Antibacterial profiling of abietane-type diterpenoids

Article abstract of DOI:10.1016/j.bmc.2016.10.019

Abietic and dehydroabietic acid are interesting diterpenes with a highly diverse repertoire of associated bioactivities. They have, among others, shown antibacterial and antifungal activity, potentially valuable in the struggle against the increasing antimicrobial resistance and imminent antibiotic shortage. In this paper, we describe the synthesis of a set of 9 abietic and dehydroabietic acid derivatives containing amino acid side chains and their in vitro antimicrobial profiling against a panel of human pathogenic microbial strains. Furthermore, their in vitro cytotoxicity against mammalian cells was evaluated. The experimental results showed that the most promising compound was 10 [methyl N-(abiet-8,11,13-trien-18-yl)-D-serinate], with an MIC₉₀of 60 μg/mL against Staphylococcus aureus ATCC 25923, and 8 μg/mL against methicillin-resistant S. aureus, Staphylococcus epidermidis and Streptococcus mitis. The IC₅₀value for compound 10 against Balb/c 3T3 cells was 45 μg/mL.

Full text of DOI:10.1016/j.bmc.2016.10.019

Accepted Manuscript
Antibacterial profiling of abietane-type diterpenoids
Andreas Helfenstein, Mikko Vahermo, Dorota A. Nawrot, Fatih Demirci,
Gökalp İ şcan, Sara Krogerus, Jari Yli-Kauhaluoma, Vânia M. Moreira, Päivi
Tammela
PII:
S0968-0896(16)30994-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.019
BMC 13346
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 July 2016
14 October 2016
15 October 2016
Please cite this article as: Helfenstein, A., Vahermo, M., Nawrot, D.A., Demirci, F., İ şcan, G., Krogerus, S., Yli-
Kauhaluoma, J., Moreira, V.M., Tammela, P., Antibacterial profiling of abietane-type diterpenoids, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.10.019
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Antibacterial profiling of abietane-type diterpenoids
Andreas Helfensteina,†, Mikko Vahermo,b,† Dorota A. Nawrot,a Fatih Demirci,c Gökalp İşcan,c Sara
Krogerus,b Jari Yli-Kauhaluoma,b Vânia M. Moreira,b,* Päivi Tammelaa,*
aCentre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, Viikinkaari 5 E (P.O. Box 56), FI-00014 University of Helsinki, Helsinki,
Finland
bDivision of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of
Helsinki, Viikinkaari 5 E (P.O. Box 56), FI-00014 University of Helsinki, Helsinki, Finland
cPharmacognosy Department, Faculty of Pharmacy, Anadolu University 26470-Eskişehir, Turkey
†These authors contributed equally to this work
*Corresponding authors: Dr. Vânia M. Moreira, vania.moreira@helsinki.fi, p. +358-504480736;
Dr. Päivi Tammela, paivi.tammela@helsinki.fi, p. +358-2941-59628

Abstract
Abietic and dehydroabietic acid are interesting diterpenes with a highly diverse repertoire of
associated bioactivities. They have, among others, shown antibacterial and antifungal activity,
potentially valuable in the struggle against the increasing antimicrobial resistance and imminent
antibiotic shortage. In this paper, we describe the synthesis of a set of 9 abietic and dehydroabietic
acid derivatives containing amino acid side chains and their in vitro antimicrobial profiling against a
panel of human pathogenic microbial strains. Furthermore, their in vitro cytotoxicity against
mammalian cells was evaluated. The experimental results showed that the most promising
compound was 10 [methyl N-(abiet-8,11,13-trien-18-yl)-D-serinate], with an MIC90 of 60 µg/mL
against Staphylococcus aureus ATCC 25923, and 8 µg/mL against methicillin-resistant S. aureus,
Staphylococcus epidermidis and Streptococcus mitis. The IC50 value for compound 10 against
Balb/c 3T3 cells was 45 µg/mL.
Keywords: abietic acid; dehydroabietic acid; amino acid derivative; bacteria; fungi; cytotoxicity

1. Introduction
The occurrence of antibiotic-resistant microorganisms is not a new phenomenon, but it has been
getting more accentuated during recent years, partly due to the emergence of multidrug-resistant
strains, together with a stagnating development of novel antibiotics. The spread of multidrug-
resistant bacteria and fungi poses a serious threat to the successful management of infections and
has as such a growing impact on both quality and cost of the health care system.1 To face this
challenge, not only improved antimicrobial stewardship, but also new antimicrobial drugs are
needed. Natural products from plants, fungi and bacteria have a strong track record for having
antimicrobial activity. Case in point, the screening of natural products for antibiotic activity has a
higher success rate than an approach based on pure combinatorial chemistry.2 A pair of natural
products that deserves our particular interest is found in many conifers around the globe: Abietic
acid (1) and dehydroabietic acid (1a) (Scheme 1) are diterpenoids that boast an intriguing and well-
studied bioactivity profile, including antiviral, antibiotic and antifungal activity.3–5
Scheme 1. Synthesis of compounds 2-10. i) EDC hydrochloride, HOBt, DIPEA, amino acid methyl
ester hydrochloride, DMF, r.t. DIPEA N,N-diisopropylethylamine; DMF = N,N-
dimethylformamide; EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt = N-
hydroxybenzotriazole; THF = tetrahydrofuran

