doublet), t (triplet), q (quartet),
quint (quintet), sext (sextuplet), sept (septuplet), m (multiplet)
and br (broad signal). High resolution mass spectrometry
(HRMS) was recorded on a quadrupole QSTAR XL spectrometer
with TOF, electrospray (ESI+). In each case, the solvent and
concentration used (mg mL−1) were indicated. TLC was
performed using Merck silica gel 60-F254 plates. Developed
chromatograms were visualised by UV absorbance (254 nm) or
3.5. General procedure for amide reduction
LiAlH4 (5 eq.) was added to a flask with dry THF at −78 °C,
and, subsequently, the corresponding amide (1 eq.) was
added, drop by drop, dissolved in dry THF. Stirring was
maintained for 12 h. After the reaction was complete,
hydrated Na2SO4 was added and allowed to react for 15
minutes until the formation of hydrogen was no longer
observed. The solution was filtered and the solvent was
removed under reduced pressure; the crude reaction was
purified by flash chromatography using Hex–AcOEt as eluent.
through application of heat to
phosphomolybdic acid (H3PMo12O40).
a
plate stained with
The manual
chromatography column was performed with silica gel (60 μm)
and with the indicated eluent in accordance with standard
techniques. The solvents and reagents used were purchased
from commercial sources and used without prior purification.
3.2. General procedure for the acylation of benzylamine with
fatty acids
3.6. General procedure for obtaining thioamides
The corresponding carboxylic acid (1 eq.) was added in a
round bottom flask and dissolved in dry CH2Cl2 (1 mL
mmol−1 carboxylic acid). Subsequently, the DCC (1.2 eq.) was
added. The mixture was kept under stirring for 15 minutes,
after which a mixture of DMAP (10% mol) and benzylamine
(1 eq.) dissolved in dry CH2Cl2 (1 mL mmol−1 benzylamine)
was added. The reaction mixture was kept stirred for 12 h at
room temperature. At the end of this time, the white solid
formed was filtered through a filter plate and the solvent was
removed from the filtrate by evaporation under reduced
pressure. The crude reaction was purified by flash
chromatography using Hex–AcOEt as eluent.
In a solution of the corresponding amide (1.0 eq.) in dry
THF, Lawesson's reagent (1.5 eq.) was added and it was
stirred at reflux for 12 h. After completion of the reaction, the
solvent was removed under reduced pressure and the
reaction crude was purified by flash chromatography using
Hex–AcOEt as the eluent.
3.7. Spectroscopic data
N-Benzylbutyramide (1a). N-Benzylbutyramide (1a) was
obtained from butanoic acid (300 mg, 3.41 mmol) and
benzylamine (3.41 mmol). 508 mg of the synthetic macamide
1a (90% yield) were obtained as a white solid. The product
was chromatographed with a Hex–AcOEt mixture (3 : 1). 1H
NMR (CDCl3, 300 MHz): δH 0.94 (t, 3H, J = 7.4 Hz), 1.66 (s, 2H,
J = 7.4 Hz), 2.17 (t, 2H, J = 7.5 Hz), 4.40 (d, 2H, J = 5.7 Hz),
6.19 (bs, 1H), 7.29–7.16 (m, 5H). 13C NMR (CDCl3, 75 MHz):
δC 13.9, 19.3, 38.7, 43.5, 127.5, 127.8, 128.7, 138.5, 173.1.
N-Benzyloctanamide (1b). N-Benzyloctanamide (1b) was
obtained from octanoic acid (443 mg, 3.07 mmol) and
benzylamine (329 mg, 3.07 mmol). 620 mg of the synthetic
macamide 1b (81% yield) were obtained as a white solid. The
3.3. Synthesis of (9Z,12Z)-N′-phenyloctadeca-9,12-
dienehydrazide
In a solution of phenylhydrazine (215 mg, 2 mmol) in dry
CH2Cl2 (3 mL mmol−1), Et3N (201 mg, 2 mmol) was added
and stirred at 0 °C. Next, (9Z,12Z)-octadeca-9,12-dienoyl
chloride (500 mg, 1.66 mmol) was added dropwise. After 10
minutes, the reaction was observed to be complete by TLC.
Water was added and extraction was carried out with CH2Cl2
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product was chromatographed with a Hex–AcOEt mixture (3 :
1). 1H NMR (CDCl3, 300 MHz): δH 0.85 (t, 3H, J = 7.1 Hz),
1.26–1.17 (m, 8H), 1.63 (q, 2H, J = 7.4 Hz) 2.18 (t, 2H, J = 7.7
Hz), 4.41 (d, 2H, J = 5.7 Hz), 5.80 (bs, 1H), 7.28–7.19 (m, 5H).
13C NMR (CDCl3, 75 MHz): δC 14.2, 22.7, 25.9, 29.1, 29.4,
31.8, 36.9, 43.7, 127.5, 127.9, 128.8, 138.5, 173.1.
(9Z,12Z)-octadeca-9,12-dienoyl chloride (736 mg, 2.47 mmol)
and aniline (230 mg, 2.47 mmol). 640 mg of the synthetic
macamide 1g (73% yield) were obtained as a yellowish liquid.
The product was chromatographed with a Hex–AcOEt mixture
1
(3 : 1). H NMR (CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 6.8 Hz),
1.34–1.17 (m, 14H), 1.73–1.60 (m, 2H), 2.05 (q, 4H, J = 6.4
Hz), 2.35 (t, 2H, J = 7.7 Hz), 2.77 (t, 2H, J = 6.0 Hz), 5.35–5.21
(m, 4H), 7.10 (t, 1H, J = 7.2 Hz), 7.18 (bs, 1H), 7.31 (t, 2H, J =
8.2 Hz), 7.51 (d, 2H, J = 7.8 Hz). 13C NMR (CDCl3, 75 MHz):
δC 14.1, 22.6, 25.6, 25.8, 27.2, 29.2, 29.3, 29.3, 29.6, 31.5, 37.6,
120.3, 124.1, 127.9, 128.0, 128.8, 130.0, 130.2, 138.3, 172.3.
N-Benzylhexadecanamide (1c). N-Benzylhexadecanamide
(1c) was obtained from hexadecanoic acid (788 mg, 1.12
mmol) and benzylamine (119 mg, 1.12 mmol). 580 mg of the
synthetic macamide 1c (60% yield) were obtained as a white
solid. The product was purified with a Hex–AcOEt mixture
1
(5 : 1). H NMR (CDCl3, 300 MHz): δH 0.85 (t, 3H, J = 7.2 Hz),
(9Z,12Z)-N-Phenethyloctadeca-9,12-dienamide
(9Z,12Z)-N-Phenethyloctadeca-9,12-dienamide
(1h).
was
1.26–1.17 (m, 24H), 1.61 (q, 2H, J = 7.7 Hz), 2.18 (t, 2H, J =
7.4 Hz), 4.41 (d, 2H, J = 5.7 Hz), 5.72 (bs, 1H), 7.32–7.20 (m,
5H). 13C NMR (CDCl3, 75 MHz): δC 14.3, 22.8, 25.9, 29.4, 29.5,
29.5, 29.6, 29.7, 29.8, 29.8, 29.8, 29.9, 29.9, 32.0, 36.9, 43.7,
127.6, 127.9, 128.8, 138.5, 173.1.
