New derivatives of dehydroabietic acid target planktonic and biofilm bacteria in S taphylococcus aureus and effectively disrupt bacterial membrane integrity

Article abstract of DOI:10.1016/j.ejmech.2015.07.038

The combination of the dehydroabietic acid scaffold with different amino acids resulted in the discovery of a new class of hybrid compounds that targets both planktonic and biofilms bacteria in Staphylococcusaureus strains and are far more potent anti-bio

Full text of DOI:10.1016/j.ejmech.2015.07.038

European Journal of Medicinal Chemistry 102 (2015) 68e79
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
New derivatives of dehydroabietic acid target planktonic and biofilm
bacteria in Staphylococcus aureus and effectively disrupt bacterial
membrane integrity
,
,
Suvi Manner a 1, Mikko Vahermo b 1, Malena E. Skogman c, Sara Krogerus b,
Pia M. Vuorela c, Jari Yli-Kauhaluoma b, Adyary Fallarero c , Vania M. Moreira
,
**
b, *
^
a Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Abo Akademi University, BioCity, Artillerigatan 6 A, FI-20520 Turku, Finland
b Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, FI-00014 Helsinki, Finland
c Pharmaceutical Biology, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, FI-00014 Helsinki, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
The combination of the dehydroabietic acid scaffold with different amino acids resulted in the discovery
of a new class of hybrid compounds that targets both planktonic and biofilms bacteria in Staphylococcus
aureus strains and are far more potent anti-biofilm agents than conventional antibiotics. Unlike dehy-
droabietic acid, these compounds can disrupt biofilms within a short time period and compromise the
integrity of the bacterial membrane. Two of the compounds identified in our study are the most potent
abietane-type anti-biofilm agents reported so far and display robust activity against pre-formed biofilms
at concentrations only 3e6-fold higher than those required to inhibit biofilm formation. Their easy
preparation based on proteolysis-resistant D- and unusual amino acids makes them useful chemical
probes to gain a deeper understanding of bacterial biofilms and outstanding candidates for further
development into new drugs to fight infections.
Received 5 May 2015
Received in revised form
13 July 2015
Accepted 20 July 2015
Available online 21 July 2015
Keywords:
Natural products
Diterpenoids
Amino acids
Dehydroabietic acid
Biofilms
© 2015 Elsevier Masson SAS. All rights reserved.
Antimicrobial
1. Introduction
chemotherapy are biofilms-related [4]. A challenging problem is
caused by biofilm involvement in nosocomial infections which will
Bacterial biofilms are multicellular structured communities of
bacteria embedded in a self-produced extracellular matrix [1,2].
This collective lifestyle typically occurs when bacteria attach to
surfaces, and allows them to be highly resilient to different envi-
ronmental and chemical stresses, when compared to single-cells
[3]. The burden of biofilms in human health derives from the fact
that they are highly tolerant to immune responses as well as to
conventional antibiotics. Indeed, in comparison to the non-biofilm
(planktonic) lifestyle, biofilms have been proven to withstand 10 to
1000-fold higher concentrations of antibiotics, which often exceed
their highest deliverable clinical concentrations. It has been esti-
mated that over 65% of all bacterial infections tolerant to
continue rising due to the increasing use of indwelling medical
devices [4].
Staphylococcus aureus is a Gram-positive bacterium and a
common cause of skin and respiratory tract infections, medical
devices-associated infections, and food contamination [5]. A great
deal of literature has been dedicated to methicillin-resistant S.
aureus (MRSA) strains and the threat they pose, but an even more
alarming fact is that also methicillin-susceptible S. aureus strains
can switch to the biofilm state and become remarkably tolerant to
antibiotics [6]. Therefore, biofilm formation by S. aureus poses a
major challenge in the treatment of infections caused by this
microorganism. This scenario calls for the development of new
compounds with broader antimicrobial effects, against both single-
cells and multicellular bacterial communities. The screening for
promising biofilm targets, such as signaling pathways, and the use
of high-throughput screening have significantly contributed to-
wards identifying relevant chemical scaffolds for the development
of anti-biofilm agents [7e9]. However, there are currently no
approved drugs targeting bacterial biofilms and only a limited
* Corresponding author.
** Corresponding author.
E-mail addresses: adyary.fallarero@helsinki.fi (A. Fallarero), vania.moreira@
helsinki.fi (V.M. Moreira).
1
These two authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.ejmech.2015.07.038
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
69
number of compounds can selectively act on biofilms and eradicate
(Scheme 1).
them at low concentrations, especially in the case of S. aureus
[10e13].
Derivatives with free carboxyl groups 4a-e were prepared from
the corresponding esters by alkaline hydrolysis. Oxidation of the
ring B position 7 of the glycine derivative 3a with chromium(VI)
trioxide gave compound 5. Compounds 6 and 7, bearing an
extended side chain, were also prepared from 1 (Scheme 1).
Screening for anti-biofilm activity of these compounds was done
using a phenotypic assay that measures cellular viability of S.
aureus biofilms with a redox dye [21]. The effects of the compounds
prior to and after biofilm formation were measured (pre- and post-
exposure assays, respectively) against two different S. aureus
strains (ATCC 25923 and Newman; Supplementary Tables 1 and 2)
to exclude strain-specific effects. Compound 1 was used as a
reference, together with penicillin G and vancomycin. We tested
Abietanes are a family of naturally occurring diterpenoids with
interesting biological activities [14]. We have recently shown that
dehydroabietic acid 1, an abietane-type diterpenoid abundant in
the resin of coniferous trees, prevents S. aureus biofilm formation in
the low micromolar range, and only 2e4-fold higher concentra-
tions of 1 are needed to significantly reduce the viability and
biomass of existing S. aureus biofilms [15]. In pursuit of more potent
anti-biofilm agents, our attention was drawn by reports on the
ability of D-amino acids to trigger biofilm disassembly in some
bacterial species [16e18]. Eradication of wild-type and multi-
resistant Pseudomonas aeruginosa biofilms has also been reported
recently with
D
-enantiomeric peptides [19]. Thus, we decided to
the compounds first at a high concentration of 400
active ones were re-tested at 100 M. Compounds displaying more
than 50% inhibitory activity against biofilms at 100 M in both pre-
and post-exposure modes were studied further. Their antimicrobial
activities (minimum inhibitory concentration, MIC) and anti-
biofilm potencies (IC50) are depicted in Table 1.
mM, and the
combine 1 with several amino acids and investigate the antimi-
crobial and anti-biofilm properties of the resulting hybrid com-
pounds [20].
m
m
2. Results and discussion
From our initial set of compounds we first identified the single
amino acid-bearing 4d and 4e as good anti-biofilm agents against S.
aureus ATCC 25923, with potencies comparable to that of 1. Like 1,
these compounds not only prevent biofilm formation (in the pre-
exposure assay) but also significantly reduce the viability of exist-
ing biofilms (in the post-exposure assay). As expected, the activity
of penicillin G and vancomycin on pre-formed biofilms was modest
2.1. Design and synthesis
Our design strategy focused on generating a library of dehy-
droabietic acid 1 derivatives by chemical modification of rings A
and B of its diterpenoid core. The synthesis was supported by
bioactivity testing and the best compounds were selected for
further mechanistic studies. A first set of compounds was synthe-
sized from 1 and ammonia 2, or the methyl ester hydrochlorides of
even at the highest concentration tested (400 mM). We found that
the free carboxyl group is important for the anti-biofilm activity of
our compounds as the methyl esters in the set 3a-e were inactive
(Supplementary Tables 1 and 2). In the pre-exposure assay,
glycine 3a and the -amino acids homoalanine 3b, valine 3c, leucine
L
3d, and phenylalanine 3e, using carbodiimide-mediated couplings
b
O
H
O
H
N
N
N
OCH2CH3
N
OH
O
O
H
H
O
O
6
7
a
C
a, c
a
b
A
B
O
NH
NH
OH
NH
O
O
O
O
O
O
O
1
R
R
OCH3
OCH3
OH
5
4a-e
3a-e
d
-H
R=
a
b
c
d
e
NH2
O
2
Scheme 1. Synthesis of compounds 2, 3aee, 4aee, and 5e7. a) EDC, HOBt, DIPEA, amino acid/dipeptide alkyl ester hydrochloride, DMF, r.t. b) NaOH (4 M, aq.), THF:MeOH, 0 ꢀC to r.t.
c) CrO3, EtOAc, HOAc, 50 ꢀC d) EDC, HOBt, NH3 (25% aq.), DMF, r.t. DIPEA ¼ N,N-diisopropylethylamine; DMF ¼ N,N-dimethylformamide; EDC ¼ 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; HOBt ¼ N-hydroxybenzotriazole; MeOH ¼ methanol; THF ¼ tetrahydrofuran.

70
S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
Table 1
Antimicrobial activity and anti-biofilm potencies of the seven most active compounds.
Cmpd
S. aureus ATCC 25923
S. aureus Newman
Biofilms (IC50
,
m
M)
POST
Planktonic bacteria
Biofilms (IC50
,
m
M)
Planktonic bacteria
PRE
Foldb
MIC (
m
M)
PRE
POST
Foldb
MIC (mM)
1
4d
4e
9a
9b
11
13
Pen G
Van
27.8
62.2
35.5
37.4
33.2
9.4
60.6
0.13
0.71
112.8
121.3
108.7
98.2
86.1
27.9
145.3
57%a
25%a
4
2
3
3
3
3
2
e
e
70
80
50
70
60
15
100
0.12
2.7
31.6
51.8
35.9
36.2
20.9
7.9
63.5
0.27
1.3
72.4
134.5
83.7
110.3
71.7
48.2
93.1
73%a
37.9
2
3
2
3
3
70
90
40
80
60
20
100
0.25
2.5
6
1
e
29
a
Inhibitory activity determined at 400 mM.
Fold ¼ IC50 (POST)/IC50 (PRE).
b
inhibition of biofilm formation can be at least partially attributed to
killing of planktonic cells. Both compounds 4d and 4e indeed have
antimicrobial activity against single-cells on both staphylococcal
strains, as revealed by their MIC values.
before (12a, 13) and producing an oxime derivative (12b). As with
most of the compounds in our previous sets, those bearing the free
carboxyl groups were the most active anti-biofilm agents (11 and
13).
We next prepared compound 9a (Scheme 2) as the
counterpart of compound 4e. We also used the unusual
acid 3-pyridylalanine to study the effect of introducing a nitrogen
atom into the aromatic ring (8e and 9e), and further oxidized it to
D
-amino acid
Compound 11 was identified as the most potent anti-biofilm and
antimicrobial derivative in comparison to the parent compound 1. It
potently prevented biofilm formation in both staphylococcal strains
and only 3e6-fold higher concentrations of compound were
needed to reduce the viability and biomass of pre-formed biofilms,
indicating that biofilms are less tolerant to 11 than to the control
antibiotics which cannot achieve such effect even at impractical
millimolar concentrations. Compound 11 is also the best antimi-
crobial agent in our study, with significantly low MIC values
D
-amino
produce the N-oxide 8f. In addition, other D-amino acids namely
tryptophan, tyrosine, and methionine were used as building blocks
in our synthesis (8b-d) as these have also been reported to affect
bacterial biofilms [16,17].
As before, the methyl esters were hydrolyzed to yield com-
pounds 9b-d. The presence of a 3-pyridyl side chain in compound
8e and the oxidation to the corresponding N-oxide 8f did not
contribute towards an overall improvement of the anti-biofilm
activity of the compounds. However, we found that like 4e, the
D-phenyl derivative 9a is a good anti-biofilm and antimicrobial
(<10 mg/mL, Supplementary Table 3) [22] for both the staphylo-
coccal strains tested. The improved antimicrobial and anti-biofilm
potencies of this compound when compared to those of the de-
rivatives 4d, 4e and 9a indicate that the size and the aromaticity of
the side chain are important factors governing the observed ac-
tivity. Although a few other abietane-type diterpenoids namely,
salvipisone and 4-epi-pimaric acid, have been reported to prevent
biofilm formation on staphylococcal strains, their activities are
modest and characterized in much less detail [23,24]. Our design
strategy starting from compound 1 gave compounds 9b and 11, the
most potent abietane-type anti-biofilm agents reported so far [20].