The main source of 1 and 1a is the resin of pine trees (Pinus sp.), but they can also be found in
many other conifers and are commercially available.6 Both 1 and 1a themselves have an intrinsic
activity against (mainly Gram-positive) bacteria and fungi.6,7 The effort to enhance these activities
has led to the synthesis of a multitude of diverse derivatives with reported antimicrobial activities,
e.g. against Staphylococcus, Pseudomonas or Cladosporium sp. 8–12
Besides their activities against bacteria and fungi, 1, 1a and their semi-synthetic derivatives also
show potential in the fight against malaria and other protozoan diseases.13–15 While 1 itself is not
toxic for human cells14, several derivatives of 1 and 1a – including some of their amino acid
conjugates - exhibit moderate anti-proliferative properties against a panel of tumor cells lines.16,17
However, their effect on microorganisms has not been elucidated yet. In this paper, we investigated
the antibacterial and antifungal properties of a series of abietane derivatives containing amino acid
side chains.
Starting from 1 and 1a, a set of 9 derivatives was synthesized using carbodiimide coupling
conditions in the presence of the corresponding amino acid methyl ester hydrochlorides. Following
the synthesis, the abietanoyl amides were evaluated in several steps: we assessed the in vitro
antimicrobial activity against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria and
fungi (C. albicans) in a two-tiered screening assay. After a primary screening, the resulting hits
served as a starting point for the synthesis of additional 2 derivatives, for which we established a
dose–response relationship. In reference to the reports about cytotoxicity of 1 and 1a against tumor
cells (see above), all active compounds were also studied for their in vitro cytotoxicity against non-
cancerous mammalian cells. In order to provide a better overview over compound bioactivity, we
supplemented the data with additional explorative studies on their effect on opportunistic
microorganisms. A database survey on known similar compounds and their reported mode of action
completes this study.
2. Materials and methods
2.1. Chemistry
All reagents were obtained from Sigma Aldrich Co or TCI Europe if not otherwise stated. Abietic
acid 1 (75% purity) was obtained from Fluka. Dehydroabietic acid (1a, 90% purity) was obtained
from Pfaltz & Bauer, USA. Compounds 1 and 1a used herein for the biological assays were

recrystallized from methanol/water. (-)-2-Aminobutyric acid methyl ester hydrochloride was
obtained from Bachem (Weil am Rhein, Germany) and β-cyclohexyl-L-alanine methyl ester
hydrochloride was obtained from Novabiochem (Läufelfingen, Switzerland). For thin layer
chromatography (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 cm-1 resolution
between 4000 and 600 cm-1 using the OPUS 5.5 software (Bruker Optics Inc., MA, USA). NMR
spectra were obtained using a Varian Mercury Plus 300 spectrometer, in CDCl3, with
tetramethylsilane (TMS) as the internal standard. The chemical shifts were reported in parts per
million (ppm) and on the  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.
Methyl N-(abieta-7,13-dien-18-yl)glycinate (2). Abietic acid 1 (188 mg; 0.622 mmol) was
dissolved in DMF (2.5 mL), at room temperature. To this solution, a mixture of 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) hydrochloride (239 mg; 1.25 mmol) and N-
hydroxybenzotriazole (HOBt) monohydrate (169 mg; 1.25 mmol) was added. The resulting mixture
was left to agitate for 30 min. Glycine methyl ester hydrochloride (157 mg; 1.25 mmol) and N,N-
diisopropylethylamine (DIPEA, 0.44 mL; 2.5 mmol) were then added, and the reaction mixture was
left to agitate for another hour after which the reaction was complete. The reaction mixture was
suspended with diethyl ether (50 mL) and water (8 mL). The aqueous phase was further extracted
with diethyl ether (2 × 50 mL). The combined organic phases were washed with a 1 M aqueous
solution of HCl (20 mL), a saturated aqueous solution of NaHCO3 (20 mL), water (20 mL), and
brine (20 mL), dried with anhydrous Na2SO4, filtered, and evaporated to dryness to give a yellowish
oil (230 mg). It was purified by FCC using ethyl acetate:n-hexane (2.5:1) to yield compound 2 as a
white solid (130 mg, 56%). Mp 68-70 C. IR (ATR) 3374, 1749, 1640, 1520, 1207 cm-1. 1H-NMR
(300 MHz, CDCl3)  0.83 (s, 3H), 0.99 (m, 6H), 1.30 (s, 3H), 3.74 (s, 3H, OCH3), 4.00 (m, 2H, -
NHCH2), 5.33 (d, J = 4.3 Hz, 1H, 7-H), 5.74 (s, 1H, 14-H), 6.23 (m, 1H, NH). 13C-NMR (75 MHz,
CDCl3)  14.3, 17.1, 18.4, 21, 21.6, 22.7, 25.4, 27.6, 34.8, 35, 37.7, 38.5, 41.7, 45.9, 46.5, 51.1,