(1h)
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (736
mg, 2.47 mmol) and phenethylamine (300 mg, 2.47 mmol).
663 mg of the synthetic macamide 1h (70% yield) were
obtained as
a
yellowish liquid. The product was
N-Benzyloctadecanamide (1d). N-Benzyloctadecanamide
(1d) was obtained from octadecanoic acid (611 mg, 2.15
mmol) and benzylamine (230 mg, 2.15 mmol). 506 mg of the
synthetic macamide 1d (63% yield) were obtained as a white
solid. The product was chromatographed with a Hex–AcOEt
chromatographed with a Hex–AcOEt mixture (4 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 6.9 Hz), 1.35–1.27 (m,
14H), 1.58–1.53 (m, 3H), 2.15–2.03 (m, 5H), 2.85–2.73 (m,
3H), 3.52 (q, 2H, J = 6.8 Hz), 5.46–5.32 (m, 4H), 7.35–7.18 (m,
5H). 13C NMR (CDCl3, 75 MHz): δC 14.0, 22.5, 25.6, 25.7, 27.1,
29.1, 29.2, 29.3, 29.6, 31.5, 35.6, 36.6, 40.6, 126.4, 127.9,
128.5, 128.7, 130.0, 130.1, 139.0, 173.4.
(9Z,12Z)-N-(3-Phenylpropyl)-octadeca-9,12-dienamide (1i).
(9Z,12Z)-N-(3-Phenylpropyl)-octadeca-9,12-dienamide (1i), 250
mg of the amine were introduced and 573 mg of the
synthetic macamide 1i were obtained (78% yield). Column
with Hex–AcOEt (6 : 1) pale yellow liquid. 1H NMR (CDCl3,
300 MHz): δH 0.89 (t, 3H, J = 7.0 Hz), 1.40–1.24 (m, 14H),
1.69–1.56 (m, 3H), 1.84 (quint, 2H, J = 7.0 Hz), 2.15–2.02 (m,
5H), 2.65 (t, 2H, J = 7.8 Hz), 2.77 (t, 2H, J = 5.9 Hz), 3.29 (q,
2H, J = 6.9 Hz), 5.42–5.28 (m, 4H), 7.22–7.15 (m, 3H), 7.30–
7.26 (m, 2H). 13C NMR (CDCl3, 75 MHz): δC 14.0, 22.5, 25.6,
25.8, 27.2, 29.1, 29.3, 29.3, 29.6, 31.2, 31.5, 33.3, 36.7, 39.2,
125.9, 127.9, 128.0, 128.3, 128.4, 130.0, 130.2, 141.5, 173.4.
1
mixture (5 : 1). H NMR (CDCl3, 300 MHz): δH 0.88 (t, 3H, J =
7.2 Hz), 1.28–1.17 (m, 28H), 1.65 (q, 2H, J = 7.7 Hz), 2.21 (t,
2H, J = 7.2 Hz), 4.45 (d, 2H, J = 5.7 Hz), 5.70 (bs, 1H), 7.32–
7.20 (m, 5H). 13C NMR (CDCl3, 75 MHz): δC 13.8, 22.4, 25.5,
29.0, 29.1, 29.1, 29.2, 29.3, 29.4, 29.4, 29.4, 29.5, 29.5, 29.5,
29.6, 31.7, 36.6, 43.3, 127.2, 127.5, 128.4, 138.2, 172.7.
(9Z)-N-Benzyloctadec-9-enamide (1e). (9Z)-N-Benzyloctadec-
9-enamide (1e) was obtained from oleic acid (500 mg, 1.77
mmol) and benzylamine (189 mg, 1.77 mmol). 209 mg of the
synthetic macamide 1e (56% yield) were obtained as a white
solid. The product was chromatographed with a Hex–AcOEt
1
mixture (5 : 1). H NMR (CDCl3, 300 MHz): δH 0.83 (t, 3H, J =
7.0 Hz), 1.32–1.23 (m, 20H), 1.62 (q, 2H, J = 6.9 Hz), 2.02–1.85
(m, 4H), 2.18 (t, 2H, J = 7.2 Hz), 4.40 (d, 2H, J = 5.7 Hz), 5.37–
5.26 (m, 2H) 5.70 (bs, 1H), 7.34–7.22 (m, 5H). 13C NMR
(CDCl3, 75 MHz): δC 14.3, 22.8, 25.9, 27.3, 27.4, 29.3, 29.4,
29.4, 29.4, 29.5, 29.7, 29.8, 29.9, 32.0, 37.0, 43.7, 127.6, 128.0,
128.8, 129.9, 130.1, 138.6, 173.0.
(9Z,12Z)-N′-Phenyloctadeca-9,12-dienehydrazide
(9Z,12Z)-N′-Phenyloctadeca-9,12-dienehydrazide
(2a).
was
(2a)
obtained from phenylhydrazine (215 mg, 2 mmol), CH2Cl2 (3
mL mmol−1), Et3N (201 mg, 2 mmol) and (9Z,12Z)-octadeca-
9,12-dienoyl chloride (500 mg, 1.66 mmol). 350 mg of synthetic
macamide 2a (70% yield) were obtained as a colourless liquid
with a yield. The product was chromatographed with a Hex–
AcOEt mixture (2: 1). 1H NMR (CDCl3, 300 MHz): δH 0.81 (t, 3H,
J = 7.1 Hz), 1.40–1.22 (m, 14H), 1.72–1.59 (m, 2H), 2.08–1.97
(m, 4H), 2.18 (t, 1H, J = 7.8 Hz), 2.69 (q, 2H, J = 5.2 Hz), 5.41–
5.27 (m, 4H), 6.94–6.75 (m, 2H), 7.38–7.19 (m, 3H). 13C NMR
(CDCl3, 75 MHz): δC 14.1, 22.6, 25.5, 25.6, 27.2, 29.2, 29.3, 29.3,
29.6, 31.5, 34.2, 113.5, 120.9, 127.9, 128.1, 129.0, 130.0, 130.2,
130.2, 148.1, 173.8.
(9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dienamide (2b).
