Expansion of the structure-activity relationship of branched chain fatty acids: Effect of unsaturation and branching group size on anticancer activity

Article abstract of DOI:10.1016/j.chemphyslip.2020.104952

Branched chain fatty acids (BCFAs) are a class of fatty acid with promising anticancer activity. The BCFA 13-methyltetradecanoic acid (13-MTD) inhibits tumour growth in vivo without toxicity but efficacy is limited by moderate potency, a property shared b

Full text of DOI:10.1016/j.chemphyslip.2020.104952

ChemistryandPhysicsofLipids232(2020)104952
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Expansion of the structure-activity relationship of branched chain fatty
acids: Effect of unsaturation and branching group size on anticancer activity
Ritik Roy1, Ariane Roseblade1, Tristan Rawling*
School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, Sydney, NSW, 2007, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Branched chain fatty acids (BCFAs) are a class of fatty acid with promising anticancer activity. The BCFA 13-
methyltetradecanoic acid (13-MTD) inhibits tumour growth in vivo without toxicity but efficacy is limited by
moderate potency, a property shared by all known BCFAs. The mechanism of action of BCFAs has not been fully
elucidated, and in the absence of a clearly defined target optimisation of BCFA potency must rely on structure-
activity relationships. Our current understanding of the structural features that promote BCFA anticancer ac-
tivity is limited by the low structural diversity of reported BCFAs.The aim of this study was to examine the effects
of two new structural modifications- unsaturation and branching group size- on BCFA activity. Thus, homo-
logous series of saturated and cis-Δ11 unsaturated BCFAs were synthesised bearing methyl, ethyl, propyl and
butyl branching groups, and were screened in vitro for activity against three human cancer cell lines. Potencies of
the new BCFAs were compared to 13-MTD and an unbranched monounstaurated fatty acid (MUFA) bearing a cis-
Δ11 double bond. The principal findings to emerge were that the anticancer activity of BCFAs was adversly
affected by larger branching groups but significantly improved by incorporation of a cis-Δ11 double bond into
the BCFA alkyl chain. This study provides new structure-activity relationship insights that may be used to de-
velop BCFAs with improved potency and therapeutic potential.
branched chain fatty acid
13-methyltetradecanoic acid
breast cancer
MCF-7
structure activity relationship
1. Introduction
D35), lymphatic (Jurkat, EL4) and pancreatic (BxPC-3) cancer with IC50
values ranging from 26–159 μM (Cai et al., 2013; Lin et al., 2012;
Breast cancer is the second leading cause of cancer-relates deaths in
women, and there is a critical need to develop novel anticancer drugs to
treat cancers that are refractory to established anticancer agents.
Accumulating evidence suggests that lipids and fatty acids possess an-
ticancer actions that can be harnessed to develop new drugs to inhibit
tumour growth, and drug design strategies to overcome their challen-
ging physicochemical properties have yielded clinically approved drugs
(Collins and Djuric, 1993; Murray et al., 2014; Murray et al., 2015; Van
& Verheij, 2013). Thus, lipids and fatty acids are viable lead compounds
for cancer drug development.
Shioiri et al., 1998; Wongtangtintharn et al., 2005; Wongtangtintharn
et al., 2004; Yang et al., 2000). 13-MTD is well tolerated by mice and
oral delivery of 13-MTD (35-70 mg/kg/day) inhibited tumour growth
in mice carrying DU 145, LCI-D35, and Jurkat xenografts (Cai et al.,
2013; Yang et al., 2000). The naturally occurring anteiso-C15 analogue
of 13-MTD, 12-methyltetradecanoic acid (12-MTA), has also been
shown to inhibit proliferation of several cancer cell lines, including
prostate (DU145, PC3), lung (H1299, A549) and breast cancer (MCF-7),
with IC50 values ranging from 71 – 146 μM, and like 13-MTD, reduced
tumour growth in vivo without any apparent toxicity (Murray et al.,
2014; Wongtangtintharn et al., 2004; Wright et al., 2005; Yang et al.,
2003).
Branched chain fatty acids (BCFAs) are a naturally occurring class of
fatty acids with anticancer actions. The widely studied BCFA, 13-me-
thyltetradecanoic acid (13-MTD, Fig. 1), is an iso-C15 branched chain
fatty acid that was first isolated from a fermented soy bean product
marketed as a nutritional and therapeutic supplement. In cell-based
studies 13-MTD inhibited proliferation and induced apoptosis in several
human cancer cell lines, including breast (MCF-7, SK-BR-3), bladder
(T24, 5637), prostate (DU-145), colon (HCT-116), liver (SNU-423, LCI-
Although 13-MTD and 12-MTA possess promising in vivo activity,
moderate potency limits their efficacy and therapeutic potential.
Medicinal chemistry-based approaches to improve drug potency typi-
cally involve understanding the steric and electronic factors that govern
the interaction of a drug with its cellular target. Several studies of 13-
MTD have identified possible mechanisms of action such as disruption
⁎ Corresponding author: School of Mathematical and Physical Sciences, Faculty of Science, University of Technology Sydney, Ultimo, NSW, 2007, Australia.
E-mail address: Tristan.Rawling@uts.edu.au (T. Rawling).
1 Co-first authors.
https://doi.org/10.1016/j.chemphyslip.2020.104952
Received 31 March 2020; Received in revised form 11 August 2020; Accepted 12 August 2020
Availableonline16August2020
0009-3084/©2020ElsevierB.V.Allrightsreserved.

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
Fig. 1. Chemical structures of branched chain fatty acids used in this study.
of mitochondrial integrity, membrane fluidity, and fatty acid bio-
synthesis (Cai et al., 2013; Lin et al., 2012; Wongtangtintharn et al.,
2006; Wongtangtintharn et al., 2005; Yang et al., 2000), however the
precise cellular target of 13-MTD remains unknown. In the absence of a
defined target, a ligand-based approach to improve BCFA anticancer
activity must be undertaken, which involves the development of a
structure-activity relationship (SAR) that describes how drug activity is
affected by structural modifications.
stationary phase in a 70 mm (ID) × 60 mm (h) column. 12 × 50 mL
fractions were collected using gradient elutions of hexane:DCM. The
first fraction was 100:0 and the percentage of DCM was increased by
5% increments to 50% DCM. 1H and 13C NMR spectra were recorded on
an Agilent 500 MHz NMR. Spectra were referenced internally to re-
sidual solvent (CDCl3; 1H δ 7.26, 13C δ 77.10. DMSO-d6; 1H δ 2.49, 13C δ
39.52). High resolution mass spectra (HRMS) were recorded on an
Agilent Technologies 6510 Q-TOF LCMS.
