Study on the synthesis, antioxidant properties, and self-assembly of carotenoid–flavonoid conjugates

Article abstract of DOI:10.3390/molecules25030636

Flavonoids and carotenoids possess beneficial physiological effects, such as high antioxidant capacity, anticarcinogenic, immunomodulatory, and anti-inflammatory properties, as well as protective effects against UV light. The covalent coupling of hydropho

Full text of DOI:10.3390/molecules25030636

molecules
Article
Study on the Synthesis, Antioxidant Properties, and
Self-Assembly of Carotenoid–Flavonoid Conjugates
1
1
1
2
2
Ildikó Línzembold , Dalma Czett , Katalin Böddi , Tibor Kurtán
, Sándor Balázs Király ,
Gergely Gulyás-Fekete , Anikó Takátsy , Tamás Lóránd , József Deli , Attila Agócs 1
1
1
1
1,3
1
,
and Veronika Nagy *
1
Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, Szigeti út 12,
H-7624 Pécs, Hungary; ildiko.szabo@aok.pte.hu (I.L.); czett.dalma@gmail.com (D.C.);
Katalin.Boddi@aok.pte.hu (K.B.); gergely.gulyas@aok.pte.hu (G.G.-F.); Aniko.Takatsy@aok.pte.hu (A.T.);
Tamas.Lorand@aok.pte.hu (T.L.); Jozsef.Deli@aok.pte.hu (J.D.); attila.agocs@aok.pte.hu (A.A.)
Department of Organic Chemistry, University of Debrecen, POB 400, H-4002 Debrecen, Hungary;
2
kurtan.tibor@science.unideb.hu (T.K.); kiraly.sandor.balazs@science.unideb.hu (S.B.K.)
3
Department of Pharmacognosy, University of P
écs, Faculty of Pharmacy, Rókus u. 2, H-7624 Pécs, Hungary
*
Correspondence: vera.nagy@aok.pte.hu; Tel.: +36-72-536-001 (ext. 31864)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Benoît Schoefs
Received: 10 January 2020; Accepted: 29 January 2020; Published: 1 February 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Flavonoids and carotenoids possess beneficial physiological effects, such as high antioxidant
capacity, anticarcinogenic, immunomodulatory, and anti-inflammatory properties, as well as protective
effects against UV light. The covalent coupling of hydrophobic carotenoids with hydrophilic
flavonoids, such as daidzein and chrysin, was achieved, resulting in new amphipathic structures.
7
-Azidohexyl ethers of daidzein and chrysin were prepared in five steps, and their azide-alkyne
0
[4 + 2] cycloaddition with pentynoates of 8 -apo-
β-carotenol, zeaxanthin, and capsanthin afforded
•
+
carotenoid–flavonoid conjugates. The trolox-equivalent antioxidant capacity against ABTS radical
cation and self-assembly of the final products were examined. The 1:1 flavonoid–carotenoid hybrids
generally showed higher antioxidant activity than their parent flavonoids but lower than that of
the corresponding carotenoids. The diflavonoid hybrids of zeaxanthin and capsanthin, however,
were found to exhibit a synergistic enhancement in antioxidant capacities. ECD (electronic circular
dichroism) and UV-vis analysis of zeaxanthin–flavonoid conjugates revealed that they form different
optically active J-aggregates in acetone/water and tetrahydrofuran/water mixtures depending on the
solvent ratio and type of the applied aprotic polar solvent, while the capsanthin derivatives showed
no self-assembly. The zeaxanthin bis-triazole conjugates with daidzein and with chrysin, differing
only in the position of a phenolic hydroxyl group, showed significantly different aggregation profile
upon the addition of water.
Keywords: carotenoids; flavonoids; click-reaction; antioxidant capacity; supramolecular chirality;
zeaxanthin–flavonoid conjugates; electronic circular dichroism
1
. Introduction
Countless natural bioactive compounds have been isolated from food products and their effects
on human health were studied. However, an increasing amount of evidence suggests that a nutrient
may have a specific action, the combination of nutrients displays different (even stronger) effects than
a sole component [
1
,
2
]. As foods are complex matrices, the bioaccessibility and bioavailability of
]. At the
a component are obviously influenced by the presence or absence of other constituents [3,4
same time, significant synergy was found in the physiological effects of many small molecules used in
combination [5].
Molecules 2020, 25, 636; doi:10.3390/molecules25030636
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Molecules 2020, 25, 636
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Our attention was directed to two natural antioxidant families, carotenoids and flavonoids. Both
are abundant in human nutrition and are connected to a beneficial influence on health. Carotenoids are
hydrophobic pigments that reduce the risks of a number of chronic diseases [6]. Especially, lutein and
zeaxanthin play an important role in the prevention of age-related macular degeneration, and were
found to be useful in the treatment of neurodegenerative disorders, such as Alzheimer’s disease [7].
Flavonoids are hydrophilic compounds and occur frequently in the form of glycosides. They are
referred to as multipotent antioxidants, as they have other favorable pharmacological effects [
8]. Daidzein,
an isoflavone, was found to be advantageous in cardiovascular diseases [ ], bone resorption [10], and
9
cell proliferation, but some adverse effects were also described [11]. Chrysin is a flavone with low
bioavailability and rapid excretion; however, it showed cancer chemopreventive activity [12], decreased
the symptoms of colitis [13], and was also found to be beneficial in connection with the insulin resistance
and type II diabetes mellitus [14].
Both carotenoids and flavonoids are rather sensitive to oxidation, but their stability and bioavailability
can be significantly increased by additives. Water-soluble carbohydrate-based nanoparticles were
described to form host–guest complexes with carotenoids, improving their properties [15]. The effect
of mixing carotenoids with flavonoids is of particular interest, as synergy arises in their protective
effect on low-density lipoprotein oxidation, which plays an important role in the development of
atherosclerosis [16]. Daidzein was found to regenerate carotenoids from their radical cation forms [17].
The carotenoid–flavonoid interactions, which happen at the water/lipid interface, were found to be of
importance for the function of both antioxidants [18].
The covalent coupling of these antioxidants with molecules of opposite polarity opens new
perspectives, as the amphiphilic structures allow an unusual location for the antioxidant molecules in
cells, and they can exert their effects in a broader range. Such modifications were found to also alter the
activity of the molecules in vitro experiments. Joining carotenoids to hydrophilic molecules resulted in
better stability and solubility, as well as increased antioxidant activity [19]. The esterification of the
flavonoid silybin with lipophilic fatty acids ameliorated its antioxidant and antiviral properties [20].
The covalent conjugation of silybin with l-ascorbic acid, trolox alcohol, or tyrosol through a lipophilic
linker provided an efficient protective action against lipid peroxidation by modifying the electron
transfer abilities of the connecting moieties [21].
There are some examples for the synthesis of flavonoid–carotenoid hybrids, which couple
a shorter than C40 apocarotenoid moiety to flavonoids directly, without any spacer. Terpenoid
C25 or C30 aldehydes were connected to hydroxylated or non-hydroxylated 6-(diethoxyphosphoryl)
methylflavones by the Wittig–Horner–Emmons reaction [22]. The resulting continuously conjugated
carotenyl flavonoids showed improved photoprotective activities, and they surpassed the individual
constituents in their antioxidant properties [22,23].
0
When 14 -apo-
β-carotenoic acid was conjugated with daidzein in position 3b by a C-C bond,
allowing the formation of a continuously conjugated
π
-system, the product exhibited a significantly
better protective effect against lipooxidation in membranes compared to the parent compounds alone
or in combination [24].
0
Daidzein (in position 7a) and quercetin (in positions 3b and 4b) were esterified by 14 -apo-
0
β-carotenoic acid using 4-dimethylaminopyridine (DMAP)/N-ethyl-N -(3-dimethylaminopropyl)
carbodiimide (EDC) coupling. Interestingly, the antioxidant activity of daidzein increased while
that of quercetin decreased after derivatization [25].
A similar DCC or EDC/DMAP esterification method was applied for the synthesis of retinoate
esters of various flavonolignans in low yields. The prepared esters showed increased radical scavenging
activity [26].
Here, we present a straightforward method for the regioselective covalent coupling of daidzein
and chrysin with various carotenoids through a long spacer, which ensures flexibility and allows the
hydrophilic and lipophilic moieties to move away or draw closer. Three different types of carotenoids
0
were studied, the C30 primary alcohol 8 -apo-
β-carotenol, the symmetric secondary diol zeaxanthin, and

Molecules 2020, 25, 636
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the asymmetric
κ-carotenoid capsanthin. The antioxidant activity and the supramolecular organization
of the prepared amphipathic compounds were also investigated.
2
. Results and Discussion
2
.1. Synthesis
Both carotenoids and flavonoids are sensitive compounds, and for the coupling of these molecules,
a very mild method was needed. Previously, azide-alkyne [4 + 2] cycloaddition (click-reaction) was
successfully applied in the synthesis of carotenoid conjugates with polyethylene glycols [27] or with
carbohydrates [28]. Since the pentynoate esters of carotenoids can be easily prepared [28], we worked
out a procedure for the synthesis of azido-derivatives of flavonoids.
The isoflavone daidzein (
preparation of 7-azidohexyl ether derivatives in five steps (Figure 1). The flavonoids (1,7
1
) and the flavone chrysin (
7
) were the molecules of choice for the
) were
acetylated by acetic anhydride in pyridine, and their position 7 were selectively deprotected using
imidazole [29]. A bromohexyl moiety was introduced in position 7 by the Mitsonobu reaction, and
after complete deacetylation, an azide substitution was applied.
Figure 1. Synthesis of 7-azidohexyl ethers of daidzein and chrysin.
The 7-azidohexyl ether of daidzein and chrysin (
6
,
12) was reacted with various carotenoid
0
pentynoates (8 -apo-
β-carotenol-pentynoate 13, zeaxanthin dipentynoate 14, capsanthin dipentynoate
15) in the presence of bis-triphenylphosphano-copper(I)-butyrate complex [30] as a catalyst,
in dichloromethane. (Figure 2). The azido-flavonoids were applied in small excess (1.3–1.5 eq.
per triple bond), providing the corresponding triazoles in satisfactory or good yields (60%–80%). In the
0
case of the 8 -apo-
β-carotenol-pentynoate 13, only monotriazoles can be formed. The other carotenoids
were used as dipentynoate esters, and during the click-reaction the formation of monotriazoles was
also observed. When the reactions were followed by TLC, the monotriazoles appeared first, and in
a longer reaction time, they were further converted to bistriazole derivatives. However, small amounts
of monotriazoles always remained at the end of the reactions, even with a higher excess of azidohexyl

Molecules 2020, 25, 636
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ethers. Yields for the monotriazoles were calculated from the starting carotenoid pentynoates, as if
they were the sole products. The capsanthin pentynoate (15) did not give a complete conversion,
as approximately 10% of the starting pentynoate was recovered.
Figure 2. Coupling of 7-azidohexyl ethers of daidzein and chrysin with carotenoid pentynoates by
1
2
azide-alkyne click-reaction. * In compound 25, the R and R groups are interchangeable.
All the prepared compounds were fully characterized by their nmR, MS, UV, and IR spectra.
As capsanthin is a non-symmetric carotenoid, it can provide two regioisomeric monotriazole derivatives.
In the case of the daidzein-capsanthin conjugates (20a and 20b), these regioisomers could be separated
1
0
and identified. In the H-NMR spectra of 20a and 20b, the chemical shifts of H-3 are slightly but
characteristically different (Table 1), and in comparison with the parent capsanthin dipentynoate (15),
0
as well as with the bistriazole 19, the substituents on C-3 can be distinguished. Similar differences in
the chemical shifts of H-3 can also be observed.
The two regioisomeric monotriazoles of 25 were also observed on TLC, but since they were formed
in low yields and their separation seemed to be extremely challenging, we used them as a mixture for
further investigations.