(9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dienamide (2b) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (593
mg, 1.99 mmol) and 1-methyl benzylamine (241 mg, 1.99
mmol). 470 mg of the synthetic macamide 2b (73% yield)
(9Z,12Z)-N-Benzyloctadeca-9,12-dienamide (1f). (9Z,12Z)-N-
Benzyloctadeca-9,12-dienamide (1f) was obtained from
(9Z,12Z)-octadeca-9,12-dienoic acid (400 mg, 1.42 mmol) and
benzylamine (151.9 mg, 1.42 mmol). 265 mg of the synthetic
macamide 1f (50% yield) were obtained as a colourless
liquid. The product was chromatographed with a Hex–AcOEt
1
mixture (5 : 1). H NMR (CDCl3, 300 MHz): δH 0.89 (t, 3H, J =
7.0 Hz), 1.31–1.20 (m, 14H), 1.67–1.62 (m, 2H), 2.07–2.01 (m,
4H), 2.21 (t, 2H, J = 7.7 Hz), 2.77 (t, 2H, J = 5.8 Hz), 4.44 (d,
2H, J = 5.7 Hz), 5.47–5.23 (m, 4H) 5.70 (bs, 1H), 7.32 (m, 5H).
13C NMR (CDCl3, 75 MHz): δC 14.2, 22.7, 25.8, 25.9, 27.3,
27.4, 29.3, 29.4, 29.5, 29.5, 29.7, 31.7, 37.0, 43.7, 127.6, 128.0,
128.1, 128.2, 128.9, 130.2, 130.4, 138.6, 173.0. HRESIMS [M +
H]+ m/z 370.3102 (calculated for C25H40NO 370.3104).
(9Z,12Z)-N-Phenyloctadeca-9,12-dienamide (1g). (9Z,12Z)-
N-Phenyloctadeca-9,12-dienamide (1g) was obtained from
were obtained as
a
yellowish liquid. The product was
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chromatographed with a Hex–AcOEt mixture (5 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 7.0 Hz), 1.36–1.25 (m,
14H), 1.49 (d, 3H, J = 6.9 Hz), 1.65–1.56 (m, 4H), 2.04–2.00
(m, 4H), 2.16 (t, 2H, J = 7.8 Hz) 2.77 (t, 2H, J = 5.9 Hz), 5.15
(quint, 1H, J = 7.1 Hz), 5.43–5.29 (m, 4H), 5.61 (bd, 1H), 7.36–
7.24 (m, 5H). 13C NMR (CDCl3, 75 MHz): δC 14.1, 21.8, 22.6,
25.7, 25.8, 27.2, 29.2, 29.3, 29.3, 29.4, 29.6, 31.5, 36.8, 48.5,
126.2, 127.2, 127.9, 128.1, 128.6, 130.1, 130.2, 143.5, 172.3.
(9Z,12Z)-N-(1-Phenylpropyl)-octadeca-9,12-dienamide (2c).
(9Z,12Z)-N-(1-Phenylpropyl)-octadeca-9,12-dienamide (2c) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (658
mg, 2.21 mmol) and 1-ethyl benzylamine (300 mg, 2.21
mmol). 608 mg of the synthetic macamide 2c (69% yield) were
were obtained as
a
yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (4 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 7.0 Hz), 1.36–1.25 (m,
14H), 1.49 (d, 3H, J = 6.9 Hz), 1.65–1.56 (m, 4H), 2.04–2.00
(m, 4H), 2.16 (t, 2H, J = 7.8 Hz) 2.77 (t, 2H, J = 5.9 Hz), 2.88
(s, 3H), 4.56 (s, 2H), 5.43–5.29 (m, 4H), 5.61 (bd, 1H), 7.36–
7.15 (m, 5H). 13C NMR (CDCl3, 75 MHz): δC 14.1, 21.8, 22.6,
25.7, 25.8, 27.2, 29.2, 29.3, 29.3, 29.4, 29.6, 31.5, 36.8, 50.6,
126.2, 127.8, 127.9, 128.4, 128.6, 129.9, 130.2, 137.4, 173.3.
(9Z,12Z)-1-(Benzyloxy)-octadeca-9,12-dien-1-olate
(2g).
(9Z,12Z)-1-(Benzyloxy)-octadeca-9,12-dien-1-olate (2g) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (581
mg, 1.94 mmol) and benzyl alcohol (200 mg, 1.94 mmol). 640
mg of the synthetic macamide 2g (80% yield) were obtained
as a yellowish liquid. The product was chromatographed with
a Hex–AcOEt mixture (4 : 1). 1H NMR (CDCl3, 300 MHz): δH
0.90 (t, 3H, J = 6.9 Hz), 1.38–1.27 (m, 14H), 1.70–1.60 (m, 2H),
2.11–2.01 (m, 4H), 2.36 (t, 2H, J = 7.6 Hz), 2.78 (t, 2H, J = 5.9
Hz), 5.12 (s, 2H), 5.40–5.30 (m, 4H), 7.38–7.35 (m, 5H). 13C
NMR (CDCl3, 75 MHz): δC 14.1, 22.6, 25.0, 25.7, 29.1, 29.2,
29.4, 29.6, 31.6, 34.3, 66.0, 128.0, 128.1, 128.1, 128.2, 128.5,
130.0, 130.2, 136.2, 173.5 HRESIMS [M + Na]+ m/z 425.2633
(calculated for C25H32O2Na 393.2764).
obtained as
a
yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (5 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.87 (t, 6H, J = 7.4 Hz), 1.34–1.23 (m,
14H), 1.62–1.57 (m, 4H), 1.85–1.74 (m, 2H), 2.06–1.98 (m, 3H),
2.15 (t, J = 7.8 Hz), 2.75 (t, 1H, J = 5.7 Hz), 4.87 (q, 1H, J = 7.3
Hz), 5.41–5.26 (m, 3H), 5.65 (d, 1H, J = 6.7 Hz), 7.34–7.22 (m,
5H). 13C NMR (CDCl3, 75 MHz): δC 10.8, 14.1, 22.6, 25.7, 25.9,
27.3, 29.2, 29.2, 29.3, 29.3, 29.4, 29.7, 31.6, 36.9, 54.8, 126.7,
127.3, 128.0, 128.1, 128.6, 130.1, 130.3, 142.4, 172.6.
(9Z,12Z)-N-(2-Hydroxy-1-phenylethyl)-octadeca-9,12-
dienamide (2d). (9Z,12Z)-N-(2-Hydroxy-1-phenylethyl)-octadeca-
9,12-dienamide (2d) was obtained from (9Z,12Z)-octadeca-9,12-
dienoyl chloride (593 mg, 2.18 mmol) and DL-2-phenylglycinol
(300 mg, 2.18 mmol). 535 mg of the synthetic macamide 2d
(77% yield) were obtained as a yellowish liquid. The product
was chromatographed with a Hex–AcOEt mixture (5: 1). 1H
NMR (CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 6.9 Hz), 1.39–1.26
(m, 14H), 1.69–1.59 (m, 4H), 2.07–2.01 (m, 3H), 2.25 (t, 2H, J =
7.4 Hz), 2.77 (t, 2H, J = 6.9 Hz), 3.91–3.87 (m, 2H), 5.07 (q, 1H, J
= 6.2 Hz), 5.41–5.29 (m, 3H), 6.1 (m, d, J = 6.6 Hz), 7.41–7.27
(m, 5H). 13C NMR (CDCl3, 75 MHz): δC 14.2, 22.7, 25.7, 25.8,
27.3, 29.3, 29.4, 29.4, 29.4, 29.7, 31.6, 36.8, 55.9, 66.5, 126.8,
127.9, 128.0, 128.2, 128.9, 130.1, 130.3, 139.3, 174.1.