52.4, 120.7, 122.6, 135.7, 145.3, 170.9 and 178.8 (COOCH3 and CONH). HRMS: calcd. for
C23H36NO3 374.2695 [M+1]+, found 374.2697.
Methyl N-(abieta-7,13-dien-18-yl)-L-valinate (3). Following the procedure for the preparation of
2, compound 3 was prepared from 1 (375 mg; 1.28 mmol), EDC hydrochloride (460 mg; 2.4
mmol), HOBt monohydrate (324 mg; 2.40 mmol), L-valine methyl ester hydrochloride (402 mg;
2.40 mmol), and DIPEA (0.90 mL; 5.1 mmol) in DMF (5 mL). The obtained yellow oil (683 mg)
was purified by FCC using ethyl acetate:n-hexane (2.5:1) to give compound 3 (230 mg, 45%). Mp
55-57 C IR (ATR) 3370, 1740, 1645, 1514, 1209 cm-1. 1H-NMR (300 MHz, CDCl3)  0.83 (s,
3H), 0.91 (m, 6H), 0.99 (m, 6H), 1.30 (s, 3H), 3.71 (s, 3H, OCH3), 4.52 (dd, 1H, J1 = 8.2 Hz and J2
= 4.7 Hz -NHCH-), 5.33 (d, J = 4.9 Hz, 1H, 7-H), 5.74 (s, 1H, 14-H), 6.20 (d, J = 8.2 Hz, 1H, NH).
13C-NMR (75 MHz, CDCl3)  14.2, 17.1, 18.1, 18.4, 19.1, 20.9, 21.5, 22.6, 25.5, 27.5, 31.3, 34.7,
35, 37.8, 38.4, 45.6, 46.6, 51, 52.1, 57.3, 120.5, 122.6, 135.6, 145.2, 172.9 and 178.2 (COOCH3 and
CONH). HRMS: calcd. for C26H42NO3 416.3165 [M+1]+, found 416.3169.
Methyl N-(abieta-7,13-dien-18-yl)-L-ethylglycinate (4). Following the procedure for the
preparation of 2, compound 4 was prepared from 1 (375 mg; 1.28 mmol), EDC hydrochloride (460
mg; 2.4 mmol), HOBt monohydrate (324 mg; 2.40 mmol), L-aminobutyric acid methyl ester
hydrochloride (369 mg; 2.40 mmol), and DIPEA (0.90 mL; 5.1 mmol) in DMF (5 mL) as a yellow
solid (671 mg). Purification by FCC using ethyl acetate:n-hexane (2.5:1) gave compound 4 (189
mg, 38%). Mp 59-71 C IR (ATR) 3357, 1742, 1639, 1520, 1212 cm-1. 1H-NMR (300 MHz, CDCl3) 
0.83 (s, 3H), 0.89 (t, J = 7.5 Hz, 3H, -CH2CH3), 0.99 (m, 6H), 1.29 (s, 3H), 3.73 (s, 3H, OCH3), 4.53 (m, 1H,
-NHCH), 5.33 (m, 1H, 7-H), 5.74 (s, 1H, 14-H), 6.23 (d, J = 7.3 Hz, 1H, NH). 13C-NMR (75 MHz, CDCl3) 
9.8, 14.3, 17.1, 18.4, 21, 21.6, 22.6, 25.4, 25.6, 27.6, 34.8, 35, 37.7, 38.4, 45.8, 46.5, 51.1, 52.14, 53.6,
120.6, 122.6, 135.7, 145.3, 173.4 and 178.2 (COOCH3 and CONH). HRMS: calcd. for C25H40NO3
402.3008 [M+1]+, found 402.3010.
Methyl N-(abieta-7,13-dien-18-yl)-L-leucinate (5). Following the procedure for the preparation of
2, compound 5 was prepared from 1 (375 mg; 1.28 mmol), EDC hydrochloride (460 mg; 2.4
mmol), HOBt monohydrate (324 mg; 2.40 mmol), L-leucine methyl ester hydrochloride (436 mg;
2.40 mmol), and DIPEA (0.90 mL; 5.1 mmol) in DMF (5 mL) as a yellowish solid (580 mg).
Purification by FCC using ethyl acetate:n-hexane (2.5:1) gave compound 5 (150 mg, 28%). Mp 88-
90 C. IR (ATR) 3325, 1745, 1631, 1518, 1200 cm-1. 1H-NMR (300 MHz, CDCl3)  0.83 (s, 3H),
0.94 (dd, J1 = 6 Hz and J2 = 3.4 Hz, 6H, -CH2(CH3)2), 1.00 (m, 6H), 1.29 (s, 3H), 3.71 (s, 3H,
OCH3), 4.59 (m, 1H, -NHCH-), 5.34 (m, 1H, 7-H), 5.75 (s, 1H, 14-H), 6.06 (d, J = 7.8 Hz, 1H,