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0
Table 1. Chemical shifts of H-3 and H-3 in capsanthin dipentynoate (15), in bistriazole 19, and in
monotriazoles 20a and 20b.
0
Compound
δ H-3 m (ppm)
δ H-3 m (ppm)
capsanthin dipentynoate 15
bistriazole 19
5.16–5.10
5.09–5.03
5.13–5.03
5.14–5.07
5.33–5.29
5.26–5.22
5.31–5.25
5.27–5.22
monotriazole 20a
monotriazole 20b
2
.2. Measurement of Antioxidant Capacity
The trolox-equivalent antioxidant capacity (TEAC) of the prepared flavonoid–carotenoid
•
+
conjugates (16–25) was investigated towards ABTS radical cation in aqueous ethanolic solution (see
the experimental section) [31]. The synthetized compounds showed significantly higher TEAC values
0
than their parent flavonoids (Figure 3, Table 2); the exceptions were the conjugates of 8 -apo-
β-carotenol
(16,21), which showed a similar activity to daidzein or chrysin.
TEAC
*
*
1
0
0
0
0
0
0
0
0
0
0
.00
.90
.80
.70
.60
.50
.40
.30
.20
.10
.00
*
*
*
*
Figure 3. TEAC values of the parent flavonoids and carotenoids, as well as those of their conjugates.
The error bars show the standard deviation. * indicates significant differences compared to the parent
carotenoid (p < 0.05).
Table 2. The TEAC values, standard deviations, and variance by the ANOVA method.
Compound
TEAC
SD%
Variance
Compound
TEAC
SD%
Variance
Daidzein
Zeaxanthin
8 monotriazole
0.2804
0.7514
0.5496
0.9353
0.6744
0.6434
0.5728
0.6643
0.7199
0.9663
0.4433
0.3057
4.9981
4.7639
4.8011
5.3524
1.8747
0.4262
3.2883
1.6416
0.0446
0.5469
3.9429
5.1589
0.0070
0.0208
0.0035
0.1123
0.0516
0.0316
0.0095
0.0095
0.0033
0.0463
0.0068
0.0157
Chrysin
Zeaxanthin
23 monotriazole
22 bistriazole
0.1458
0.7514
0.4636
0.8313
4.5581
4.7639
1.3732
3.7065
0.0132
0.0208
0.0066
0.0064
1
1
7 bistriazole
1
7
bistriazole in water
Zea:daidzein 1:2 mixt.
Capsanthin
Capsanthin
25 monotriazole
24 bistriazole
0.5728
0.5026
0.3698
3.2883
2.1947
4.4718
0.0095
0.0091
0.0242
2
2
0a monotriazole
0b monotriazole
9 bistriazole
-apo-β-carotenal
6 triazole
1
0
0
8
8 -apo-β-car.
0.4433
0.1256
3.9429
0.2122
0.0068
0.0284
1
21 triazole

Molecules 2020, 25, 636
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Zeaxanthin conjugated with one daidzein (18) or chrysin (23) was found to be weaker antioxidants
than zeaxanthin; however, its bistriazole derivatives (17 and 22) exceeded the parent zeaxanthin.
The antioxidant capacity of capsanthin did not significantly change on conjugation with one daidzein,
and no differences in the TEAC values of monotriazoles 20a and 20b were found. The bistriazole
derivative 19, however, surpassed capsanthin in antioxidant capacity. The conjugation with chrysin
0
lowered the TEAC value in small extents. The 8 -apo-
than its flavonoid derivatives.
β-carotenol was found to be a better antioxidant
The daidzein-bistriazole derivatives of zeaxanthin and capsanthin were the best antioxidants
under the examined conditions. The TEAC value of a simple zeaxanthin:daidzein 1:2 mixture is
much lower than that of the bistriazole conjugate 17, which indicates a synergistic enhancement in
antioxidant capacity.
•
+
The above results were obtained in ethanolic solution of ABTS (see the experimental section).
The antioxidant property of compound 17 was also determined in aqueous ABTS + solution, where
the TEAC value was found to drop by ca. 20%. The reason for this phenomenon can be explained by
the water-induced aggregation of the carotenoid derivative, which is an accordance with literature
data [32].
•
2
.3. Supramolecular Assembly
When the coupling click-reactions were followed by TLC, and samples were taken directly from
the dichloromethane reaction mixtures, the formation of an intense orange-red spot on the start-line
was observed in the hexane-acetone eluent while the spots of the products remained faint. This
phenomenon generally suggests a salt formation; however, it was hardly possible in these reactions.
After the disappearance of the starting materials, the dichloromethane solvent was evaporated from
0
the reaction mixture of 8 -apo-
β-carotenol-pentynoate and 7-azidohexyl daidzein, then the reaction
mixture was taken up in acetone. Interestingly, when the acetonic solution was applied on the TLC
plate and eluted by the same eluent (hexane-acetone), no start-point substance remained, but the whole
amount was eluted with the R value of the product. This finding was rather unusual, as the retention
f
properties of a substance depend on the quality/polarity of the eluent but independent of the solvent in
which it is applied on the plate, since the plate is dried before elution. We repeated the TLC experiment
using different solvents for the application of the sample, but the same hexane-acetone eluent for the
development. The product triazole 16 in methanolic, chloroformic, or ethyl acetate solutions was
found to behave similarly as in dichloromethane, i.e., it gave a start-spot, while it was eluted when
it was applied in acetonic solution. From these observations, we suspected the formation of some
aggregates, which formed in the presence of certain solvents on drying and did not moved on the
chromatographic plate.
Carotenoids are known to form self-assemblies among certain conditions even in nature. These
supramolecular organizations frequently have ordered structures (H- or J-type aggregates), which
results in significant changes in the UV-Vis spectra of carotenoids [33–35]. Moreover, the aggregates
can exhibit a so-called supramolecular exciton chirality due to the optically active helical structures,
which can be studied by electronic circular dichroism (ECD) spectroscopy [36,37]. Aggregation of
amphipathic carotenoid derivatives was also examined thoroughly [38].
0
As 16 daidzein-8 -apo-
β-carotenol hybrid is not chiral, its aggregation can only be examined by the
change in its UV-Vis properties. The absorption spectrum of the ethanolic solution of 16 upon aqueous
dilution showed an intense hypsochromic and a small bathochromic shift that suggest self-assembly of
1
6 with the formation of J-aggregate (Figure S1).
The aggregation properties of the optically active bistriazoles 17, 19, 22, and 24, as well as
monotriazole 23 were investigated by ECD spectroscopy. The acetone or tetrahydrofuran (THF) solution
of these derivatives were diluted with water, and the ECD spectra were recorded with the different
dilutions. Upon dilution with water, the optically active capsanthin derivatives (19
, 24) did not show
any significant increase in the ECD transitions that would have suggested supramolecular organization.

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In contrast, intense ECD bands appeared in the spectra of the bistriazole daidzein-zeaxanthin conjugate
7 in acetone when the water content was increased above 25% (Figure 4a).
1
(
a)
(b)
Figure 4.
(a) The ECD spectra of bistriazole daidzein-zeaxanthin conjugate 17 in acetone/water mixtures
1
:0–1:3 (b) in THF/water mixtures 1:1–1:10.
The absorption spectra showed a characteristic bathochromic shift with a new band at 519 nm,
which indicated the formation of a weakly-coupled J-aggregate (Figure S2). Zeaxanthin and zeaxanthin
derivatives modified at the sec-hydroxyl group can form both J- and H-aggregates depending on the
applied pH, initial concentration, and solvent/water ratio [39,40]. The intense 480 nm positive cotton
effect (CE) with shoulders at 456 and 519 nm, the broad and intense negative CE at 412 nm, and the
weak negative CE at 547 nm are characteristic of a J-aggregate also observed in the EtOH/water solution
of zeaxanthin under certain conditions [39
,40]. Interestingly, a similar ECD profile was observed in
sergeant-soldier aggregate of 5% - and 95%
α
β-carotene [37], in which weak van der Waals interactions
also prevail. The ECD spectra recorded in the range of 1:0–1:3 acetone/water ratio showed strong
characteristic ECD bands from a 2:1 ratio and the largest amplitudes were observed at a 1:2 ratio.
A further increase of the water content to a 1:3 ratio decreased both the ECD and absorption intensity.
The intensities of the five CEs did not show a monotonous change with the increase of the water
content; the 547 nm CE had maximum intensity at the 1:1 ratio, while the 480 and 412 CEs had the
largest amplitude at the 1:2 ratio.
In THF, a similar ECD feature appeared with the increase of the water ratio, and the maximum
ECD intensity was reached at the 1:5 ratio, while a further increase of the water content decreased
the amplitude of the CEs (Figure 4b). The absorption and ECD spectra of 17 at the 2:3 and 1:2
ratio differed from those at the 1:3, 1:5, and 1:10 ratios, which indicated the presence of at least two
different J-aggregates.
The bistriazole chrysin-zeaxanthin conjugate (22) also formed a J-aggregate with the increasing
amount of water in acetone, which was, however, different from that of 17. The conjugate 22 had
a bisignate ECD curve even in pure acetone, indicating aggregate formation in the absence of water
(
Figure 5a,b). In acetone/water 5:1, the high-wavelength positive CE shifted to the red with increased
intensity. The 545 nm positive CE with shoulders at 508 and 464 nm and the negative CE at 397 nm
represented an ECD pattern similar to that of 17, with a considerable red shift. In acetone/water
4
:1, the intensity increased significantly and reached the maximum, while further addition of water
flattened the ECD curve and changed the absorption maxima. The diminishing ECD intensity in
acetone/water ratio 2:1–1:3 suggested the deterioration of the J-aggregate.
In the THF solution, 22 had a low-intensity ECD spectrum (Figure 5c,d), different from that
in acetone, which did not change much up to the 2:3 THF/water ratio. At the 1:2 THF/water ratio,
an intense negative ECD couplet centered at 425 nm appeared, which was red shifted with the 1:3
ratio. A further significant change occurred at the 1:5 and 1:10 ratios, the ECD curves of which differed

Molecules 2020, 25, 636
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only in the amplitudes and they had a similar ECD pattern to that of 17, with opposite signs of CEs.
The ECD spectra of 22 at the high water ratio were very different from those in acetone, and the
mirror-image relationship with the ECD spectrum of 17 acetone/water 1:2 indicated their opposite
sense of supramolecular chirality. The three different ECD and absorption spectra suggested the
presence of at least three different types of J-aggregate.
(
a)
(b)
(
c)
(
d)
Figure 5. ECD spectra of bistriazole chrysin–zeaxanthin conjugate 22 in (a) acetone/water 1:0–5:1;
(
b) acetone/water 4:1–1:3 (c) THF/water 1:0–2:3. (d) THF/water 2:3–1:10.
The zeaxanthin conjugates 17 and 22 differ only in the attached daidzein/chrysin flavonoid
moiety, but they produced markedly different aggregation profiles upon the addition of water in both
acetone and THF. The aggregate formation was also sensitive to the type of non-protic solvent, acetone
versus THF.
In the acetone/water mixtures, the monotriazole chrysin–zeaxanthin conjugate (23) showed
the same characteristic changes in both the ECD and absorption spectra as the bistriazole
daidzein–zeaxanthin conjugate 17, indicating the formation of the same J-aggregate (Figure 6a,b).
The ECD transitions above 400 nm reached the maximum intensities at the 1:1 ratio and there was
no significant decrease in the intensity up to the 1:3 ratio.
In the THF/water mixtures, a completely different aggregation process could be observed
(Figure 7a,b). Aggregation started at the 1:1 ratio and the negative ECD couplet reached the maximum
intensity at the 1:2 ratio. The intensity of the ECD bands was so high at the initial concentration that
a sixfold dilution had to be applied to record the ECD bands properly. According to the characteristic
5
30 nm absorption band, a J-aggregate formed at the 2:3 and 1:2 ratios.
By increasing the ratio of water further, the negative couplet was inverted to a positive one at
the 1:5 and 1:10 ratios. At these ratios, both hypsochromic and bathochromic shifts of the absorption
bands took place compared to the bands at the 1:1 ratio (Figure 8). The inversion of the ECD couplet
with the increasing amount of water in THF suggests the inversion of chirality in the aggregate, which
could not be observed in the acetone/water mixtures.