(9Z,12Z)-1-(Benzylthio)-octadeca-9,12-dien-1-olate
(2h).
(9Z,12Z)-1-(Benzylthio)-octadeca-9,12-dien-1-olate (2h) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (357
mg, 1.20 mmol) and benzyl mercaptan (150 mg, 1.20 mmol).
355 mg of the synthetic macamide 2h (76% yield) were
obtained as
a
yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (4 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.88 (t, 3H, J = 6.9 Hz), 1.29 (m, 14H),
1.64 (m, 2H), 2.02 (m, 4H), 2.54 (t, 2H, J = 7.7 Hz), 2.76 (t,
2H, J = 5.9 Hz), 4.10 (s, 2H), 5.38–5.25 (m, 4H), 7.33–7.23 (m,
5H) 13C NMR (CDCl3, 75 MHz): δC 14.1, 22.6, 25.6, 25.7, 27.2,
27.3, 28.9, 29.1, 29.2, 29.4, 29.6, 31.6, 33.1, 43.8, 127.2, 128.0,
128.1, 128.6, 128.8, 130.0, 130.2, 137.8, 198.6.
(9Z,12Z)-N-(2-Phenylpropan-2-yl)-octadeca-9,12-dienamide
(2e). (9Z,12Z)-N-(2-Phenylpropan-2-yl)-octadeca-9,12-dienamide
(2e) was obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride
(658 mg, 2.21 mmol) and 1-methyl-1-phenyl ethylamine (300
mg, 2.21 mmol). 704 mg of the synthetic macamide 2e (80%
yield) were obtained as a yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (7 : 1). 300 mg of
the amine were introduced and 704 mg of product were
obtained (80% yield). 1H NMR (CDCl3, 300 MHz): δH 0.89 (t,
3H, J = 7.9 Hz), 1.39–1.25 (m, 14H), 1.66–1.56 (m, 4H), 1.70 (s,
6H), 2.08–2.02 (m, 3H), 2.14 (t, 2H, J = 7.4 Hz), 2.77 (t, 1H, J =
(9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dien-1-amine (2i).
(9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dien-1-amine (2i) was
obtained from derivative 1f (150 mg, 0.40 mmol) and LiAlH4
(76 mg, 2 mmol). 119 mg of the synthetic macamide 2i (83%
yield) were obtained as a yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (1 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.85 (t, 3H, J = 6.9 Hz), 1.33–1.20 (m,
14H), 1.32 (d, 3H, J = 10.4 Hz), 2.06–1.96 (m, 4H), 2.49–2.34
(m, 2H), 2.73 (t, 2H, J = 5.8 Hz), 3.71 (q, 1H, J = 6.6 Hz), 5.41–
5.26 (m, 4H), 7.16–7.28 (m, 5H) 13C NMR (CDCl3, 75 MHz): δC
14.2, 22.7, 24.4, 25.8, 27.3, 27.3, 27.5, 29.4, 29.5, 29.6, 29.8,
30.4, 31.7, 48.0, 58.5, 126.7, 126.9, 128.1, 128.1, 128.5, 130.2,
130.3, 146.0. HRESIMS [M + H]+ m/z 370.3468 (calculated for
C26H44N 370.3468).
(9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dienothiamide
(2j). (9Z,12Z)-N-(1-Phenylethyl)-octadeca-9,12-dienothiamide
(2j) was obtained from derivative 1f (130 mg, 0.34 mmol) and
Lawesson's reagent (206 mg, 0.51 mmol). 135 mg of the
synthetic macamide 2j (99% yield) were obtained as a
yellowish liquid. The product was chromatographed with a
5.9 Hz), 5.39–5.29 (m, 4H), 5.66 (bs, 1H), 7.41–7.19 (m, 5H). 13
C
NMR (CDCl3, 75 MHz): δC 14.1, 22.6, 25.7, 25.8, 27.2, 29.2, 29.2,
29.3, 29.3, 29.4, 29.7, 31.5, 37.3, 55.7, 124.8, 126.5, 128.0, 128.1,
128.3, 130.1, 130.2, 147.1, 172.5.
(9Z,12Z)-N-Benzyl-N-methyloctadeca-9,12-dienamide (2f).
(9Z,12Z)-N-Benzyl-N-methyloctadeca-9,12-dienamide (2f) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (572
mg, 1.92 mmol) and N-benzyl methylamine (233 mg, 1.92
mmol). 477 mg of the synthetic macamide 2f (65% yield)
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1
Hex–AcOEt mixture (15 : 1). 1H NMR (CDCl3, 300 MHz): δH
0.83 (t, 3H, J = 6.9 Hz), 1.32–1.18 (m, 14H), 1.50 (d, 3H, J =
6.9 Hz), 1.72–1.62 (m, 2H), 2.03–1.93 (m, 2H), 2.54 (t, 2H, J =
7.8 Hz), 2.71 (t, 2H, J = 5.9 Hz), 5.36–5.25 (m, 4H), 5.70
(quint, 1H, J = 7.1 Hz), 7.27–7.17 (m, 5H), 7.78 (bd, 1H, J =
8.0 Hz). 13C NMR (CDCl3, 75 MHz): δC 14.0, 19.9, 22.4, 25.5,
27.1, 28.7, 29.0, 29.1, 29.2, 29.4, 29.5, 31.4, 47.0, 54.2, 126.4,
127.5, 127.8, 127.9, 128.6, 129.8, 130.0, 141.3, 204.2.
(9Z,12Z)-N-(1-Cyclohexylethyl)-octadeca-9,12-dienamide
(3a). (9Z,12Z)-N-(1-Cyclohexylethyl)-octadeca-9,12-dienamide
(3a) was obtained from (9Z,12Z)-octadeca-9,12-dienoyl
chloride (187 mg, 0.63 mmol) and cyclohexyl ethylamine (80
mg, 0.63 mmol). 178 mg of the synthetic macamide 3a (73%
yield) were obtained as a yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (5 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.88 (t, 3H, J = 6.9 Hz), 1.29 (m, 14H),
1.64 (m, 2H), 2.02 (m, 4H), 2.54 (t, 2H, J = 7.7 Hz), 2.76 (t,
2H, J = 5.9 Hz), 4.10 (s, 2H), 5.38–5.25 (m, 4H), 7.33–7.23 (m,
5H). 13C NMR (CDCl3, 75 MHz): δC 14.2, 18.2, 22.7, 25.8, 26.0,
26.3, 26.3, 26.6, 27.3, 29.2, 29.3, 29.4, 29.4, 29.5, 29.7, 31.7,
37.3, 43.2, 49.3, 128.1, 128.2, 130.2, 130.3, 172.4.