Some SAR insights on the BCFA scaffold have emerged. A series of
C12-20 iso-BCFAs, which are structural analogues of 13-MTD but with
elongated or contracted alkyl chains, were assessed for activity against
MCF-7 breast cancer cells. The iso-C16 BCFA was the most potent in the
series and inhibited MCF-7 cell proliferation with an IC50 of ∼ 80 μM,
and potency decreased with an increase or decrease in chain length
(IC50 concentrations from ∼ 150 – 300 μM) (Wongtangtintharn et al.,
2004). Iso-BCFAs exhibit greater anticancer activity relative to their
anteiso-counterparts. In a direct comparison, 13-MTD exerted greater
anti-proliferative and pro-apoptotic effects than 12-MTA in MCF-7 cells
(Vahmani et al., 2019). Similarly, Iso-C17 inhibited MCF-7 cell pro-
liferation with an IC50 of ∼190μM, whereas the anteiso-C17 analogue
only reduced cell growth to ∼80% at the highest concentration tested
(400 μM) (Vahmani et al., 2019; Wongtangtintharn et al., 2004). Me-
thyl branching beyond the iso- and anteiso-positions, as well as multiple
methyl branching groups, have also been explored, however cytotoxic
Gas chromatography-mass spectrometry (GC-MS) was performed on
a
Shimadzu GCMS-QP2020 gas chromatograph-mass spectrometer
fitted with an SH-Rxi-5Sil MS silica fused capillary column (30.0 m ×
0.25 mm × 0.25 μm). Automated injections were performed on a
Shimadzu AOC-20i auto injector. The injection port temperature was
280 °C and 1.0 μL injections were made in splitless mode with a purge
time of 1 min. Helium was used as a carrier gas, at a flow rate of 1.42
mL/min. The column temperature was as follows: initial temperature of
40 °C for 2 min, then ramped at 6 °C/min to 320 °C, and held for 1 min.
Eluted compounds were subjected to methane chemical ionisation and
detection was made in full scan mode.
The purity of all test compounds was confirmed by absolute quan-
titative NMR (qNMR). Briefly, DMSO-d6 was spiked with the internal
calibrant (IC) 1,3,5-trioxane (99.5%) to give a final IC concentration of
2.92 mg/mL. Test compounds were accurately weighed on a 5-decimal
place analytical balance, dissolved in 600 μL of IC spiked DMSO-d6 and
transferred to 5 mm NMR tubes. 1H NMR spectra was recorded and the
average integration of one proton from the IC and test compound was
determined. The purity of the test compound (t) was then calculated
using the following equation:
activity against SK-BR-3 breast cancer cells remained modest (IC50
∼
300–350 μM) (Oku & Yanagita, 2009; Wongtangtintharn et al., 2006).
The SAR for BCFA anticancer activity is limited by the lack of
structural diversity in the BCFAs tested to date. All tested BCFAs possess
methyl branching groups at various positions along saturated alkyl
chains, and to our knowledge the effect of extended alkyl branching and
unsaturation on anticancer activity has not been investigated. In this
paper we report on the synthesis of a homologous series of saturated
and unsaturated BCFAs, in which terminally branched methyl groups
are replaced with ethyl, propyl and butyl moieties (Fig. 1). The antic-
ancer activity of these new BCFAs were assessed against MCF-7, MDA-
MB-231 and HeLa cancer cell lines and compared to 13-MTD, and the
results were used to gain SAR insights that we anticipate will assist in
the design of new and more potent BCFAs.
Integralareat × MW × masst
t
× Purity
IC
IntegralareaIC × MW × massIC
IC
2.2. Methyl 11-bromoundecanoate (2)
To a mixture of 1 (6.00 g, 22.63 mmol) in methanol (300 mL) was
added AcCl (8.69 g, 113.13 mmol). The resulting solution was left to
stir at room temperature for 4 h. Excess methanol was removed under
pressure and the residue was dissolved in DCM (80 mL). The organic
phase was washed with saturated NaHCO3 in water (100 mL), water
(100 mL) and brine (100 mL) and dried over MgSO4. The solution was
concentrated in vacuo affording methyl ester 2 as a yellow oil (5.88 g,
93%) that was used without further purification. HRMS (ESI) m/z [M
+H]+ calcd for C12H23O2Br 279.0954; found 279.0946. 1H and 13C
NMR spectra were in agreement with previously reported data (Jana
et al., 2018).
2. Material and methods
2.1. General chemistry
11-Bromoundecanoic acid was purchased from Fluorochem
(Hadfield, Derbyshire, UK). All other reagents and anhydrous solvents
were purchased from Sigma Aldrich (Castle Hill, NSW, Australia).
Reactions were monitored by thin-layer chromatography (TLC) using
silica gel 60 F254 plates. TLC plates were visualised with UV light and
potassium permanganate TLC stain. Reaction products were purified by
dry column vacuum chromatography with Merck silica gel 60H as the
2.3. (11-methoxy-11-oxoundecyl)(triphenyl)phosphonium bromide (3)
Ester 2 (5.88 g, 21.06 mmol) was left to stir with PPh3 (5.52 g,
21.06 mmol) at 140 °C for 20 h. The resulting mixture was dissolved in
2

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
DCM (5 mL) and cold diethyl ether (15 mL) was added to form a pre-
cipitate. The solid was collected by vacuum filtration and washed with
cold diethyl ether (2 × 15 mL) and hexane (3 × 15 mL). The product
was dried in vacuo, affording 3 as a white solid (10.41 g, 91%) that was
used without further purification. Mp = 111-113 °C. HRMS (ESI) m/z
[M]+ calcd for C30H38O2P 461.2598; found 461.2599. 1H and 13C NMR
spectra were in agreement with previously reported data (Dejarlais &
Emken, 1978).