Molecules 2020, 25, 636
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(
a)
(b)
Figure 6. ECD spectra of monotriazole chrysin–zeaxanthin conjugate 23 in (
b) acetone/water 1:1–1:3.
a) acetone/water 1:0–1:1;
(
(
a)
(b)
Figure 7. ECD spectra of monotriazole chrysin–zeaxanthin conjugate 23 in (a) THF/water 1:0–2:1.
(
b) THF/water 1:1–1:10.
(
a)
(b)
(
c)
(d)
Figure 8. UV-vis spectra of chrysin–zeaxanthin conjugates of (
:0–1:3; ( ) bistriazole 22 in THF/water 1:1–1:10; ( ) monotriazole 23 in acetone/water 1:0–1:3;
d) monotriazole 23 in THF/water 1:0–1:1.
a) the bistriazole 22 in acetone/water
1
b
c
(

Molecules 2020, 25, 636
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3
. Conclusions
A straightforward method was worked out for the synthesis of novel carotenoid–flavonoid
conjugates and their antioxidant capacities were measured. Some of these amphipathic compounds
containing a flexible linker were found to show self-aggregation in the presence of water. The antioxidant
capacity was measured in ethanolic solutions. Determination of the TEAC values in aqueous medium
seems to be more relevant biologically, but most probably, the antioxidant property under such
conditions rather belongs to the aggregates than to the individual molecules. A further study is
needed to clarify how the aggregation influences the antioxidant capacity. In the future, the solvent
dependence of the antioxidant activity, and the structure of the aggregates will be examined with
atomic force microscopy.
4
. Experimental Section
4
.1. Materials and Methods
Melting points were measured on a Stuart SMP30 apparatus (Cole-Parmer, Staffordshire, UK) and
are uncorrected. The UV spectra were implemented on a Jasco spectrophotometer model V-550 UV/Vis
(
(
JASCO, Tokyo, Japan). ECD spectra were recorded at room temperature with a J-810 spectropolarimeter
JASCO, Tokyo, Japan). The IR spectra were run on an Impact 400 FT-IR spectrophotometer (Nicolet,
Germany) in KBr pellets using a KBr pellet as the background reference spectrum. nmR spectra were
1
13
recorded with a Bruker Avance III Ascend 500 spectrometer (500/125 MHz for H/ C) (Bruker BioSpin
13
1
GmbH, Rheinstetten, Germany). The C and H-NMR assignments for 6, 12, 16, 17, 19, and 23 were
1
13
made on the basis of 1D ( H, C APT) and 2D (HSQC, HMBC) experiments, and the assignments for
the other compounds were based on structural similarities with the above molecules. Chemical shifts
1
were referenced to the residual solvent signals, or to Me Si ( H). A special numbering of H and C
4
atoms, indicated in the formulas (Figure 9), for the flavonoid moieties was applied, since the structure
of the final coupled products was rather complex.
Figure 9. Numbering of the flavonoid derivatives.
Molar masses were obtained by an Autoflex II MALDI instrument (Bruker Daltonics, Bremen,
Germany). 2,5-Dihydroxy-benzoic acid (DHB) was used for the ionization of the samples. Mass
spectra were monitored either in positive or in negative mode (depending on the chemical structure)
with pulsed ionization (
λ = 337 nm; nitrogen laser). Spectra were measured in reflectron mode using
a delayed extraction of 120 ns. The elemental analysis measurements were performed on a Fisons EA
1
110 CHNS apparatus (Thermo Scientific, Waltham, MA, US).
Thin layer chromatography was performed on TLC Silica gel 60 F254 on Al sheets (Merck,
Darmstadt, Germany), and the spots were visualized under UV light. Preparative layer chromatography
was executed on PLC Silica gel 60 F254 1 mm on glass plate (Merck). For column chromatography,
a Kieselgel 60 (Merck, particle size 0.063–0.200 mm) was used. All reagents used for synthesis were
commercial and of analytically pure quality and all organic solvents were of HPLC grade. Organic
◦
solutions were dried over anhydrous Na SO and concentrated under reduced pressure at 40 C (bath
2
4
temperature). Carotenoid pentynoates were prepared by a published method [28].

Molecules 2020, 25, 636
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4
.2. Assay for the Antioxidant Activity of the Carotenoids/TEAC Assay
A literature procedure [31] was applied with modifications. The 2,2 -azino-bis(3-ethylbenzothiazoline-
0
•
+
•
+
6-sulfonic acid) radical cation (ABTS ) was produced in aqueous solution by reacting ABTS in 7 mM and
•+
potassium persulfate in 2.45 mM concentrations. The stock ABTS solution was prepared 12–16 h before
•+
the experiments, and stored at room temperature in dark. The absorbance of the ABTS solution was set
to 0.70 0.05 at 734 nm by dilution with ethanol. Trolox was dissolved in ethanol, and the carotenoids and
the carotenoid conjugates in tetrahydrofuran (THF). The antioxidants (in final concentrations of 1.25, 2.5,
±
•+ ◦
3.75, and 5 µM) were incubated with ABTS solution at 37 C for 6 min, respectively. For the statistical
analysis, the measurements of absorbance were carried out at each concentration in triplicate (n = 3), and
each measurement was repeated 2 times. SD% was lower than 5% in each case. The percentage inhibition
•
Aantioxidant)/A , where A is the absorbance of the ABTS
0 0
+
of absorbance at 734 nm was calculated as (A0
−
solution and Aantioxidant is the absorbance measured after the addition of the antioxidant. The calculated
values were plotted against the final concentration of the antioxidants. The slopes of the curves were
compared with that for trolox, and the trolox equivalent antioxidant capacity (TEAC) value is the ratio of
the slopes for the antioxidant and for trolox.
Statistical analysis of the TEAC values was performed by the ANOVA method (Microsoft Excel
2016) (Table 2). To examine the relationship among the TEAC values of the carotenoids, flavonoids,
and those of their conjugates, two-tailed t tests were calculated. An F test was performed in each case
to determine the type of the t test to be used. Less than 5% in the t test was regarded as a significant
difference (Table 3). For the calculations, the functions of Microsoft Excel (2016) were used.
Table 3. Statistical analysis of the TEAC values.
Comparison
F Test
T Test
Comparison
F Test
T Test
Daidzein:18
Daidzein:17
Daidzein:17 in water
Daidzein:Zea-daid. mixt.
Zeaxanthin:18
Zeaxanthin:17
Zeaxanthin:17 in water
Zeaxanthin: Zea-daid. mixt.
74.28%
0.32%
45.15%
9.80%
30.03%
6.57%
29.05%
55.19%
0.04%
4.18%
0.00%
0.00%
4.99%
1.05%
15.51%
74.42%
Daidzein:16
8 -apo-β-car:16
27.71%
67.89%
94.39%
4.93%
0
Chrysin:23
Chrysin:22
Zeaxanthin:23
Zeaxanthin:22
23.74%
65.04%
52.52%
50.99%
3.63%
0.86%
2.10%
27.87%
Chrysin:25
Chrysin:24
Capsanthin:25
Capsanthin:24
81.78%
80.59%
88.47%
34.33%
0.67%
3.33%
7.40%
22.00%
Daidzein:20a
Daidzein:20b
Daidzein:19
Capsanthin:20a
Capsanthin:20b
Capsanthin:19
62.55%
41.90%
4.06%
86.17%
27.09%
13.29%
0.51%
0.00%
0.00%
30.11%
22.39%
4.94%
Chrysin:21
8 -apo-β-car:21
69.35%
41.10%
32.28%
3.86%
0
4
.3. Acetylation of Flavonoids
Daidzein (
1
) or chrysin (
7
) (8 mmol) was dissolved in acetic anhydride (30 mL), and was stirred
◦
◦
at 60 C for 10 min. Dry pyridine (8 mL) was added dropwise to the stirred mixture at 60 C. After
5
min, a white precipitate appeared. After 2 h, the reaction mixture was poured onto ice and stirred
for an hour. The formed white precipitate was filtered out, and washed with water thoroughly. After
drying above potassium hydroxide for 2 days, the crude products were recrystallized from ethanol.
◦
7
-Acetoxy-3-(4-acetoxyphenyl)-4H-chromen-4-one (
2
): white crystals (98%); mp 188–189 C; UV (THF)
−1 1
λmax 243, 277, 301 nm; IR (KBr) νmax 1752, 1617, 1646, 1224 cm ; H-NMR (CDCl , 500 MHz)
δ
8.32
3
(
1H, d, J = 8.7 Hz, H-5a), 8.00 (1H, s, H-2c), 7.58 (2H, d, J = 8.6 Hz, 2 H-2b), 7.31 (1H, d, J = 1.9 Hz,
13
H-8a), 7.16–7.19 (3H, m, 2 H-3b, H-6a), 2.36 (3H, s, CH ), 2.32 (3H, s, CH ); C-NMR (CDCl , 125 MHz)
3
3
3
δ
175.4 (C, C-4c), 169.3, 168.4 (C, 2 Ac C=O), 156.7 (C, C-8c), 154.6 (C, C-7a), 153.1 (CH, C-2c), 150.8
(
C, C-4b), 130.0 (CH, 2 C-2b), 129.2 (C, C-1b), 127.9 (CH, C-5a), 124.8 (C, C-3c), 122.3 (C, C-4a), 121.6

Molecules 2020, 25, 636
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(
CH, 2 C-3b), 119.6 (CH, C-6a), 110.9 (CH, C-8a), 21.1 (2 Ac CH ); MS (MALDI-TOF, positive mode,
3
+
+
+
with DHB matrix) m/z 339.0 [M + H] 360.9 [M + Na] 376.9 [M + K] ; Anal. calcd. for C H O : C
19
14
6
6
7.45, H 4.17, found C 67.05, H 4.03.
◦
5
2
7
,7-Diacetoxy-2-phenyl-4H-chromen-4-one (
92 nm; IR (KBr) νmax 1769, 1630 cm ; H-NMR (CDCl , 500 MHz)
8
): white crystals (95%); mp 195–196 C; UV (THF) λmax 243,
7.85 (2H, d, J = 8.0 Hz, 2 H-2b),
H-3b, H-4b), 7.35 (1H, d, J = 2.1 Hz, H-6a), 6.85 (1H, d, J = 2.1 Hz, H-8a), 6.65 (1H,
3H, 2 s, 2 x CH3); 13C-NMR (CDCl , 125 MHz)
176.3 (C, C-4c), 169.3, 167.9 (C,
Ac C=O), 162.5 (C, C-2c), 157.7 (C, C-7a), 153.9 (C, C-8c), 150.2 (C, C-5a), 131.7 (CH, C-4b), 131.1
−
1 1
δ
×
3
.55-7.49 (3H, m, 2
×
s, H-3c ), 2.44, 2.35 (2
2
(
×
δ
3
×
C, C-1b), 129.1, 126.2 (CH, C-3b, C-2b), 115.0 (C, C-4a), 113.6 (CH, C-6a), 109.0 (CH, C-8a), 108.6 (CH,
C-3c), 21.1, 21.0 (2 Ac CH ); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 339.5 [M + H] ,
61.0 [M + Na] 377.0 [M + K] ; Anal. calcd. for C H O : C 67.45, H 4.17, found C 67.23, H 4.16.
19 14 6
+
3
+
+
3
4
7
.4. Selective Deacetylation of Flavonoid Diacetates
-Hydroxy-3-(4-acetoxyphenyl)-4H-chromen-4-one (3): Daidzein diacetate (2) (0.412 g, 1.21 mmol) was
dissolved in 20 mL CH Cl , imidazole (0.248 g, 3.6 mmol, 3 eq.) in 10 mL CH Cl was added dropwise
2
2
2
2
◦
15 C in ice/salt bath. The reaction mixture was stirred at room temperature for 2 h under N .
2
at
−
A white precipitate appeared. The reaction mixture was diluted with CH Cl (100 mL), was washed
2
2
with 3M HCl (3
×
100 mL) and water (100 mL), dried (Na SO ), and the solvent was evaporated under
2 4
reduced pressure. The crude products (0.41 g) were crystallized from EtOH giving white crystals
◦
(
1
0.264 g, 73%); mp 231–232 C; UV (THF) λmax 243, 277(sh), 297, 307 nm; IR (KBr) νmax 3102, 1751, 1618,
258 cm ; H-NMR (DMSO-d , 500 MHz)
−1 1
δ
10.82 (1H, s, OH), 8.41 (1H, s, H-2c), 8.00 (1H, d, J = 8.8 Hz,
6
H-5a), 7.61 (2H, d, J = 8.6 Hz, H-3b), 7.18 (2H, d, J = 8.6 Hz, H-2b), 6.95 (1H, dd, J = 8.6, 2.2 Hz, H-6a),
.89 (1H, d, J = 2.13 Hz, H-8a), 2.29 (3H, s, CH3); 13C nmR (DMSO-d , 125 MHz)
174.2 (C, C-4c), 169.1
C, Ac C=O), 162.5, 157.3 (C, C-8c, C-7a), 153.7 (CH, C-2c), 150.0 (C, C-4b), 129.9 (CH, 2 C-2b), 129.5 (C,
6
δ
6
(
C-1b), 127.2 (CH, C-5a), 122.7 (C, C-3c), 121.4 (CH, 2 C-3b), 116.5 (C, C-4a), 115.2 (CH, C-6a), 102.1 (CH,
+
C-8a), 20.7 (CH , Ac-CH ); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 297.0 [M + H]
3
+
3
+
3
18.9 [M + Na] 336.9 [M + K] ; Anal. calcd. for C H O : C 68.92, H 4.08, found C 68.90, H 4.05.
17 12 5
5-Acetoxy-7-hydroxy-2-phenyl-4H-chromen-4-one (9): Chrysin diacetate (8) (1.0 g, 2.96 mmol) was
dissolved in 50 mL CH Cl , imidazole (0.603g, 8.87 mmol, 3 eq.) in 40 mL CH Cl was added dropwise
2
2
2
2
◦
at
−
15 C in ice/salt bath. The reaction mixture was stirred at room temperature for 2 hours under
N . A white precipitate appeared, which was filtered out and washed with cold CH Cl . The crude
2
2
2
◦
product (0.79 g) was recrystallized from EtOH and yielded white crystals (0.42 g, 48%). mp 185–186 C;
UV (THF) λmax 208, 243, 301 nm; IR (KBr) νmax 1770, 1628, 1282 cm ; H nmR (DMSO-d , 500 MHz)
−
1 1
6
δ
11.10 (1H, s, OH), 8.02 (2H, d, J = 6.5 Hz, 2
×
H-2b), 7.59-7.53 (3H, m, 2
×
H-3b, H-4b), 6.94 (1H, d,
J = 2.3 Hz, H-6a*), 6.75 (1H, s, H-3c ), 6.57 (1H, d, J = 2.3 Hz, H-8a*), 2.30 (3H, s, CH ) *interchangeable;
3
1
3
C nmR (DMSO-d „ 125 MHz)
δ
175.2 (C, C-4c), 168.7 (C, Ac C=O), 162.2 (C, C-2c), 160.9 (C, C-7a),
58.2 (C, C-8a), 150.0 (C, C-5a), 131.5 (CH, C-4b), 130.8 (C, C-1b), 129.0, 126.1 (CH, C-2b, C-3b), 109.4 (C,
C-4a), 108.6 (CH, C-6a*), 107.4 (CH, C-3c), 100.8 (CH, C-8a*), 20.9 (CH , Ac CH ) *interchangeable; MS
6
1
3
3
+
+
+
(
MALDI-TOF, positive mode, with DHB matrix) m/z 297.0 [M + H] 319.9 [M + Na] 336.9 [M + K] ;
Anal. calcd. for C H N O : C 68.92, H 4.08, found C 68.80, H 3.86.
17
12
3
5
4
.5. Mitsunobu Reactions
Monoacetates of daidzein (
3
) or chrysin (9) (6.76 mmol) and triphenylphosphine (7.44 mmol,
1
1
.1 eq.) were suspended in freshly distilled THF (100 mL). Then 6-bromo-1-hexanol (0.87 mL, 6.76 mmol,
eq.) was added, and diisopropyl azodicarboxylate (1.44 mL, 7.44 mmol, 1.1 eq.) were dropped to
the stirred solution. After the dissolution of the starting materials, the yellow solution was stirred
overnight under nitrogen, at room temperature. The reaction mixture was evaporated under reduced
pressure, and the residue was purified by column chromatography (DCM-MeOH, 150:1).