Hex–AcOEt mixture (4 : 1). H NMR (CDCl3, 300 MHz): δH 0.89
(t, 3H, J = 6.8 Hz), 1.44–4.17 (m, 14H), 1.47 (d, 3H, J = 6.9
Hz), 1.66–1.58 (m, 2H), 1.79–1.70 (m, 2H), 2.11–2.02 (m, 4H),
2.15 (t, 2H, J = 7.9 Hz), 2.54 (t, 2H, J = 7.6 Hz), 2.82–2.74 (m,
4H), 5.16 (quint, 1H, J = 7.3 Hz), 5.44–5.28 (m, 4H), 5.61 (bd,
1H, J = 7.9 Hz), 7.03 (d, 2H, J = 8.6 Hz), 7.32 (d, 2H, J = 8.6
Hz). 13C NMR (CDCl3, 75 MHz): δC 14.2, 22.7, 25.8, 27.3, 29.2,
29.2, 29.3, 29.3, 29.4, 29.5, 29.7, 31.7, 121.9, 127.5, 128.1,
128.2, 130.2, 130.4, 140.8, 150.0, 172.2.
(9Z,12Z)-N-(1-(4-Methoxyphenyl)-ethyl)-octadeca-9,12-
dienamide
(3e).
(9Z,12Z)-N-(1-(4-Methoxyphenyl)-ethyl)-
octadeca-9,12-dienamide (3e) was obtained from (9Z,12Z)-
octadeca-9,12-dienoyl chloride (197 mg, 0.66 mmol) and 1-(4-
methoxyphenyl)-ethylamine (100 mg, 0.66 mmol). 226 mg of
the synthetic macamide 3e (83% yield) were obtained as a
yellowish liquid. 1H NMR (CDCl3, 300 MHz): δH 0.87 (t, 3H, J =
7.0 Hz), 1.40–1.27 (m, 14H), 1.42 (d, 3H, J = 7.0 Hz), 1.54–1.61
(m, 2H), 2.02–2.10 (m, 4H), 2.15 (t, 2H, J = 7.8 Hz), 2.75 (t, 2H,
J = 5.9 Hz), 3.74 (s, 3H), 5.05 (quint, 1H, J = 10.7 Hz), 5.44–
5.32 (m, 4H), 6.12 (bd, 1H, J = 7.9 Hz), 6.82 (d, 2H, J = 8.7 Hz),
7.20 (d, 2H, J = 8.7 Hz). 13C NMR (CDCl3, 75 MHz): δC 14.0,
21.7, 22.5, 25.6, 25.8, 27.2, 29.1, 29.3, 29.3, 29.3, 29.6, 31.5,
36.8, 47.9, 55.2, 113.9, 127.3, 130.0, 130.2, 135.6, 158.7, 172.2.
(9Z,12Z)-N-(1-(p-Tolyl)-ethyl)-octadeca-9,12-dienamide (3f).
(9Z,12Z)-N-(1-(p-Tolyl)-ethyl)-octadeca-9,12-dienamide (3f) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (289
mg, 0.97 mmol) and 1-(4-methylphenyl)-ethylamine (130 mg,
0.97 mmol). 331 mg of the synthetic macamide 3f (80% yield)
(9Z,12Z)-N-(3,3-Dimethylbutan-2-yl)-octadeca-9,12-
dienamide (3b). (9Z,12Z)-N-(3,3-Dimethylbutan-2-yl)-octadeca-
9,12-dienamide (3b) was obtained from (9Z,12Z)-octadeca-
9,12-dienoyl chloride (294 mg, 0.98 mmol) and 2-amino-3,3-
dimethylbutane (100 mg, 0.98 mmol). 310 mg of the synthetic
macamide 3b (77% yield) were obtained as a yellowish liquid.
The product was chromatographed with a Hex–AcOEt mixture
1
(7 : 1). H NMR (CDCl3, 300 MHz): δH 0.83 (s, 3H), 0.83 (t, 9H,
were obtained as a yellowish liquid. The product was
J = 8.0 Hz), 0.98 (d, 3H, J = 7.8 Hz), 1.24 (m, 14H), 1.57 (m,
2H), 1.98 (q, 4H, J = 6.4 Hz), 2.11 (t, 2H, J = 7.9 Hz), 2.70 (t,
2H, J = 5.9 Hz), 3.83 (m, 1H), 5.30 (m, 4H), 5.52 (bd, 1H, J =
9.5 Hz). 13C NMR (CDCl3, 75 MHz): δC 14.0, 16.2, 22.6, 25.6,
26.0, 26.2, 27.2, 29.3, 29.3, 29.3, 29.6, 31.5, 34.1, 37.1, 52.4,
127.9, 128.0, 130.0, 130.1, 172.3.
chromatographed with a Hex–AcOEt mixture (6 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.90 (t, 3H, J = 6.9 Hz), 1.25–1.40 (m,
14H), 1.45 (d, 3H, J = 6.9 Hz), 1.66–1.58 (m, 2H), 2.01–2.08
(m, 4H), 2.15 (t, 2H, J = 8.0 Hz), 2.32 (s, 3H), 2.78 (t, 2H, J =
5.9 Hz), 5.10 (quint, 1H, J = 7.0 Hz), 5.27–5.43 (m, 4H), 6.02
(bd, J = 7.8 Hz), 7.12 (d, 2H, J = 8.0 Hz), 7.20 (d, 2H, J = 8.1
Hz) 13C NMR (CDCl3, 75 MHz): δC 14.1, 21.04, 21.8, 22.6,
25.7, 25.8, 27.2, 29.2, 29.3, 29.31, 29.4, 31.6, 36.8, 48.3, 126.1,
128.0, 128.1, 129.3, 130.1, 130.2, 136.8, 140.5, 172.2.
(9Z,12Z)-N-(1-((3r,5r,7r)-Adamantan-1-yl)-ethyl)-octadeca-
9,12-dienamide (3c). (9Z,12Z)-N-(1-((3r,5r,7r)-Adamantan-1-yl)-
ethyl)-octadeca-9,12-dienamide (3c) was obtained from
(9Z,12Z)-octadeca-9,12-dienoyl chloride (250 mg, 0.84 mmol)
and 1-adamantylamine (150 mg, 0.84 mmol). 240 mg of the
synthetic macamide 3c (65% yield) were obtained as a
yellowish liquid. The product was chromatographed with a
(9Z,12Z)-N-(1-(4-(Trifluoromethyl)-phenyl)-ethyl)-octadeca-
9,12-dienamide
(3g).