These two compounds and also compounds 3b, 4b, 4d, 5e7, 8b,
8def, 9aec, 10, 11, 12aeb, and 13 are new.
agent, with similar potency to 1 (Table 1). We also identified the D-
tryptophan derivative 9b as a potent anti-biofilm and antimicrobial
agent with a modest activity improvement, particularly in pre-
formed biofilms of S. aureus ATCC 25923, when compared to 1
(Table 1).
Next we investigated the importance of the aromaticity of the
amino acid side chain of compound 4e for its activity by using the
unusual amino acid cyclohexyl-L-alanine for the synthesis of com-
pound 11 (Scheme 3). A set of derivatives of this compound was
also prepared by introducing a carbonyl group at position 7 as
The most active compounds identified from our sets were
followed-up in selectivity studies, by determining their inhibitory
Scheme 2. Synthesis of compounds 8aef and 9aee. a) EDC, HOBt, DIPEA, amino acid alkyl ester hydrochloride, DMF, r.t. b) NaOH (4 M, aq.), THF:MeOH, 0 ꢀC to r.t. c) m-CPBA, CHCl3,
0
ꢀC to r.t. m-CPBA ¼ m-chloroperoxybenzoic acid.

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
71
Scheme 3. Synthesis of cyclohexyl-L-alanine derivatives 10, 11, 12aeb, and 13. a) EDC, HOBt, DIPEA, cyclohexyl-L-alanine$HCl, DMF, r.t. b) NaOH (4 M, aq.), THF:MeOH, 0 ꢀC to r.t. c)
CrO3, EtOAc, HOAc, 50 ꢀC d) H2NOH$HCl, pyridine, EtOH, 100 ꢀC.
activities against three other biofilm-forming strains. For this study,
we tested the compounds at a concentration corresponding to their
IC50 values, earlier determined on S. aureus ATCC 25923 biofilms
(Table 2, pre-exposure assay). Like compound 1, the selected
compounds were overall inactive against the Gram-negative
Escherichia coli showing less than 15% inhibition at the concentra-
tion tested. For the other staphylococcal strains tested, the inhibi-
tion values were comparable to those determined on the S. aureus
ATCC 25923 biofilms, indicating that all compounds act on a
broader representation of Gram-positive bacterial biofilms.
acting, reaching nearly 70% inhibition (Fig. 1A) within the first
hour. As the action of the compounds was found to involve rapid
events, we reasoned that one plausible target would be the bac-
terial membrane or the peptidoglycan (PG) layer.
Thus, we next measured the changes occurring in the bacterial
membrane potential after 1 h of treatment with 1, 9b and 11.
Whereas 1 did not alter the membrane potential of pre-formed S.
aureus biofilms, a statistically significantly higher fluorescence
emission of the potential sensitive probe DiBAC4(3) was detected in
9b and 11-treated cells, when compared to control (untreated)
cells, clearly correlating with a dissipation of the membrane po-
tential (Fig. 1B). This prompted us to further investigate the
occurrence of ATP leakage. Using a luciferase-based assay, efflux of
ATP from 9b and 11-treated biofilms to the extracellular medium
was shown to be significantly higher than in untreated biofilms
(Fig. 1C), confirming that both compounds are able to destabilize
the biofilm integrity leading to a lethal metabolic collapse. It is
known that ATP efflux correlates with rapid changes in cellular
viability, and typically it occurs as result of the formation of large
and stable membrane pores (>1.5 nm in diameter) [25]. We found
that unlike the parent dehydrobietic acid 1, the amino acid-
containing derivatives 9b and 11 were shown to evoke a fast and
very effective destabilization of the membrane integrity [25].
Furthermore, membrane perturbation was directly followed by
2.2. Studies of the mechanistic action
To shed light into the possible mechanism of actions of our
compounds, we first followed the changes in cellular viability after
the exposure of pre-formed S. aureus ATCC 25923 biofilms to
compounds 1, 9b and 11, for a short period of time (1 h). The
viability of the biofilms remained unaltered upon exposure to 1, in
agreement with our previous findings that its anti-biofilm effects
are preferentially elicited upon binding to intracellular targets and
require longer response times [15]. However, in sharp contrast, a
very rapid and sudden drop in the viable biofilm core (more than
50% of inhibition of viable biofilms) was induced by both com-
pounds 9b and 11, with 9b being particularly effective and fast-
Table 2
Anti-biofilm activity of the seven most active compounds against three other biofilm forming strains.
Cmpda
% Inhibition
E. coli XL1 Blue
S. epidermidis ATCC 12228
S. epidermidis ATCC 35984
1
4d
4e
9a
9b
11
13
Pen G
Van
7.4 ( 3.3)
14.2 ( 4.5)
7.4 ( 2.7)
N.A.
8.7 ( 5.6)
5.2 ( 11.3)
N.A.
49.4 ( 1.0)
62.4 ( 2.2)
57.3 ( 4.6)
92.3 ( 1.3)
70.3 ( 5.6)
42.3 ( 9.5)
66.6 ( 12.6)
28.0 ( 2.3)
N.A.b
23.2 ( 8.0)
53.4 ( 4.8)
58.1 ( 4.1)
55.2 ( 1.5)
66.9 ( 0.9)
48.8 ( 4.8)
52.9 ( 6.5)
15.5 ( 15.0)
N.A.b
9.6 ( 6.2)
3.2 ( 4.1)
a
IC50 value determined on S. aureus ATCC 25923.
N.A. ¼ Not active.
b

72
S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
acids in the stationary phase of growth. In the best study species
(Bacillus subtilis), these amino acids have been thought to play
regulatory functions by modulating the synthesis of the PG layer
(acting as substrates of the PG synthesis and causing structural
changes on it) as well as by regulating periplasmic enzymes, such as
penicillin-binding proteins (PBP) [26]. It has been demonstrated
that treatment of S. aureus biofilms with
D-amino acids, among
them -tryptophan, causes disorganization of several proteins that
D
surround the cell wall, although the exact proteins involved have
not yet been identified [17]. It seems thus plausible to hypothesize
that a short-period exposure to the compounds may trigger a
similar disruption of the PG layer, ultimately compromising the
membrane integrity and overall architectural stability of the S.
aureus biofilms, as we have demonstrated here. A common concern
related to antibacterial and anti-biofilm compounds is unspecific
cytotoxicity. We tested the activity of 9b against HL cells (origi-
nating from the human respiratory tract) and found that it causes
unspecific cytotoxicity as only 23% ( 5.9) of the cells remained
viable upon exposure to 100 mM of this compound (Supplementary
Table 4). However, no such effect was observed for compound 11 as
no statistically significant reduction in the viability of HL cells was
observed at any of the tested concentrations (up to 100 mM).
3. Conclusion
This study highlights the value of abietane-type diterpenoids
such as 1 as starting materials for the development of new anti-
microbial and anti-biofilm agents useful against staphylococcal
infections. New, potent compounds were prepared using not only
the naturally occurring L-amino acids as side chains but also D- and
unusual amino acids, which have the competitive advantage of
resistance to enzymatic proteolysis. The most active compounds
identified in our study, 9b and 11, are the most potent abietane-
type anti-biofilm agents reported so far and display robust activ-
ity against pre-formed biofilms at concentrations only 3e6-fold
higher than those required to inhibit biofilm formation. Thus, our
design strategy rendered faster-acting anti-biofilm agents, in
comparison to the parent compound 1, and more importantly, to-
wards which biofilms are less tolerant than to standard antibiotics.
Our compounds seem to act preferentially on Gram-positive spe-
cies and effectively disrupt bacterial biofilms with a mode of action
that resembles that of many antimicrobial peptides (AMPs). How-
ever, while practical applications of many AMPs are hampered by
their large sizes, susceptibility to enzymatic degradation, and high
production costs, our compounds overcome these limitations, as
they can easily be prepared in high yield and purity, in few reaction
steps. In addition, their fast-acting kinetics can be exploited for the
preparation of more active biocides, cleaning agents or other
microbicidal products requiring short contact times with patho-
genic microoorganisms. Taken together, our findings provide a very
promising role for the abietane-type diterpenoids in the search of
new antimicrobial and anti-biofilm agents against Gram-positive
bacteria. We are currently working towards the synthesis of addi-
tional amino acid derivatives of 1 to explore the full potential of our
hybrid design strategy against several other relevant biofilm-
forming bacterial strains.
Fig. 1. Changes occurring in biofilms exposed to 1, 9b and 11 (100 mM) during 1 h
*p < 0.05; ***p < 0.001 indicate statistically significant differences when compared to
untreated biofilms (unpaired t-test with Welch's correction).
fluorescence imaging using Syto9 and propidium iodide (PI)
staining (Fig. 2). After 1 h of treatment with 9b and 11, the green
fluorescence of Syto9 was quenched by the red fluorescence of PI (B
and C images), in comparison to intact biofilms (A). Since PI can
only enter cells with damaged membranes, this confirms that
biofilms exposure to 9b or 11 indeed results in a rapid loss of
membrane integrity. The fact that the membrane-targeting effect is
observed in 9b and 11, but it is absent in the parent compound 1,
lacking the amino acid motif, is a noticeable mechanistic aspect.
4. Experimental procedures
4.1. Chemistry
All reagents were obtained from Sigma Aldrich Co or TCI Europe.
Dehydroabietic acid (1, 90% purity) was obtained from Pfaltz &
Bauer, USA. (e)-2-Aminobutyric acid methyl ester hydrochloride,
H-Gly-Gly-OEt$HCl, and H-Ala-Ala-Ala-OMe acetate salt were
Various bacterial species, among them S. aureus, produce D-amino

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
73
Fig. 2. Membrane permeability studies in biofilms exposed to the following treatments for 1 h. A: untreated biofilms; B: biofilms þ9b (100
m
M); C: biofilms þ11 (100
mM); D:
biofilms þ PenG (400
mM); E: biofilms þ Van (400
mM). Scale bars correspond to 100 mm.
obtained from Bachem (Weil am Rhein, Germany).
alanine methyl ester hydrochloride was obtained from Nova-
b
-Cyclohexyl-
L
-
after which the reaction was completed. The reaction mixture was
suspended by addition of diethyl ether (200 mL) and water (40 mL).
The aqueous phase was further extracted with diethyl ether
(2 ꢂ 100 mL). The resulting organic phase was washed with a 1 M
aqueous solution of HCl (50 mL), a saturated solution of NaHCO3
(50 mL), water (50 mL), and brine (50 mL), dried with Na2SO4,
filtered, and evaporated to dryness to give 3a as a white solid (1.2 g,
€
biochem (Laufelfingen, Switzerland). For thin layer chromatog-
raphy (TLC) analysis, Kieselgel 60HF254/Kieselgel 60G was used
(Merck). Flash column chromatography (FCC) was made with a
Biotage High-Performance Flash Chromatography Sp4-system
(Uppsala, Sweden) using a 0.1-mm pathlength flow cell UV-
detector/recorder module (fixed wavelength: 254 nm), and 12-
mm or 25-mm flash cartridges. Melting points were recorded
with an Electrothermal capillary tube melting point apparatus and
are uncorrected. IR spectra were obtained using a Vertex 70 (Bruker
Optics Inc., MA, USA) FTIR instrument. The FTIR measurements
were made directly in solids with a horizontal attenuated total
reflectance (ATR) accessory (MIRacle, Pike Technology, Inc, WI,
USA). The transmittance spectra were recorded at a 4 cmꢁ1 reso-
lution between 4000 and 600 cmꢁ1 using the OPUS 5.5 software
(Bruker Optics Inc., MA, USA). NMR spectra were obtained using a
Varian Mercury Plus 300 spectrometer, in CDCl3 or DMSO-d6, with
tetramethylsilane (TMS) as the internal standard. The chemical
97%). 1H NMR (300 MHz, CDCl3)
d 1.21 (s, 3H), 1.23 (s, 6H), 1.32 (s,
3H), 2.14 (dd, J ¼ 12.4, 2.1 Hz, 1H), 2.31 (d, J ¼ 12.9 Hz, 1H), 2.86 (m,
3H), 3.76 (s, 3H), 4.04 (d, J ¼ 5.1 Hz, 2H), 6.32 (brs, 1H), 6.86 (s, 1H),
6.98 (d, J ¼ 8.1 Hz, 1H), 7.16 (d, J ¼ 8.1 Hz, 1H). 13C NMR (75 MHz,
CDCl3)
d 16.6, 18.8, 21.2, 24.1, 24.1, 25.4, 30.1, 33.6, 37.2, 37.4, 38.1,
41.7, 45.7, 47.5, 52.4, 124.0, 124.2, 127.0, 134.8, 145.8, 147.0, 170.9,
178.9. IR (ATR) 3376, 1758, 1640, 1519, 1205, 1179, 820 cmꢁ1. HRMS
m/z: calcd. for C23H34NO3 372.2539 [MþH]þ, found 372.2538.