Molecules 2020, 25, 636
13 of 21
◦
4): white crystals (71%); mp 164–165 C; UV
7
(
-(6-Bromohexyloxy)-3-(4-acetoxyphenyl)-4H-chromen-4-one (
−1 1
THF) λmax 243, 277, 296, 306 nm; IR (KBr) νmax 1759, 1633, 1266 cm ; H-NMR (CDCl , 500 MHz)
δ
3
8
2
.20 (1H, d, J = 8.7 Hz, H-5a), 7.94 (1H, s, H-2c), 7.58 (2H, d, J = 7.6 Hz, 2 H-3b), 7.16 (2H, d, J = 7.2 Hz,
H-2b), 7.00 (1H, d, J = 8.8 Hz, H-6a), 6.84 (1H, s, H-8a), 4.07 (2H, t, J = 6.3 Hz, O-CH ), 3.44 (2H,
2
t, J = 6.3 Hz, Br-CH ), 2.32 (3H, s, Ac CH ), 1.92–1.86 (4H, m, CH -
β
,
γ
), 1.54–1.58 (4H, m, CH2-
α,δ);
2
3
2
13
C-NMR (CDCl , 125 MHz)
δ 175.4 (C, C-4c), 169.3 (C, Ac C=O), 163.6 (C, C-7a), 157.9 (C, C-8c), 152.6
3
(
CH, C-2c), 150.6 (C, C-4b), 130.0 (CH, 2 C-3b), 129.6 (C, C-1b), 127.8 (CH, C-5a), 124.58 (C, C-3c), 121.6
(
CH, 2 C-2b), 118.3 (C, C-4a), 114.9 (CH, C-6a), 100.7 (CH, C-8a), 68.5 (O-CH ), 33.6 (Br-CH ), 32.6, 28.8,
2
2
2
4
6
7.9, 25.2 (CH ,
α
,
β
,
γ
,
δ
-CH ), 21.1 (Ac CH ); MS (MALDI-TOF, positive mode, with DHB matrix) m/z
2
+
2
3
+
+
58.9 [M + H] , 480.9 [M + K] , 498.7 [M + K] ; Anal. calcd. for C H BrO : C 60.14, H 5.05, found C
0.15, H 4.98.
23
23
5
◦
5
(
-Acetoxy-7-(6-bromohexyloxy)-2-phenyl-4H-chromen-4-one (10): white crystals (68%); mp 102–103 C; UV
−1 1
THF) λmax 205, 216, 242, 300 nm; IR (KBr) νmax 1775, 1635, 1207 cm ; H-NMR (CDCl , 500 MHz)
δ
3
7
.85 (2H, d, J = 7.1 Hz, 2 x H-2b), 7.53-7.50 (3H, m, 2
×
H-3b, H-4b), 6.86 (1H, s, H-3c*), 6.60 (2H, ps,
H-6a*, H-8a*), 4.06 (2H, t, J = 6.2 Hz, O-CH ), 3.43 (2H, t, J = 6.6 Hz, Br-CH ), 2.44 (3H, s, Ac CH ),
2
2
3
13
1
1
1
.92–1.84 (4H, m, 2
×
CH ), 1.52 (4H, ps, 2
×
CH ) *interchangeable; C-NMR (CDCl , 125 MHz) δ
2 3
2
76.5 (C, C-4c), 169.6 (C, Ac C=O), 162.9 (C, C-2c), 161.9 (C, C-7a), 158.8 (C, C-8c), 150.5 (C, C-5a),
31.4 (CH, C-4b), 129.0, 126.1 (CH, C-2b, C-3b), 111.1 (C, C-4a), 108.6 (CH, C-6a*), 108.4 (CH, C-3c*),
9
9.4 (CH, C-8a), 68.6 (CH , O-CH ), 33.6 (CH , Br-CH -), 32.6, 28.7, 27.8, 25.2 (CH , 4 CH ), 21.1 (Ac
2 2 2 2 2 2
+
CH ) *interchangeable; MS (MALDI-TOF, positive mode, without matrix) m/z 458.6 [M + H] 480.6
3
+
+
[M + Na] 498.6 [M + K] ; Anal. calcd. for C H BrO : C 60.14, H 5.05, found C 60.44, H 4.94.
23 23 5
4
.6. Deprotection
The acetylated 7-bromohexyl ether of daidzein (
4) or chrysin (10) (5.0 mmol) was dissolved in
dry MeOH (80 mL), and freshly prepared sodium methylate solution (10 mL) was added dropwise to
the mixture, which was stirred at room temperature for 2 h. The pH of the mixture was controlled
and the amount of NaOMe was replaced due to the formation of phenolates. After the complete
disappearance of the starting material, the solution was neutralized by Amberlite IRC 86 acidic resin,
and was evaporated in vacuum.
◦
7
(
-(6-Bromohexyloxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one (
5
): white crystals (97%); mp 151–152 C; UV
−
1 1
THF) λmax 243, 277, 292, 306 nm; IR (KBr) νmax 3210, 1628, 1260 cm ; H-NMR (CDCl , 500 MHz)
δ
3
8
8
.21 (1H, d, J = 8.9 Hz, H-5a), 7.92 (1H, s, H-2c), 7.37 (2H, d, J = 8.6 Hz, 2 H-3b), 6.99 (1H, dd, J = 2.3,
.9 Hz, H-6a), 6.82-6.85 (3H, m, H-8a, 2 H-2b), 6.04 (1H, s, OH), 4.07 (2H, t, J = 6.4 Hz, O-CH ), 3.44 (2H,
2
t, J = 6.7 Hz, Br-CH ), 1.92 (2H, dt, J = 6.9, 13.9 Hz, CH ), 1.87 (2H, dt, J = 6.7, 13.1 Hz, CH ), 1.54 (4H,
2
2
2
m, 2 CH2); 13C nmR (CDCl , 125 MHz)
δ
176.3 (C, C-4c), 163.6 (C, C-7a), 158.1 (C, C-8c), 156.0 (C, C-4b),
3
1
1
52.3 (CH, C-2c), 130.3 (CH, C-3b), 127.8 (CH, C-5a), 125.1 (C, C-1b), 123.9 (C, C-3c), 118.3 (C, C-4a),
15.7 (CH, C-2b), 115.0 (CH, C-6a), 100.6 (CH, C-8a), 68.5 (O-CH ), 33.7 (Br-CH ), 32.7, 28.8, 27.9, 25.3
2
2
+
+
(4 CH ); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 417,3 [M + H] , 438.6 [M + Na] ,
2
+
4
55.1 [M + K] ; Anal. calcd. for C H BrO : C 60.44, H 5.07, found C 60.38, H 5.09.
21
21
4
◦
-Hydroxy-7-(6-bromohexyloxy)-2-phenyl-4H-chromen-4-one (11): white powder (96%); mp 129–130 C;
5
−1 1
UV (THF) λmax 243, 290, 308 nm; IR (KBr) νmax 3064, 1660, 1615, 1174 cm ; H-NMR (CDCl , 500 MHz)
3
δ
12.70 (1H, s, OH), 7.88 (2H, dd, J = 1.3 Hz, J = 7.8 Hz, 2
×
H-2b), 7.55–7.50 (3H, m, 2
×
H-3b, H-4b), 6.66
(
1H, s, H-3c), 6.49 (1H, d, J = 2.2 Hz, H-8a), 6.35 (1H, d, J = 2.2 Hz, H-6a), 4.04 (2H, t, J = 6.4 Hz, O-CH ),
2
3
.44 (2H, t, J = 6.7 Hz, Br-CH ), 1.94–1.81 (4H, m,2
×
CH ), 1.53–1.52 (4H, m, 2
×
CH2); 13C-NMR
176.3 182.4 (C, C-4c), 165.1 (C, C-7a), 163.91 (C, C-2c), 162.2 (C, C-5a), 157.8 (C,
C-8c), 131.8 (CH, C-4b), 131.4 (C, C-1b), 129.1 (CH, C-3b), 126.3 (CH, C-2b), 105.9 (CH, C-3c), 105.6 (C,
C-4a), 98.6 (CH, C-6a), 93.1 (CH, C-8a), 68.4 (CH , O-CH ), 33.7 (CH , Br-CH -), 32.6, 28.8, 27.8, 25.2
2
2
(CDCl , 125 MHz) δ
3
2
2
2
2
+
(
CH , 4
×
CH ); MS (MALDI-TOF, positive mode, without matrix) m/z 417.9 [M + H] , 456.4 [M + K];
2
2
Anal. calcd. for C H BrO : C 60.44, H 5.07, found C 60.29, H 4.96.
21
21
4