NH). 13C-NMR (75 MHz, CDCl3)  14.3, 17.1, 18.4, 21, 21.6, 22.2, 22.6, 23, 25.2, 25.4, 27.6, 34.8,
35, 37.8, 38.5, 41.8, 45.8, 46.5, 51.1, 52.3, 120.7, 122.6, 135.7, 145.3, 174 and 178.3 (COOCH3 and
CONH). HRMS: calcd. for C27H44NO3 430.3321 [M+1]+, found 430.3320.
Methyl N-(abieta-7,13-dien-18-yl)-L-phenylalaninate (6). Following the procedure for the
preparation of 2, compound 6 was prepared from 1 (375 mg; 1.28 mmol), EDC hydrochloride (460
mg; 2.4 mmol), HOBt monohydrate (324 mg; 2.40 mmol), L-phenylalanine methyl ester
hydrochloride (518 mg; 2.40 mmol), and DIPEA (0.90 mL; 5.1 mmol) in DMF (5 mL) as a yellow
solid (740 mg). Purification by FCC using ethyl acetate:n-hexane (2.5:1) gave compound 6 (170
mg, 30%). Mp 67-69 C. IR (ATR) 3361, 1749, 1628, 1521, 1211 cm-1. 1H-NMR (300 MHz,
CDCl3)  0.83 (s, 3H), 1.00 (m, 6H), 1.19 (s, 3H, 19-CH3), 3.10 (ddd, J1 = 20.7 Hz, J2 = 13.4 Hz,
and J3 = 6.3 Hz, 2H, CH2Ph), 3.71 (s, 3H, OCH3), 4.83 (m, 1H, NHCH), 5.26 (m, 1H, 7-H), 5.74 (s,
1H, 14-H), 6.09 (d, J1 =7.3 Hz, 1H, NH), 7.11 (m, 2H, aromatic-H), 7.28 (m, 3H, aromatic-H). 13C-
NMR (75 MHz, CDCl3)  14.3, 17, 18.4, 21, 21.6, 22.6, 25.3, 27.6, 34.8, 35, 37.7, 38.1, 38.4, 45.7,
46.5, 51.1, 52.4, 53.4, 120.7 (aromatic-C), 122.6 (aromatic-C), 127.3 (aromatic-C), 128.8 (aromatic-
C), 129.4 (aromatic-C), 135.6 (aromatic-C), 136.3 (aromatic-C), 145.2 (aromatic-C), 172.6 and
178.1 (COOCH3 and CONH). HRMS: calcd. for C30H42NO3 464.3165 [M+1]+, found 464.3166.
Methyl N-(abieta-7,13-dien-18-yl)cyclohexyl-L-alaninate (7). Following the procedure for the
preparation of 2, compound 7 was prepared from 1 (188 mg; 0.622 mmol), EDC hydrochloride (239
mg; 1.25 mmol), HOBt monohydrate (169 mg; 1.25 mmol), β-cyclohexyl-L-alanine methyl ester
hydrochloride (266 mg; 2.40 mmol), and DIPEA (0.44 mL; 2.5 mmol) in DMF (2.5 mL) as a
yellow oil (380 mg). Purification by FCC using ethyl acetate:n-hexane (2.5:1) gave compound 7
(141 mg, 48%). Mp 108-110 C. IR (ATR) 3374, 1744, 1634, 1521, 1202 cm-1. 1H-NMR (300
MHz, CDCl3)  0.84 (s, 3H), 1.00 (m, 6H), 1.29 (s, 3H), 3.71 (s, 3H, OCH3), 4.63 (m, 1H, NHCH-),
5.35 (m, 1H, 7-H), 5.75 (s, 1H, 14-H), 6.04 (d, J = 7.7Hz, 1H, NH). 13C-NMR (75 MHz, CDCl3) 
14.3, 17.1, 18.4, 21, 21.6, 22.6, 25.4, 26.3, 26.4, 26.5, 27.6, 32.7, 33.7, 34.6, 34.8, 35, 37.7, 38.5,
40.1, 45.8, 46.5, 50.4, 51.1, 52.4, 120.6, 122.6, 135.7, 145.3, 174.1 and 178.2 (COOCH3 and
CONH). HRMS: calcd. for C30H48NO3 470.3634 [M+1]+, found 470.3636.
Methyl N-(abieta-7,13-dien-18-yl)-L-serinate (8). Following the procedure for the preparation of
2, compound 8 was prepared from 1 (188 mg; 0.622 mmol), EDC hydrochloride (239 mg; 1.25
mmol), HOBt monohydrate (169 mg; 1.25 mmol), L-serine methyl ester hydrochloride (195 mg;
2.40 mmol), and DIPEA (0.44 mL; 2.5 mmol) in DMF (2.5 mL) as a yellow solid (239 mg).
Purification by FCC using ethyl acetate:n-hexane (2.5:1) gave compound 8 (80 mg, 32%). Mp 64-