4.1.1.3. Methyl N-(abiet-8,11,13-trien-18-oyl)
L
-ethylglycinate (3b).
Following the procedure for compound 3a, compound 3b was
prepared from 1 (250 mg, 0.83 mmol), EDC hydrochloride (239 mg,
1.25 mmol), HOBt monohydrate (169 mg, 1.25 mmol), (e)-2-
aminobutyric acid methyl ester hydrochloride (276 mg,
1.79 mmol), and DIPEA (0.44 mL, 2.5 mmol) in DMF (2.5 mL).
Compound 3b: white solid (307 mg, 92%). 1H NMR (300 MHz,
shifts were reported in parts per million (ppm) and on the
d scale
from TMS as an internal standard. The coupling constants J are
quoted in Hertz (Hz). ESI-MS was performed by direct injection
using a Synapt G2 HDMS (Waters, MA, USA) instrument.
CDCl3)
d
0.92 (t, J ¼ 7.5 Hz, 3H), 1.21 (s, 3H), 1.23 (s, 3H), 1.24 (s, 3H),
4.1.1. Compound synthesis
1.31 (s, 3H), 2.11 (dd, J ¼ 12.4, 1.8 Hz, 1H), 2.32 (d, J ¼ 12.9 Hz, 1H),
2.86 (m, 3H), 3.74 (s, 3H), 4.58 (m, 1H), 6.25 (d, J ¼ 7.5 Hz, 1H), 6.88
(s, 1H), 7.00 (d, J ¼ 8.1 Hz, 1H), 7.17 (d, J ¼ 8.1 Hz, 1H). 13C NMR
4.1.1.1. Dehydroabietyl amide (2). Compound 1 (0.500 g, 1.66 mmol)
was dissolved in DMF (5 mL) at room temperature. EDC hydro-
chloride (351 mg, 1.83 mmol) and HOBt monohydrate (247 mg,
1.83 mmol) were added and the mixture was stirred for 1 h before
the dropwise addition of a 25% aqueous ammonia solution (1.2 mL).
After 48 h the reaction was completed. The reaction mixture was
suspended by addition of diethyl ether (100 mL) and water (20 mL).
The aqueous phase was further extracted with diethyl ether
(2 ꢂ 100 mL). The resulting organic phase was washed with a 1 M
aqueous solution of HCl (2 ꢂ 50 mL), and water (50 mL), dried with
Na2SO4, filtered, and evaporated to dryness to give a white solid
(440 mg) which was purified by FCC using ethyl acetate:n-hexane
(2:1) to give 2 as a white solid (357 mg, 72%). 1H NMR (300 MHz,
(75 MHz, CDCl3) d 9.8,16.6,18.9, 21.3, 24.1, 24.1, 25.4, 25.7, 30.2, 33.6,
37.3, 37.6, 38.2, 45.9, 47.5, 52.4, 53.5, 124.0, 124.2, 127.1, 134.8, 145.9,
147.1, 173.4, 178.2. IR (ATR) 3361, 1743, 1637, 1517, 1207, 821 cmꢁ1
.
HRMS m/z: calcd. for
C
25H38NO3 400.2852 [MþH]þ, found
400.2853.
4.1.1.4. Methyl
N-(abiet-8,11,13-trien-18-oyl)
L
-valinate
(3c).
Following the procedure for compound 3a, compound 3c was
prepared from 1 (250 mg, 0.83 mmol), EDC hydrochloride (239 mg,
1.25 mmol), HOBt monohydrate (169 mg, 1.25 mmol), valine methyl
ester hydrochloride (209 mg, 1.25 mmol), and DIPEA (0.44 mL,
2.5 mmol) in DMF (2.5 mL). Compound 3c: white solid (309 mg,
CDCl3)
d 1.21 (s, 3H), 1.23 (s, 6H), 1.29 (s, 3H), 2.11 (m, 1H), 2.32 (m,
1H), 2.85 (m, 3H), 5.73 (brs, 2H), 6.87 (s, 1H), 6.99 (d, J ¼ 8.2 Hz, 1H),
90%). 1H NMR (300 MHz, CDCl3)
d
0.93 (d, J ¼ 7.0 Hz, 3H), 0.95 (d,
7.16 (d, J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d
16.8, 18.9, 21.3,
J ¼ 6.4 Hz, 3H), 1.21 (s, 3H), 1.23 (s, 3H), 1.24 (s, 3H), 1.31 (s, 3H), 2.11
(dd, J ¼ 12.4, 2.0 Hz, 1H), 2.18 (m, 1H), 2.33 (d, J ¼ 13 Hz, 1H), 2.88
(m, 3H), 3.74 (s, 3H), 4.58 (dd, J ¼ 8.4, 4.8 Hz, 1H), 6.23 (d, J ¼ 8.4 Hz,
1H), 6.89 (s, 1H), 7.00 (dd, J ¼ 8.1, 1.8 Hz, 1H), 7.17 (d, J ¼ 8.1 Hz, 1H).
24.1, 24.1, 25.3, 30.1, 33.6, 37.2, 37.5, 38.2, 45.7, 47.5, 124.0, 124.2,
127.0, 134.7, 145.9, 147.1, 181.4. IR (ATR) 3433, 3362, 1628, 1606, 823,
632 cmꢁ1. HRMS m/z: calcd. for C20H30NO 300.2327 [MþH]þ, found
300.2329.
13C NMR (75 MHz, CDCl3)
d 16.6, 18.1, 18.9, 19.2, 21.4, 24.1, 24.1, 25.5,
30.2, 31.4, 33.6, 37.4, 37.8, 38.2, 45.9, 47.7, 52.2, 57.3, 124.0, 124.3,
127.1, 134.9, 145.9, 147.0, 173.0, 178.4. IR (ATR) 3454, 1737, 1658,
1498, 1305, 821 cmꢁ1. HRMS m/z: calcd. for C26H40NO3 414.3008
[MþH]þ, found 414.3009.
4.1.1.2. Methyl
N-(abiet-8,11,13-trien-18-oyl)
glycinate
(3a).
Compound 1 (1.00 g, 3.33 mmol) was dissolved in DMF (10 mL), at
room temperature. EDC hydrochloride (956 mg, 4.99 mmol) and
HOBt monohydrate (676 mg, 5.00 mmol) were added and the
mixture was left to agitate for 1 h. Glycine methyl ester hydro-
chloride (628 mg, 5.00 mmol) and DIPEA (1.76 mL, 10.1 mmol) were
then added and the mixture was left to agitate for another hour
4.1.1.5. Methyl N-(abiet-8,11,13-trien-18-oyl)
L
-leucinate (3d).
Following the procedure for compound 3a, compound 3d was
prepared from 1 (250 mg, 0.83 mmol), EDC hydrochloride (239 mg,

74
S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
1.25 mmol), HOBt monohydrate (169 mg, 1.25 mmol), leucine
methyl ester hydrochloride (226 mg, 1.24 mmol), and DIPEA
(0.44 mL, 2.5 mmol) in DMF (2.5 mL). Compound 3d: white solid
4.1.1.9. N-(Abiet-8,11,13-trien-18-oyl)
L
-valine (4c). Following the
procedure for compound 4a, compound 4c was prepared from 3c
(118 mg, 0.29 mmol), using THF:MeOH 1:1 (4 mL), and 4 M NaOH
(2.4 mL). Compound 4c: white solid (108 mg, 94%). 1H NMR
(305 mg, 86%). 1H NMR (300 MHz, CDCl3)
d
0.95 (d, J ¼ 5 Hz, 6H),
1.21 (s, 3H), 1.23 (s, 3H), 1.24 (s, 3H), 1.30 (s, 3H), 2.07 (dd, J ¼ 12.4,
2.0 Hz, 1H), 2.33 (d, J ¼ 13.3 Hz, 1H), 2.87 (m, 3H), 3.73 (s, 3H), 4.63
(m,1H), 6.07 (d, J ¼ 8 Hz,1H), 6.89 (s,1H), 7.0 (dd, J ¼ 8.2,1.8 Hz,1H),
(300 MHz, CDCl3)
d
0.96 (d, J ¼ 7.0 Hz, 3H) 0.99 (d, J ¼ 6.4 Hz, 3H),
1.21 (s, 3H), 1.24 (s, 3H), 1.25 (s, 3H), 1.32 (s, 3H), 2.11 (d, J ¼ 12.3 Hz,
1H), 2.28 (m, 2H), 2.85 (m, 3H), 4.58 (m, 1H), 6.27 (d, J ¼ 8.2 Hz, 1H),
6.88 (s, 1H), 7.0 (d, J ¼ 8.2 Hz, 1H), 7.17 (d, J ¼ 8.2 Hz, 1H). 13C NMR
7.17 (d, J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.5, 18.9, 21.3,
22.1, 23.0, 24.1, 24.1, 25.2, 25.4, 30.1, 33.6, 37.3, 37.6, 38.2, 41.7, 46.0,
47.4, 51.0, 52.3, 124.0, 124.2, 127.1, 134.8, 145.9, 147.1, 173.9, 178.3. IR
(ATR) 3346, 1755, 1629, 1527, 1155, 821 cmꢁ1. HRMS m/z: calcd. for
(75 MHz, CDCl3) d 16.6, 18.0, 18.9, 19.3, 21.4, 24.1, 24.1, 25.5, 30.1,
31.0, 33.6, 37.4, 37.7, 38.2, 45.9, 47.8, 57.5, 124.1, 124.3, 127.1, 134.8,
145.9,147.0,176.2,179.2. IR (ATR) 3435, 3076,1724,1637,1525,1406,
1207, 821 cmꢁ1. HRMS m/z: calcd. for C25H38NO3 400.2852 [MþH]þ,
found 400.2852.
C
27H42NO3 428.3165 [MþH]þ, found 428.3169.
4.1.1.6. Methyl N-(abiet-8,11,13-trien-18-oyl) -phenylalaninate (3e).
L
4.1.1.10. N-(Abiet-8,11,13-trien-18-oyl) -leucine (4d). Following the
L
Following the procedure for compound 3a, compound 3e was
prepared from 1 (250 mg, 0.83 mmol), EDC hydrochloride (239 mg,
1.25 mmol), HOBt monohydrate (169 mg, 1.25 mmol), phenylala-
nine methyl ester hydrochloride (269 mg, 1.25 mmol), and DIPEA
(0.44 mL, 2.5 mmol) in DMF (2.5 mL). Compound 3e: white solid
procedure for compound 4a, compound 4d was prepared from 3d
(150 mg, 0.35 mmol), using THF:MeOH 1:1 (3.7 mL), and 4 M NaOH
(3 mL). Compound 4d: white solid (120 mg, 83%). 1H NMR
(300 MHz, CDCl3)
d 0.96 (m, 6H), 1.21 (s, 3H), 1.24 (s, 6H), 1.30 (s,
3H), 2.09 (dd, J ¼ 12.3, 1.7 Hz, 1H), 2.33 (d, J ¼ 12.8 Hz, 1H), 2.85 (m,
3H), 4.60 (m, 1H), 6.12 (d, J ¼ 7.7 Hz, 1H), 6.88 (s, 1H), 7.00 (d,
J ¼ 8.2 Hz, 1H), 7.17 (d, J ¼ 8.2 Hz, 1H), 10.49 (s, 1H). 13C NMR
(372 mg, 97%). 1H NMR (300 MHz, CDCl3)
d 1.19 (s, 3H), 1.20 (s, 6H),
1.22 (s, 3H), 2.01 (d, J ¼ 12.2 Hz, 1H), 2.27 (d, J ¼ 12.8 Hz, 1H), 2.78
(m, 3H), 3.05 (dd, J ¼ 14.1, 7.0 Hz, 1H), 3.17 (dd, J ¼ 13.5, 5.9 Hz, 1H),
3.71 (s, 3H), 4.90 (m, 1H), 6.12 (d, J ¼ 7.6 Hz,1H), 6.84 (s, 1H), 6.97 (d,
J ¼ 8.2 Hz, 1H), 7.12 (m, 3H), 7.25 (m, 3H). 13C NMR (75 MHz, CDCl3)
(75 MHz, CDCl3)
d 16.5, 18.9, 21.3, 21.9, 24.1, 24.1, 25.2, 25.4, 30.0,
33.6, 37.3, 37.5, 38.2, 41.0, 45.9, 47.5, 51.3, 124.1, 124.2, 127.1, 134.8,
146.0, 147.0, 177.3, 179.3. IR (ATR) 3359, 1733, 1627, 1523, 1232,
819 cmꢁ1. HRMS m/z: calcd. for C26H40NO3 414.3008 [MþH]þ,
found 414.3007.
d
16.5, 18.9, 21.1, 24.1, 25.4, 30.1, 33.6, 37.2, 37.4, 38.1, 45.7, 47.4, 52.4,
53.2, 124.0, 124.2, 127.0, 127.2, 128.7, 129.3, 134.8, 136.2, 145.8, 147.0,
172.5, 178. IR (ATR) 3388, 1743, 1639, 1517, 1515, 1215, 821 cmꢁ1
.