2.4.5. Methyl (Z)-tetradec-11-enoate (5e)
Yellow oil, yield: 86%. 1H NMR (500 MHz, CDCl3) δ 5.39-5.31 (m, 2
H), 3.66 (s, 3 H), 2.30 (t, J =7.5 Hz, 2 H), 2.06-1.99 (m, 4 H), 1.62
(quint, J =7 Hz, 2 H), 1.37-1.25 (m, 12 H), 0.95 (t, J =7.5 Hz, 3 H). 13
C
NMR (125 MHz, CDCl3) δ 175.83, 131.34, 128.85, 51.38, 33.98, 29.75,
29.46, 29.35, 29.23, 29.22, 29.06, 27.02, 24.68, 20.55, 14.48. Z:E ratio
= 100.0 : 0.0. HRMS (ESI) m/z [M+H]+ calcd for C15H28O2 241.2162;
found 241.2168.
2.5. General synthesis of the monounsaturated branched fatty acids 6a-d
2.4. General synthesis of the monounsaturated methyl esters 5a-d
To a solution of the unsaturated methyl ester (5a-d, 0.20 mmol) in
EtOH (6 mL) was added 1.5 M NaOH (8 mL). The reaction was heated
to 40 °C and stirred for four hours. The volume of the reaction mixture
was reduced to 10 mL by rotary evaporation and the solution was ad-
justed to pH 1 with 1.0 M HCl. The solution was extracted with EtOAc
(3 × 50 mL) and the combined extracts were dried over MgSO4. The
EtOAc was removed in vacuo to afford unsaturated fatty acids 6a-d in
high purity (> 95%) without further purification.
To a suspension of 3 (1.500 g, 2.77 mmol) in anhydrous THF (10
mL) at 0 °C under a nitrogen atmosphere was added NaN(TMS)2 (1.0 M
in THF, 2.77 mmol). The resulting bright orange mixture was stirred for
20 minutes at room temperature. The reaction mixture was then cooled
to −78 °C, and the aldehyde (4a-d, 1.85 mmol) in THF (3 mL) was
added dropwise. The mixture was stirred for 30 minutes at −78 °C after
which the reaction was allowed to warm to room temperature for two
hours. After further stirring for two hours at room temperature the
reaction was quenched with saturated aqueous NH4Cl (30 mL) and
extracted with EtOAc (3 × 50 mL). The combined extracts were dried
over MgSO4 and the EtOAc was removed in vacuo. The residue was
2.5.1. (Z)-13-methyltetradec-11-enoic acid (6a)
Yellow oil, yield = 93%. 1H NMR (500 MHz, CDCl3) δ 5.25-5.15 (m,
2 H), 2.61-2.54 (m, 2 H), 2.35 (t, J =7.5 Hz, 2 H), 2.01 (q, J =7.5 Hz, 2
H), 1.63 (quint, J =7.5 Hz, 2 H), 1.37-1.23 (m, 12 H), 0.94 (d, J =7.0
Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 179.65, 137.49, 127.46, 33.99,
29.89, 29.44, 29.38, 29.24, 29.21, 29.04, 27.28, 26.42, 24.67, 23.22.
FT-IR vmax (neat) 2923 (O-H stretch), 2853 (C-H stretch), 1707 (C = O
stretch), 1463 (C-H bend), 1411 (O-H bend), 722 (C = C bend) cm-1.
HRMS (ESI) m/z [M+H]+ calcd for C15H29O2 241.2162; found
241.2163. qNMR purity = 95%.
purified on silica gel using
a stepwise gradient elution with
hexane:DCM (100:0 to 50:50) to yield the unsaturated methyl esters 5a-
d.
2.4.1. Methyl (Z)-13-methyltetradec-11-enoate (5a)
Yellow oil, yield = 42%. 13C NMR (125 MHz, CDCl3) δ 174.31,
137.46, 127.46, 51.40, 34.09, 29.88, 29.43, 29.39, 29.24, 29.22, 29.12,
27.27, 26.41, 24.93, 23.21. Z:E ratio = 97.8 : 2.2. HRMS (ESI) m/z [M
+H]+ calcd for C16H31O2 255.2319; found 255.2318. The 1H spectrum
was in agreement with previously reported data (Foglia and Vail,
1993).
2.5.2. (Z)-13-ethylpentadec-11-enoic acid (6b)
Yellow oil, yield = 97%. 1H NMR (500 MHz, CDCl3) δ 5.40 (dt, J =
10.5, 2.5 Hz, 1 H), 5.01 (t, J =7.5 Hz, 1 H), 2.35 (t, J =7.5 Hz, 2 H),
2.16-2.07 (m, 1 H), 2.01 (q, J =7.5 Hz, 2 H), 1.63 (quint, J =7.5 Hz, 2
H), 1.44-1.37 (m, 2 H), 1.37-1.21 (m, 12 H), 1.20-1.09 (m, 2 H), 0.83 (t,
J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 178.74, 134.45, 130.06,
40.63, 33.82, 29.89, 29.69, 29.45, 29.39, 29.32, 29.21, 29.05, 28.31,
27.69, 24.69. 22.69, 14.12, 11.87. FT-IR vmax (neat) 2922 (O-H stretch),
2853 (C-H stretch), 1708 (C = O stretch), 1456 (C-H bend), 1412 (O-H
bend), 723 (C = C bend) cm-1. HRMS (ESI) m/z [M+H]+ calcd for
2.4.2. Methyl (Z)-13-ethylpentadec-11-enoate (5b)
Yellow oil, yield = 75%. 1H NMR (500 MHz, CDCl3) δ 5.39 (dt, J =
10.5, 2.5 Hz, 1 H), 5.00 (t, J =7.5 Hz, 1 H), 3.66 (s, 3 H), 2.30 (t, J
=7.5 Hz, 2 H), 2.16-2.06 (m, 1 H), 2.00 (q, J =7.5 Hz, 2 H), 1.61
(quint, J =7.5 Hz, 2 H), 1.43-1.36 (m, 2 H), 1.35-1.24 (m, 12 H), 1.19-
1.10 (m, 2 H), 0.82 (t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ
174.32, 134.43, 130.04, 51.42, 40.62, 34.10, 29.88, 29.44, 29.40,
29.30, 29.22, 29.13, 28.30, 27.68, 24.94, 11.86. Z:E ratio = 100.0 : 0.0.