66 C. IR (ATR) 3404, 1743, 1634, 1516, 1207 cm-1. 1H-NMR (300 MHz, CDCl3)  0.83 (s, 3H),
0.99 (m, 6H), 1.32 (s, 3H), 3.77 (s, 3H, OCH3), 3.92 (d, J = 4.1 Hz, 2H, CH2OH), 4.63 (m, 1H,
NHCH-), 5.34 (m, 1H, 7-H), 5.74 (s, 1H, 14-H), 6.71 (d, J = 5.9 Hz, 1H, NH). 13C-NMR (75 MHz,
CDCl3)  14.3, 17.1, 18.4, 21, 21.6, 22.6, 25.5, 27.6, 34.8, 35.1, 37.5, 38.4, 45.9, 46.6, 51.1, 53,
55.3, 63.9 (CH2OH), 120.5, 122.5, 135.7, 145.5, 171.3 and 179.4 (COOCH3 and CONH). HRMS:
calcd. for C24H38NO4 404.2801 [M+1]+, found 404.2803.
Methyl N-(abiet-8,11,13-trien-18-yl)-L-serinate (9). Following the procedure for the preparation
of 2, compound 9 was prepared from 1a (250 mg; 0.83 mmol), EDC hydrochloride (239 mg; 1.25
mmol), HOBt monohydrate (169 mg; 1.25 mmol), L-serine methyl ester hydrochloride (194 mg;
1.25 mmol), and DIPEA (0.44 mL; 2.5 mmol) in DMF (2.5 mL). Compound 9 (304 mg, 91%): Mp
103-104 C. IR (ATR) 3404, 1745, 1639, 1515, 1500, 1209, 821 cm-1. 1H-NMR (CDCl3)  1.21 (s,
3H), 1.23 (s, 3H), 1.24 (s, 3H), 1.32 (s, 3H), 2.14 (dd, J1 = 12.4 Hz and J2 = 2.1 Hz, 1H), 2.32 (d, J
= 11.4 Hz, 1H), 2.85 (m, 3H), 3.78 (s, 3H, OCH3), 3.93 (m, 2H, -CH2OH), 4.68 (m, 1H, -NHCH-),
6.73 (d, J = 7.1 Hz, 1H, NH), 6.87 (s, 1H, 14-H), 6.99 (dd, J1 = 8.2 Hz and J2 = 1.8 Hz, 1H,
aromatic-H), 7.16 (d, J = 8.2 Hz, 1H, aromatic-H). 13C-NMR (75 MHz, CDCl3)  16.3, 18.6, 21.1,
23.9, 25.2, 30, 33.4, 37.1, 37.2, 37.9, 45.6, 47.4, 52.6, 54.8, 63.6, 123.9 (aromatic-C), 124
(aromatic-C), 126.8 (aromatic-C), 134.5 (aromatic-C), 145.7 (aromatic-C), 146.7 (aromatic-C),
171.1 and 179.1 (COOCH3 and CONH). HRMS: calcd. for C24H36NO4 402.2644 [M+1]+, found
402.2641.
Methyl N-(abiet-8,11,13-trien-18-yl)-D-serinate (10). Following the procedure for the preparation
of 2, compound 10 was prepared from 1a (700 mg; 2.33 mmol), EDC hydrochloride (670 mg; 3.49
mmol), HOBt monohydrate (472 mg; 3.49 mmol), D-serine methyl ester hydrochloride (544 mg;
3.49 mmol) and DIPEA (1.22 mL; 6.93 mmol) in DMF (7 mL). Compound 10 (911 mg, 97%): Mp
52-53 C. IR (ATR) 3400, 1741, 1639, 1517, 1515, 1211, 821 cm-1. 1H-NMR (CDCl3)  1.20 (s,
3H), 1.23 (s, 3H), 1.23 (s, 3H), 1.32 (s, 3H), 2.13 (dd, J1 = 12.4 Hz and J2 = 1.9 Hz, 1H), 2.32 (d, J
= 12.7 Hz, 1H), 2.86 (m, 3H), 3.78 (s, 3H, OCH3), 3.92 (ddd, J1 = 24 Hz, J2 = 11.1, and J3 = 3.8 Hz,
2H, -CH2OH), 4.68 (m, 1H, -NHCH-), 6.73 (d, J = 7.1 Hz, 1H, NH), 6.86 (s, 1H, 14-H), 6.99 (dd,
J1 = 8.2 Hz and J2 = 1.6 Hz, 1H, aromatic-H), 7.16 (d, J = 8.2 Hz, 1H, aromatic-H). 13C-NMR (75
MHz, CDCl3)  16.3, 18.6, 21, 23.9, 23.9, 25.2, 30, 33.4, 37.1, 37.2, 37.9, 45.6, 47.4, 52.6, 54.8,
63.5, 123.9 (aromatic-C), 124 (aromatic-C), 126.8 (aromatic-C), 134.5 (aromatic-C), 145.7
(aromatic-C), 146.8 (aromatic-C), 171.2 and 179.1 (COOCH3 and CONH). HRMS: calcd. for
C24H36NO4 402.2644 [M+1]+, found 402.2643.