HRMS m/z: calcd. for
C
30H40NO3 462.3008 [MþH]þ, found
4.1.1.11. N-(Abiet-8,11,13-trien-18-oyl)
L
-phenylalanine
(4e).
462.3004.
Following the procedure for compound 4a, compound 4e was
prepared from 3e (150 mg, 0.32 mmol), using THF:MeOH 1:1
(3.7 mL), and 4 M NaOH (3 mL). Compound 4e: white solid (135 mg,
4.1.1.7. N-(Abiet-8,11,13-trien-18-oyl) glycine (4a). Compound
(0.200 g, 0.54 mmol) was dissolved in THF:MeOH 1:1 (4.8 mL), at
ꢀC, under magnetic stirring. A 4 M aqueous solution of NaOH
2
93%). 1H NMR (300 MHz, DMSO-d6)
d 1.10 (s, 3H),1.15 (s, 3H), 1.20 (s,
0
3H), 1.22 (s, 3H), 2.08 (d, J ¼ 10.9 Hz, 1H), 2.28 (d, J ¼ 12.3 Hz, 1H),
2.80 (m, 3H), 2.94 (dd, J ¼ 13.5, 10.5 Hz, 1H), 3.08 (dd, J ¼ 13.5,
4.1 Hz, 1H) 4.54 (m, 1H), 6.87 (d, J ¼ 1.2 Hz, 1H), 7.01 (d, J1 ¼ 8.2 Hz,
1H), 7.26 (m, 6H), 7.80 (m, 1H), 12.55 (s, 1H). 13C NMR (75 MHz,
(4.4 mL) was added dropwise and after the addition the mixture
was left to agitate at room temperature for 1 h, after which the
reaction was suspended by careful addition of a 4 M aqueous so-
lution of HCl dropwise until the pH reached 6e7. The mixture was
concentrated and extracted with diethyl ether (3 ꢂ 75 mL) after the
addition of water (25 mL). The resulting organic phase was washed
with a 1 M aqueous solution of HCl (50 mL), water (50 mL), and
brine (50 mL), dried with Na2SO4, filtered, and evaporated to dry-
ness to give 4a as a white solid (183 mg, 95%). 1H NMR (300 MHz,
DMSO-d6)
d 16.2, 18.5, 20.0, 23.9, 24.0, 24.9, 29.2, 32.9, 35.8, 36.2,
36.6, 37.3, 44.4, 46.3, 53.5, 123.4, 123.9, 126.1, 126.3, 127.9, 129.1,
134.6, 138.2, 145.0, 147.1, 173.4, 177.3. IR (ATR) 3442, 1755, 1598,
1537, 1261, 1091, 1020, 798 cmꢁ1. HRMS m/z: calcd. for C29H38NO3
448.2852 [MþH]þ, found 448.2851.
CDCl3)
d
1.20 (s, 3H), 1.23 (s, 6H), 1.32 (s, 3H), 2.13 (dd, J1 ¼ 12.4,
4.1.1.12. Methyl N-(7-oxoabiet-8,11,13-trien-18-oyl) glycinate (5).
Compound 3a (0.20 g, 0.54 mmol) was dissolved in glacial acetic
acid (3.1 mL) and added dropwise to a cooled (0 ꢀC) solution of
chromium (VI) oxide (0.060 g, 0.60 mmol) in glacial acetic acid
(0.8 mL) and ethyl acetate (1.7 mL), over a period of about 10 min.
The reaction mixture was then warmed to 50 ꢀC, under argon. After
4 h more chromium (VI) oxide (0.060 g, 0.60 mmol) was added and
after 1 h the reaction was completed. The reaction mixture was
suspended by cooling in an ice bath and adding ice and dichloro-
methane (150 mL). The aqueous phase was further extracted with
dichloromethane (2 ꢂ 75 mL). The combined organic phases were
washed with water (50 mL), a saturated aqueous solution of
NaHCO3 (50 mL), water (50 mL), and brine (50 mL), dried with
Na2SO4, filtered, and evaporated to dryness. Purification by FCC
using ethyl acetate:n-hexane (2:1) gave 5 as a white solid (116 mg,
2.1 Hz, 1H), 2.31 (d, J ¼ 12.6 Hz, 1H), 2.84 (m, 3H), 4.06 (d, J1 ¼ 5.0,
2.9 Hz, 2H), 6.45 (brs, 1H), 6.87 (s, 1H), 6.99 (d, J ¼ 8.2, 1.8 Hz, 1H),
7.15 (d, J ¼ 8.2 Hz,1H), 8.06 (bs,1H). 13C NMR (75 MHz, CDCl3)
d 16.6,
18.8, 21.2, 24.1, 25.4, 30.0, 33.6, 37.2, 37.3, 38.1, 42.0, 45.7, 47.6, 124.1,
124.2, 127.1, 134.8, 145.9, 146.9, 173.3, 180. IR (ATR) 3380, 1730, 1641,
1522, 1197, 820 cmꢁ1. HRMS m/z: calcd. for C22H32NO3 358.2382
[MþH]þ, found 358.2383.
4.1.1.8. N-(Abiet-8,11,13-trien-18-oyl)
L
-ethylglycine
(4b).
Following the procedure for compound 4a, compound 4b was
prepared from 3b (150 mg, 0.38 mmol), using THF:MeOH 1:1
(3.7 mL), and 4 M NaOH (3 mL). Compound 4b: white solid (133 mg,
92%). 1H NMR (300 MHz, CDCl3)
d
0.97 (t, J ¼ 7.4 Hz, 3H),1.21 (s, 3H),
1.23 (s, 3H), 1.24 (s, 3H), 1.31 (s, 3H), 2.11 (dd, J1 ¼ 12.4, 2 Hz, 1H),
2.33 (d, J ¼ 12.1 Hz, 1H), 2.85 (m, 3H), 4.55 (dd, J ¼ 7.2, 5.5 Hz, 1H),
6.29 (d, J ¼ 7.2 Hz, 1H), 6.88 (s, 1H), 7.00 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.17
56%). 1H NMR (300 MHz, CDCl3)
d
1.22 (d, J ¼ 1 Hz, 3H), 1.24 (d,
J ¼ 1 Hz, 3H), 1.26 (s, 3H), 1.39 (s, 3H), 2.35 (d, J ¼ 11.7 Hz, 1H), 2.58
(m, 3H), 2.91 (m, 1H), 3.76 (s, 3H), 4.01 (m, 2H), 6.33 (brs, 1H), 7.27
(d, J ¼ 8.2 Hz, 1H), 7.39 (dd, J ¼ 8.2 Hz, 1.2 Hz, 1H), 7.83 (d, J ¼ 1.2 Hz,
(d, J ¼ 8.2 Hz, 1H), 10.5 (bs, 1H). 13C NMR (75 MHz, CDCl3)
d 9.9, 16.5,
18.9, 21.3, 24.1, 24.1, 25.2, 25.4, 30.1, 33.6, 37.3, 37.5, 38.1, 45.8, 47.6,
53.9, 124.1, 124.2, 127.1, 134.8, 146.0, 147.0, 176.3, 179.3. IR (ATR)
3435, 1716, 1623, 1529, 1224, 821 cmꢁ1. HRMS m/z: calcd. for
1H). 13C NMR (75 MHz, CDCl3)
d 16.5, 18.4, 23.9, 24.0, 33.7, 36.9, 37.1,
37.4, 37.4, 41.8, 44.2, 46.6, 52.6,123.5,125.2,130.9,132.7,147.0,153.1,
C
24H36NO3 386.2695 [MþH]þ, found 386.2691.
170.6, 177.5, 198.7. IR (ATR) 3392, 1755, 1678, 1645, 1526, 1198 cmꢁ1
.

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
75
HRMS m/z: calcd. for C23H32NO4 386.2331 [MþH]þ, found
738 cmꢁ1. HRMS m/z: calcd. for C32H41N2O3 501.3117 [MþH]þ,
386.2333.
found 501.3115.
4.1.1.13. Ethyl N-(abiet-8,11,13-trien-18-oyl) glycyl-glycinate (6).
Following the procedure for compound 3a, compound 6 was pre-
pared from 1 (0.500 g, 1.66 mmol), EDC hydrochloride (478 mg,
2.49 mmol), HOBt monohydrate (338 mg, 2.49 mmol), H-Gly-Gly-
OEtꢃHCl (472 mg, 2.40 mmol), and DIPEA (0.88 mL, 5.0 mmol) in
DMF (5 mL). Compound 6: white solid (600 mg, 81%). 1H NMR
4.1.1.17. Methyl N-(abiet-8,11,13-trien-18-oyl)
Following the procedure for compound 3a, compound 8c was
prepared from 1 (0.500 g, 1.66 mmol), EDC hydrochloride (478 mg,
2.49 mmol), HOBt monohydrate (338 mg, 2.49 mmol), D-tyrosine
methyl ester hydrochloride (578 mg, 2.49 mmol), and DIPEA
D
-tyrosinate (8c).
(0.88 mL, 5.0 mmol) in DMF (5 mL). Compound 8c: white solid
(300 MHz, CDCl3)
d
1.20 (s, 3H), 1.21 (s, 3H), 1.22 (s, 3H), 1.27 (m,
(623 mg, 79%). 1H NMR (300 MHz, CDCl3)
d 1.19 (s, 3H), 1.20 (s, 3H),
3H), 1.31 (s, 3H), 2.15 (m, 1H), 2.29 (m, 1H), 2.83 (m, 3H), 4.01 (m,
4H), 4.18 (m, 2H), 6.75 (m, 1H), 6.85 (s, 1H), 6.98 (m, 2H), 7.14 (m,
1.21 (s, 3H), 1.22 (s, 3H), 2.07 (dd, J ¼ 12.4, 2 Hz, 1H), 2.28 (d,
J ¼ 12.9 Hz, 1H), 2.79 (m, 3H), 2.96 (m, 1H), 3.09 (dd, J ¼ 13.8, 5.9 Hz,
1H), 3.72 (s, 3H), 4.82 (m, 1H), 6.21 (d, J ¼ 7.6 Hz, 1H), 6.71 (m, 2H),
6.84 (s, 1H), 6.92 (m, 2H), 6.97 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.13 (d,
1H). 13C NMR (75 MHz, CDCl3)
d 14.3, 16.6, 18.8, 21.4, 24.1, 24.1, 25.3,
30.1, 33.6, 37.2, 37.4, 38.1, 41.5, 43.6, 45.5, 47.5, 61.6, 124.0, 124.1,
127.0, 134.7, 145.8, 147.0, 169.6, 169.7, 179.5. IR (ATR) 3335, 1747,
1637, 1520, 1196, 1024. HRMS m/z: calcd. for C26H39N2O4 443.2910
[MþH]þ, found 443.2910.
J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.5, 18.8, 21.1, 24.1, 25.3,
30.0, 33.6, 37.2, 37.2, 38.0, 45.6, 47.5, 52.5, 53.5, 115.8, 124.0, 124.1,
127.0, 127.2, 130.3, 134.7, 145.8, 146.9, 155.7, 172.7, 178.7. IR (ATR)
3354, 1743, 1633, 1514, 1220, 821 cmꢁ1. HRMS m/z: calcd. for
4.1.1.14. N-(Abiet-8,11,13-trien-18-oyl)
glycyl-glycine
(7).
C
30H40NO4 478.2957 [MþH]þ, found 478.2954.