Molecules 2020, 25, 636
14 of 21
4
.7. Azide Substitutions
The 7-bromohexyl ether of daidzein (
0 mmol, 10 eq.) were dissolved in dry DMF. The reaction mixture was stirred at 60 C for 2 days under
5
) or chrysin (11) (4.0 mmol) and sodium azide (2.6 g,
◦
4
N . When the TLC showed the disappearance of the starting material, the excess sodium-azide and
2
byproducts were filtered out. The mother liquor was diluted with CHCl (600 mL) and washed with
3
water (3
×
200 mL). The organic phase was dried on Na SO and filtered. The solvent was evaporated
2 4
under reduced pressure, and the crude product was recrystallized from EtOH.
◦
7
(
-(6-Azidohexyloxy)-3-(4-hydroxyphenyl)-4H-chromen-4-one ( ): white flakes (65%); mp 144–145 C; UV
6
−
1 1
THF) λmax 243, 277 (sh), 291 nm; IR (KBr) νmax 3217, 2104, 2150, 1628 cm ; H nmR (DMSO-d ,
6
5
00 MHz)
δ 9.53 (1H, s, OH), 8.34 (1H, s, H-2c), 8.00 (1H, d, J = 8.8 Hz, H-5a), 7.40 (2H, d, J = 8.6 Hz,
H-3b), 7.11 (1H, d, J = 2.3 Hz, H-8a), 7.05 (1H, dd, J = 2.3, 8.9 Hz, H-6a), 6.81 (2H, d, J = 8.6 Hz, H-2b),
4
.10 (2H, t, J = 6.4 Hz, O-CH ), 3.33 (2H, t, J = 6.9 Hz, N -CH ), 1.76 (2H, pddd, J = 6.6, 13.4 Hz,
α
-CH2),
2
3
2
1
1
.56 (2H, pddd, J = 7.0, 14.2 Hz,
74.5 (C, C-4c), 162.9 (C, C-7a), 157.2 (C, C-8c), 157.1 (C, C-4b), 152.8 (CH, C-2c), 129.9 (CH, C-3b), 126.7
δ
-CH ), 1.36-1.48 (4H, m,
β,γ
-CH2); 13C-NMR (DMSO-d , 125 MHz)
δ
2
6
(
(
(
CH, C-5a), 123.5 (C, C-1b), 122.2 (C, C-3c), 117.4 (C, C-4a), 114.8 (CH, C-2b), 114.7 (CH, C-6a), 100.8
CH, C-8a), 68.2 (O-CH ), 50.5 (N -CH ), 28.1 (
α
-CH ), 28.0 (
δ
-CH ), 25.7(
γ
-CH ), 24.8 (
β
-CH ); MS
2
3
2
2
2
2
2
+
+
MALDI-TOF, positive mode, with DHB matrix) m/z 379.7 [M + H] , 417.5 [M + K] ; Anal. calcd. for
C H N O : C 66.48, H 5.58, N 11.08 found C 66.23, H 5.53 N 10.82.
12
21
3
4
◦
-Hydroxy-7-(6-azidohexyloxy)-2-phenyl-4H-chromen-4-one (12): white flakes (52%); mp 97–98 C; UV
5
(
−
THF) λmax 243, 291, 309(sh) nm; IR (KBr) νmax 3060, 2131, 2085, 1653, 1603 cm ; H-NMR (CDCl ,
3
1 1
5
00 MHz)
δ
12.70 (1H, s, OH), 7.87 (2H, d, J = 7.0 Hz, 2
×
H-2b), 7.56-7.50 (3H, m, 2
×
H-3b, H-4b),
6
.65 (1H, s, H-3c), 6.48 (1H, d, J = 1.8 Hz, H-8a), 6.35 (1H, d, J = 1.8 Hz, H-6a), 4.03 (2H, t, J = 6.4 Hz,
O-CH ), 3.30 (2H, t, J = 6.8 Hz, N -CH ), 1.86–1.80 (2H, m, CH ), 1.68–1.62 (2H, m, CH ), 1.55–1.45 (4H,
2
3
2
2
2
1
3
m, 2 CH2); C nmR (CDCl , 125 MHz)
δ
182.40 (C, C-4c), 165.07 (C, C-7a), 163.89 (C, C-2c), 162.17
3
(
C, C-5a), 157.78 (C, C-8c), 131.75 (CH, C-4b), 131.36 (C, C-1b), 129.04 (CH, C-3b), 126.24 (CH, C-2b),
1
05.85 (CH, C-3c), 105.63 (C, C-4a), 98.56 (CH, C-6a), 93.07 (CH, C-8a), 68.39 (CH , O-CH -), 51.35 (CH ,
2 2 2
N -CH -), 28.82, 28.77, 26.44, 25.57 (CH , 4 CH ); MS (MALDI-TOF, positive mode, with DHB matrix)
3
2
2
2
+
+
m/z 380.1 [M + H] , 417.9 [M + K] ; Anal. calcd. for C H N O : C 66.48, H 5.58, N 11.08 found C
21
21
3
4
6
6.57, H 5.06 N 10.99.
4
.8. Azide-Alkyne Click-Reactions
The carotenoid pentynoates (13
and the 7-azidohexyl ether derivatives of flavonoids (6,
,
14
,
15) (0.1 mmol) were dissolved in dry dichloromethane (4 mL),
12) (1.5 or 2.5 eq.) and bistriphenylphosphano-
copper(I)-butyrate (C H COOCu(PPh ) ) (6 mg) were added. The reaction mixtures were stirred under
3
7
3 2
nitrogen, in darkness. When TLC showed the disappearance of the starting materials (typically in
4 h), the solvent was evaporated in vacuum, and the residue was purified by column chromatography.
2
After crystallization, the products were stored in closed ampoules under argon. Yields are given
for the bistriazoles and monotriazoles separately. The analytical samples were further purified by
preparative TLC.
0
0
8
-(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-8 -apo-
◦
β
-carotene (16): red crystals (62%); mp 118–119 C; UV (THF) λmax 242, 278, 289, 375, 402, 429, 455 nm;
−
1 1
IR (KBr) νmax 3414, 1734, 1626 cm ; H-NMR (CDCl , 500 MHz)
δ
8.18 (1H, dd, J = 1.3, 8.9 Hz, H-5a),
3
7
.88 (1H, d, J = 1.3 Hz, H-2c), 7.37–7.39 (2H, m, H-3b), 7.34 (1H, s, triazole CH), 6.95 (1H, d, J = 8.9 Hz,
0
H-6a), 6.87 (2H, d, J = 8.6 Hz, H-2b), 6.80 (1H, s, H-8a), 6.68–6.57 (3H, m, H-11, H-15, H-15 ), 6.43 (1H,
dd, J = 11.0, 15.0 Hz, H-11 ), 6.34 (1H, d, J = 9.9 Hz, H-12), 6.31 (1H, d, J = 10.1 Hz, H-12 ), 6.25-6.22
0
0
0
0
(
2H, m, H-14, H-14 ), 6.20–6.11 (4H, m, H-7, H-8, H-10, H-10’), 4.56 (2H, s, H-8 ), 4.32 (2H, t, J = 7.1 Hz,
N-CH ), 4.01 (2H, t, J = 4.0 Hz, O-CH ), 3.06 (2H, t, J = 7.2 Hz, CH -C(=C)N), 2.78 (2H, t, J = 7.2 Hz,
2
2
2
CH -C=O), 2.02 (2H, t, J = 6.0 Hz, 2 H-4), 1.97 (6H, s, 2 CH ), 1.93 (3H, s, CH ), 1.80 (3H, s, CH ),
2
3
3
3

Molecules 2020, 25, 636
15 of 21
1
1
.71 (3H, s, CH ), 1.64-1.60 (2H, m, 2 H-3), 1.55-1.45 (4H, m, CH -
β
, 2 H-2), 1.42-1.36 (2H, m, CH2-
γ),
3
2
.26 (2H, ps, CH2-
α
), 1.03 (6H, s, 2 CH3); 13C-NMR (CDCl , 125 MHz)
δ
176.1 (C, C-4c), 172.6 (C,
3
OC=O), 163.4 (C, C-7a), 158.0 (C, C-4b), 156.4 (C, C-8c), 152.1 (CH, C-2c), 146.3 (C, triazole NC=C),
0
1
38.5 (CH, C-12 ), 137.9 (C, C-6), 137.7 (CH, C-8), 137.1 (CH, C-12), 136.8 (C, C-13), 136.2, 135.7 (C,
0
0
0
C-9, C-13 interchangeable), 133.0, 132.2 (CH, C-14, C-14 interchangeable), 131.7 (C, C-9 ), 130.7 (CH,
C-10), 130.5, 129.6 (CH, C-15, C-15 interchangeable), 130.2 (CH, C-3b), 129.4 (C, C-5), 129.0 (CH, C-10’),
27.7 (CH, C-5a), 126.8 (CH, C-7), 125.3 (CH, C-11), 125.0 (C, C-1b), 123.7 (C, C-3c), 123.3 (CH, C-11 ),
0
0
1
1
21.2 (CH, triazole), 118.3 (C, C-4a), 115.7 (CH, C-2b), 114.8 (CH, C-6a), 100.6 (CH, C-8a), 69.9 (CH ,
2
0
C-8 ), 68.3 (CH -O), 50.1 (CH -N), 39.7 (CH , C-2), 34.3 (C, C-1), 33.7 (CH -CO), 33.1 (CH , C-4), 30.1
2
2
2
2
2
(
(
CH , CH -
δ
), 29.0 (CH , C-16, C-17), 28.8 (CH , CH -
α
), 26.2 (CH , CH -
γ
), 25.4 (CH , CH -
β
), 21.7
2
2
3
2
2
2
2
2
2
0
CH , CH -18), 21.0 (CH , CH -C(=C)N), 19.3 (CH , C-3), 14.7 (CH , CH -19 ), 12.81, 12.75 (CH ,
3
3
2
2
2
3
3
3
+
CH -19,20,20’); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 878.7 [M + H] ; Anal. calcd.
3
for C H N O : C 76.59, H 7.69, N 4.79 found C 76.12, H 7.47 N 4.23.
56
67
3
6
0
3
β
,3 -bis(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-
◦
,
β
-carotene (17): red crystals (42%); mp 177–178 C; UV (THF) λmax 243, 278, 429(sh), 456, 486 nm; IR
−1 1
(
KBr) νmax 3414, 2919, 1730, 1624, 1253 cm ; H-NMR (CDCl , 500 MHz)
δ
8.16 (2H, dd, J = 5.3, 8.9 Hz,
3
2
×
H-5a), 7.88 (2H, d, J = 3.1 Hz, 2 x H-2c), 7.37 (4H, d, J = 3.8 Hz, 4
×
H-3b), 7.36 (2H, s, 2 triazole
×
CH), 6.94 (2H, dd, J = 1.9 Hz, J = 8.8 Hz, 2
2
m, H-14,14 ), 6.14 (2H, d, J = 11.4 Hz, H-10,10’), 6.11–6.05 (4H, m, H-7,7 , H-8,8 ), 5.09-5.03 (2H, m,
H-3,3 ), 4.34 (4H, t, J = 7.0 Hz, 2
×
H-6a), 6.87 (4H, d, J = 7.9 Hz, 4
H-8a), 6.67–6.59 (4H, m, H-11,11 , H-15,15 ), 6.34 (2H, d, J = 14.8 Hz, H-12,12 ), 6.27–6.21 (2H,
×
H-2b), 6.80 (2H, s,
0
0
0
×
0
0
0
0
×
N-CH ), 4.01 (4H, t, J = 5.5 Hz, 2
×
O-CH ), 3.05 (4H, t, J = 7.1 Hz,
2
2
2
×
CH -C(=C)N), 2.73 (4H, t, J = 7.0 Hz, 2
×
CH -C=O), 2.39 (2H, dd, J = 5.3 Hz, J = 16.9 Hz, H-4eq
,
2
2
0
0
0
H-4 eq), 2.11–2.04 (2H, m, H-4ax, H-4 ax), 1.95 (12H, s, CH -19,19 , CH -20,20’,), 1.82–1.74 (10H, m,
H-2eq, H-2 eq, 2
3
3
0
0
0
×
2 CH ), 1.70 (6H, s, CH -18,18 ), 1.58–1.48 (6H, m, H-2ax, H-2 ax, 2
×
CH ), 1.42–1.35
2
2
3
0 0
CH ), 1.09, 1.05 (12H. 2 s, CH -16,16 , CH -17,17 ); C-NMR (CDCl , 125 MHz) δ 176.2
2 3 3 3
13
(
(
4H, m, 2
×
C, C-4c), 172.2 (C, OC=O), 163.4 (C, C-7a), 158.0, 156.6 (C, C-4b, C-8c interchangeable), 152.2 (CH,
0
0
C-2c), 146.5 (C, triazole NC=C), 138.71, 137.7 (CH, C-12,12 and C-8,8 ), 137.9, 136.5, 135.5, 125.5 (C,
C-5,5 , C-6,6 , C-9,9 , C-13,13 interchangeable), 132.7 (CH, C-14,14 ), 131.5, 130.1 (CH, C-10,10’ and
C-15,15 interchangeable), 130.2 (CH, C-3b), 127.7 (CH, C-5a), 125.2, 124.9 (CH, C-7,7 and C-11,11
interchangeable), 125.1 (C, C-1b), 123.4 (C, C-3c), 121.2 (CH, triazole), 118.2 (C, C-4a), 115.7 (CH, C-2b),
0
0
0
0
0
0
0
0
0
0
1
14.9 (CH, C-6a), 100.6 (CH, C-8a), 68.7 (CH, C-3,3 ), 68.3 (CH -O), 50.1 (CH -N), 44.0 (CH , C-2,2 ), 38.4
2
2
2
0
0
(
CH , C-4,4 ), 36.7 (C, C-1,1 ), 34.0 (CH -CO), 30.1, 28.8, 26.2, 25.4 (CH , CH -
α
,
β
,
γ
,
δ
interchangeable),
2
2
2
2
0
0
0
3
0.0, 28.5 (CH , CH -16,16 and CH -17,17 interchangeable), 21.5 (CH , CH -18,18 ), 21.0 (CH ,
3
3
3
3
3
2
0
CH -C(=C)N), 12.8, 12.7 (CH , CH -19,19 and CH -20,20’ interchangeable); MS (MALDI-TOF, positive
mode, with DHB matrix) m/z 1509.794 [M + Na] ; Anal. calcd. for C H N O : C 74.27, H 7.18, N
2
3
3
3
+
92
106
6
12
5
.65 found C 74.13, H 7.79 N 5.56.
0
3
-(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-3 -
◦
(4-pentynoyloxy)-
β
,
β
-carotene (18): red crystals (7%); mp 155–156 C; UV (THF) λmax 242, 279, 340,
−1 1
4
28(sh), 455, 484 nm; IR (KBr) νmax 3420, 1730, 1624, 1253 cm ; H-NMR (CDCl , 500 MHz)
δ
8.19 (1H,
3
d, J = 8.9 Hz, H-5a), 7.90 (1H, s, H-2c), 7.42 (2H, d, J = 8.5 Hz, H-3b), 7.36 (1H, s, triazole CH), 6.96 (1H,
dd, J = 2.2, 8.9 Hz, H-6a), 6.88 (2H, d, J = 8.5 Hz, H-2b), 6.81 (1H, d, J = 2.1 Hz, H-8a), 6.70–6.60 (4H, m,
0
0
0
0
H-11,11 , H-15,15 ), 6.36 (2H, dd, J = 2.8, 14.7 Hz, H-12,12 ), 6.25 (2H, d, J = 7.5 Hz, H-14,14 ), 6.16
(
0 0 0
2H, dd, J = 4.0, 11.6 Hz, H-10,10’), 6.12–6.09 (4H, m, H-7,7 and H-8,8 ), 5.13–5.03 (2H, m, H-3,3 ),
4
2
2
.34 (2H, t, J = 7.1 Hz, O-CH ), 4.03 (2H, t, J = 6.2 Hz, N-CH ), 3.06 (2H, t, J = 7.3 Hz, CH -C(=C)N),
2 2 2
0
.73 (2H, t, J = 7.2 Hz, CH -C=O), 2.56–2.46 (4H, m, 2 CH pentynoate), 2.47–2.38 (2H, m, H-4,4 eq),
2
2
0
0
.17–2.07 (2H, m, H-4,4 ax), 1.98 (1H, s, C
≡
CH), 1.97 (12H, ps, 4 CH -19,19 ,20,20’), 1.86–1.76 (6H, m,
3
0
0
0
H-2,2 eq, a linker CH ), 1.72, 1.71 (6H,2 s, CH -18,18 ), 1.56–1.51 (2H, m, H-2,2 ax), 1.42–1.38 (4H, m,
2
2
3
0
0
linker CH ), 1.27–1.20 (2H, m, a linker CH ), 1.11, 1.10, 1.08, 1.06 (12H, 4 s, CH -16,16 ,17,17 ); MS
2
2
3