(9Z,12Z)-N-(1-(4-(Trifluoromethyl)-
phenyl)-ethyl)-octadeca-9,12-dienamide (3g) was obtained
from (9Z,12Z)-octadeca-9,12-dienoyl chloride (141 mg, 0.47
mmol) and 1-(4-trifluoromethylphenyl)-ethylamine (90 mg,
0.47 mmol). 156 mg of the synthetic macamide 3g (73%
yield) were obtained as a yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (6 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.88 (t, 3H, J = 6.8 Hz), 1.25–1.40 (m,
14H), 1.48 (d, 3H, J = 6.9 Hz), 1.66–1.58 (m, 2H), 2.01–2.08
(m, 4H), 2.18 (t, 3H, J = 8.1 Hz), 2.77 (t, 2H, J = 6.2 Hz), 5.17
(quint, 1H, J = 7.2 Hz), 5.27–5.43 (m, 4H), 5.74 (bd, 1H, J =
7.1 Hz), 7.41 (d, 2H, J = 8.1 Hz), 7.58 (d, 2H, J = 8.1 Hz). 13C
NMR (CDCl3, 75 MHz): δC 14.2, 22.0, 22.7, 25.8, 25.8, 27.3,
27.3, 25.8, 25.8, 29.3, 29.4, 29.5, 29.7, 31.7, 36.9, 48.5, 175.7
(q, J = 3.7 Hz), 126.6, 128.0, 128.2, 130.1, 130.1, 130.4.
1
Hex–AcOEt mixture (7 : 1). H NMR (CDCl3, 300 MHz): δH 0.88
(t, 3H, J = 6.8 Hz), 1.00 (d, 3H, J = 6.9 Hz), 1.42–1.25 (m, 14H),
1.60–1.40 (m, 6H), 1.75–1.57 (m, 8H), 2.10–1.93 (m, 6H), 2.16
(t, 2H, J = 7.8 Hz), 2.77 (t, 2H, J = 5.9 Hz), 3.77–3.67 (m, 1H),
5.42–5.22 (m, 4H). 13C NMR (CDCl3, 75 MHz): δC 14.2, 14.8,
22.7, 25.8, 26.1, 27.3, 28.5, 29.3, 29.4, 29.5, 29.5, 29.8, 31.7,
35.9, 37.2, 37.4, 38.6, 52.8, 128.1, 128.2, 130.2, 130.4, 172.5.
(9Z,12Z)-N-(1-(4-Hydroxyphenyl)-ethyl)-octadeca-9,12-
dienamide
(3d).
(9Z,12Z)-N-(1-(4-Hydroxyphenyl)-ethyl)-
octadeca-9,12-dienamide (3d) was obtained from (9Z,12Z)-
octadeca-9,12-dienoyl chloride (271 mg, 0.91 mmol) and 1-(4-
hydroxyphenyl)-ethylamine (125 mg, 0.91 mmol). 251 mg of
the synthetic macamide 3d (69% yield) were obtained as a
yellowish liquid. The product was chromatographed with a
(9Z,12Z)-N-(1-(4-Fluorophenyl)-ethyl)-octadeca-9,12-
dienamide (3h). (9Z,12Z)-N-(1-(4-Fluorophenyl)-ethyl)-octadeca-
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9,12-dienamide (3h) was obtained from (9Z,12Z)-octadeca-9,12-
dienoyl chloride (246 mg, 0.82 mmol) and 4-fluoro-α-methyl
benzylamine (115 mg, 0.82 mmol). 255 mg of the synthetic
macamide 3h (79% yield) were obtained as a yellowish liquid.
The product was chromatographed with a Hex–AcOEt mixture
1H, J = 8.0 Hz), 7.40–7.22 (m, 4H). 13C NMR (CDCl3, 75 MHz):
δC 14.2, 19.3, 21.2, 22.7, 25.8, 25.9, 29.3, 29.4, 29.5, 29.7, 31.7,
36.9, 45.3, 124.8, 126.4, 127.4, 128.0, 128.2, 130.2, 130.4, 130.9,
136.2, 141.3, 172.1.
(9Z,12Z)-N-(1-(m-Tolyl)-ethyl)-octadeca-9,12-dienamide (3l).
(9Z,12Z)-N-(1-(m-Tolyl)-ethyl)-octadeca-9,12-dienamide (3l) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (176
mg, 0.59 mmol) and m-tolyl ethylamine (80 mg, 0.59 mmol).
162 mg of the synthetic macamide 3l (77% yield) were
1
(3 : 1). H NMR (CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 6.7 Hz),
1.42–1.25 (m, 14H), 1.45 (d, 3H, J = 6.9 Hz), 1.54–1.61 (m, 2H),
2.04–2.10 (m, 4H), 2.16 (t, 2H, J = 8.1 Hz), 2.16 (t, 2H, J = 8.1
Hz), 2.77 (t, 2H, J = 6.1 Hz), 5.11 (quint, 1H, J = 7.2 Hz), 5.42–
5.34 (m, 4H), 5.92 (bd, 1H), 6.9–7.1 (m, 2H), 7.26–7.36 (m, 2H).
13C NMR (CDCl3, 75 MHz): δC 14.1, 21.9, 22.7, 25.7, 25.8, 27.3,
29.2, 29.3, 29.3, 29.4, 29.7, 31.6, 36.9, 48.0, 115.5 (d, J = 79.9
Hz), 127.9 (d, J = 8.0 Hz), 128.1 (d, J = 12.5 Hz), 130.2 (d, J =
obtained as
a
yellowish liquid. The product was
chromatographed with a Hex–AcOEt mixture (7 : 1). 1H NMR
(CDCl3, 300 MHz): δH 0.89 (t, 3H, J = 6.9 Hz), 1.41–1.25 (m,
14H), 1.45 (d, 2H, J = 6.9 Hz), 1.63 (m, 3H), 2.08–2.01 (m,
4H), 2.16 (t, 2H, J = 7.9 Hz), 2.35 (s, 3H), 2.77 (t, 2H, J = 5.9
Hz), 5.10 (quint, 1H, J = 7.2 Hz), 5.43–5.28 (m, 4H), 5.60 (d,
1H, J = 7.8 Hz), 7.26–7.06 (m, 4H) 13C NMR (CDCl3, 75 MHz):
δC 14.2, 21.6, 21.9, 22.7, 25.8, 25.9, 27.4, 29.3, 29.4, 29.5, 29.8,
31.7, 37.1, 48.7, 123.3, 127.2, 128.1, 128.2, 128.2, 128.7, 130.2,
130.4, 138.5, 143.4, 172. HRESIMS [M + H]+ m/z 398.3354
(calculated for C27H44NO 398.3359).
16.0 Hz), 139.3 (d, J = 3.1 Hz), 162.1 (d, J = 245.4 Hz), 172.3. 19
NMR (CDCl3, 75 MHz): δC −115.5.