Following the procedure for compound 4a, compound 7 was pre-
pared from 6 (0.600 g, 1.35 mmol), using THF:MeOH 1:1 (12 mL),
and 4 M NaOH (11 mL). Compound 7: white solid (270 mg, 48%). 1H
4.1.1.18. Methyl N-(abiet-8,11,13-trien-18-oyl)
D-methioninate (8d).
Following the procedure for compound 3a, compound 8d was
prepared from 1 (0.500 g, 1.66 mmol), EDC hydrochloride (478 mg,
NMR (300 MHz, CDCl3)
d 1.19 (s, 6H), 1.22 (s, 3H), 1.28 (s, 3H), 2.12
(d, J ¼ 12.4 Hz,1H), 2.27 (m, 1H), 2.81 (m, 3H), 3.99 (m, 4H), 6.85 (m,
2.49 mmol), HOBt monohydrate (338 mg, 2.49 mmol), D-methio-
nine methyl ester hydrochloride (497 mg, 2.49 mmol), and DIPEA
1H), 6.98 (m, 1H), 7.13 (m, 2H), 7.29 (s, 1H), 8.86 (bs, 1H). 13C NMR
(75 MHz, CDCl3)
d
16.5, 18.8, 21.3, 24.1, 25.3, 30.0, 33.6, 37.2, 37.2,
(0.88 mL, 5.0 mmol) in DMF (5 mL). Compound 8d: white solid
38.0, 41.5, 43.5, 45.4, 47.6, 124.0, 124.2, 127.0, 134.7, 145.9, 147.0,
(620 mg, 84%). 1H NMR (300 MHz, CDCl3)
d 1.21 (s, 3H), 1.23 (s, 6H),
170.4, 172.4, 180.4. IR (ATR) 3358, 1733, 1634, 1520, 1209, 820 cmꢁ1
.
1.31 (s, 3H), 2.00 (m, 1H), 2.09 (s, 3H), 2.17 (m, 2H), 2.32 (d,
J ¼ 12.7 Hz,1H), 2.49 (m, 2H), 2.85 (m, 3H), 3.76 (s, 3H), 4.72 (m,1H),
6.49 (d, J ¼ 7.5 Hz, 1H), 6.87 (s, 1H), 6.99 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.16
HRMS m/z: calcd. for
415.2597.
C
24H35N2O4 415.2597 [MþH]þ, found
(d, J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 15.7, 16.6, 18.9, 21.3,
4.1.1.15. Methyl N-(abiet-8,11,13-trien-18-oyl)
(8a). Compound 1 (1.00 g, 3.33 mmol), -phenylalanine methyl
D-phenylalaninate
24.1, 25.4, 30.2, 30.2, 31.7, 33.6, 37.3, 37.5, 38.1, 45.9, 47.5, 51.9, 52.6,
124.1, 124.2, 127.0, 134.7, 145.9, 147.0, 172.9, 178.5. IR (ATR) 3356,
1743, 1523, 1213, 823 cmꢁ1. HRMS m/z: calcd. for C26H40NO3S
446.2729 [MþH]þ, found 446.2727.
D
ester hydrochloride (1.08 g, 5.00 mmol), EDC (0.96 g, 5.0 mmol),
HOBt (0.68 g, 5.0 mmol) were dissolved in dry DMF (11 mL). The
reaction mixture was stirred until all the solids were dissolved.
DIPEA (1.74 mL, 10.0 mmol) was added. After stirring the mixture at
room temperature for 105 min, it was poured into water (100 mL).
The resulting precipitate was filtered and purified by FCC (silica
column, 15 / 25% EtOAc in n-hexane). Compound 8a: white solid
4.1.1.19. Methyl N-(abiet-8,11,13-trien-18-oyl) H-b-(3-pyridyl)-D-ala-
ninate (8e). Compound 1 (540 mg, 1.80 mmol), H-
b
-(3-pyridyl)-D-
Ala-OMe hydrochloride (0.500 g, 1.98 mmol), EDC (380 mg,
1.98 mmol), HOBt (270 mg, 1.98 mmol) were dissolved in dry DMF
(11 mL). DIPEA (1.74 mL, 10.0 mmol) was added. After stirring the
mixture at room temperature for 16 h, it was poured into cold H2O
(80 mL). The precipitated solid was filtered and purified by FCC
(silica column, 50% EtOAc in n-hexane) Compound 8e: white solid
(0.65 g, 42%). 1H NMR (300 MHz, CDCl3)
d 1.19 (m, 9H), 1.22 (m, 3H),
2.04 (d, J ¼ 12.4 Hz, 1H), 2.28 (d, J ¼ 13.5 Hz, 1H), 2.76 (m, 3H), 3.05
(dd, J ¼ 14.1, 6.4 Hz, 1H), 3.17 (dd, J ¼ 14.1, 5.3 Hz, 1H), 3.73 (s, 3H),
4.88 (m, 1H), 6.15 (d, J ¼ 7.6 Hz, 1H), 6.83 (s, 1H), 6.97 (d, J ¼ 8.2 Hz,
1H), 7.10 (m, 3H), 7.25 (m, 3H). 13C NMR (75 MHz, CDCl3)
d
16.5,18.9,
(0.70 g, 84%). 1H NMR (300 MHz, CDCl3)
d 1.20 (s, 6H), 1.23 (s, 3H),
21.2, 24.1, 24.1, 25.4, 30.0, 33.6, 37.2, 37.4, 38.0, 38.1, 45.6, 47.5, 52.4,
53.3, 124.0, 124.2, 127.0, 127.3, 128.7, 129.3, 134.8, 136.2, 145.8, 147.1,
172.6, 178.1. IR (ATR) 3360, 1742, 1639, 1497, 1213, 700 cmꢁ1. HRMS
m/z: calcd. for C30H40NO3 [MþH]þ 462.3008, found 462.3009.
1.23 (s, 3H), 2.09 (dd, J ¼ 12.3, 2.3 Hz, 1H), 2.29 (d, J ¼ 11.7 Hz, 1H),
2.80 (m, 3H), 3.07 (dd, J ¼ 14.1, 6.5 Hz, 1H), 3.24 (dd, J ¼ 14.1, 5.3 Hz,
1H), 3.77 (s, 3H), 4.93 (m, 1H), 6.30 (d, J ¼ 7.0 Hz, 1H), 6.86 (d,
J ¼ 1.8 Hz, 1H), 6.99 (dd, J ¼ 8.2, 2.3 Hz, 1H), 7.15 (d, J ¼ 8.2 Hz, 1H),
7.24 (m,1H), 7.49 (dt, J ¼ 8.2, 2.3 Hz,1H), 8.35 (d, J ¼ 1.8 Hz,1H), 8.51
4.1.1.16. Methyl N-(abiet-8,11,13-trien-18-oyl)
D
-tryptophanate (8b).
(dd, J ¼ 5.3, 1.8 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.5, 18.8, 21.3,
Following the procedure for compound 3a, compound 8b was
prepared from 1 (0.500 g, 1.66 mmol), EDC hydrochloride (478 mg,
24.1, 25.4, 30.0, 33.6, 35.3, 37.2, 37.5, 38.0, 45.4, 47.5, 52.7, 53.0,
123.6, 124.0, 124.2, 127.0, 132.1, 134.7, 137.1), 145.9), 146.9, 148.3,
2.49 mmol), HOBt monohydrate (338 mg, 2.49 mmol),
D-trypto-
150.2, 172.1, 178.4. IR (ATR) 3252, 1732, 1653, 1539, 1234, 708 cmꢁ1
.
phan methyl ester hydrochloride (634 mg, 2.49 mmol), and DIPEA
HRMS m/z: calcd. for
463.2957.
C
29H39N2O3 [MþH]þ 463.2961, found
(0.88 mL, 5.0 mmol) in DMF (5 mL). Compound 8b: white solid
(655 mg, 79%). 1H NMR (300 MHz, CDCl3)
d 1.15 (s, 3H), 1.18 (s, 3H),
1.22 (s, 3H), 1.24 (s, 3H), 2.06 (m, 1H), 2.29 (m, 1H), 2.70 (m, 1H),
2.83 (m, 1H), 3.33 (d, J ¼ 5.8 Hz, 2H), 3.72 (s, 3H), 4.96 (m, 1H), 6.31
(d, J ¼ 7.4 Hz, 1H), 6.84 (d, J ¼ 1.3 Hz, 1H), 6.97 (m, 1H), 7.04 (m, 1H),
7.08 (m, 1H), 7.17 (m, 2H), 7.35 (d, J ¼ 8.2 Hz, 1H), 7.55 (d, J ¼ 8.2 Hz,
4.1.1.20. Methyl N-(abiet-8,11,13-trien-18-oyl) H-
b-(3-pyridyl-N-ox-
ide)- -alaninate (8f). Compound 8e (0.10 g, 0.22 mmol) was dis-
D
solved in CHCl3 (2 mL). This solution was cooled on an ice bath and
m-CPBA (0.10 g, 0.45 mmol) was added in small portions. The re-
action mixture was stirred at room temperature for 17 h. It was
transferred to a silica gel column and purified by FCC (10% MeOH in
EtOAc). Compound 8f: white solid (50 mg, 47%). 1H NMR (300 MHz,
1H), 8.31 (bs, 1H). 13C NMR (75 MHz, CDCl3)
d 16.4, 18.8, 21.0, 24.1,
24.1, 25.3, 27.6, 29.9, 33.6, 37.1, 38.1, 45.6, 47.4, 52.4, 53.3, 110.3,
111.4, 118.7, 119.8, 122.4, 122.7, 123.9, 124.1, 127.0, 127.7, 134.8, 136.3,
145.7, 147.0, 172.9, 178.4. IR (ATR) 3315, 1743, 1635, 1498, 1213,
CDCl3)
d 1.20 (s, 3H), 1.20 (s, 3H), 1.22 (s, 3H), 1.26 (s, 3H), 2.11 (dd,

76
S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
J ¼ 12.3, 2.1 Hz, 1H), 2.29 (d, J ¼ 11.7 Hz, 1H), 2.80 (m, 3H), 3.03 (dd,
J ¼ 14.4, 6.2 Hz,1H), 3.21 (dd, J ¼ 14.4, 5.6 Hz,1H), 3.79 (m, 3H), 4.88
(q, J ¼ 6.5 Hz, 1H), 6.48 (d, J ¼ 7.0 Hz, 1H), 6.86 (s, 1H), 6.99 (dd,
J ¼ 8.2, 1.8 Hz, 1H), 7.15 (d, J ¼ 8.2 Hz, 2H), 7.24 (m, 1H), 8.05 (s, 1H),
25.4, 30.1, 30.2, 30.8, 33.6, 37.2, 37.4, 38.1, 45.9, 47.6, 52.3, 124.1,
124.2, 127.1, 134.7, 145.9, 146.9, 175.6, 179.7. IR (ATR) 3373, 1737,
1631, 1541, 1228, 821 cmꢁ1. HRMS m/z: calcd. for C25H38NO3S
432.2572 [MþH]þ, found 432.2573.
8.14 (d, J ¼ 6.5 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.6, 18.8, 21.4,
24.1, 24.1, 25.4, 30.0, 33.6, 35.0, 37.2, 37.5, 38.0, 45.4, 47.6, 52.7, 53.0,
124.1, 124.2, 125.8, 127.0, 127.6, 134.6, 136.3, 137.9, 139.8, 145.9,
146.9, 171.6, 178.8. IR (ATR) 3254, 1742, 1649, 1261, 1213, 1159,
681 cmꢁ1. HRMS m/z: calcd. for C29H38N2O4Na [MþNa]þ 501.2729,
found 501.2731.
4.1.1.25. Methyl N-(abiet-8,11,13-trien-18-oyl) H-b-(3-pyridyl)-D-
alanine (9e). Following the procedure for compound 4a, compound
9e was prepared from 8e (240 mg, 0.52 mmol), using THF:MeOH
1:1 (4.8 mL) and 4 M NaOH (4.3 mL). The reaction mixture was
acidified with 1 M HCl and the water phase was extracted with
diethyl ether. The organic phase was dried with anhydrous Na2SO4
and evaporated. The crude product was purified by FCC (DCM/
MeOH). Compound 9e: white solid (102 mg, 44%). 1H NMR
4.1.1.21. N-(Abiet-8,11,13-trien-18-oyl)
D-phenylalanine
(9a).