Molecules 2020, 25, 636
16 of 21
+
(
MALDI-TOF, positive mode, with DHB matrix) m/z 1130.7 [M + Na] ; Anal. calcd. for C H N O :
71
85
3
8
C 76.93, H 7.73, N 3.79 found C 76.18, H 7.35 N 3.62.
0
3
,3 -bis(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-
β
,
κ
-
◦
carotene (19): dark red crystals (40%); mp 86 C decomp.; UV (THF) λmax 242, 288, 360, 475 nm; IR
KBr) νmax 3414, 1734, 1626, 1252 cm ; H-NMR (CDCl , 500 MHz)
−
1 1
(
2
(
(
(
δ
8.17 (2H, dd, J = 2.5, 8.9 Hz,
3
0
×
H-5a), 7.88 (2H, d, J = 2.2 Hz, 2
×
H-2c), 7.37–7.35 (7H, m, 4
×
H-3b, H-8 , 2
H-2b), 6.79 (2H, d, J = 2.1 Hz, 2
×
triazole CH), 6.94
H-8a), 6.70–6.47
2H, d, J = 8.9 Hz, 2 H-6a), 6.89–6.87 (4H, m, 2
×
×
×
0
0
0
0
0
6H, m, H-10’,11,11 ,12 ,15,15 ), 6.42–6.32 (3H, m, H-7 ,12,14 ), 6.24 (1H, d, J = 11.3 Hz, H-14), 6.14
1H, d, J = 11.3 Hz, H-10), 6.12-6.09 (2H, m, H-7, H-8), 5.26-5.23 (1H, m, H-3 ), 5.09-5.04 (1H, m, H-3),
0
4
.33 (4H, dd, J = 7.0 Hz, J = 13.7.0 Hz, 2
×
N-CH ), 4.00 (4H, t, J = 4.6 Hz, 2
×
O-CH ), 3.04 (4H, dd,
2
2
0
J = 6.8 Hz, J = 12.9 Hz, CH -C(=C)N), 2.95 (1H, dd, J = 8.8 Hz, J = 14.9 Hz, H-4
α
0α
), 2.74–2.69 (4H, m,
2
2
×
CH -C=O), 2.39 (1H, dd, J = 5.3 Hz, J = 16.8 Hz, H-4eq), 2.11–2.02 (2H, m, H-2 , H-4ax), 1.96, 1.95,
2
0
.94, 1.93 (12H, 4s, CH -19,19 ,20,20’), 1.82–1.74 (6H, m, H-2eq, H-2 , 2 CH ), 1.70 (3H, s, CH -18),
3 2 3
0
1
1
β
0
0
.58–1.47 (6H, m, H-2ax, H-4 , 2 CH ), 1.42–1.36 (4H, m, 2 CH ), 1.28 (3H, s, CH -18 ), 1.26–1.22 (4H,
β
2
0
2
3
0
13
m, 2 CH ), 1.15, 1.09 (6H, 2 s, CH -16 ,17 ), 1.06, 0.84 (6H, 2 s, CH -16,17); C nmR (CDCl , 125 MHz)
δ
2
3
3
3
0
202.5 (C, C-6 ), 176.2 (C, 2
×
C-4c), 172.5 (C, 2
×
OC=O), 163.4 (C, 2
×
C-7a), 158.0 (C, 2
×
C-4b),
0
1
1
56.5 (C, 2
×
C-8c), 152.2 (CH, 2 x C-2c), 147.2 (CH, C-8 ), 146.5, 146.42 (C, 2 triazoles NC=C), 142.1,
41.0, 138.6, 137.5, 135.4, 132.5, 131.7, 131.4, 129.8, 125.5, 124.0, 121.3, 121.2 (CH, 2
×
triazoles CH,
C-3c,
0
0
0
0
C-7,8,10,10’,11,11 ,12,12 ,14,14 ,15,15 ), 137.9, 137.6, 136.0, 135.9, 133.5, 125.6, 125.1, 123.6, (C, 2
2
2
C-3), 68.4 (CH , O-CH ), 58.6 (C, C-5 ), 50.1 (CH , N-CH ), 47.6 (CH , C-2 ), 44.0 (CH , C-2), 43.7 (CH ,
C-4 ), 42.3 (CH , C-4), 38.4, 36.7 (C, C-1,1 ), 34.0, 33.9 (CH , 2
CH -16,16 ,17,17 ), 30.2, 28.8, 26.2, 25.5 (CH , 8
2
DHB matrix) m/z 1525.8 [M + Na] ; Anal. calcd. for C H N O : C 73.48, H 7.10, N 5.59 found C
×
0
0
0
C-1b, C-5,6,9,9 ,13,13 ), 130.2 (CH, 4
×
C-3b), 127.73, 127.71 (CH, 2
C-2b), 114.9, 114.8 (CH, 2
×
C-5a), 120.6 (CH, C-7 ), 118.2 (C,
0
×
C-4a), 115.7 (CH, 4
×
×
C-6a), 100.6 (CH, C-8a), 73.8 (CH, C-3 ), 68.7 (CH,
0
0
2
2
2
2
2
2
2
0
0
×
CH -CO), 30.0, 28.6, 25.6, 24.8 (CH ,
2 3
2
2
0
0
0
CH linker), 21.5, 20.7 (CH , CH -18,18 ), 21.1 (CH ,
2 3 3 2
×
3
2
0
CH -C=C), 12.9, 12.81, 12.80, 12.7 (CH , CH -19,19 ,20,20’); MS (MALDI-TOF, positive mode, with
2 3 3
×
+
92
106
6
13
7
2.92, H 6.68 N 5.49.
0
3
-(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-3 -
◦
(4-pentynoyloxy)-
β
,
κ
-carotene (20a): dark red crystals (22%); mp 86–87 C decomp.; UV (THF) λmax
42, 288, 476 nm; IR (KBr) νmax 3100–3400 (br), 3295, 2922, 1729, 1663, 1629, 1253 cm ; H-NMR
−1 1
2
(
CDCl , 500 MHz)
δ
8.19 (1H, d, J = 8.9 Hz, H-5a), 7.90 (1H, s, H-2c), 7.44 (2H, d, J = 8.6 Hz, H-3b),
3
0
7
6
6
.36 (1H, s, triazole CH), 7.34 (1H, d, J = 14.8 Hz, H-8 ), 6.96 (1H, dd, J = 2.1 Hz, J = 8.9 Hz, H-6a),
.89 (2H, d, J = 8.6 Hz, H-2b), 6.81 (1H, d, J = 2.2 Hz, H-8a), 6.72–6.51 (6H, m, H-10’,11,11 ,12 ,15,15 ),
.43 (1H, d, J = 15.1 Hz, H-7 ), 6.38–6.35 (2H, m, H-12,14 ), 6.26 (1H, d, J = 11.2 Hz, H-14), 6.15 (1H,
0
0
0
0
0
0
d, J = 12.5 Hz, H-10), 6.13–6.10 (2H, m, H-7, H-8), 5.31–5.25 (1H, m, H-3 ), 5.13–5.03 (1H, m, H-3),
4
2
.34 (2H, t, J = 7.1 Hz, N-CH ), 4.04 (2H, t, J = 6.3 Hz, O-CH ), 3.05 (4H, t, J = 7.3 Hz, CH -C(=C)N),
2
2
2
0
.99 (1H, dd, J = 8.4 Hz, J = 14.7 Hz, H-4
α
), 2.73 (2H, t, J = 7.4 Hz, CH -C=O), 2.52–2.50 (4H, m,
2
0
CH -pentynoate), 2.40 (1H, dd, J = 5.8 Hz, J = 17.9 Hz, H-4eq), 2.12–2.05 (2H, m, H-2
α
, H-4ax), 1.98,
2
0
0
0
1.972, 1.965, 1.95 (13H, 4s, CH
≡
C, CH -19,19 ,20,20’), 1.85–1.73 (4H, m, H-2eq, H-2 , CH ), 1.71 (3H, s,
3
β
2
CH -18), 1.69–1.51 (m, 2 CH H-2ax, H-4 , overlaid with the signal of water), 1.45–1.37 (4H, m,
2
×
3
2
β
0 0 0
CH ), 1.28 (3H, s, CH -18 ), 1.18, 1.10 (6H, 2 s, CH -16 ,17 ), 1.06, 0.86 (6H, 2 s, CH -16,17); MS
2 3 3 3
+
(
MALDI-TOF, positive mode, with DHB matrix) m/z 1146.7 [M + Na] ; Anal. calcd. for C H N O :
71 85 3 9
C 75.84, H 7.62, N 3.74 found C 75.23, H 7.36 N 3.40.
0
3
-(4-pentynoyloxy)-3 -(1-(6-(3-(4-hydroxyphenyl)-4H-chromen-4-one-7-yloxy)hex-1-yl)-1,2,3-triazol-4-yl)-
◦
propanoyloxy)-
2
β
,κ
-carotene (20b): dark red crystals (23%); mp 130–131 C decomp.; UV (THF) λmax
05, 242, 288, 476 nm; IR (KBr) νmax 3100–3400 (br), 3305, 2921, 1727, 1663, 1623, 1253 cm 1; 1H-NMR
−
(
7
6
CDCl , 500 MHz) δ 8.19 (1H, d, J = 9.0 Hz, H-5a), 7.90 (1H, s, H-2c), 7.44 (2H, d, J = 8.6 Hz, H-3b),
3
0
.36 (1H, s, triazole CH), 7.33 (1H, d, J = 16.4 Hz, H-8 ), 6.96 (1H, dd, J = 2.2 Hz, J = 8.9 Hz, H-6a),
0
0
0
.89 (2H, d, J = 8.5 Hz, H-2b), 6.82 (1H, d, J = 2.1 Hz, H-8a), 6.72–6.50 (6H, m, H-10’,11,11 ,12 ,15,15 ),