Compounds 2, 4, 7 and 10 are novel. For the bioactivity testing, all compounds were initially
dissolved in DMSO and, whenever possible, used as freshly prepared solutions to prevent
degradation.
2.2. Evaluation of antimicrobial activity
Antimicrobial activity was evaluated in a two-tiered screening set-up. In a primary screen, we
exposed three strains (E. coli, S. aureus and C. albicans) to the initial set of compounds.
Compounds that inhibited the growth by at least 75% were then confirmed in a secondary
evaluation, together with compounds 9 and 10. This step provided information about the dose–
response relationship. Both steps followed the CLSI and NCCLS broth microdilution protocols for
antimicrobial testing,18-19 and the assays were carried out at least in triplicate.
E. coli ATCC 25922, S. aureus ATCC 25923 and C. albicans ATCC 90028 were obtained from
Microbiologics Inc. (St. Cloud, MN). For the assays, bacteria were grown in Mueller-Hinton broth
(MHB, Becton Dickinson, Sparks, MD) overnight at 37 °C; C. albicans was cultured on Sabouraud
dextrose agar (SDA, Becton Dickinson) for 48 h at 30 °C. Compounds and controls were added to
the plate, and the microorganisms were then seeded into 96-well plates (Thermo Fisher Scientific,
Roskilde, DK) with inocula of 5×105 CFU/mL in MHB for bacteria and 1×104 CFU/mL in
RPMI 1640 (Gibco, Paisley, UK) for fungi. Each plate carried a negative control (media and
DMSO) and antimicrobial agent as positive control at MIC90 (S. aureus: ciprofloxacin 1 µg/mL;
E. coli: gentamicin 2 µg/mL; C. albicans: amphotericin B 16 µg/mL.
In the primary screening, all compounds were tested at a concentration of 50 µM and dose-response
experiments with a dilution series were carried out as secondary evaluation.
Plates were incubated for 24 h (C. albicans: 48 h), and absorbance was measured at 620 nm on a
Victor 1420 plate reader (PerkinElmer Life Sciences, Turku, Finland). The percentage of inhibition
was calculated by comparing the optical density at 620 nm (OD620) of the bacterial suspension with
and without the compound (negative control).
Since the compounds 9 and 10 showed activity against S. aureus, we carried out additional
experiments to determine the minimum inhibitory concentrations (MIC90) against S. aureus
ATCC 4330 (MRSA) and an array of microorganisms that are commonly responsible for food
poisoning and spoilage or dermatological and dental problems (

Cladosporium
cladosporioides ATCC 16022).
Assays were carried out in similar manner as described above following the CLSI and NCCLS
guidelines for broth microdilution methods.18–19. Dilution series of antimicrobial standards
chloramphenicol, tetracycline, ampicillin, clotrimazole, fluconazole, and nystatin were used as
positive controls in the assays (MIC90 values are presented in Table 2).
2.3. Cytotoxicity assays on mammalian cells
Cytotoxicity was assessed against the BALB/c 3T3 mouse embryonic fibroblast cell line, obtained
from ECACC (UK) and grown in DMEM (Sigma-Aldrich, USA), supplemented with 5% fetal
bovine serum (FBS, Gibco, USA), 5% new-born calf serum (NCS, Gibco, USA), 292 µg/mL L-
glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were incubated at 37 °C, 5%
of CO2, and 95% humidity. For the assays, cells were seeded into clear Nunc 96-well microplates
(Thermo Fisher Scientific, Roskilde, DK) at a density of 10,000 cells/well. After overnight
incubation, cells were exposed to the compounds for 24 h. Polymyxin B sulphate (15,000 IU/mL),
which is toxic for BALB/c 3T3 cells, was used as a positive control on every assay plate. The
compounds were diluted with assay media with 5% serum and 1% DMSO. Cytotoxicity of the
optimized compounds was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) and lactate dehydrogenase (LDH) assays. In the MTT assay, viable cells reduce MTT to the
purple-colored formazan, which can then be quantified.20 10 µL of MTT solution (5 mg/mL in
sterile PBS, pH 7.4) were added to each well, and the plate was incubated for 3 h. After removing
the media, the formazan crystals were dissolved in DMSO, and absorbance was measured at
550 nm and 655 nm (background). The LDH assay measures the release of LDH from cells with a
damaged cell membrane. The assay was carried out using CytoTox-ONE™ Homogeneous
Membrane Integrity Assay (Promega, Madison, WI) according to the manufacturer's instructions.21
Both assays were first carried out with four compound concentrations between 10-40 µg/mL, and
compounds exhibiting cytotoxicity in that range were then re-tested with eight concentrations
ranging from 10-80 µg/mL to determine IC50 values. The values were computed with OriginPro 8.6
(OriginLab Corp, Northampton, MA, USA).

2.4. Search for related compounds in online databases
In order to survey supplementary information about bioactivity of similar compounds, as well as to
postulate a possible mode of action, we searched the ChEMBL and PubChem databases for
chemically similar structures (similarity threshold of 90). This was done using ChEMBL web
services, but due to the technical limitations of the interface, some stereochemical properties of the
molecules could not be submitted. We examined the returned literature lists to gather information
about bioactivities reported for similar compounds and, if applicable, for their mode of action.
We also compared molecular fingerprints of the active compounds with known antibiotics. The
structures were retrieved from ChEMBL by searching for ATC code "J". We then calculated the
Tanimoto similarity between the optimized compound set and the known anti-infectives using
RDKit.
3. Results and discussion
Abietic acid (1) has been widely studied for its bioactivity, and our findings reinforce its position as
a promising parent structure for the synthesis of novel, bioactive compounds. They contribute to the
body of knowledge related to compound 1 and deepen the understanding of the potential and
properties of 1 and its derivatives.
3.1. Antimicrobial screening
As a response to the upcoming shortage of potent antibiotics, this study first and foremost aimed to
evaluate the antimicrobial potential of amino acid derivatives of abietic acid (1). Although the anti-
proliferative activity of amino acid-based derivatives of 1 has been previously reported,17,22 there is
yet no detailed study to address their potential antimicrobial properties. Moreover, our previous
work showed that the combination of amino acids with an abietane core, such as that of 1a, resulted
in potent antimicrobial and anti-biofilm agents,11,12 which suggests that this type of modification is
worth pursuing when designing compounds with potential antimicrobial effects. In previous
publications, abietic acid 1 and related compounds have been shown to be effective mostly against
Gram-positive bacteria.3,23 Some Gram-negative bacteria – including certain strains of E. coli –
have, however, been reported to show capability to degrade both 1 and dehydroabietic acid (1a).24
Based on these findings, it was not surprising that 1 itself revealed an – albeit modest – inhibitory

