Selected Compounds Structurally Related to Acyclic Sesquiterpenoids and Their Antibacterial and

25 Pages·2015·2.74 MB·English

Molecules 2015, 20, 11272-11296; doi:10.3390/molecules200611272
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Selected Compounds Structurally Related to Acyclic
Sesquiterpenoids and Their Antibacterial and Cytotoxic Activity
Radosław Bonikowski 1,*, Paulina Świtakowska 1, Monika Sienkiewicz 2 and
Małgorzata Zakłos-Szyda 3
1 Institute of General Food Chemistry, Łódź University of Technology, Stefanowskiego St. 4/10,
90-924 Łódź, Poland; E-Mail: [email protected]
2 Environmental Biology Department, Medical University of Łódź, Żeligowskiego St. 7/9,
90-752 Łódź, Poland; E-Mail: [email protected]
3 Institute of Technical Biochemistry, Łódź University of Technology, Stefanowskiego St. 4/10,
90-924 Łódź, Poland; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: [phone]; Fax: [phone].
Academic Editor: Jean Jacques Vanden Eynde
Received: 30 April 2015 / Accepted: 15 June 2015 / Published: 18 June 2015

Abstract: By implementing a common and industrially used method, 30 compounds which
are structurally related to geranyl acetone, nerolidol, farnesal, farnesol and farnesyl acetate were
obtained. Their antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis,
Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii
bacteria was investigated. Pharmacophore models were proposed based on the obtained
results and 3D QSAR modelling. Cytotoxic effects against mainly human immortalised and
normal cell lines of different origin (malignant melanoma MeWo, colorectal adenocarcinoma
HT29, promyelocytic leukemia HL60, gingival fibroblasts HFIG, skin keratinocytes HaCaT
and rat small intestine epithelium IEC6) were examined. The odour descriptions of newly
synthesised compounds are given.
Keywords: terpenoids; geranyl acetone; nerolidol; farnesol; farnesyl acetate; structurally
related compound; antimicrobial activity; cytotoxicity
Molecules 2015, 20 11273

1. Introduction
Most of the natural acyclic sesquiterpenoids are used in the fragrance and flavour industry as odorants
and flavourings. This group includes geranyl acetone (1), nerolidol (2), farnesal (3), farnesol (4) and
farnesyl acetate (5) (Figure 1).
O OH
O
1 2 3
O
OH O
4 5

Figure 1. Structures of geranyl acetone (1); nerolidol (2); farnesal (3); farnesol (4) and
farnesyl acetate (5).
Nerolidol and farnesol, in addition to their flavouring characteristics, also possess antibacterial
properties. Farnesol, which is present in many creams and nourishing agents, has a bacteriostatic effect
against bacteria that can cause acne (Staphylococcus aureus, Staphylococcus epidermidis) and does not
affect naturally occurring microflora on the skin [1]. It blocks the formation of biofilm by the drug-resistant
strains of Staphylococcus aureus and acts synergistically with antibiotics [2–4]. Brilhante et al. showed
that farnesol had an inhibiting activity alone and in combination with antifungal agents against strains
of dimorphic fungi from the genus Coccidioides and against Candida spp. clinical isolates [4,5].
Nerolidol shows a toxic effect against, inter alia, Salmonella enterica, Staphylococcus aureus,
Escherichia coli and Aspergillus niger, and increases the antibacterial activity of antibiotics [6,7].
Nagaki et al. examined farnesal activity against S. aureus finding that it showed the highest activity
among the analysed terpenoids [8]. Shin et al. evaluated antimicrobial activity of farnesyl acetate
which inhibited the growth of S. aureus and Pseudomonas aeruginosa [9]. Geranyl acetone has also
antimicrobial properties as shown by the examination of its food preservative ability [10]. Moreover,
natural sesquiterpenoids in vivo and in vitro have chemopreventive and chemotherapeutic properties
against human cancer cell lines (inter alia: human malignant melanoma, promyelocytic leukemia and
lung cancer) [11–14].
In our previous studies [15–17], we described the synthesis and mainly the odour activity of the
analogues of acyclic terpenoids where isobutenyl moiety was replaced by a furan or thiophene ring.
This time we decided to examine the bioactivity (scent, antibacterial and cytotoxic) of compounds
structurally related to acyclic sesquiterpenoids where the osmophore and the 6-methylheptenone moiety
will be maintained, and the changes will be introduced into the middle part of the chain as shown in
Figure 2.
Molecules 2015, 20 11274