Molecules 2020, 25, 636
17 of 21
0 0
.41 (1H, d, J = 15.1 Hz, H-7 ), 6.38–6.34 (2H, m, H-12,14 ), 6.26 (1H, d, J = 11.7 Hz, H-14), 6.16 (1H,
d, J = 11.3 Hz, H-10), 6.12–6.08 (2H, m, H-7, H-8), 5.27–5.22 (1H, m, H-3 ), 5.14–5.07 (1H, m, H-3),
6
0
4
2
.33 (2H, t, J = 7.1 Hz, N-CH ), 4.04 (2H, t, J = 6.2 Hz, O-CH ), 3.03 (4H, t, J = 7.1 Hz, CH -C(=C)N),
2
2
2
0
.96 (1H, dd, J = 8.5 Hz, J = 14.5 Hz, H-4
α
), 2.71 (2H, t, J = 7.2 Hz, CH -C=O), 2.60–2.50 (4H, m,
2
0
CH -pentynoate), 2.45 (1H, dd, J = 6.4 Hz, J = 17.7 Hz, H-4eq), 2.07–2.03 (2H, m, H-2
α
, H-4ax), 1.99,
2
0
0
1
.97, 1.96, 1.95, 1.94 (13H, 5s, CH
≡
C, CH -19,19 ,20,20’), 1.86–1.76 (4H, m, H-2eq, H-2 , CH ), 1.72 (3H,
3
β
2
0
s, CH -18), 1.69–1.65 (2H, m, CH ), 1.61–1.51 (4H, m, H-2ax, H-4
water), 1.43–1.33 (4H, m, 2 CH ), 1.29 (3H, s, CH -18 ), 1.15, 1.11 (6H, 2 s, CH -16 ,17 ), 1.08, 0.84 (6H,
2
β
, CH overlayed with the signal of
3
2
2
0
0
0
2
3
3
+
s, CH -16,17); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 1146.8 [M + Na] ; Anal. calcd.
3
for C H N O : C 75.84, H 7.62, N 3.74 found C 75.32, H 7.75 N 3.63.
71
85
3
9
0
0
8
-(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-8 -apo-
◦
β
-carotene (21): red crystals (75%); mp 113–114 C; UV (THF) λmax 242, 286, 407, 432, 456 nm; IR (KBr)
−
1 1
νmax 3341, 2923, 1736, 1614 cm ; H nmR (CDCl , 500 MHz) δ 12.71 (1H, s, OH), 7.88 (2H, d, J = 6.7 Hz,
3
2
×
H-2b), 7.56–7.50 (3H, m, 2
×
H-3b, H-4b), 7.33 (1H, s, triazole CH), 6.66 (1H, s, H-3c), 6.67–6.56
0
0
(3H, m, H-11, H-15, H-15 ), 6.48 (1H, d, J = 2.0 Hz, H-8a), 6.45 (1H, dd, J = 11.0, 15.0 Hz, H-11 ), 6.35
0
0
(1H, d, J = 1.8 Hz, H-6a), 6.34–6.30 (2H, m, H-12,12 ), 6.25–6.20 (2H, m, H-14, H-14 ), 6.16–6.11 (4H,
0
m, H-7, H-8, H-10, H-10’), 4.56 (2H, s, H-8 ), 4.32 (2H, t, J = 7.1 Hz, N-CH ), 4.01 (2H, t, J = 6.3 Hz,
2
O-CH ), 3.06 (2H, t, J = 7.1 Hz, CH -C(=C)N), 2.78 (2H, t, J = 7.2 Hz, CH -C=O), 2.06–2.01 (2H, m,
2
2
2
2
H-4), 1.96, 1.94 (6H+3H, 2 s, CH -19,20,20’), 1.91 (2H, m, CH -
δ
, overlayed with s at 1.94 ppm), 1.80
3
2
0
(3H, s, CH -19 ), 1.72 (3H, s, CH -18), 1.64-1.59 (2H, m, 2 H-3), 1.54–1.50 (2H, m, CH -
β), 1.48-1.46 (2H,
3
3
2
13
m, 2 H-2), 1.38–1.23 (4H, m, CH2-
α
, CH2-
γ
), 1.03 (6H, s, CH -16,17); C nmR (CDCl , 125 MHz)
δ
3
3
1
82.4 (C, C-4c), 172.6 (C, C=O), 165.0 (C, C-7a), 164.0 (C, C-2c), 162.2 (C, C-5a), 157.8 (C, C-8c), 146.4 (C,
0
triazole NC=C), 138.5 (CH, C-12 ), 137.9 (C, C-6), 137.7 (CH, C-8), 137.1 (CH, C-12), 136.8 (C, C-13),
1
C-1b), 130.8 (CH, C-10), 130.5, 129.6 (CH, C-15,15 ), 129.4 (C, C-5), 129.1 (CH, C-3b), 129.0 (CH, C-10’),
1
0
0
0
36.2 (C, C-9), 135.7 (C, C-13 ), 133.0, 132.2 (CH, C-14,14 ), 131.8 (CH, C-4b), 131.7 (C, C-9 ), 131.4 (C,
0
0
26.8 (CH, C-7), 126.3 (CH, C-2b), 125.3 (CH, C-11), 123.3 (CH, C-11 ), 121.1 (CH, triazole), 105.9 (CH,
0
C-3c), 105.7 (C, C-4a), 98.6 (CH, C-6a), 93.1 (CH, C-8a), 69.9 (CH , C-8 ), 68.3 (CH , O-CH ), 50.0 (CH ,
2
2
2
2
N-CH ), 39.7 (CH , C-2), 34.3 (C, C-1), 33.7 (CH -CO), 33.1 (CH , C-4), 31.9 (CH , CH -
δ), 29.0 (CH3,
2
2
2
2
2
2
CH -16,17), 28.8 (CH , CH -
α
), 26.2 (CH , CH -
γ
), 25.5 (CH , CH -
β
), 21.7 (CH , CH -18), 21.1 (CH ,
3
2
2
2
2
2
2
3
3
2
0
CH -C(=C)N), 19.3 (CH , C-3), 14.1 (CH , C-19 ), 12.81, 12.76 (CH , CH -19,20,20’); MS (MALDI-TOF,
2
2
3
3
3
+
positive mode, with DHB matrix) m/z 916.441 [M + K] ; Anal. calcd. for C H N O : C 76.59, H 7.69,
56
67
3
6
N 4.79 found C 76.21, H 7.41, N 4.37.
0
3
β
,3 -bis(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-
◦
,
β-carotene (22): orange-red crystals (59%); mp 92–93 C; UV (THF) λmax 243, 277, 432, 458, 486 nm;
−
1 1
IR (KBr) νmax 2922, 1729, 1614 cm ; H nmR (CDCl , 500 MHz)
δ 12.71 (2H, s, 2 OH), 7.87 (4H, d,
3
J = 6.6 Hz, 2
×
H-2b), 7.53–7.50 (6H, m, 4
×
H-3b, 2
×
H-4b), 7.35 (2H, s, 2 triazole CH), 6.65 (2H, s,
0
0
0
H-3c), 6.63–6.61 (4H, m, H-11,11 ,15,15 ), 6.47 (2H, s, 2
×
H-8a), 6.35 (4H, pd, J = 12.4 Hz, H-12,12 ,
0
2
×
H-6a), 6.24 (2H, d, J = 5.5 Hz, H-14,14 ), 6.14 (2H, d, J = 11.1 Hz, H-10,10’), 6.10–6.05 (4H, m,
0
0
0
H-7,7 ,8,8 ), 5.06 (4H, ps, H-3,3 ), 4.34 (4H, t, J = 6.5 Hz, 2
×
N-CH ), 4.01 (4H, ps, 2
×
O-CH ), 3.04
2
2
(4H, t, J = 6.8 Hz, 2 CH -C(=C)N), ), 2.72 (4H, t, J = 6.6 Hz, 2
×
×
CH -C=O), 2.40 (2H, dd, J = 3.7 Hz,
2
2
0
0
0
J = 16.8 Hz, H-4eq, H-4 eq), 2.11–2.02 (2H, m, H-4ax, H-4 ax), 1.95 (12H, s, CH -19,19 ,20,20’), 1.80 (2H,
3
0
0
CH ), 1.71 (6H, s, CH -18,18 ), 1.58-1.52 (6H, m,
2 3
pt, J = 6.4 Hz, H-2eq, H-2 eq), 1.76–1.73 (8H, m, 4
×
0
0
0
13
H-2ax, H-2 ax, 2 x CH ), 1.42–1.33 (4H, m, 2
×
CH ), 1.10, 1.06 (12H, 2s, CH -16,16 ,17,17 ); C-NMR
2
2
3
(
CDCl , 125 MHz)
δ
182.4 (C, 2
×
C-4c), 172.4 (C, 2
×
C=O), 165.0 (C, 2
×
C-7a), 163.9 (C, 2
×
C-2c), 162.2
3
0
0
(
1
C, 2
×
C-5a), 157.8 (C, 2
×
C-8c), 146.5 (C, 2
×
triazole NC=C), 138.7 (CH, C-8, 8 ), 137.6 (CH, C-12,12 ),
0
0
0
0
37.9, 136.4, 135.5 (C, C-5,5 ,6,6 ,9,9 *), 132.6 (C-14,14 ), 131.8 (CH, 2
×
C-4b) 131.5 (CH, C-10,10’), 131.3
0
0
(
1
C, 2
×
C-1b), 130.1 (CH, C-15,15 ), 129.0 (CH, 2
×
C-3b), 126.3 (CH, 2
×
×
C-2b), 125.5 (C, C-13,13 *),
0
0
25.2 (CH, C-7,7 ), 124.8 (CH, C-11,11 ), 121.0 (CH, 2
triazole CH), 105.9 (CH, 2
×
C-3c), 105.7 (C, 2
×
0
C-4a), 98.6 (CH, 2
×
C-6a), 93.1 (CH, 2 C-8a), 68.6 (CH, C-3,3 ), 68.3 (CH , 2
×
×
CH -O), 50.0 (CH , 2
×
2
2
2