HRMS (ESI) m/z [M+H]+ calcd for C18H35O2 283.2632; found
283.2634.
C17H33O2 269.2475; found 269.2472. qNMR purity = 97%.
2.5.3. (Z)-13-propylhexadec-11-enoic acid (6c)
Yellow oil, yield = 90%. 1H NMR (500 MHz, CDCl3) δ 5.34 (dt, J =
10.5, 2.5 Hz, 1 H), 5.01 (t, J =7.5 Hz, 1 H), 2.35 (t, J =7.5 Hz, 2 H),
2.33-2.27 (m, 1 H), 2.01 (q, J =7.5 Hz, 2 H), 1.63 (quint, J =7.5 Hz, 2
H), 1.39-1.25 (m, 16 H), 1.24-1.19 (m, 2 H), 1.18-1.09 (m, 2 H), 0.86 (t,
J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 179.17, 135.08, 129.47,
38.28, 36.69, 33.89, 29.92, 29.70, 29.62, 29.51, 29.45, 29.40, 29.34,
29.22, 29.16, 29.07, 28.95, 27.71, 24.69, 20.46, 14.32. FT-IR vmax
(neat) 2922 (O-H stretch), 2853 (C-H stretch), 1708 (C = O stretch),
1465 (C-H bend), 1413 (O-H bend), 722 (C = C bend) cm-1. HRMS
(ESI) m/z [M+H]+ calcd for C19H37O2 297.2788; found 297.2786.
qNMR purity = 97%.
2.4.3. Methyl (Z)-13-propylhexadec-11-enoate (5c)
Yellow oil, yield = 21%. 1H NMR (500 MHz, CDCl3) δ 5.34 (dt, J =
10.5, 2.5 Hz, 1 H), 5.01 (t, J =7.5 Hz, 1 H), 3.66 (s, 3 H), 2.30 (t, J
=7.5 Hz, 2 H), 2.00 (q, J =7.5 Hz, 2 H), 1.61 (quint, J =7.5 Hz, 2 H),
1.34-1.25 (m, 12 H), 1.24-1.17 (m, 2 H), 1.16-1.08 (m, 2 H), 0.86 (t, J
=7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 174.33, 135.06, 129.47,
51.42, 38.26, 36.68, 34.11, 29.91, 29.44, 29.40, 29.33, 29.23, 29.15,
27.69, 24.95, 20.45, 14.30. Z:E ratio = 100.0 : 0.0. HRMS (ESI) m/z [M
+H]+ calcd for C20H39O2 311.2945; found 311.2947.
2.5.4. (Z)-13-butylheptadec-11-enoic acid (6d)
2.4.4. Methyl (Z)-13-butylheptadec-11-enoate (5d)
Yellow oil, yield = 81%. 1H NMR (500 MHz, CDCl3) δ 5.35 (dt, J
=10.5 Hz, 2.5 Hz, 1 H), 5.02 (t, J =7.5 Hz, 1 H), 2.35 (t, J =7.5 Hz, 2
H), 2.30-2.21 (m, 1 H), 2.01 (q, J =7.5 Hz, 2 H), 1.63 (quint, J =7.5
Hz, 2 H), 1.38-1.22 (m, 20 H), 1.21-1.09 (m, 4 H), 0.87 (t, J =7.5 Hz, 6
H). 13C NMR (125 MHz, CDCl3) δ 179.34, 135.19, 129.44, 37.08, 35.68,
33.91, 29.89, 29.65, 29.62, 29.47, 29.40, 29.34, 29.23, 29.06, 27.70,
24.67, 22.92, 14.12. FT-IR vmax (neat) 2922 (O-H stretch), 2853 (C-H
stretch), 1708 (C = O stretch), 1465 (C-H bend), 1411 (O-H bend), 724
(C = C bend) cm-1. HRMS (ESI) m/z [M+H]+ calcd for C21H41O2
Yellow oil, yield = 43%. 1H NMR (500 MHz, CDCl3) δ 5.34 (dt, J =
10.5, 2.5 Hz, 1 H), 5.01 (t, J =7.5 Hz, 1 H), 3.66 (s, 3 H), 2.30 (t, J
=7.5 Hz, 2 H), 2.28-2.21 (m, 1 H), 2.00 (q, J =7.5 Hz, 2 H), 1.61
(quint, J =7.5 Hz, 2 H), 1.36-1.21 (m, 20 H), 1.20-1.09 (m, 4 H), 0.91-
84 (m, 6 H). 13C NMR (125 MHz, CDCl3) δ 174.32, 135.18, 129.44,
51.42, 37.08, 35.68, 34.11, 29.90, 29.62, 29.47, 29.41, 29.34, 29.24,
29.15, 27. 70, 24.95, 22. 92, 14.12. Z:E ratio = 99.7 : 0.3. HRMS (ESI)
m/z [M+H]+ calcd for 339.3258; found 339.3255.
3

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
325.3101; found 325.3103. qNMR purity = 96%.
2.7.2. 13-ethylpentadecanoic acid (8b)
Yellow oil, yield = 96%. 1H NMR (500 MHz, CDCl3) δ 2.35 (t, J
=7.5 Hz, 2 H), 1.63 (quint, J =7.5 Hz, 2 H), 1.37-1.20 (m, 21 H), 1.19-
1.11 (m, 2 H), 0.83 (t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ
178.57, 40.38, 33.79, 32.76, 30.13, 29.70, 29.65, 29.59, 29.43, 29.24,
29.06, 26.77, 25.44, 24.69. FT-IR vmax (neat) 2922 (O-H stretch), 2853
(C-H stretch), 1708 (C = O stretch), 1460 (C-H bend), 1412 (O-H bend)
cm-1. HRMS (ESI) m/z [M+H]+ calcd for C17H35O2 271.2632; found
271.2635. qNMR purity = 96%.