Figure 2. The structural relationship of the examined compounds to the natural sesquiterpenoids.
We reported compounds structurally related to geranyl acetone and nerolidol (Figure 3) before [18]
due to their olfactory and antibacterial properties as potential fragrance components which, according
to current trends, should possess added values, especially preserving properties, thus allowing for a
reduction or elimination of traditional cosmetic preservatives [19,20].
O
O O O O
R
6 R = CH3 8 9 10 11
7 R = H
OH
OH OH OH OH
R
12 R = CH3 14 15 16 17
13 R = H
Figure 3. Compounds structurally related to geranyl acetone (6 to 11) and nerolidol (12 to 17).
2. Results and Discussion
2.1. Synthetic Methodology
Starting from the alcohols 12–17 [18], we obtained a series of compounds structurally related to
farnesal (18–23) (Figure 4) by an oxidative rearrangement of tertiary allylic alcohols [21].
We observed that the obtained aldehydes exhibited high lability, which could somehow be confirmed
by the dependence of the yield on the isolation method. The yields of the distilled products were 36%
to 67%, while for products purified by column chromatography on silica gel, the yields were 71% to
83%. Furthermore, we found that the aldehydes 18–23 were rather stable in the solutions. The E/Z
geometric isomer ratio, confirmed by GC and NMR analyses, of the rearranged C-C double bond was
2:1 for 18, 19, 20, 23, while for 21 and 22 it was 1:1 and 3:2, respectively. The most characteristic and
diagnostic signal on the NMR spectra was the signal of the γ methyl substituent. In the case of the
E isomer, the 1H chemical shift, due to deshielding by the carbonyl group, was higher (ca. 2.1 ppm and
ca. 1.9 ppm for the Z isomer). On the other hand, in the E isomer, the signal of the carbon atom, due to
the steric effect, was shifted upfield (ca. 17 ppm and ca. 25 ppm for the Z isomer).
The scent of the compounds structurally related to farnesal, or rather its absence, was surprising. The
only exception was the odour of 21, which was intense, floral with fatty and melon notes, and of 23,
which was geranyl acetone-like (green, fruity, waxy and woody).
Molecules 2015, 20 11275