effect against S. aureus, but not against C. albicans or E. coli (Table 1). In the primary screen at
50 µM (20 µg/mL) concentration, 1 inhibited the growth of S. aureus by 44%, a value surpassed
only by compound 8 (76% inhibition). Compound 8 moderately inhibited also the growth of
C. albicans (39%). Based on the activity of 8 against S. aureus, we subsequently synthesized two
additional compounds, 9 and 10, starting from 1a instead of 1, in an attempt to generate potentially
more chemically stable compounds by replacing the conjugated diene system of 1 with the aromatic
ring of 1a. In addition, compound 9 bears the same L-serine based side-chain as compound 8
whereas compound 10 bears a D-serine based side-chain.
Table 1. Primary screening results for compounds 1-8. The table shows the inhibition of growth
(%) ± SD (n = 3) at 50 µM concentration after 24 h incubation (48 h for C. albicans). The growth
was determined by measuring the optical density at 620 nm. Antimicrobial agents were used as
positive controls at MIC90: S. aureus, ciprofloxacin 1 µg/mL; E. coli, gentamicin 2 µg/mL;
C. albicans, amphotericin B 16 µg/mL.
Compound
S. aureus
E. coli
C. albicans
ATCC 90028
-53 ± 0.31
ATCC 25923
45 ± 0.03
ATCC 25922
0 ± 0.05
1
2
3
4
5
6
7
8
13 ± 0.28
6 ± 0.02
8 ± 0.04
6 ± 0.06
11 ± 0.04
6 ± 0.04
6 ± 0.03
3 ± 0.03
9 ± 0.06
20 ± 0.13
0 ± 0.01
-12 ± 0.03
-9 ± 0.05
-1 ± 0.24
-8 ± 0.19
76 ± 0.04
23 ± 0.11
-8 ± 0.08
-13 ± 0.17
-35 ± 0.06
39 ± 0.07
MIC90 and IC50 values against S. aureus were determined for compounds 1, 8, 9, and 10. Compound
9 had a weaker antimicrobial activity (MIC90 >125 µg/mL) than compounds 1 and 8 (MIC90 = 63
and 61 µg/mL, respectively) against S. aureus, suggesting that the conjugated diene system of 1 is
important for activity. However, compound 10, the 1a derivative containing a D-serine methyl ester

side chain, had similar MIC90 (60 µg/mL) as 1 and 8. We also measured the activity against
methicillin-resistant S. aureus (MRSA), and it is noteworthy that compound 10 showed a 7.5-fold
stronger inhibition against the resistant strain (MIC90 8 µg/mL) than against S. aureus ATCC
25923. The MIC90 values of compounds 1 and 8 against MRSA were 31 µg/mL and 62 µg/mL,
respectively.
The demonstrated activity against S. aureus lead us to determine MIC90 values against an extended
set of microorganisms. The set included pathogens usually found in spoiled food, infected skin
and/or teeth, such as
. These additional screening results are summarized in Table 2. It shows
noteworthy activity of 10 against
and
90 8 µg/mL). Previous work has
described derivatives of 1 and 1a with comparable potency (MIC against S. aureus between 5 and
20 µM corresponding to 2-8 µg/mL,23,25), and it has been suggested that compounds isolated from
natural products could be considered interesting if their MIC lies below 10 µg/mL.26
Table 2. MIC90 values (µg/mL) against extended set of bacteria and fungi for compounds 1, 8, and
10 and antimicrobial agents used as positive controls.
S.
S.
P.
S.
R.
C.
Compound
typhimurium epidermidis acnes mitis mucilaginosa cladosporoides
31
8
4
16
31
63
1
>125
31
16
8
>125 >125 31
>125
>125
8
16
8
31
10
Ampicillin
0.5
2
0.125 2
Chloramphenicol 8
8
4
4
Tetracyclin
Clarithromycin
Fluconazole
Nystatin
16
1
2
1
0.5
0.25
0.5
520
64
0.125
In this study, the most promising candidate in terms of antimicrobial activity was compound 10,
which suggests beneficial influence of aromatic ring structure of 1a and D-serine methyl ester side
chain on bioactivity. Compound 10 shows good activity especially against S. aureus (MRSA),
S. epidermidis and S. mitis, but further studies would be required to elucidate in detail the observed
biological activities. Detailed mode of action studies for this particular type of derivatives have not