F
(9Z,12Z)-N-(1-(4-Bromophenyl)-ethyl)-octadeca-9,12-
dienamide (3i). (9Z,12Z)-N-(1-(4-Bromophenyl)-ethyl)-octadeca-
9,12-dienamide (3i) was obtained from (9Z,12Z)-octadeca-9,12-
dienoyl chloride (201 mg, 0.67 mmol) and 4-bromo-α-methyl
benzylamine (135 mg, 0.67 mmol). 255 mg of the synthetic
macamide 3i (82% yield) were obtained as a yellowish liquid.
The product was chromatographed with a Hex–AcOEt mixture
(9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-ethyl)-octadeca-9,12-
dienamide (3m). (9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-ethyl)-
octadeca-9,12-dienamide (3m) was obtained from (9Z,12Z)-
octadeca-9,12-dienoyl chloride (239 mg, 0.80 mmol) and
1-(3,5-dimethylphenyl)-ethylamine (120 mg, 0.80 mmol). 162
mg of the synthetic macamide 3m (75% yield) were obtained
as a yellowish liquid. The product was chromatographed with
a Hex–AcOEt mixture (7 : 1). 1H NMR (CDCl3, 300 MHz): δH
0.89 (t, 3H, J = 6.7 Hz), 1.41–1.25 (m, 14H), 1.45 (d, 2H, J =
6.9 Hz), 1.63 (m, 2H), 2.08–2.01 (m, 4H), 2.15 (t, 2H, J = 7.9
Hz), 2.24 (s, 3H), 2.26 (s, 3H), 2.77 (t, 2H, J = 5.9 Hz), 5.10
(quint, 1H, J = 7.2 Hz), 5.43–5.28 (m, 4H), 5.60 (d, 1H, J = 7.8
Hz), 7.12–7.03 (m, 3H) 13C NMR (CDCl3, 75 MHz): δC 14.2,
19.5, 20.0, 21.9, 22.7, 25.8, 25.9, 27.4, 29.3, 29.4, 29.5, 29.8,
31.7, 37.1, 48.7, 123.6, 127.8, 128.0, 128.2, 130.0, 130.2, 130.4,
1
(4 : 1). H NMR (CDCl3, 300 MHz): δH 0.87 (t, 3H, J = 4.9 Hz),
1.42–1.25 (m, 14H), 1.45 (d, 3H, J = 7.0 Hz), 1.56–1.61 (m, 2H),
2.05 (q, 4H, J = 6.4 Hz), 2.12 (t, 2H, J = 8.1 Hz), 2.75 (t, 2H, J =
5.9 Hz), 5.00 (quint, 1H, J = 7.3 Hz), 5.42–5.32 (m, 4H), 6.45
(bd, 1H, J = 7.8 Hz), 7.12 (d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.4
Hz). 13C NMR (CDCl3, 75 MHz): δC 14.1, 21.7, 22.6, 25.6, 25.8,
27.2, 29.2, 29.3, 29.3, 29.3, 29.6, 31.5, 36.6, 48.0, 120.9, 128.0,
127.9, 128.1, 130.0, 130.2, 131.5, 142.8, 172.4.
(9Z,12Z)-N-(1-(4-Ethylphenyl)-ethyl)-octadeca-9,12-
dienamide (3j). (9Z,12Z)-N-(1-(4-Ethylphenyl)-ethyl)-octadeca-
9,12-dienamide (3j) was obtained from (9Z,12Z)-octadeca-
9,12-dienoyl chloride (159 mg, 0.53 mmol) and 1-(4-
ethylphenyl)-ethylamine (80 mg, 0.53 mmol). 157 mg of the
synthetic macamide 3j (73% yield) were obtained as a
yellowish liquid. The product was chromatographed with a
137.0, 140.9, 172.1. HRESIMS [M
(calculated for C28H46NO 412.3574).
+
H]+ m/z 412.3566
1
Hex–AcOEt mixture (4 : 1). H NMR (CDCl3, 300 MHz): δH 0.89
(9Z,12Z)-1-(1-(3,5-Dimethylphenyl)-ethoxy)-octadeca-9,12-
dien-1-olate (4a). (9Z,12Z)-1-(1-(3,5-Dimethylphenyl)-ethoxy)-
octadeca-9,12-dien-1-olate (4a) was obtained from (9Z,12Z)-
octadeca-9,12-dienoyl chloride (139 mg, 0.46 mmol) and
1-(3,5-dimethylphenyl)-ethan-1-ol (70 mg, 0.46 mmol). 128
mg of the synthetic macamide 4a (80% yield) were obtained
as a yellowish liquid. The product was chromatographed with
a Hex–AcOEt mixture (6 : 1). 1H NMR (CDCl3, 300 MHz): δH
0.89 (t, 3H, J = 6.7 Hz), 1.41–1.25 (m, 14H), 1.45 (d, 2H, J =
6.9 Hz), 1.63 (m, 2H), 2.08–2.01 (m, 4H), 2.15 (t, 2H, J = 7.9
Hz), 2.26 (s, 6H), 2.77 (t, 2H, J = 5.9 Hz), 4.08 (q, 1H, J = 6.9
Hz) 5.10 (quint, 1H, J = 7.2 Hz), 5.43–5.28 (m, 4H), 5.60 (d,
1H, J = 7.8 Hz), 6.92 (s, 2H), 6.84 (s, 1H). 13C NMR (CDCl3, 75
MHz): δC 14.2, 19.5, 20.0, 21.9, 22.7, 25.8, 25.9, 27.4, 29.3,
29.4, 29.5, 29.8, 31.7, 37.1, 72.1, 123.8, 127.8, 128.0, 129.5,
130.0, 130.2, 130.4, 138.2, 141.8, 173.1.
(t, 3H, J = 6.8 Hz), 1.23 (t, 3H, J = 7.6 Hz), 1.40–1.26 (m, 14H),
1.47 (d, 3H, J = 6.9 Hz), 1.68–1.59 (m, 2H), 2.06 (m, 4H), 2.15
(t, 2H, J = 7.9 Hz), 2.63 (q, 2H, J = 7.6 Hz), 2.77 (t, 2H, J = 5.7
Hz), 2.12 (quint, 1H, J = 7.0 Hz), 5.47–5.28 (m, 4H), 5.63 (bd,
1H, J = 7.9 Hz), 7.17 (d, 2H, J = 8.1 Hz), 7.25 (d, 2H, J = 8.1
Hz). 13C NMR (CDCl3, 75 MHz): δC 13.8, 15.2, 21.3, 22.3, 25.4,
25.4, 26.9, 28.2, 28.9, 29.0, 29.1, 29.3, 31.2, 36.7, 48.0, 126.0,
127.6, 127.8, 127.8, 129.8, 130.0, 140.2, 143.1, 171.8.
(9Z,12Z)-N-(1-(o-Tolyl)-ethyl)-octadeca-9,12-dienamide (3k).