O OH OAc
1123 PCC R NaBH4 R AEct32NO R
18 R = CH3 24 R = CH3 30 R = CH3
19 R = H 25 R = H 31 R = H
O OH OAc
14 PCC NaBH4 Ac2O
Et3N
20 26 32
O OH OAc
15 PCC NaBH4 Ac2O
Et3N
21 27 33
O OH OAc
16 PCC NaBH4 Ac2O
Et3N
22 28 34
O OH OAc
17 PCC NaBH4 Ac2O
Et3N
23 29 35
Figure 4. Synthesis of compounds structurally related to farnesal (18 to 23), farnesol (24 to 29)
and farnesyl acetate (30 to 35).
The obtained aldehydes reduced with sodium borohydride in the usual manner yielded compounds
structurally related to farnesol (47% to 97%). The scent of the products was faint and floral, except for
29, which was odourless.
Subsequent esterification with acetic anhydride in the presence of triethylamine gave compounds
structurally related to farnesyl acetate (yield 69% to 90%) which, just as the compounds structurally
related to farnesal, were generally odourless but with one exception. Compound 35 had a very faint
quince-like scent.
For all compounds which had the scent, we performed additionally GC-olfactometry experiments.
For the compounds structurally related to both geranyl acetone and nerolidol [18], we did not observe
significant differences in the odour description of the isomers.
2.2. Antibacterial Activity of the Terpenoids and Their Structurally Related Compounds
All of the obtained compounds and the parent terpenes were subjected to an evaluation of their
antibacterial activity (Table 1). Data for geranyl acetone, nerolidol, ketones 6 to 11 and alcohols 12 to
17 were presented by us before [18].
Generally, the ketones 6 to 11 demonstrated the highest antibacterial activity against both standard
strains from the Enterococcus genus and all of the tested Gram-negative bacteria with a Minimal
Inhibitory Concentrations (MIC) between 12 µg/mL and 32 µg/mL. Parent terpenoid-farnesol and some
alcohols related to it and their acetates showed slightly similar activity. The most resistant to all of the
tested compounds was the standard strain of Staphylococcus aureus ATCC 43300. Although Togashi et al.
found that farnesol was active against Staphylococcus aureus FDA209P at concentrations of 10 μg/mL
and higher, in their investigations the bacterial suspension contained 1 × 105 CFU and the culture was
incubated for 48 h with shaking and monitored turbidimetrically [2]. On the other hand, Jin et al. stated
that farnesol had an inhibiting activity against Mycobacterium smegmatis mc2 155 ATCC 700084 with
Molecules 2015, 20 11276

a MIC value at 64 µg/mL by micro-dilution and in accordance with NCCLS guidelines [22]. The MIC
values obtained in our research for farnesol were from 16 µg/mL to 52 µg/mL against all of the tested
standard strains. The other tested terpenoids and their structurally related compounds had an inhibiting
activity against standard bacteria at a concentration between 12 µg/mL and 54 µg/mL.
For synthesised compounds we also obtained by column chromatography (E)-(Z)-isomer enriched
fractions containing ≥ 85% of the appropriate isomer according to the GC, but the determined bioactivity
(antimicrobial and cytotoxicity) did not differ significantly from that obtained for the mixtures (data not
presented). Ciproflaxicin and gentamicin were used as the control for the reference strains.
Table 1. Antibacterial activity of structurally related compounds and parent terpenes against
Gram-positive and Gramnegative reference strains.
Minimum Inhibitory Concentrations MIC (µg/mL)
Gram-Positive Bacteria Gram-Negative Bacteria
Compound Staphylococcus Enterococcus Enterococcus Escherichia Klebsiella Acinetobacter
aureus ATCC faecalis ATCC faecium ATCC coli ATCC pneumoniae baumannii
43300 51299 35667 25922 ATCC 700603 ATCC 19606
Geranyl acetone (1) 48 36 28 26 18 18
6 26 18 16 14 14 12
7 26 16 18 16 16 14
8 38 26 28 18 18 22
9 28 18 14 28 18 16
10 46 26 18 32 18 16
11 26 22 18 28 28 14
Nerolidol (2) 48 38 32 46 16 26
12 48 46 42 40 28 26
13 46 42 38 40 20 22
14 48 38 36 38 18 28
15 46 48 38 28 16 16
16 48 38 36 32 24 22
17 48 38 38 46 18 16
Farnesal (3) 24 22 36 46 44 36
18 18 22 28 52 46 46
19 28 34 38 44 36 42
20 22 20 32 48 38 38
21 32 38 42 54 48 52
22 26 24 46 44 36 36
23 28 26 36 46 34 36
Farnesol (4) 26 18 16 28 16 18
24 22 18 32 52 44 40
25 28 32 34 50 48 44
26 32 30 36 48 42 38
27 28 26 18 36 18 34
28 26 18 16 28 16 18
29 28 22 18 38 36 32
Molecules 2015, 20 11277