Compound 8a (550 mg, 1.2 mmol) was dissolved in 1:1 THF/MeOH
(15 mL). A 4 M aqueous solution of NaOH (13 mL) was added. After
stirring the mixture at room temperature for 3 h, it was cooled on
an ice bath and acidified with 4 M HCl. The precipitate was filtered
and dried in vacuo. Compound 9a: white solid (490 mg, 91%). 1H
(300 MHz, DMSO-d6)
d 1.08 (s, 3H), 1.09 (s, 3H), 1.13 (s, 3H), 1.16 (s,
3H), 1.88 (d, J ¼ 11.7 Hz, 1H), 2.25 (d, J ¼ 12.3 Hz, 1H), 2.70 (m, 3H),
3.03 (dd, J ¼ 13.5, 5.3 Hz, 1H), 3.14 (dd, J ¼ 13.5, 5.3 Hz, 1H), 4.14 (m,
1H), 6.79 (m, 1H), 6.94 (d, J ¼ 8.21 Hz, 1H), 7.12 (m, 2H), 7.21 (dd,
J ¼ 7.62, 4.69 Hz,1H), 7.49 (d, J ¼ 7.62 Hz, 1H), 8.30 (m, 1H), 8.34 (dd,
NMR (300 MHz, CDCl3)
d 1.19 (s, 6H), 1.22 (s, 3H), 1.24 (s, 3H), 2.03
(d, J ¼ 12.3 Hz, 1H), 2.29 (d, J ¼ 12.3 Hz, 1H), 2.78 (m, 3H), 3.11 (dd,
J ¼ 14.1, 7.0 Hz, 1H), 3.29 (dd, J ¼ 14.1, 5.8 Hz, 1H), 4.87 (q, J ¼ 6.4 Hz,
1H), 6.17 (d, J ¼ 7.0 Hz, 1H), 6.85 (s, 1H), 6.99 (d, J ¼ 8.2 Hz, 1H), 7.16
J ¼ 4.69, 1.76 Hz, 1H). 13C NMR (75 MHz, DMSO-d6)
d 16.2, 18.3, 20.4,
23.9, 24.9, 29.3, 32.8, 34.0, 36.5, 36.7, 37.7, 44.8, 46.2, 54.5, 122.7,
123.6, 123.9, 126.3, 134.2, 134.2, 136.8, 144.9, 146.9, 150.4, 173.3,
176.1. IR (ATR) 3316, 1594, 1497, 1415, 821, 712 cmꢁ1. HRMS m/z:
calcd. for C28H37N2O3 449.2804 [MþH]þ, found 449.2805.
(m, 3H), 7.27 (m, 3H). 13C NMR (75 MHz, CDCl3)
d 16.4, 18.8, 21.2,
24.2, 25.3, 29.9, 33.6, 37.2, 37.2, 37.3, 38.0, 45.6, 47.6, 53.5, 124.0,
124.1, 127.1, 127.4, 128.9,129.4,134.8, 135.9, 145.9,146.9, 175.1, 179.5.
IR (ATR) 3445, 1747, 1600, 1539, 1205, 700 cmꢁ1. HRMS m/z: calcd.
for C29H38NO3 [MþH]þ 448.2852, found 448.2856.
4.1.1.26. Methyl N-(abiet-8,11,13-trien-18-oyl) cyclohexyl-L-alaninate
(10). Following the procedure for compound 3a, compound 10 was
prepared from 1 (250 mg, 0.83 mmol), EDC hydrochloride (239 mg,
4.1.1.22. N-(Abiet-8,11,13-trien-18-oyl)
D
-tryptophan
(9b).
1.25 mmol), HOBt monohydrate (169 mg, 1.25 mmol),
-alanine methyl ester hydrochloride (277 mg, 1.25 mmol), and
DIPEA (0.44 mL, 2.5 mmol) in DMF (2.5 mL). Compound 10: white
solid (374 mg, 96%). 1H NMR (300 MHz, CDCl3)
1.21 (s, 3H), 1.23 (s,
b-cyclohexyl-
Following the procedure for compound 4a, compound 9b was
prepared from 8b (250 mg, 0.50 mmol), using THF:MeOH 1:1
(5.0 mL), and 4 M NaOH (4.2 mL). Compound 9b: white solid
L
d
(226 mg, 93%). 1H NMR (300 MHz, CDCl3)
d
1.05 (s, 3H), 1.13 (s, 3H),
3H), 1.24 (s, 3H), 1.30 (s, 3H), 2.09 (dd, J ¼ 12.4, 2.1 Hz, 1H), 2.32 (d,
J ¼ 12.9 Hz, 1H), 2.88 (m, 3H), 3.72 (s, 3H), 4.67 (m, 1H), 6.07 (d,
J ¼ 8.2 Hz, 1H), 6.89 (s, 1H), 7.0 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.17 (d,
1.22 (s, 3H), 1.24 (s, 3H), 1.89 (d, J ¼ 12.3 Hz, 1H), 2.23 (d, J ¼ 12.6 Hz,
1H), 2.81 (m, 3H), 3.36 (m, 2H), 4.83 (m, 1H), 6.25 (m, 1H), 6.79 (m,
1H), 7.07 (m, 5H), 7.29 (m, 2H), 7.56 (d, J ¼ 7.8 Hz), 8.11 (s, 1H). 13
C
J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.4, 18.7, 21.1, 23.9, 24.0,
NMR (75 MHz, CDCl3)
d
16.3, 18.7, 21.0, 24.1, 24.1, 25.3, 26.9, 29.8,
25.3, 26.1, 26.3, 26.4, 30.0, 32.4, 33.4, 33.6, 34.4, 37.2, 37.3, 38.0, 40.0,
45.8, 47.3, 50.1, 52.2, 123.9, 124.1, 126.9, 134.7, 145.7, 146.9, 173.9,
178.1. IR (ATR) 3344, 1751, 1627, 1525, 1448, 1172, 819 cmꢁ1. HRMS
m/z: calcd. for C30H46NO3 468.3478 [MþH]þ, found 468.3477.
31.0 33.6, 37.0, 37.1, 38.0, 45.7, 47.5, 53.8, 109.7, 111.6, 118.5, 119.9,
122.4, 123.2, 123.9, 124.1, 127.0, 127.7, 134.8, 136.3, 145.8, 146.9,
175.5, 179.8. IR (ATR) 3402, 3257, 1728, 1629, 1529, 740 cmꢁ1. HRMS
m/z: calcd. for C31H39N2O3 487.2961 [MþH]þ, found 487.2961.
4.1.1.27. N-(Abiet-8,11,13-trien-18-oyl) cyclohexyl- -alanine (11).
L
4.1.1.23. N-(Abiet-8,11,13-trien-18-oyl)
D
-tyrosine
(9c).
Following the procedure for compound 4a, compound 11 was
prepared from 10 (0.10 g, 0.21 mmol), using THF:MeOH 1:1 (3 mL),
and 4 M NaOH (1.8 mL). Compound 11: white solid (95 mg, 98%). 1H
Following the procedure for compound 4a, compound 9c was
prepared from 8c (250 mg, 0.52 mmol), using THF:MeOH 1:1
(7.3 mL), and 4 M NaOH (4.4 mL). Compound 9c: white solid
NMR (300 MH, CDCl3) d 1.21 (s, 3H),1.24 (s, 6H),1.31 (s, 3H), 2.10 (m,
(217 mg, 89%). 1H NMR (300 MHz, CDCl3)
d
1.20 (s, 3H), 1.21 (s, 3H),
1H), 2.33 (d, J ¼ 11.8 Hz, 1H), 2.86 (m, 3H), 4.64 (m, 1H), 6.11 (d,
1.24 (s, 6H), 2.08 (dd, J ¼ 11.1, 2.0 Hz, 1H), 2.30 (d, J ¼ 12.6 Hz, 1H),
2.80 (m, 3H), 3.00 (dd, J ¼ 13.9, 6.6 Hz, 1H), 3.14 (dd, J ¼ 13.9, 5.1 Hz,
1H), 4.87 (m, 1H), 6.37 (d, J ¼ 7.5 Hz, 1H), 6.72 (d, 1H, J ¼ 8.2 Hz),
J ¼ 7.7 Hz, 1H), 6.89 (s, 1H), 7.00 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.17 (d,
J ¼ 8.2 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d 16.5, 18.9, 21.3, 24.1, 24.1,
25.4, 26.2, 26.4, 26.5, 30.1, 32.5, 33.6, 33.7, 34.6, 37.3, 37.4, 38.2, 39.5,
45.9, 47.5, 50.6, 124.1, 124.2, 127.1, 134.8, 145.9, 147.0, 177.1, 179.3. IR
(ATR) 3346, 1732, 1625, 1525, 1232, 819 cmꢁ1. HRMS m/z: calcd. for
6.86 (s, 1H), 6.98 (m, 3H), 7.15 (d, J ¼ 8.2 Hz, 1H), 7.36 (bs, 1H). 13
C
NMR (75 MHz, CDCl3)
d 16.4, 18.8, 21.2, 24.1, 24.1, 25.4, 30.0, 33.6,
36.8, 37.2, 37.2, 38.0, 45.3, 47.6, 53.7, 115.9, 124.0, 124.2, 127.1, 127.3,
130.4, 134.7, 145.9, 146.9, 155.4, 175.3, 179.7. IR (ATR) 3280, 1718,
1616, 1515, 1220, 821 cmꢁ1. HRMS m/z: calcd. for C29H38NO4
464.2801 [MþH]þ, found 464.2801.
C
29H44NO3 454.3321 [MþH]þ, found 454.3322.
4.1.1.28. Methyl N-(7-oxoabiet-8,11,13-trien-18-oyl) cyclohexyl-L-
alaninate (12a). Following the procedure for compound 5, com-
pound 12a was prepared from 10 (0.20 g, 0.43 mmol), chro-
mium(VI) oxide (133 mg, 1.34 mmol), glacial acetic acid (3.9 mL),
and ethyl acetate (1.7 mL). Compound 12a: white solid (98 mg,
4.1.1.24. N-(Abiet-8,11,13-trien-18-oyl)
D
-methionine
(9d).
Following the procedure for compound 4a, compound 9d was
prepared from 8d (250 mg, 0.56 mmol), using THF:MeOH 1:1
(7.9 mL), and 4 M NaOH (4.7 mL). Compound 9d: white solid
47%). 1H NMR (CDCl3)
d 1.23 (s, 3H), 1.25 (s, 3H), 1.27 (s, 3H), 1.39 (s,
3H), 2.38 (m, 2H), 2.67 (m, 2H), 2.92 (sept. J ¼ 7 Hz, 1H), 3.72 (s, 3H),
(234 mg, 96%). 1H NMR (300 MHz, CDCl3)
d 1.21 (s, 3H), 1.24 (s, 6H),
4.64 (m, 1H), 6.10 (d, J ¼ 8.1 Hz, 1H), 7.28 (d, J ¼ 8.1 Hz, 1H), 7.39 (d,
1.32 (s, 3H), 2.14 (m, 3H), 2.10 (s, 3H), 2.33, (m, 1H), 2.57 (t, 2H,
J ¼ 7.3 Hz), 2.85 (m, 3H), 4.72 (m,1H), 6.70 (d, J ¼ 7.2 Hz,1H), 6.88 (d,
J ¼ 1.6 Hz, 1H), 7.00 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.17 (d, J ¼ 8.2 Hz, 1H),
J ¼ 8.1 Hz), 7.85 (s, 1H). 13C NMR (75 MHz, CDCl3)
d 16.6, 18.5, 23.9,
23.9, 24.0, 26.1, 26.3, 26.5, 32.7, 33.6, 33.7, 34.6, 37.1, 37.2, 37.4, 37.5,
40.2, 44.0, 46.5, 50.6, 52.4, 123.4, 125.2, 131.0, 132.6, 147.0, 153.1,
173.8, 176.9, 198.4. IR (ATR) 3394, 1745, 1681, 1676, 1521, 1450, 1251,
9.66 (brs, 1H). 13C NMR (75 MHz, CDCl3)
d 15.6, 16.5, 18.8, 21.3, 24.1,

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
77
1197, 835 cmꢁ1. HRMS m/z: calcd. for C30H44NO4 482.3270 [MþH]þ,
Corning Incorporated, NY, USA), 5 mL per well depending on the
type of assay and plates were incubated in 37 ꢀC, 200 rpm for 18 h.
found 482.3271.