2.5.5. (Z)-tetradec-11-enoic acid (6e)
Yellow oil, yield: 85%. HRMS (ESI) m/z [M+H]+ calcd for
C
14H26O2 227.2006; found 227.2004. qNMR purity = 99%. 1H and 13
C
NMR spectra were in agreement with previously reported data (Wube
et al., 2011).
2.6. General synthesis of the saturated methyl esters 7a-d
To a mixture of the monounsaturated methyl ester (5a-d, 0.65
mmol) in EtOH (10 mL) under a hydrogen atmosphere was added Pd/C
(10 wt. %, 0.20 mmol). The suspension was stirred for 20 hours at room
temperature, after which the mixture was filtered over celite and wa-
shed with EtOH (2 × 25 mL). The EtOH was removed in vacuo and the
residue was purified on silica gel with a stepwise gradient elution with
hexane:DCM (100:0 to 50:50) to yield 7a-d.
2.7.3. Characterization of 13-propylhexadecanoic acid (8c)
Yellow oil, yield = 96%. 1H NMR (500 MHz, CDCl3) δ 2.35 (t, J
=7.5 Hz, 2 H), 1.63 (quint, J =7.5 Hz, 2 H), 1.40-1.17 (m, 27 H), 0.87
(t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 1.78.73, 36.94,
36.10, 33.81, 33.67, 30.14, 29.69, 29.65, 29.61, 29.58, 29.50, 29.42,
29.35, 29.23, 29.15, 29.06, 29.67, 24.69, 19.80, 14.54. FT-IR vmax
(neat) 2923 (O-H stretch), 2853 (C-H stretch), 1710 (C = O stretch),
1465 (C-H bend), 1418 (O-H bend) cm-1. HRMS (ESI) m/z [M+H]+
calcd for C19H39O2 299.2945; found 299.2946. qNMR purity = 97%.
2.6.1. Methyl 13-methyltetradecanoate (7a)
Yellow oil, yield = 87%. 1H NMR (500 MHz, CDCl3) δ 3.66 (s, 3 H),
2.30 (t, J =7.5 Hz, 2 H), 1.61 (quint, J =7.5 Hz, 2 H), 1.58-1.44 (m, 1
H), 1.36-1.25 (m, 16 H), 1.17-1.12 (m, 2 H), 0.85 (d, J =7.5 Hz, 6 H).
13C NMR (125 MHz, CDCl3) δ 174.34, 51.42, 34.12, 29.92, 29.69,
29.63, 29.58, 29.44, 29.25, 29.14, 27.96, 27.40, 24.95, 22.65. HRMS
(ESI) m/z [M+H]+ calcd for C16H33O2 252.2475; found 252.2478.
2.7.4. Characterization of 13-butylheptadecanoic acid (8d)
Yellow oil, yield = 89%. 1H NMR (500 MHz, CDCl3) δ 2.35 (t, J
=7.5 Hz, 2 H), 1.63 (quint, J =7.5 Hz, 2 H), 1.41-1.17 (m, 31 H), 0.88
(t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 178.77, 37.35, 33.81,
33.70, 33.38, 30.14, 29.69, 29.65, 29.58, 29.43, 29.32, 29.05, 28.96,
26.70, 24.68, 23.16, 14.17. FT-IR vmax (neat) 2921 (O-H stretch), 2852
(C-H stretch), 1709 (C = O stretch), 1465 (C-H bend), 1412 (O-H bend)
cm-1. HRMS (ESI) m/z [M+H]+ calcd for C21H43O2 327.3258; found
327.3256. qNMR purity = 96%.
2.6.2. Methyl 13-ethylpentadecanoate (7b)
Yellow oil, yield = 68%. 1H NMR (500 MHz, CDCl3) δ 3.67 (s, 3 H),
2.30 (t, J =7.5 Hz, 2 H), 1.61 (quint, J =7.5 Hz, 2 H), 1.35-1.19 (m, 21
H), 1.18-1.11 (m, 2 H), 0.83 (t, J =7.5 Hz, 6 H). 13C NMR (125 MHz,
CDCl3) δ 174.34, 51.41, 40.36, 34.11, 32.75, 30.13, 29.69, 29.64,
29.59, 29.44, 29.25, 29.14, 26.76, 25.43, 24.95, 10.91. HRMS (ESI) m/
z [M+H]+ calcd for C18H37O2 285.2788; found 285.2787.
2.8. General cell culture
Fetal bovine serum and trypsin/EDTA were purchased from
(Invitrogen, Life Technologies, Victoria, Australia). Dulbecco's Modified
Eagle Medium (DMEM), penicillin and streptomycin, phosphate-buf-
fered saline (PBS), DMSO and general biochemicals were purchased
from Sigma-Aldrich (Castle Hill, NSW, Australia). Human tumour cell
lines were obtained from ATCC (Manassas, VA) and were grown at 37
°C in a humidified atmosphere of 5% CO2 in air in DMEM supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin.
Confluent cells (80–90%) were harvested using Trypsin/EDTA after
washing in PBS.
2.6.3. Methyl 13-propylhexadecanoate (7c)
Yellow oil, yield = 83%. 1H NMR (500 MHz, CDCl3) δ 3.66 (s, 3 H),
2.30 (t, J =7.5 Hz, 2 H), 1.61 (quint, J =7.5 Hz, 2 H), 1.28-1.15 (m, 27
H), 0.87 (t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 174.34,
51.42, 36.94, 36.10, 34.12, 33.67, 30.15, 29.69, 29.65, 29.59, 29.44,
29.25, 29.15, 26.67, 24.95, 19.79, 14.54. HRMS (ESI) m/z [M+H]+
calcd for C20H41O2 313.3101; found 313.3103.
2.6.4. Methyl 13-butylheptadecanoate (7d)
Yellow oil, yield = 44%. 1H NMR (500 MHz, CDCl3) δ 3.66 (s, 3 H),
2.30 (t, J =7.5 Hz, 2 H), 1.61 (quint, J =7.5 Hz, 2 H), 1.33-1.15 (m, 31
H), 0.89 (t, J =7.5 Hz, 6 H). 13C NMR (125 MHz, CDCl3) δ 174.32,
51.41, 37.34, 33.70, 33.38, 30.14, 29.69, 29.65, 29.59, 29.44, 29.25,
29.15, 28.96, 26.70, 24.95, 23.16, 14.16. HRMS (ESI) m/z [M+H]+
calcd for C22H45O2 341.3414; found 341.3415.