Table 1. Cont.
Minimum Inhibitory Concentrations MIC (µg/mL)
Gram-Positive Bacteria Gram-Negative Bacteria
Compound Staphylococcus Enterococcus Enterococcus Escherichia Klebsiella Acinetobacter
aureus ATCC faecalis faecium coli ATCC pneumoniae baumannii
43300 ATCC 51299 ATCC 35667 25922 ATCC 700603 ATCC 19606
Farnesyl acetate (5) 28 20 20 28 22 22
30 30 24 18 30 24 24
31 24 20 20 26 22 18
32 28 22 20 28 18 22
33 26 18 16 26 18 22
34 32 20 18 18 20 18
35 34 16 14 16 18 12
Ciprofloxacin 8 2 8 4 2 4
Gentamicin 16 >64 * 32 4 32 32
* The maximum examined concentration was 64 µg/mL.
Pharmacophores
Having numerous data regarding the antimicrobial activity (MICs), we determined the possible
pharmacophore models that define the activity of the described compounds (Figures 5 to 10).
The aldehyde 18 shows the highest activity against Staphylococcus aureus ATCC 43300 and the
pharmacophore is defined by one hydrogen-bond acceptor (HBA, green) which is surrounded by the
four excluded volumes (EV, grey) and two hydrophobes (H, cyan) comprising the chain (Figure 4).
One of them is in the line designated by the hydrogen-bond acceptor at a distance 5.7 Å from it, and
the other is at a distance 12.4 Å from HBA. The least active compounds (1, 2, 12, 14, 16 and 17),
in their probable and favourable energy conformations, do not fill one hydrophobe, more distant
from hydrogen-bond acceptor. Geranyl acetone (1) is too short. In the case of nerolidol (2) and its
structurally related alcohols (12, 14, 16 and 17) closer to HBA, hydrophobe is filled with vinyl moiety
which makes it impossible to further fill hydrophobe.

Figure 5. Aldehyde 18 aligned to the pharmacophore model of Staphylococcus aureus
active compounds (on the left), the least active ones 1, 2, 12, 14, 16 and 17 (on the right).
The pharmacophore model of Enterococcus faecalis active compounds (Figure 6) consists of one
HBA, two hydrophobes and five excluded volumes. Near the hydrogen bond acceptor there should be
no steric hindrance, which indicates the presence of four excluded volumes nearby. The distances
Molecules 2015, 20 11278

from HBA to the hydrophobes are 5.8 and 7.2 Å. In this case, as in the pharmacophore model of
Staphylococcus aureus active compounds, the least active compounds do not fill one hydrophobe, the
cause are the steric and conformational factors. Furthermore, in the case of the least active ones
structurally related to nerolidol 12 and 15, it can be assumed that the short length of the molecule in
conjunction with the position of the functional group which is more in the middle of the chain causes
lowering of the activity of these compounds. In turn, in the case of the most active ketone 7 and acetate 35,
connection of suitable chain length and exposition of the functional group makes these compounds
more active.

Figure 6. Compounds structurally related to geranyl acetone and farnesyl acetate (7 and 35)
aligned to the pharmacophore model of Enterococcus faecalis active compounds (on the left),
the least active ones 12 and 15 (on the right).
In the set of analysed compounds, the most potent against Enterococcus faecium are ketone 9 and
acetate 35. The pharmacophore comprises one hydrogen-bond acceptor, two hydrophobes and five
excluded volumes (Figure 7). Both hydrophobes are in the line perpendicular to the line determined by
HBA and the distances are 6.9 and 10.0 Å. The lack of filling of one hydrophobe is the cause of low
activity of compounds 12 and 22. This is due to the same considerations as in the two previous cases.