Molecules 2020, 25, 636
18 of 21
0
0
0
CH -N), 44.0 (CH , C-2,2 ), 38.4 (CH , C-4,4 ), 36.7 (C, C-1,1 ), 34.0 (CH , 2
×
CH -CO), 30.1, 28.5 (CH ,
2
2
2
2
2
3
0
0
0
CH -16,16 ,17,17 ), 30.2, 28.8, 26.2, 25.5 (CH , 2
CH -C(=C)N), 12.8, 12.7 (CH , CH -19,19 ,20,20’) *interchangeable; MS (MALDI-TOF, positive mode,
with DHB matrix) m/z 1509.913 [M + Na] ; Anal. calcd. for C H N O : C 74.27, H 7.18, N 5.65
×
4 CH linker), 21.5 (CH , CH -18,18 ), 21.1 (CH , 2
×
3
2
2
3
3
2
0
2
3
3
+
92
106
6
12
found C 74.13, H 7.83 N 5.09.
0
3
-(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-3 -
◦
(
4-pentynoyloxy)-
β
,
β
-carotene (23): orange-red crystals (60%); mp 122–123 C; UV (THF) λmax 277, 429,
−1 1
458, 486 nm; IR (KBr) νmax 3308, 2922, 1727, 1613 cm ; H-NMR (CDCl , 500 MHz)
δ
12.71 (1H,
H-3b, H-4b), 7.35 (1H, s, triazole
CH), 6.66 (1H, s, H-3c), 6.65-6.60 (4H, m, H-11,11 ,15,15 ), 6.48 (1H, d, J = 2.1 Hz, H-8a), 6.36 (2H, dd,
J = 5.5 Hz, J = 14.7 Hz, H-12,12 ), 6.35 (1H, s, H-6a), 6.25 (2H, pd, J = 7.4 Hz, H-14,14 ), 6.15 (2H, dd,
J = 6.4 Hz, J = 11.5 Hz, H-10,10’), 6.12–6.05 (4H, m, H-7,7 ,8,8 ), 5.13–5.03 (2H, m, H-3,3 ), 4.34 (2H, t,
J = 7.1 Hz, N-CH ), 4.02 (2H, t, J = 6.3 Hz, O-CH ), 3.05 (2H, t, J = 7.2 Hz, CH -C(=C)N), 2.72 (4H, t,
3
s, OH), 7.88 (2H, d, J = 6.6 Hz, 2
×
H-2b), 7.56–7.50 (3H, m, 2
×
0
0
0
0
0
0
0
2
2
2
0
J = 7.3 Hz, CH -C=O), 2.56–2.50 (4H, m, CH pentynoate), 2.47–2.37 (2H, m, H-4eq,4 eq), 2.15–2.06 (2H,
2
2
0
0
m, H-4ax,4 ax), 1.98, 1.96, 1.95 (3s, 13H, HC
with the singlet at 1.95 ppm), 1.84–1.76 (4H, m, H-2eq, H-2 eq, CH2-
≡
C, CH -19,19 ,20,20’), 1.94–1.91 (2H, m, CH -
γ
, overlayed
), 1.72, 1.71 (6H, 2 s, CH -18,18 ),
δ), 1.11, 1.10, 1.07, 1.06 (12H, 4s,
3
2
0
0
α
3
0
1.61–1.50 (4H, m, H-2ax, H-2 ax, CH2-
β
), 1.44–1.38 (2H, m, CH2-
0
0
13
CH -16,16 ,17,17 ); C nmR (CDCl , 125 MHz)
δ
182.4 (C, C-4c), 172.5, 171.5 (C, 2
×
OC=O), 165.0 (C,
3
3
0
C-7a), 164.0 (C, C-2c), 162.2 (C, C-5a), 157.8 (C, C-8c), 146.5 (C, triazole NC=C), 138.7 (CH, C-8,8 ),
37.9, 136.5, 135.6, 135.5 (C, C-5,5 ,6,6 ,9,9 *), 137.7 (CH, C-12,12 ), 132.6 (CH, C-14,14 ), 131.8 (CH,
0
0
0
0
0
1
0
C-4b), 131.5 (CH, C-10,10’), 131.4 (C, C-1b), 130.1 (CH, C-15,15 ), 129.1 (CH, 2
×
C-3b), 126.3 (CH, 2
×
0
0
0
C-2b), 125.5 (C, C-13,13 *), 125.3, 125.2 (CH, C-7,7 ), 124.9 (CH, C-11,11 ), 121.0 (CH, triazole CH), 105.9
CH), 68.9, 68.6 (CH, C-3,3 ),
8.3 (CH , CH -O), 50.1 (CH -N), 44.04, 44.03 (CH , C-2,2 ), 38.4 (CH , C-4,4 ), 36.72, 36.70 (C, C-1,1 ),
), 30.0, 28.53, 28.51 (CH , CH -16,16 ,17,17 ), 28.8 (CH ,
), 21.5 (CH , CH -18,18 ), 21.1 (CH , CH -C(=C)N), 14.4
CH), 12.8, 12.7 (CH , CH -19,19 ,20,20’) *interchangeable; MS (MALDI-TOF, positive
mode, with DHB matrix) m/z 1130.7 [M + Na] ; Anal. calcd. for C H N O : C 76.93, H 7.73, N 3.79
0
0
(CH, C-3c), 105.7 (C, C-4a), 98.6 (CH, C-6a), 93.1 (CH, C-8a), 82.6 (C, C
≡
0
0
6
3
2
2
2
2
2
0
0
4.0, 33.7 (CH , 2
×
CH -CO), 30.2 (CH , CH -
γ
2
2
2
2
3
3
2
0
CH2-
α
), 26.2 (CH , CH -
δ
), 25.5 (CH , CH -
β
2
2
2
2
3
3
2
2
0
(CH , CH -C
≡
2
2
3
3
+
71
85
3
8
found C 76.32, H 7.45 N 3.50.
0
3
β
3
,3 -bis(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-
◦
,
κ
-carotene (24): dark red crystals (88%); mp 88–90 C; UV (THF) λmax 243, 278, 476 nm; IR (KBr) νmax
−1 1
414, 1728, 1641, 1624, 1252 cm ; H nmR (CDCl , 500 MHz)
J = 6.9 Hz, 4
.71–6.46 (6H, m, H-10’,11,11 ,12 ,15,15 ), 6.65 (2H, s, 2
m, H-7 ,12,14 ), 6.34 (2H, s, 2
δ
12.70 (2H, s, 2
×
OH), 7.88 (4H, d,
triazole CH, H-8 ),
3
0
×
H-2b), 7.52 (6H, pd, J = 7.6 Hz, 4
×
H-3b, 2
×
H-4b), 7.34 (3H, brs, 2
H-3c), 6.48 (2H, s, 2 x H-8a), 6.42-6.32 (3H,
H-6a), 6.25 (1H, d, J = 11.1 Hz, H-14), 6.15 (1H, d, J = 11.0 Hz, H-10),
×
0
0
0
6
×
0
0
×
0
6
2
.12-6.07 (2H, m, H-7, H-8), 5.30-5.21 (m, 1H, H-3 ), 5.11–5.02 (m, 1H, H-3), 4.33 (4H, pd, J = 4.9 Hz,
×
N-CH ), 4.01 (4H, t, J = 5.6 Hz, 2
×
O-CH ), 3.06–3.02 (4H, m, 2
×
CH -C(=C)N), 2.96 (1H, dd,
2
2
2
0
J = 9.0 Hz, J = 14.8 Hz, H-4
α
), 2.74-2.69 (4H, m, 2
×
CH -C=O), 2.40 (dd, 1H, J = 5.1 Hz, J = 16.9 Hz,
2
0
0
H-4eq), 2.11–2.03 (2H, m, H-4ax, H-2
m, H-2eq, H-2 , 2 CH linker), 1.71 (3H, s, CH -18), 1.58–1.47 (6H, m, H-2ax, H-4 , 2 CH linker),
.43–1.35 (4H, m, 2 CH linker), 1.29 (3H, s, CH -18 ), 1.28–1.21 (4H, m, 2 CH linker, overlayed with
the s at 1.29 ppm), 1.15, 1.10 (6H, 2s, CH -16,17) 1.06, 0.84 (6H, 2s, CH -16 ,17 ); C-NMR (CDCl3,
25 MHz)
C, 2 C-2c), 162.2 (C, 2
NC=C), 142.1, 140.9, 138.6, 137.5, 135.3, 132.4, 131.7, 129.7 (CH, C-8,10,10’,12,12 ,14,14 ,15,15 ), 137.9,
37.6, 136.0, 135.9, 133.6 (C, C-6,9,9 ,13,13 ), 131.8 (CH, 2
partially overlayed with C-3b), 126.3 (CH, 4 C-2b), 125.6 (C, C-5), 125.5, 124.0, 121.1, 121.0 (CH, 2
triazole CH, C-7,11,11 ), 120.6 (CH, C-7 ), 105.9 (CH, 2
α
), 1.98, 1.96, 1.94 (12H, 3s, CH -19,19 ,20,20’), 1.85–1.73 (6H,
3
0
0
β
2
3
β
2
0
1
2
3
2
0
0
13
3
3
0
1
(
δ
202.4 (C, C-6 ), 182.4 (C, 2
×
C-4c), 172.5, 172.4 (C, 2
×
OC=O), 165.0 (C, 2
×
C-7a), 163.9
triazole
0
×
×
C-5a), 157.8 (C, 2
×
C-8c), 147.1 (CH, C-8 ), 146.5, 146.4 (C, 2
×
0
0
0
0
0
1
×
C-4b), 129.1 (CH, 4
×
3b), 128.9 (C, 2 C-1b,
×
×
0
0
×
C-3c), 105.7 (C, 2
×
C-4a), 98.6 (CH, 2
×
0
0
C-6a), 93.1 (CH, 2
×
C-8a), 73.7 (CH, C-3 ), 68.6 (CH, C-3), 68.3 (CH , 2
×
CH -O), 58.6 (C, C-5 ), 50.1
2
2

Molecules 2020, 25, 636
19 of 21
0
0
(
CH , 2
×
CH -N), 47.6 (CH , C-2 ), 44.0 (CH , C-2), 43.7 (CH , C-4 ), 42.3 (CH , C-4), 38.4, 36.7 (C,
2
2
2
2
2
2
0
C-1,1 ), 34.0, 33.9 (CH , 2
×
CH -CO), 30.2, 28.7, 26.2, 25.5 (CH , 8
×
CH ), 30.2, 28.5, 25.6, 24.8 (CH ,
2
2
2
2
3
0
0
0
CH -16,16 ,17,17 ), 21.5, 20.7 (CH , CH -18,18 ), 21.1 (CH , 2x CH -C(=C)N), 12.9, 12.8, 12.7, (CH ,
3
3
3
2
2
3
0
+
CH -19,19 ,20,20’); MS (MALDI-TOF, positive mode, with DHB matrix) m/z 1526.1 [M + Na] ; Anal.
3
calcd. for C H N O : C 73.48, H 7.10, N 5.59 found C 73.05, H 6.98 N 55.46.
92
106
6
13
0
3
-(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-3 -
0
(4-pentynoyloxy)-
β
,
κ
-carotene and 3-(4-pentynoyloxy)-3 -(1-(6-((5-hydroxy-2-phenyl-4H-chromen-4-one-7-
◦
yl)oxy)hex-1-yl)-1,2,3-triazol-4-yl)-propanoyloxy)-
β
,
κ
-carotene (25): dark red crystals (21%); mp 139–140 C;
−1 1
UV (THF) λmax 242, 288, 476 nm; IR (KBr) νmax 3313, 1609, 1663, 2924 cm ; H nmR (CDCl , 500 MHz)
3
δ
12.71 (H, s, OH), 7.89 (2H, d, J = 6.9 Hz, 2
×
H-2b), 7.55–7.51 (3H, m, 2
×
H-3b, H-4b), 7.35–7.33
0
0
0
0
(2H, m, triazole CH, H-8 ), 6.67 (1H, s, H-3a), 6.73–6.51 (6H, m, H-10’,11,11 ,12 ,15,15 ), 6.49 (1H, ps,
0
0
H-8a), 6.45–6.33 (3H, m, H-7 ,12,14 ), 6.35 (1H, s, H-6a), 6.26 (1H, d, J = 11.1 Hz, H-14), 6.18–6.16
0
(3H, m, H-7,8,10), 5.31–5.24 (m, 1H, H-3 ), 5.14–5.05 (m, 1H, H-3), 4.36–4.32 (2H, m, N-CH ), 4.03
2
(
2H, t, J = 6.0 Hz, O-CH ), 3.07–3.03 (2H, m, CH -C(=C)N), 2.98 (1H, dd, J = 5.6 Hz, J = 14.3 Hz,
2
2
0
H-4
2
α
), 2.75–2.70 (2H, m, CH -C=O), 2.55–2.51 (2H, m, 2
×
CH pentynoate), 2.43–2.38 (m, 1H, H-4eq),
2
2
0
0
.15–2.06 (2H, m, H-2
α
, H-4ax), 1.99, 1.98, 1.97, 1.95 (13H, 4s, CH
≡
C, CH -19,19 ,20,20’), 1.83–1.78 (4H,
3
0
0
m, H-2eq, H-2 , CH ), 1.73, 1.72 (3H, 2 s, CH -18), 1.62–1.50 (6H, m, 2
×
CH H-2ax, H-4 β), 1.45–1.38
β
2
3
2
0
0
0
(4H, m, 2 CH ), 1.29, 1.30 (3H, 2 s, CH -18 ), 1.19, 1.16, 1.12, 1.11 (6H, 4 s, CH -16 ,17 ), 1.08, 1.07, 0.89,
2
3
3
0
0
0
0
.85 (2 s, CH -16,17, overlaid with the signal of grease). The methyl groups of 16,16 ,17,17 ,18 and 18
3
13
0
give separate signals for the two regioisomers; C nmR (CDCl , 125 MHz) δ 202.4 (C, C-6 ), 182.4 (C,
3
C-4c), 172.5, 172.4 (C, 2
×
OC=O), 165.0 (C, C-7a), 163.9 (C, C-2c), 162.2 (C, C-5a), 157.8 (C, C-8c), 147.1
0
(
CH, C-8 ), 146.5 (C, triazole NC=C), 142.1, 140.9, 138.6, 137.5, 135.3, 132.4, 131.8, 131.4, 129.7 (CH,
0
0
0
0
0
C-8,10,10’,12,12 ,14,14 ,15,15 ), 137.9, 137.6, 136.0, 135.9, 133.6 (C, C-6,9,9 ,13,13 ), 131.8 (CH, C-4b),
29.1 (CH, 2 3b), 128.3 (C, C-1b), 126.3 (CH, 2 C-2b), 125.6 (C, C-5), 125.5, 124.1, 121.0, 121.0 (CH,
1
×
×
0
0
triazole CH, C-7,11,11 ), 120.6 (CH, C-7 ), 105.9 (CH, C-3c), 105.7 (C, C-4a), 98.6 (CH, C-6a), 93.1 (CH,
0
C-8a), 82.5 (C, C
≡
CH), 74.0, 73.8 (CH, C-3 ), 69.0 (CH, HC
≡
C), 68.9, 68.6 (CH, C-3), 68.3 (CH , CH -O),
2
2
0
0
0
5
8.6 (C, C-5 ), 50.0 (CH , CH -N), 47.7, 47.6 (CH , C-2 ), 44.1 (CH , C-2), 43.7 (CH , C-4 ), 42.3 (CH ,
2
0
2
2
2
2
2
C-4), 38.5, 36.7 (C, C-1,1 ), 34.1, 34.0, 33.8, 33.7 (CH , 2
×
CH -CO), 30.2, 28.8, 26.2, 25.5 (CH , 4
×
CH2),
2
2
2
0
0
0
3
1
0.2, 28.5, 25.6, 24.8 (CH , CH -16,16 ,17,17 ), 21.5, 20.7 (CH , CH -18,18 ), 21.1 (CH , CH -C(=C)N),
3
3
3
3
2
2
0
4.5 (CH -C C), 12.9, 12.8, 12.75, 12.70, (CH , CH -19,19 ,20,20’); MS (MALDI-TOF, positive mode,
3 3
≡
2
+
with DHB matrix) m/z 1124.3 [M + H] .
Supplementary Materials: The nmR and UV spectra of the prepared compounds are available online.
Author Contributions: Conceptualization, G.G.-F., J.D. and V.N.; Data curation, D.C., K.B., T.K., S.B.K., G.G.-F.,
A.T. and T.L.; Formal analysis, D.C., K.B., T.K., A.T., T.L. and V.N.; Funding acquisition, T.K., J.D. and A.A.;
Investigation, I.L., D.C., K.B., T.K. and S.B.K.; Methodology, D.C., K.B., A.T. and V.N.; Project administration, V.N.;
Supervision, V.N.; Validation, I.L., D.C. and K.B.; Visualization, V.N.; Writing—original draft, V.N.; Writing—review
&
editing, A.A. and V.N. All authors have read and agree to the published version of the manuscript.
Funding: This research was funded by Hungarian Scientific Research Fund (grants: NKFI K 115931, 131493 and
1
20181).
Acknowledgments: The authors thank Cec
í
lia Sár for the elemental analysis measurements. The authors are
grateful to Erika Jámbor, Krisztina Sajti, Judit Rigó and Roland Lukács for their skillfull assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of all the compounds (1–25) are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).