2.9. Cell viability assay
The anticancer activity of the synthesized compounds was de-
termined using the MTS assay. Cells were seeded in 96-well plates (5 ×
103 cells per well), 24 h later serum was removed and cells were treated
with various concentrations of the test compounds, in DMSO (final
concentration 0.1%), for 48 h. Control cells received serum-free DMEM
and DMSO at 0.1%.
2.7. General procedure for synthesis of saturated branched fatty acids 8b-d
Cell viability was determined using the CellTiter 96® AQueous One
Solution Cell Proliferation Assay (Promega, USA) according to the
manufacturer’s recommendation. IC50 values were defined as the drug
concentration that prevented cell growth of more than 50%
(relative to the vehicle control) and were determined using non-
linear regression analysis with Prism 7.0 (GraphPad Software, CA,
USA).
To a mixture of the saturated methyl ester (7a-d, 0.20 mmol) in
EtOH (6 mL) was added 1.5 M NaOH (8 mL). The reaction was heated
to 40 °C and stirred for four hours. The volume of the reaction mixture
was reduced to 10 mL by rotary evaporation and the solution was ad-
justed to pH 1 with 1.0 M HCl. The white solid was extracted with
EtOAc (3 × 50 mL) and the combined extracts were dried over MgSO4.
EtOAc was removed in vacuo to afford 13-MTD and 8b-d in high purity
(> 95%) without further purification.
2.10. Statistical analysis
2.7.1. 13-methyltetradecanoic acid (13-MTD)
For in vitro assays, data were analysed using one-way analysis of
variance (ANOVA) followed by Tukey’s post hoc analysis. P < 0.05
were accepted as being statistically significant. All statistical analyses
were performed using GraphPad Prism version 7.0 (GraphPad Software,
CA, USA). The results are expressed as the means SEM unless
White solid, yield = 89%. Mp = 47-49 °C (lit. Mp = 46.5-47 °C)
(Shioiri et al., 1998). FT-IR, HRMS, 1H and 13C NMR spectra were in
agreement with previously reported data (Khan et al., 2018). qNMR
purity = 98%.
4

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ChemistryandPhysicsofLipids232(2020)104952
Scheme 1. Synthesis of the fatty acids 13-MTD 8b-d and 6a-e. Reagents and conditions (i) AcCl, methanol, rt, 4 h; (ii) PPh3, 140 °C, 20 h; (iii) NaN(TMS)2, THF, -78 °C
to rt, 2 h; (iv)1.5 M NaOH, ethanol, 40 °C, 4 h; (v) Pd/C, hydrogen, rt, 20 h.
otherwise specified.
mass spectra. Based on the elution order (Aro et al., 1998; Rawling
et al., 2010), the first compounds to elute were identified as the trans-
isomers (Rt ∼ 31.88 min), which were baseline separated from the cis-
isomers (Rt ∼ 31.98 min). The peak areas were then used to calculate
the Z:E ratio, which in all cases exceed 97:3.
3. Results
3.1. Synthesis
Olefins 5a-d were then divided into two sets of aliquots, and alka-
line hydrolysis of the ester groups in one of the sets afforded un-
saturated BCFAs 6a-d (Fig. 1). The double bonds in the remaining 5a-d
aliquots were reduced with palladium and hydrogen to give saturated
BCFA esters 7a-d. Finally, removal of the ester protecting groups
yielded BCFAs 13-MTD and 8b-d (see Supplementary Material for 1H
and 13C NMR spectra).
Saturated (13-MTD and 8b-d) and unsaturated (6a-d) BCFAs were
synthesised as shown in Scheme 1. In the first step the carboxylic acid
group in bromo fatty acid 1 was ester-protected using acetyl chloride
(AcCl) and methanol. Under these mild reaction conditions acetyl
chloride generates HCl in situ, which catalyses the esterification reac-
tion. Methyl ester 2 was then reacted with triphenyl phosphine in a
solvent-free melt to generate triphenylphosphonium bromide 3, which
was then used in Wittig reactions with commercially available alde-
hydes 4a-d to yield unsaturated branched fatty acid esters 5a-d with cis-
double bonds in the Δ11 positions. To produce the desired cis-olefins
the Wittig reactions were conducted in THF at -78 °C with sodium bis
(trimethylsilyl)amide as the base. These reaction conditions were
chosen because they have been shown to favour formation of cis-olefins
(Z:E ratio ≥ 97:3) (Ainai et al., 2003; Ito et al., 2006; Rawling et al.,
2010). To confirm that the Wittig reactions proceeded with the desired
selectivity, the Z:E ratio of 5a-d was determined by GC–MS. The gas-
chromatograms of these compounds contained two peaks with identical
To assess the role of the methyl branching group and cis-Δ11 double
bond in the anticancer activity of 6a, we also prepared the mono-
unsaturated fatty acid (MUFA) 6e, which is the straight chain analogue
of 6a. 6e was synthesised following the synthetic procedure outlined in
Scheme 1. The Wittig reaction of phosphonium bromide 3 and straight
chain aldehyde 4e affording the ester protect MUFA 5e with a cis-Δ11
double bond (Z:E ratio confirmed by GC-MS analysis). Base-catalysed
hydrolysis of the 5e with sodium hydroxide provided 6e in high purity.