Figure 7. Compounds structurally related to geranyl acetone and farnesyl acetate 9 and 35
aligned to the pharmacophore model of Enterococcus faecium active compounds (on the
left), the least active ones 12 and 22 (on the right).
Ketone 6 shows the highest activity against the Gram-negative bacteria strains: Escherichia coli,
Klebsiella pneumoniae and Acinetobacter baumannii. Acetate 35 shows equal activity towards the
last-mentioned strain. The pharmacophores are defined by one hydrogen-bond acceptor, three
(Escherichia coli) or two (two other strains) hydrophobes and five (Klebsiella pneumoniae) or two
excluded volumes (Figures 8–10, respectively). In the case of E. coli, the hydrophobes include methyl
Molecules 2015, 20 11279

substituents at C-6 and C-7 and an isobutenyl moiety; in the case of K. pneumoniae the methyl groups
seem to play an important role and they fit into the hydrophobes. In turn, the pharmacophore
describing the spatial requirement for A. baumannii active molecules indicates the important role of
methyl substituent at C-7 and the isobutenyl moiety. The distances from HBA to the hydrophobes
are 5.5, 7.2, 9.0 and 3.4, 4.2 and 5.8, 6.1 Å (Escherichia coli and Klebsiella pneumoniae and
Acinetobacter baumannii, respectively). In the case of pharmacophores with regard to the compounds
least active against Gram-negative bacteria, the problem seems to be the position of the functional
group in the chain. While both hydrophobes are filled, in each case the functional group passes the
hydrogen-bond acceptor. While for compounds active against Gram-positive bacteria the exposition of
the functional group is beneficial, here it does not bring the desired effect.
Figure 8. Compound structurally related to geranyl acetone 6 aligned to the pharmacophore
model of Escherichia coli active compounds (on the left), the least active ones 18 and 21
(on the right).

Figure 9. Ketone 6 aligned to the pharmacophore model of Klebsiella pneumoniae active
compounds (on the left), the least active ones 18 and 21 (on the right).
Figure 10. Compounds structurally related to geranyl acetone and farnesyl acetate 6
and 35 aligned to the pharmacophore model of Acinetobacter baumannii active compounds
(on the left), the least active ones 21 (on the right).
Molecules 2015, 20 11280

The pharmacophore modelling was conducted additionally only for the most active substances
(Figure 11). The result obtained confirmed the previously described observations regarding the required
length of the chain and exposition of the functional group.
Compared to the more general models obtained by using all of the compounds described herein, the
above can be considered as more restrictive. Although the appearance of these models is different, their
analysis leads to identical conclusions. The compounds with longer chains have higher antimicrobial
activity. Additionally, exposition of the functional group is once the favourable factor (activity against
Gram-positive bacteria) while it is unfavourable at another time (activity against Gram-negative bacteria).
Moreover, in the above described steric conditions, it can be seen that the β-methyl substituent also
plays an important role (in the case of S. aureus).

(a) (b) (c)

(d) (e) (f)
Figure 11. The pharmacophore models of the most active compounds against
Gram-positive: S. aureus (a); E. faecalis (b); E. faecium (c) and most active compounds
against Gram-negative strains: E. coli (d); K. pneumoniae (e); A. baumannii (f).
2.3. Cytotoxicity of the Parent Terpenes and Structurally Related Compounds
All of the obtained compounds and parent terpenes as positive controls were additionally subjected
to cytotoxicity (MTT test) using a popular in vitro model of human leukaemia HL-60 cells. We
obtained the cytotoxic activities of geranyl acetone and nerolidol that were close to those reported
elsewhere [11], although they possessed lower biological effectiveness. The IC level (interpolated),
50
which means the compound concentration required to suppress cell viability by 50%, was ca. 135 µM
and 295 µM for nerolidol (2) and geranyl acetone (1), respectively. Our studies showed that nerolidol
and synthetic structurally related compounds (12 to 17) were more active inhibitors of cellular
dehydrogenases as compared to compounds based on the geranyl acetone structure (Table 2). The IC
50
values for the alcohols 12 to 17 were in the range of ca. 500 µM to 750 µM, except for 16 (ca. 1000 µM),
while for geranyl acetone and synthetic ketones 6 to 11, they were higher than 1000 µM.
Molecules 2015, 20 11281