(9Z,12Z)-N-(1-(o-Tolyl)-ethyl)-octadeca-9,12-dienamide (3k) was
obtained from (9Z,12Z)-octadeca-9,12-dienoyl chloride (176 mg,
0.59 mmol) and o-tolyl ethylamine (80 mg, 0.59 mmol). 162 mg
of the synthetic macamide 3k were obtained (77% yield) as a
yellowish liquid. The product was chromatographed with a
1
Hex–AcOEt mixture (7: 1). H NMR (CDCl3, 300 MHz): δH 0.97
(t, 3H, J = 6.7 Hz), 1.46–1.34 (m, 14H), 1.55 (d, 3H, J = 6.8 Hz),
1.73–1.66 (m, 2H), 2.16–2.10 (m, 2H), 2.22 (t, 2H, J = 8.1 Hz),
2.46 (s, 3H), 2.85 (t, 2H, J = 6.0 Hz), 5.52–5.36 (m, 4H), 5.69 (bd,
(9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-ethyl)-octadeca-9,12-
dien-1-amine (4b). (9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-ethyl)-
octadeca-9,12-dien-1-amine (4b) was obtained from derivative
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7c (80 mg, 0.20 mmol) and LiAlH4 (38 mg, 1 mmol). 59 mg of
the synthetic macamide 4b (77% yield) were obtained as a
yellowish liquid. The product was chromatographed with a
was used as a positive control at a concentration of 0.008 μM,
showing cell death.
1
Hex–AcOEt mixture (1 : 1). H NMR (CDCl3, 300 MHz): δH 0.85
3.10. TNF-α inhibition assay
(t, 3H, J = 6.9 Hz), 1.33–1.20 (m, 14H), 1.32 (d, 3H, J = 10.4
Hz), 2.06–1.96 (m, 4H), 2.49–2.34 (m, 2H), 2.73 (t, 2H, J = 5.8
Hz), 3.71 (q, 1H, J = 6.6 Hz), 5.41–5.26 (m, 4H), 6.91 (s, 1H),
7.00 (s, 2H). 13C NMR (CDCl3, 75 MHz): δC 14.2, 21.5, 22.7,
25.8, 27.3, 27.4, 29.4, 29.5, 29.6, 29.8, 30.4, 31.7, 48.0, 58.7,
124.8, 126.9, 128.1, 128.1, 130.1, 130.3, 130.4, 138.3.
THP-1 cell (5 × 104 cells per well) were seeded on a 96-well
culture plate and incubated for 12 h. The cells were then pre-
treated with various concentrations of the synthetic
macamides for 2 h, before stimulation with LPS (0.1 μg mL−1)
with or without synthetic macamides samples for 12 h.
Supernatants were then collected and the protein expression
levels of TNF-α were measured by using an enzyme-linked
immunosorbent assay kit (ELISA), according to the
manufacturer's instructions (Diaclone Company, Besancon,
(9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-ethyl)-octadeca-9,12-
dienethioamide
(4c).
(9Z,12Z)-N-(1-(3,5-Dimethylphenyl)-
ethyl)-octadeca-9,12-dienethioamide (4c) was obtained from
derivative 3m (70 mg, 0.17 mmol) and Lawesson's reagent
(103 mg, 0.25 mmol). 62 mg of the synthetic macamide 4c
(93% yield) were obtained as a yellowish liquid. The product
was chromatographed with a Hex–AcOEt mixture (15 : 1). 1H
NMR (CDCl3, 300 MHz): δH 0.83 (t, 3H, J = 6.9 Hz), 1.32–1.18
(m, 14H), 1.50 (d, 3H, J = 6.9 Hz), 1.72–1.62 (m, 2H), 2.03–1.93
(m, 2H), 2.54 (t, 2H, J = 7.8 Hz), 2.71 (t, 2H, J = 5.9 Hz), 5.36–
5.25 (m, 4H), 5.70 (quint, 1H, J = 7.1 Hz), 6.95 (s, 2H), 7.11 (s,
1H). 13C NMR (CDCl3, 75 MHz): δC 14.0, 19.9, 22.4, 25.5, 27.1,
28.7, 29.0, 29.1, 29.2, 29.4, 29.5, 31.4, 47.0, 54.2, 124.5, 128.0,
128.2, 129.6, 130.1, 130.4, 138.6, 138.9, 141.4, 204.2.
France). Absorbance was read at 450 nm on
a
spectrophotometric ELISA plate reader (Anthos 2020, Version
2.0.5, Biochrom Ltd., UK). The percentage of TNF-α inhibition
was calculated from the ratio between the observed TNF-α
amount secreted by treated cells (μM) and the baseline
secretion of TNF-α (pg mL−1). C87 (Sigma-Aldrich; 332420-90-
3) was used as positive control at a concentration of 0.11 μM.
Results were normalised to the DMSO solvent control (1%).
3.11. Statistical analysis
CC50 and IC50 values were determined by non-linear
regression. All the experiments were performed in triplicate.
One-way ANOVA statistical analysis (Tukey's multiple
comparisons test, *p < 0.05; ***p < 0.001) was performed to
evaluate the significant differences among values. All the
analysis was performed using GraphPad Prism, version 8.4.0.
3.8. Cell culture
The THP-1 cell line (human peripheral blood monocyte, TIB-
202) was used in this study. Cells were grown in specific
media according to ATCC recommendations. The incubation
condition for the cells was in an atmosphere of 95% air and
5% CO2 at 37 °C. DMEM (Sigma-Aldrich, St. Louis, MO, Author contribution
USA), FBS (Summit Biotechnology; Ft. Collins, CO) and PBS
VTP and JAG experimental design of the chemistry study;
(SAFC Biosciences, Inc. Andover-Hampshire, UK) were used
as culture mediums. L-Glutamine was obtained from
Applichem. Penicillin and streptomycin were purchased from
Fisher Scientific (Pittsburgh, PA). For cytotoxicity and activity
assays, the synthetic macamides were dissolved in DMSO
(Merck) at a concentration of 10 mM.
ARS and LAT contributed to the analysis of the spectral data;
LAT and AMS contributed to the conception and
experimental design of the pharmacological study and
analysis of the measurements of the clog P values; LAT
contributed to the writing and review of the manuscript.
Conflicts of interest
3.9. MTT-cytotoxicity assay
There are no conflicts to declare.
THP-1 cells were seeded in 96-well plates at a density of 5 ×
104 cells per well and incubated overnight at 37 °C in a
humidified atmosphere of 5% CO2. Subsequently, cells were
treated with test synthetic macamides at the indicated
concentrations and DMSO as control for 72 h. An MTT
solution (50 μL, 7 mg mL−1 in PBS) was added to each well,
and plates were incubated for an additional 2 h. Next, the
medium was removed and 100 μL of DMSO was added to
each well to dissolve the formazan crystals that were formed.
Acknowledgements
This work was supported by the Fundación de la Universidad
Autónoma de Madrid (FUAM).
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