4.1.1.29. Methyl N-(7-hydroxyiminoabiet-8,11,13-trien-18-oyl) cyclo-
4.2.3. Exposure to compounds and biofilm quantification
hexyl-
L
-alaninate (12b). Compound 12a (250 mg, 0.52 mmol) and
Anti-biofilm activity of the compounds in DMSO was assessed
against both S. aureus strains and in two exposure conditions, prior-
to and post-biofilm formation, as recently described [15,28]. In the
pre-exposure mode of the assay, compounds and bacterial sus-
pensions were added simultaneously prior to biofilm formation
and plates were incubated for 18 h in 37 ꢀC with shaking (200 rpm).
During the post-exposure mode, the compounds were added after
biofilms were first formed as described earlier (37 ꢀC, 200 rpm for
18 h) and plates were incubated for additional 24 h at 37 ꢀC,
200 rpm. Penicillin G (potassium salt, Fluka Biochemika, Buchs,
Switzerland) and vancomycin (hydrochloride hydrate, Sigma
Aldrich, St. Louis, MO, USA) prepared in Mueller Hinton Broth
(MHB, Fluka Biochemika, Buchs, Switzerland) were used as control
hydroxylamine hydrochloride (0.060 g, 0.88 mmol) were dissolved
in EtOH (1.5 mL) and pyridine (63
mL) was added. The reaction
mixture was stirred in a closed vial at 100 ꢀC for 3 h. Solvents were
evaporated and the residue was purified by FCC (silica column,
15 / 50% EtOAc in n-hexane). Compound 12b: white solid
(200 mg, 78%). 1H NMR (300 MHz, CDCl3)
d 1.12 (s, 3H), 1.23 (s, 3H),
1.25 (s, 3H), 1.42 (s, 3H), 2.27 (m, 2H), 2.67 (m, 2H), 2.88 (sept,
J ¼ 6.9 Hz,1H), 3.71 (s, 3H), 4.66 (m, 1H), 6.13 (d, J ¼ 8.2 Hz, 1H), 7.20
(m, 2H), 7.68 (s,1H). 13C NMR (75 MHz, CDCl3)
d 16.6,18.4, 23.1, 23.5,
23.9, 24.2, 26.0, 26.3, 26.5, 32.5, 33.7, 33.8, 34.4, 36.7, 37.3, 37.4, 40.1,
42.1, 46.5, 50.5, 52.3, 122.4, 122.9, 128.0, 129.0, 146.6, 148.8, 155.6,
174.0, 177.4. IR (ATR) 3360, 1738, 1641, 1508, 1447, 1204, 951,
729 cmꢁ1. HRMS m/z: calcd. for C30H45N2O4 [MþH]þ 497.3379,
found 497.3379.
antibiotics and tested always at 400 mM. Untreated biofilms, TSB-
media controls (cell-free) and wells containing biofilms and 2%
DMSO were included as controls. Initial screening of the compound
collection was performed at final compound concentrations of 400
4.1.1.30. N-(7-Oxoabiet-8,11,13-trien-18-oyl)
cyclohexyl-L-alanine
(13). Compound 12a (350 mg, 0.73 mmol) was dissolved in THF/
MeOH 1:1 (10 mL) and a 4 M aqueous solution of NaOH (8.5 mL)
was added. The color of the reaction mixture changed to yellow.
The reaction mixture was then stirred at room temperature for 2 h
45 min. The mixture was acidified with a 4 M aqueous solution of
HCl and concentrated. Water was added and the mixture was
extracted three times with ethyl acetate. The organic phase was
then washed with a 1 M aqueous solution of HCl, water and brine
and dried with Na2SO4 and evaporated. The crude product was
purified by FCC (silica column, 50% EtOAc in n-hexane and 2% acetic
acid). Compound 13: white solid (0.23 g, 68%). 1H NMR (300 MHz,
and 100 mM. Resazurin assay was used for the quantification of
biofilms viability as previously described [21,29]. Planktonic sus-
pension was carefully removed from plates and biofilms were
stained with 20 mM resazurin (Sigma Aldrich, St. Louis, MO, USA)
solution prepared in sterile 0.01 M phosphate buffered saline PBS
(pH ¼ 7.3e7.5, Lonza, Verviers, Belgium) and incubated at room
temperature (200 rpm shaking, darkness, 20 min) followed by
fluorescence measurements (
l
ex ¼ 560 nm, em ¼ 590 nm) using
l
Varioskan Flash Multimode Plate Reader operated with SkanIt®
software, version 2.4.3 (ThermoFisher Scientific, Vantaa, Finland).
CDCl3)
d
1.23 (s, 3H), 1.25 (s, 3H), 1.26 (s, 3H), 1.38 (s, 3H), 2.40 (m,
4.2.4. Determination of anti-biofilm potencies and effects on
planktonic bacteria
Anti-biofilm potencies (IC50 and IC90) of the most active com-
pounds 1, 4d, 4e, 9a, 9b, 11, 13 and reference antibiotics were
determined in both exposure modes on both strains of S. aureus. At
2H), 2.65 (m, 2H), 2.92 (sept, J ¼ 6.9 Hz, 1H), 4.62 (m, 1H), 6.15 (d,
J ¼ 7.6 Hz, 1H), 7.28 (d, J ¼ 9.4 Hz, 1H), 7.40 (dd, J ¼ 8.2, 1.8 Hz, 1H),
7.85 (d, J ¼ 2.3 Hz, 1H), 9.21 (bs, 1H). 13C NMR (75 MHz, CDCl3)
d
16.5, 18.4, 23.9, 24.0, 24.0, 26.1, 26.3, 26.5, 32.6, 33.7, 33.7, 34.6,
37.0, 37.1, 37.4, 39.6, 43.9, 46.6, 50.6, 123.5, 125.3, 130.9, 132.8, 147.1,
153.2, 176.7, 177.8, 198.8. IR (ATR) 3348, 1732, 1681, 1636, 1531,1232,
1194, 833 cmꢁ1. HRMS m/z: calcd. for C29H41NO4Na [MþNa]þ
490.2933, found 490.2932.
least 12 concentrations ranging from 0.001 to 400 mM were tested
in duplicates for the inhibitory activity using the resazurin-based
viability assay. Potencies were calculated via a non-linear regres-
sion analysis (sigmoidal doseeresponse with variable slope) using
Microsoft Excel 2010 and GraphPad Prism v. 4.0 software (Graph-
Pad software Inc., US). To estimate compounds effects on planktonic
bacteria minimal inhibitory concentrations (MIC) of the most active
compounds were determined. Minimal concentrations causing no
turbidity on suspended bacteria were measured. For that purpose,
after exposing biofilms to the compounds (37 ꢀC, 200 rpm, 18 h),
planktonic phase was transferred to a sterile 96-microtiter well
4.2. Biology
4.2.1. Bacterial strains and growth conditions
Biofilm-producing, clinical strains of S. aureus (ATCC 25923 and
Newman), Staphylococcus epidermidis (ATCC 12228, non-clinical
and ATCC 35984, clinical) and E. coli (XL1 Blue, non-clinical) were
provided by the Faculty of Pharmacy, University of Helsinki,
Finland. Freezer stocks of bacterial strains containing 20% glycerol
were maintained at ꢁ70 ꢀC. Bacteria were pre-cultured in 30 g/L
tryptic soy broth (TSB, Fluka Biochemika, Buchs, Switzerland)
overnight in 37 ꢀC with 220 rpm shaking. Cultures were prepared
by diluting the overnight cultures 1000 times (S. aureus, E. coli) and
100 times (S. epidermidis) in TSB and bacteria were grown under
aerobic conditions in 37 ꢀC, 200 rpm shaking approximately for 4 h
to an OD595 of ~0.4 which corresponds to ~108 colony forming units
per milliliter (CFU/mL).
plate and quantitative readouts were detected at
Varioskan Plate Reader (2.4.3 software) [15,28].
l
¼ 620 nm using
4.2.5. Anti-biofilm activity against other strains
The most active compounds (1, 4d, 4e, 9a, 9b, 11, 13) were
further tested for the anti-biofilm activity against two strains of S.
epidermidis and E. coli. Control antibiotics at a concentration of
400 mM were also included. Compounds were tested in four bio-
logical replicates at concentrations at which they caused 50% in-
hibition of biofilm viability in pre-exposure mode of assay on S.
aureus ATCC 25923. Biofilm viability was quantified using resazurin
staining assay, as described above, but in case of E. coli, the incu-
bation period was prolonged to 35 min [29].
4.2.2. Biofilm formation
Biofilms were formed according to the conditions previously
optimized and described by Refs. [21,27]. Fresh, exponentially
grown cultures were diluted to be 106 CFU/mL and added to flat-
4.2.6. Time-killing studies
bottomed 96-well plates (Nunclon™
D
surface, Nunc, Roskilde,
Kinetics of the anti-biofilm activity of the two most active
compounds (9b and 11) and reference compound 1 in the post-
Denmark), 200 L per well, or flat-bottomed 6-well plates (Costar®,
m

78
S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
exposure mode of the assay were monitored on S. aureus ATCC
25923 biofilms. Biofilms were formed as described above and after
an incubation period of 18 h, planktonic suspension was removed
and biofilms were exposed to the compounds at a final concen-
suspension was removed from the wells and biofilms were incu-
bated with 1 mL of compound solutions 9b and 11, prepared in
sterile PBS at a final concentration of 100
mM. Control antibiotics
penicillin G and vancomycin were tested at 400
m
M. Untreated
tration of 100
Penicillin G and vancomycin were included as control antibiotics
and tested at a concentration of 400 M. Plates were further
incubated in 37 ꢀC, 200 rpm and biofilms were quantified with
m
M and fresh TSB was added into the control wells.
biofilms and negative controls wells were incubated only with
sterile PBS. After 1 h, the solutions were then removed and a
mixture of probes (34 mL) containing 5 mM of Syto9 and 30 mM of
m
propidium iodide (LIVE/DEAD® BacLight™, Molecular Probes, Life
Technologies, Eugene, OR, US) was added and incubated for 15 min
at room temperature in the darkness. Afterwards, coverslips were
flipped and mounted with a drop of Mounting Oil (LIVE/DEAD®
BacLight™, Molecular Probes, Life Technologies, Eugene, OR, US)
into slides that were imaged using an Inverted Evos FL microscope
with a 40ꢂ coverslip-corrected objective. Images were obtained
using cubes for GFP and RFP (for Syto9 and PI fluorescence,
respectively) and directly acquired with the microscope; over-
lapping of the green and red fluorescence was done directly on the
microscope while taking the pictures.
resazurin staining assay after
described.
1 h incubation, as previously
4.2.7. Determination of membrane depolarization
Membrane depolarization caused by compounds 1, 9b and 11
was detected using a membrane-potential-sensitive probe, bis(1,3-
dibutylbarbituric acid) trimethine oxonol, DiBAC4(3) (Molecular
Probes®, Invitrogen, CA, US) according to the protocol originally
described [25] with some modifications. S. aureus ATCC 25923
biofilms were formed on flat bottomed 96-well plates (Nunclon™
D
surface, Nunc, Roskilde, Denmark) for 18 h. After finishing the 18 h
incubation, planktonic suspension was removed and biofilm were
washed with PBS (0.01 M, pH ¼ 7.3e7.5, Lonza, Verviers, Belgium)
4.2.10. Cytotoxicity studies
Human lung (HL) epithelial cells [30] were grown in RPMI 1640
(Biowhittaker, Lonza, Walkersville, USA) supplemented with inac-
followed by pre-incubation with 5
in PBS (100
removed and biofilms were exposed to solutions containing
DiBAC4(3) at a concentration of 5 M and compounds at a final
concentration of 100 M. Control antibiotics, penicillin G and
vancomycin were included at a concentration of 400 M. Plates
mM DiBAC4(3) solution prepared
mL) at room temperature for 30 min. Then, solution was
tivated FBS 7%, L-glutamine 2 mM and gentamicin 20 mg/mL. Cells
were routinely cultured in 75 cm2 cell culture flasks at 37 ꢀC in 5%
CO2 in an air-ventilated humidified incubator to around 90%
confluence. The harvesting was done by adding 0.25% trypsin in
m
m
m
PBS. Cell suspensions (200
m
L) of 4 ꢂ 105 cells/ml were added into
were incubated at room temperature for 1 h. Biofilms were washed
twice with PBS and followed by DiBAC4(3) fluorescence was
plates and incubated at 37 ꢀC for 24 h. After that, 20
mL of culture
media was removed and replaced with a similar volume of com-
measured at
l
ex ¼ 485 nm and em ¼ 535 nm using Varioskan
l
pounds solutions (9b and 11) at different concentrations
Flash Multimode Plate Reader (2.4.3 software, ThermoFisher, Van-
taa, Finland).