been reported. In general abietane-type compounds have been reported to interact with phospholipid
membranes and membrane-bound enzymes, particularly effectively disrupting cell membranes of
Gram-positive bacteria.11, 27-30
3.2. Cytotoxicity against mammalian cells
The quality of an antibiotic candidate is not only determined by its capability to affect bacteria, but
the compound must also be innocuous to human cells. Therefore, we conducted cytotoxicity
experiments against mammalian cells to estimate the selectivity of the compound effects between
microbial and mammalian cells.
The cytotoxicity of compounds 8-10 against non-cancerous BALB/c 3T3 cells was measured by
MTT and LDH assays, and the IC50 values were determined based on the results from the MTT
assay. IC50 values against BALB/c 3T3 cells were very similar: compound 8, 36 µg/mL; compound
9, 48 µg/mL; and compound 10, 45 µg/mL. With the IC50 values against bacterial cells (S. aureus)
in the same order of magnitude (21-30 µg/mL), there is no clear selectivity of the compounds
between microbial and mammalian cells.
Using the LDH assay, we measured the increase in leakage of intracellular LDH as compared to the
spontaneous release in untreated cells, which indicates the cell wall-damaging effect of the
compounds. At higher compound concentrations (9 and 10 > 50 µg/mL, 8 > 36 µg/mL), LDH
leakage starts to increase in a concentration-dependent manner, suggesting that the observed effects
for these compounds are very likely related to membrane disturbance (Supplementary material, Fig.
A.1.).
The reported cytotoxicity of abietic acid 1 derivatives varies, and cytotoxic compounds with IC50
values below 10 µM as well as relatively innocuous compounds with IC50 values above 100 µM
against both skin fibroblasts and human metastatic melanoma cells have been described.14 Within
this context, the cytotoxicity of our derivatives seems to be at moderate level. Still, cytotoxicity
against mammalian cells can be a serious unwanted effect of derivatives of 1, which highlights the
need for evaluating it as early as possible.
3.3. Bioactivity of related compounds
Knowing that a broad spectrum of bioactivities has been described for abietic acid 1 and related
compounds, we looked for entries of the compounds in this study in the online databases ChEMBL,

PubChem and ChemSpider. We also crawled ChEMBL for chemically related compounds in an
attempt to find background information on similar compounds. At the time of writing, neither
ChEMBL, PubChem nor ChemSpider held a record for any of the compounds. ChEMBL lists three
compounds with a similarity coefficient >90: two of them are similar to 2-7 or 8 and one to 9 and
10 (Table 3). Within the similar compounds, bioactivity has been reported only for entry
ChEMBL496686 (see Supplementary material, Fig. A.2.), described as a neuroprotective agent that
inhibits the activation of p38 MAPK and JNK in murine HT22 cells.31
In addition, based on our selection criteria, we downloaded the structures of 323 antibiotics from
ChEMBL. However, none of them showed a similar fingerprint to abietic acid 1 or its derivatives
(max value: 0.22). While a high similarity would have been useful for the elucidation of the mode
of action, the absence is also encouraging; 1 and/or its derivatives may act in a novel way to be
explored in future studies.
Table 3. Bioactivity of similar compounds retrieved from ChEMBL.a
ChEMBL ID/
PubChem CID
Similar to
Activity
PubChem Assay ID
397977
Neuroprotective effect against
iodoacetic acid-induced
ischemia
CHEMBL496686/
44592777
2, 3-7
Inhibition of p38 MAPK
activation in mouse HT22 cells 397981
Inhibition of JNK activation
397982
Inhibition of p38 MAPK
activation
397983
397984
Inhibition of JNK activation
CHEMBL496869/
44592778
2, 3-5, 8,
None

CHEMBL3289363/
90681715
None
9-10
aCompounds with a similarity >90 are listed.
4. Conclusions
In this study, we have identified the serine derivatives (8 and 10) of 1 and 1a with good
antimicrobial properties against Gram-positive bacteria. However, the compounds were poorly
selective towards bacterial cells and caused cell membrane damage at high concentrations by
inducing intracellular LDH leakage. The activity of compound 10 against important pathogens such
as MRSA, S. epidermidis and S. mitis was nonetheless noteworthy. Our results highlight the
potential of 1 and 1a as a starting point for the development of new antimicrobial agents. The risk to
induce resistance is low due to apparently fast-killing membrane targeting effects. Future efforts
should focus on optimization of their potency and selectivity, and on studying their activity against
a broader microbial spectrum as well as other pathologic targets.
Acknowledgements
This work was supported by the European Union Seventh Framework Programme FP7
WoodWisdom-Net+ under Academy of Finland grant agreement no. 256515 (GREASE) and
TUBITAK 110O951. AH and PT (Grant no. 277001 and 284477) and JYK (Grants no. 264020 and
265481) were also supported by the Academy of Finland. VMM thanks the Magnus Ehrnrooth
Foundation (project ME2012n44) for financial support.

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*Graphical Abstract (for review)
S. aureus MIC90
ATCC 25923: 60 µg/mL
MRSA: 8 µg/mL
Compound 10

Highlights



Novel derivatives of abietic and dehydroabietic acid were synthesized
Biological activity was evaluated against a panel of bacteria and fungi
Compound 10 is active against certain Gram-positive bacteria, including MRSA