5

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
3.2. Cell culture conditions
Prior to testing the all synthetic BCFAs against the full panel of
cancer cell lines, we first developed assay conditions using 13-MTD,
which is reported to reduce MCF-7 cell viability with IC50 concentra-
tions of 10.03 μg/ml (∼41 μM) and ∼150 μM in media containing 10%
fetal bovine serum (FBS) (Wongtangtintharn et al., 2004; Yang et al.,
2000). In our hands, 13-MTD failed to affect MCF-7 cell viability at
concentrations up to 500 μM after 72 h under these conditions. The
same result was found with 13-MTD synthesised by us or purchased
commercially. One important difference between our assay conditions
and those used for 13-MTD, and indeed all other previously reported
anticancer BFCAs, was our choice of DMSO as the drug delivery vehicle
rather than Tween 80 (Cai et al., 2013; Lin et al., 2012; Shioiri et al.,
1998; Wongtangtintharn et al., 2006; Wongtangtintharn et al., 2005;
Wongtangtintharn et al., 2004; Yang et al., 2000). Tween 80 has been
shown to potentiate the in vitro effects of some anticancer agents by
permeabilising the cell membrane (Riehm and Biedler, 1972), and it is
possible Tween 80 also potentiates BFCA anticancer activity in vitro. It
is important to note that these observations do not invalidate the an-
ticancer actions of 13-MTD and other BCFAs, as indeed 13-MTD is ac-
tive in vivo (Yang et al., 2000). They do however indicate that Tween 80
is not an ideal vehicle for in vitro anticancer assays.
We next attempted cell viability assays with DMSO as vehicle but in
serum free media (SFM), as SFM has been used previously in assessing
the anticancer activity of the BCFA 12-methyltetradecanoic acid (Yang
et al., 2003). In SFM we found that 13-MTD significantly reduced MCF-
7 cell viability at concentrations ≥ 100 μM (Fig. 2a). To confirm that
the cells were viable and proliferative SFM we conducted cell counts
from the time of serum withdrawal to the end of the 48 h treatment
period. We found that MCF-7 cell number increased 1.5 fold over the
treatment period. Similarly, the other two cell lines used in this study,
MDA-MB-231 and HeLa cells, grew 1.9 fold and 2.2 fold, respectively in
SFM over 48 h. Thus, all cell viability assays were performed in SFM for
48 h with DMSO as delivery vehicle.
3.3. In vitro anticancer activity
The in vitro anticancer activities of the test fatty acids were first
assessed against MCF-7 cells. Dose-response curves were constructed
(Fig. 2) and IC50 concentrations were determined (Table 1). We also
calculated Hill slopes (HS) and maximum effects (Emax) for each com-
pound as these metrics can provide mechanistic information (Fallahi-
Sichani et al., 2013).
Fig. 2. Dose-response curves showing the effects of synthetic BCFAs on the
viability of MCF-7 breast cancer cells after 48 h treatment. A) 13-MTD, B) sa-
turated BCFAs 8b-d, C) unsaturated BCFAs 6a-d. Data represents the
mean SEM of at least
3 independent experiments. ** P < 0.01, ****
P < 0.0001, comparison of the indicated compound to the vehicle control.
In MCF-7 cells, 13-MTD was the most potent in the series of satu-
rated BCFAs (13-MTD, 8b-d) and reduced MCF-7 viability with an IC50
of 135 13 μM (Table 1, Fig. 2a). IC50 concentrations for the re-
maining saturated BCFAs were not able to be calculated due to lim-
itations in the solubilities of these compounds at concentrations greater
than 900 μM. From the dose-response curves obtained (Fig. 2b), the
ethyl-branched analogue 8b displayed modest activity and was able to
reduce MCF-7 cellular viability to 49 7% at 900 μM. Propyl-branched
BCFA 8c only affected cellular viability at 900 μM (76 4%), and the
butyl-branched analogue 8d did not affect cellular viability at any of
the test concentrations.
We also assessed the anticancer activity of the BCFAs against a
wider panel of cancer cell lines (Table 1 and Fig. 3). In HeLa cervical
cancer cells the unsaturated and methyl branched BCFA 6a was the
most active in the series and reduced cell viability with an IC50 of
80.7 6.1 μM. All remaining BCFAs including 13-MTD were much less
active that 6aand were only able to reduce cell viability to 70–90% of
control at 900 μM (Fig S1 and Table S1 in Supplementary Material).
A similar pattern of activity emerged when the BFCAs were tested
against MDA-MB-231 breast cancer cells. Thus 6a fully abolished cell
viability (Emax = 0) with an IC50 of 534 39 μM (Fig. 3) while 13-MTD
and the other BCFAs had no effect on cell viability at concentrations up
to 900 μM (Table S2 in Supplementary Material).
Unlike the saturated BCFAs 8b-d, all of the unsaturated BCFA’s 6a-d
were active against MCF-7 cells. Although 6a-d reduced MCF-7 viability
with similar IC50 values, the shapes of the dose-response curves re-
vealed differences between these compounds (Fig. 2c). Methyl, ethyl
and propyl branched compounds 6a-c displayed relatively steep dose-
MUFA 6e, the straight-chain analogue of 6a, was also assessed for
anticancer activity in all three cell lines (Fig. 4). It was observed that 6e
reduced the viability of all cell lines with IC50 concentrations ranging
from 86–158 μM. Comparisons of the IC50 concentrations of 6a and 6e
in each cell line revealed that 6a was approximately 1.5–2 fold more
active that its unbranched counterpart in MCF-7 and HeLa cells, while
in MDA-MB-231 cells 6e was 3.4 fold more active. Viability of MCF-7
cells was fully abolished by 6e (Emax = 0.0), however Emax values for
response curves with Hill slopes (HS) > 2.8 and maximum effect (Emax
)
values of 0, reflecting the ability of 6a-c to completely abolish MCF-7
cell viability (Table 1). In contrast, the dose-response curve of the butyl-
branched analogue 6d was shallow (HS = -1.6) and the cell viability
did not drop below 32% as the concentration of 6d was increased from
150-450 μM (Emax = 0.3).
6

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
Table 1
IC50 concentrations, Hill Slopes (HS) and maximum effect (Emax) of fatty acids against the MCF-7, MDA-MB-231 and HeLa cancer cell linesa.