Table 2. Concentration-response effects (viability) of the studied compounds on cancer
and regular cells metabolic activity after 24 h.
Cancer Cell Lines Normal Cell Lines
Malignant Colorectal Promyelocytic Epithelium of
Compound Fibroblasts Keratinocytes
Melanoma Adenocarcinoma Leukaemia the Small
HFIG HaCaT
MEWO HT29 HL-60 Intestine IEC6
Geranyl acetone (1) 93% ± 4% 98% ± 8% 295 82% ± 7% 89% ± 3% 79% ± 6%
6 102% ± 5% 102% ± 6% 67% ± 5% 83% ± 2% 118% ± 5% 95% ± 7%
7 99% ± 2% 98% ± 2% 67% ± 5% 92% ± 8% 101% ± 7% 82% ± 3%
8 94% ± 5% 111% ± 3% 72% ± 5% 81% ± 9% 90% ± 2% 97% ± 5%
9 83% ± 9% 107% ± 8% 69% ± 0% 85% ± 6% 89% ± 4% 76% ± 2%
10 93% ± 5% 103% ± 9% 62% ± 8% 98% ± 7% 112% ± 8% 78% ± 4%
11 112% ± 4% 92% ± 1% 59% ± 7% 102% ± 3% 100% ± 5% 81% ± 5%
Nerolidol (2) 99% ± 3% 103% ± 8% 135 75% ± 6% 102 ± 5% 76% ± 9%
12 97% ± 5% 97% ± 3 500 110% ± 7% 101% ± 6% 73% ± 8%
13 83% ± 5% 99% ± 6% 400 87% ± 7% 87% ± 6% 96% ± 5%
14 99% ± 6% 112% ± 5% 750 99% ± 4% 106% ± 5% 65% ± 8%
15 104% ± 6% 104% ± 7% 750 96% ± 5% 90% ±7% 110% ± 3%
16 100% ± 7% 102% ± 3% 950 106% ± 5% 89% ± 4% 113% ± 2%
17 112% ± 0% 108% ± 4% 750 87% ± 5% 95% ± 0% 98% ± 5%
Farnesal (3) 467 110% ± 9% 363 650 702 85% ± 2%
18 630 98% ± 9% 502 690 800 79% ± 6%
19 600 96% ± 2% 550 750 850 94% ± 4%
20 690 82% ± 6% 480 610 850 64% ± 7%
21 640 98% ± 6% 600 830 830 91% ± 3%
22 700 93% ± 3% 600 740 880 76% ± 3%
23 730 87% ± 7% 630 800 900 82% ± 5%
Farnesol (4) 378 930 54 350 489 58% ± 8%
24 512 78% ± 6% 100 390 686 85% ± 5%
25 500 108% ± 11% 167 454 667 82% ± 2%
26 495 96% ± 5% 145 531 620 76% ± 3%
27 456 106% ± 2% 143 489 687 93% ± 5%
28 512 93% ± 8% 198 687 698 101% ± 4%
29 587 82% ± 2% 252 756 946 103% ± 2%
Farnesyl acetate (5) 734 105% ± 3% 121 98% ± 7% 102% ± 3% 78% ± 7%
30 850 96% ± 4% 289 105% ± 3% 900 86% ± 6%
31 930 107% ± 8% 350 850 98% ± 7% 94% ± 5%
32 800 103% ± 7% 278 980 950 92% ± 4%
33 103% ± 5% 110% ± 9% 400 100% ± 6% 101% ± 6% 95% ± 5%
34 98% ± 6% 98% ± 3% 312 99% ± 3% 110% ± 5% 79% ± 3%
35 112% ± 5% 83% ± 5% 343 87% ± 8% 102% ± 4% 71% ± 4%
The half maximal inhibitory concentrations (IC ) after 24 h, interpolated values (bolded). Range of the
50
concentrations 10–1000 µM. If the IC exceeded 1000 µM viability [%] ± standard deviation at 1000 µM is given.
50
The very high cytotoxic activity of farnesol, and thus its potential use as an anticancer agent, was
confirmed previously in studies performed on non-small cell lung cancer cells (H460 and A549),