(1e100
Then, culture media was replaced with a solution of resazurin in
PBS at a concentration of 20
M and maintained for 2 h at 37 ꢀC in
m
M) and plates were incubated for additional 24 h at 37 ꢀC.
m
4.2.8. Measurement of ATP efflux from biofilms
the incubator conditions [31]. Reduced resazurin signal as indica-
tion of cell viability was measured using a Varioskan Flash multi-
ATP efflux from the S. aureus ATCC 25923 biofilms after expo-
sure to compounds 9b and 11 was quantified. S. aureus ATCC 25923
biofilms were formed on flat bottomed 6-well plates (Corning®
Costar®, NY, US) in 37 ꢀC with 200 rpm shaking for 18 h. Negative
control wells containing 5 mL of cell-free TSB were also included.
After the incubation period, the planktonic suspension was
removed from the wells and biofilms were incubated with 1.25 mL
of compound solutions 9b and 11, prepared in sterile PBS at a final
mode plate reader (
l
ex ¼ 570 nm and em ¼ 590 nm) and results
l
expressed as relative fluorescence units (RFUs). Cells without test
compounds were included as positive controls, wells containing
only media as negative controls and 0.5% DMSO as solvent controls.
Percentages of cell viability were calculated in relation to the un-
treated cells.
concentration of 100
comycin were tested at 400
controls wells were incubated only with PBS. After incubation in
37 ꢀC for 1 h suspensions were filtered using 0.22
m centrifugal
filter (Jet Biofil, China) and diluted 10-fold with sterile PBS in
Eppendorf tubes. Then, 100 L of each filtrate in duplicates was
transferred to a clear-bottom 96-well plate (Isoplate-TC) (Perkin
Elmer, Waltham, MA, US). CellTiter-Glo® reagent (Promega, Madi-
son, WI, US) was prepared, used according to the manufacturer's
protocol and luciferin luminescence was measured at 560 nm using
Varioskan Flash Multimode Plate Reader (2.4.3 software, Thermo-
Fisher, Vantaa, Finland).
m
M. Control antibiotics penicillin G and van-
mM. Untreated biofilms and negative
4.2.11. Statistical analysis
Assay performance was evaluated using statistical parameters:
screening window coefficient Z’-factor, signal-to-noise (S/N) and
signal-to-background (S/B) eratios [27]. In each assay, control wells
containing only bacteria and TSB were included to establish
maximal and minimum signals, respectively. For the analysis of
data from ATP efflux and membrane depolarization experiments
unpaired t-test with Welch's correction was applied using Graph-
Pad Prism v. 4.0 esoftware (GraphPad software Inc., US). p < 0.05
was considered in all cases as statistically significant.
m
m
Acknowledgments
4.2.9. Membrane permeability studies using fluorescence
microscopy
Financial support for this work was provided by Academy of
Finland (decisions 264020, 272266, and 282981) and Magnus
Ehrnrooth Foundation (ME2012n44).
Membrane permeability on S. aureus ATCC 25923 biofilms upon
exposure to compounds 9b and 11 for 1 h was also measured. S.
aureus ATCC 25923 biofilms were first formed on flat-bottomed 24-
well plates (Nunc, Roskilde, Denmark) in which coverslips have
been placed on the bottom (total growth area was estimated to be
1.9 cm2) in the same manner as described earlier (exponentially
grown cultures of 106 CFU/mL incubated at 37 ꢀC with 200 rpm
shaking for 18 h). After the incubation period, the planktonic
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.07.038.

S. Manner et al. / European Journal of Medicinal Chemistry 102 (2015) 68e79
79
[17] A.I. Hochbaum, I. Kolodkin-Gal, L. Foulston, R. Kolter, J. Aizenberg, R. Losick,
Inhibitory effects of -amino acids on Staphylococcus aureus biofilm devel-
opment, J. Bacteriol. 193 (2011) 5616e5622.
[18] S. Sarkar, M.M. Pires, -amino acids do not inhibit biofilm formation in
Staphylococcus aureus, PLoS One 10 (2015) e0117613.
[19] C. de la Fuente-Nunez, F. Reffuveille, S.C. Mansour, S.L. Reckseidler-Zenteno,
References
D
[1] J. Costerton, P. Stewart, E. Greenberg, Bacterial biofilms: a common cause of
persistent infections, Science 284 (1999) 1318e1322.
[2] L. Hall-Stoodley, J.W. Costerton, P. Stoodley, Bacterial biofilms: from the nat-
ural environment to infectious diseases, Nat. Rev. Microbiol. 2 (2004) 95e108.
[3] H.C. Flemming, J. Wingender, The biofilm matrix, Nat. Rev. Microbiol. 8 (2010)
623e633.
[4] D. Lebeaux, A. Chauhan, O. Rendueles, C. Beloin, From in vitro to in vivo models
of bacterial biofilm-related infections, Pathogens 2 (2013) 288e356.
[5] M. Otto, Staphylococcal infections: mechanisms of biofilm maturation and
detachment as critical determinants of pathogenicity, Annu. Rev. Med. 64
(2013) 175e188.
D
D. Hernandez, G. Brackman, T. Coenye, R.E. Hancock, D-enantiomeric peptides
that eradicate wild-type and multidrug-resistant biofilms and protect against
lethal Pseudomonas aeruginosa infections, Chem. Biol. 22 (2015) 196e205.
[20] V.M. Moreira, A. Fallarero, J. Yli-Kauhaluoma, P. Vuorela, Abietane-type
diterpenoids, Finn. Pat. Appl. no FI20145858, 2014.
[21] M.E. Sandberg, D. Schellmann, G. Brunhofer, T. Erker, I. Busygin, R. Leino,
P.M. Vuorela, A. Fallarero, Pros and cons of using resazurin staining for
quantification of viable Staphylococcus aureus biofilms in a screening assay,
J. Microbiol. Methods 78 (2009) 104e106.
[22] J.L. Rios, M.C. Recio, Medicinal plants and antimicrobial activity,
J. Ethnopharmacol. 100 (2005) 80e84.
[23] F. Ali, P.L. Sangwan, S. Koul, A. Pandey, S. Bani, S.T. Abdullah, P.R. Sharma,
S. Kitchlu, I.A. Khan, 4-epi-Pimaric acid: a phytomolecule as a potent anti-
bacterial and anti-biofilm agent for oral cavity pathogens, Eur. J. Clin.
Microbiol. Infect. Dis. 31 (2012) 149e159.
[24] L. Kuzma, M. Rozalski, E. Walencka, B. Rozalska, H. Wysokinska, Antimicrobial
activity of diterpenoids from hairy roots of Salvia sclarea L.: salvipisone as a
potential anti-biofilm agent active against antibiotic resistant Staphylococci,
Phytomedicine 14 (2007) 31e35.
[25] K. Okuda, T. Zendo, S. Sugimoto, T. Iwase, A. Tajima, S. Yamada, K. Sonomoto,
Y. Mizunoe, Effects of bacteriocins on methicillin-resistant Staphylococcus
aureus biofilm, Antimicrob. Agents Chemother. 57 (2013) 5572e5579.
ꢀ
ꢀ
[6] B. Amorena, E. Gracia, M. Monzon, J. Leiva, C. Oteiza, M. Perez, J.L. Alabart,
ꢀ
J. Hernandez-Yago, Antibiotic susceptibility assay for Staphylococcus aureus in
biofilms developed in vitro, J. Antimicrob. Chemother. 44 (1999) 43e55.
[7] R.J. Worthington, J.J. Richards, C. Melander, Small molecule control of bacterial
biofilms, Org. Biomol. Chem. 10 (2012) 7457e7474.
[8] N. Barraud, B.G. Kardak, N.R. Yepuri, R.P. Howlin, J.S. Webb, S.N. Faust,
S. Kjelleberg, S.A. Rice, M.J. Kelso, Cephalosporin-3'-diazeniumdiolates: tar-
geted NO-donor prodrugs for dispersing bacterial biofilms, Angew. Chem. Int.
Ed. Engl. 51 (2012) 9057e9060.
[9] C. Lu, C.K. Maurer, B. Kirsch, A. Steinbach, R.W. Hartmann, Overcoming the
unexpected functional inversion of a PqsR antagonist in Pseudomonas aeru-
ginosa: an in vivo potent antivirulence agent targeting pqs quorum sensing,
Angew. Chem. Int. Ed. Engl. 53 (2014) 1109e1112.
[10] A. Garrison, F. Bai, Y. Abouelhassan, N.G. Paciaroni, S. Jin, R.W. Huigens Iii,
Bromophenazine derivatives with potent inhibition, dispersion and eradica-
tion activities against Staphylococcus aureus biofilms, RSC Adv.
1120e1124.
[11] Z. Su, L. Peng, R.J. Worthington, C. Melander, Evaluation of 4,5-disubstituted-
2-aminoimidazole-triazole conjugates for antibiofilm/antibiotic resensitiza-
tion activity against MRSA and Acinetobacter baumannii, ChemMedChem 6
(2011) 2243e2251.
[12] T.J. Opperman, S.M. Kwasny, J.D. Williams, A.R. Khan, N.P. Peet, D.T. Moir,
T.L. Bowlin, Aryl rhodanines specifically inhibit staphylococcal and entero-
coccal biofilm formation, Antimicrob. Agents Chemother. 53 (2009)
4357e4367.
[13] Y. Ma, Y. Xu, B.D. Yestrepsky, R.J. Sorenson, M. Chen, S.D. Larsen, H. Sun, Novel
inhibitors of Staphylococcus aureus virulence gene expression and biofilm
formation, PLoS One 7 (2012) e47255.
[26] H. Lam, D.C. Oh, F. Cava, C.N. Takacs, J. Clardy, M.A. de Pedro, M.K. Waldor, D-
5 (2015)
amino acids govern stationary phase cell wall remodeling in bacteria, Science
325 (2009) 1552e1555.
€€
€
[27] M. Sandberg, A. Maattanen, J. Peltonen, P.M. Vuorela, A. Fallarero, Automating
96-well microtitre plate model for Staphylococcus aureus biofilms: an
a
approach to screening of natural antimicrobial compounds, Int. J. Antimicrob.
Agents 32 (2008) 233e240.
[28] S. Manner, M. Skogman, D. Goeres, P. Vuorela, A. Fallarero, Systematic
exploration of natural and synthetic flavonoids for the inhibition of Staphy-
lococcus aureus biofilms, Int. J. Mol. Sci. 14 (2013) 19434e19451.
[29] M.E. Skogman, P.M. Vuorela, A. Fallarero, Combining biofilm matrix mea-
surements with biomass and viability assays in susceptibility assessments of
antimicrobials against Staphylococcus aureus biofilms, J. Antibiot. (Tokyo) 65
(2012) 453e459.
[30] C.C. Kuo, J.T. Grayston, A sensitive cell line, HL cells, for isolation and propa-
gation of Chlamydia pneumoniae strain TWAR, J. Infect. Dis. 162 (1990)
755e758.
[14] M.A. Gonzalez, Aromatic abietane diterpenoids: their biological activity and
synthesis, Nat. Prod. Rep. 32 (2015) 684e704.
[15] A. Fallarero, M. Skogman, J. Kujala, M. Rajaratnam, V.M. Moreira, J. Yli-Kau-
haluoma, P. Vuorela, (þ)-Dehydroabietic acid, an abietane-type Diterpene,
inhibits Staphylococcus aureus biofilms in vitro, Int. J. Mol. Sci. 14 (2013)
12054e12072.
[31] D. Karlsson, A. Fallarero, G. Brunhofer, C. Mayer, O. Prakash, C.G. Mohan,
P. Vuorela, T. Erker, The exploration of thienothiazines as selective butyr-
ylcholinesterase inhibitors, Eur. J. Pharm. Sci. 47 (2012) 190e205.
[16] I. Kolodkin-Gal, D. Romero, S. Cao, J. Clardy, R. Kolter, R. Losick, D-amino acids
trigger biofilm disassembly, Science 328 (2010) 627e629.