MCF-7
MDA-MB-231
HeLa
Fatty Acid
IC50 [μM]
HS
Emax
IC50 [μM]
HS
Emax
IC50 [μM]
HS
Emax
13-MTD
8b
135 13
> 750
−3.1
–
0.0
–
Inactivec
Inactivec
Inactivec
Inactivec
534 39
Inactivec
Inactivec
Inactivec
158 42
–
–
> 900
> 900
–
–
–
–
–
–
8c
> 900
–
–
–
–
> 900
–
–
8d
> 900
–
–
–
–
> 900
–
–
6a
49.4 4.8
58.9 9.2
51.8 6.1
47.6 3.1
86.4 9.3
−6.4
−3.2
−2.8
−1.6
−2.3
0.0
0.0
0.0
0.3
0.0
−2.6
0.0
80.7 6.1
> 900
−1.5
0.0
6b
–
–
–
–
6c
–
–
> 900
–
–
6d
–
–
> 900
–
–
6e
−2.2
N/Ab
119 13
−1.2
N/Ab
a
b
c
Cell viability was measured using the MTS assay after 48 h of treatment.
N/A: not applicable. Emax values for 6e in these cell lines could not be calculated because 6e was not soluble in cell media at concentration above 200 μM.
No effect on cell viability at concentrations up to 900 μM.
In considering this, we developed two homologous series of BCFAs
based on the structure of 13-MTD- one fully saturated series, and a
corresponding unsaturated series possessing cis-Δ11 double bonds- and
assessed their anticancer activity against three cancer cell lines.
4.1. Effects on MCF-7 cell viability
13-MTD, a widely studied iso-BCFA, inhibits cell growth in various
cancer cell lines both in vitro and in vivo (Cai et al., 2013; Lin et al.,
2012; Wongtangtintharn et al., 2005; Yang et al., 2000). 13-MTD has
previously been reported to reduce MCF-7 cell viability with an IC50
concentrations of ∼41 μM and ∼150 μM (Wongtangtintharn et al.,
Fig. 3. Dose-response curves showing the effects of unsaturated BCFA 6a on the
viability of MDA-MB-231 and HeLa cancer cells after 48 h treatment. Data re-
presents the mean SEM of at least 3 independent experiments.
2004), and using the assay conditions developed for this study we found
similar levels of cytotoxicity (cf. IC50 135 μM, Table 1). In contrast, 13-
MTD was essentially inactive in both MDA-MB-231 and HeLa cells.
Within the saturated series, 13-MTD was the only BCFA to significantly
affect cellular viability at or below 300 μM. As the size of the branching
group was increased to ethyl (8b) and propyl (8c) chains, the associated
activity decreased (Fig. 2b). Indeed, the butyl-branched analogue 8d
was inactive at all test concentrations. These findings clearly illustrate a
preference for smaller methyl-branching groups in saturated BCFAs.
The unsaturated BCFAs 6a-d were all active and reduced MCF-7
viability. This activity pattern indicates that the activity of unsaturated
BCFAs are not as sensitive to branching group size as their saturated
counterparts. Interestingly, while 6a-d displayed similar IC50 con-
centrations, differences in the dose-response curve of butyl-branched
6d were observed. The HS values for 6a-d were within the range re-
ported for a wide panel of anticancer drugs against MCF-7 cells (Fallahi-
Sichani et al., 2013), however 6d had a relatively low HS and high Emax
relative to 6a-c. We saw no evidence that the high Emax value of 6d
resulted from precipitation in the assays. Indeed, similar shallow dose-
response curves with Emax values > 0 have been reported for the clin-
ical anticancer drugs docetaxel and geldanamycin in cell viability as-
says using the MCF-7 cell line (Fallahi-Sichani et al., 2013). Thus, the
differences in HS and Emax values calculated for 6d suggest that 6d
reduces MCF-7 cell viability by a different mechanism of action to 6a-c
(Fallahi-Sichani et al., 2013), which may arise from the increased bulk
provided by the butyl branching-group in 6d causing it to interact with
different cellular target/s to that of 6a-c. Follow-up mechanistic studies
could assess the ability of 6a-d to inhibit COX-2 activity as MUFAs with
anticancer activity have been shown to reduce COX-2 mediated pros-
taglandin E2 (PGE2) synthesis (Cui et al., 2012).
Fig. 4. Dose-response curves showing the effects of MUFA 6e on the viability of
MCF-7, MDA-MB-231 and HeLa cancer cells after 48 h treatment. Data re-
presents the mean SEM of at least 3 independent experiments.
6e against MDA-MB-231 and HeLa cells could not be determined be-
cause 6e was not soluble in cell media at concentrations above 200 μM.
4. Discussion
The development of new anticancer drugs with novel mechanisms
of action and low toxicity remains an ongoing need in cancer treatment.
BCFAs are structurally distinct from current oncology drugs, well-tol-
erated in mice and inhibit tumour growth in vivo, and thus present an
interesting opportunity for drug development (Murray et al., 2015). The
precise cellular target/s of anticancer BCFAs are not known, and
medicinal chemistry approaches to improve potency must rely on the
synthesis and testing of BCFA analogues, and derivation of an SAR to
guide drug design. To date, antiproliferative studies have exclusively
examined saturated BCFAs with methyl branching-groups and reported
gains in potency have been moderate (Wongtangtintharn et al., 2004).
4.2. Effects on MDA-MB-231 and HeLa cell viability
13-MTD has been shown to reduce the viability of over 10 cancer
cell lines, however the anticancer activity of 13-MTD and other BCFAs
has not been studied using MDA-MB-231 and HeLa cancer cell lines. In
7

R. Roy, et al.
ChemistryandPhysicsofLipids232(2020)104952
this study we found that these two cell lines are much more resistant
than MCF-7 cells to BCFA anticancer activity. 13-MTD was essentially
inactive against both cell lines, and this lack of activity was also ob-
served for all saturated BCFAs (8b-d) and unsatutared BCFAs 6b-d. The
only exception was the unsaturated and methyl-branched BCFA 6a,
which was active across all three cell lines. Comparison of the IC50
concentrations showed that MCF-7 cells were most sensitive to 6a,
followed by HeLa and then MDA-MB-231 cells. Consistent with this
pattern of activity, HeLa cell viability was moderately reduced by the
remaining BCFAs at 900 μM, while MDA-MB-231 cells were unaffected
at all test concentrations.
online version, at doi:https://doi.org/10.1016/j.chemphyslip.2020.
104952.
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Author Contributions
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TR conceived of the study. RR, AR and TR designed experiments,
analysed data and wrote the manuscript. RR synthesised the branched
fatty acids and AR carried out cell culture and anticancer testing.
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Acknowledgements
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This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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