Novel 2-arylthiopropanoyl-CoA inhibitors of α-methylacyl-CoA racemase 1A (AMACR; P504S) as potential anti-prostate cancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103263

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity (IC₅₀ = 22–100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively correlates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy)phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity against LnCaP, Du145 and PC3 cells at 500 μM, but lacked activity at 100 μM. This is the first extensive structure-activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.103263

BioorganicChemistry92(2019)103263
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel 2-arylthiopropanoyl-CoA inhibitors of α-methylacyl-CoA racemase 1A
(AMACR; P504S) as potential anti-prostate cancer agents
Maksims Yevglevskisa, Amit Nathubhaia,b, Katty Waddaa,c, Guat L. Leea, Suzanne Al-Rawia,
Tingying Jiaoa,d, Paul J. Mitchella, Tony D. Jamesc, Michael D. Threadgilla,
⁎
Timothy J. Woodmana, Matthew D. Lloyda,
a Drug & Target Discovery, Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
b University of Sunderland, School of Pharmacy and Pharmaceutical Sciences, Sciences Complex, Sunderland SR1 3SD, UK1
c Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
d School of Pharmaceutical Sciences, Shandong University, Jinan, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain
fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target
and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report
the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity
(IC50 = 22–100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively cor-
relates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy)
phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid
precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent
Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity
against LnCaP, Du145 and PC3 cells at 500 µM, but lacked activity at 100 µM. This is the first extensive structure-
activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed
AMACR inhibitors.
α-Methylacyl-CoA racemase (AMACR, P504S)
Branched-chain fatty acid metabolism
Castrate-resistant prostate cancer (CRPC)
Drug lipophilicity
Enzyme inhibitors
Ibuprofen
Mixed competitive inhibition
Rational drug design
Structure-activity relationships
1. Introduction
methylacyl-CoAs are generated. Oxidation of cholesterol to bile acids
also results in the formation of R-2-methylacyl-CoAs (with the chiral
Branched-chain fatty acids, e.g. phytanic and pristanic acids, are
common components of the human diet [1–3]. Degradation of these
fatty acids occurs as the corresponding acyl-CoA, initially within per-
oxisomes and subsequently within mitochondria [3,4]. Phytanic acid
possesses a 3-methyl group, which hinders degradation by β-oxidation
[1–4]. Therefore, initial metabolism is via the α-oxidation pathway,
resulting in the formation of pristanic acid, which possesses a 2-methyl
group, which is subsequently converted into pristanoyl-CoA. The ste-
reochemical configuration of chiral centres in phytanic acid are re-
tained in the α-oxidation pathway [3] and this means that R-2-
centre at carbon-25 in the standard numbering system for bile acids)
[1,2]. The acyl-CoA oxidases catalysing the first step in the β-oxidation
degradative pathway requires S-2-methylacyl-CoAs [5–7] and cannot
accept R-2-methylacyl-CoAs. This means that 2R-pristanoyl-CoA, its β-
oxidation pathway products and the acyl-CoA derivatives of bile acids
cannot be directly metabolized by β-oxidation.
The enzyme α-methylacyl-CoA racemase (AMACR; P504S; E.C.
5.1.99.4) catalyses conversion of these R-2-methylacyl-CoAs to S-2-
methylacyl-CoAs, thus enabling β-oxidation. The reaction appears to
occur by a deprotonation/reprotonation mechanism [8–10], probably
Abbreviations: AD, androgen-dependent; AI, androgen-independent; AR, androgen receptor; AMACR, α-methylacyl-CoA racemase (a.k.a. P504S, E.C. 5.1.99.4);
CRPC, castrate-resistant prostate cancer; CoA, coenzyme A; DIAD, Diisopropyl azodicarboxylate; DMF, dimethylformamide; DPBS, Dulbecco’s phosphate buffered
saline; EDTA, ethylenediaminetetraacetic acid; EtOAc, ethyl acetate; FBS, foetal bovine serum; MeOH, methanol; MtMCR, M. tuberculosis 2-methylacyl-CoA racemase;
MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCa, prostate cancer; PE, petroleum ether; S.E.M., Standard
Error of the Mean
⁎ Corresponding author.
E-mail address: M.D.Lloyd@bath.ac.uk (M.D. Lloyd).
1 Current address.
https://doi.org/10.1016/j.bioorg.2019.103263
Received 20 March 2019; Received in revised form 4 September 2019; Accepted 5 September 2019
Availableonline07September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
through an enolate intermediate [11], and a near 1:1 mixture of S- and
R-2-methylacyl-CoA epimers is produced (‘racemization’) [9,10,12].
The resulting S-2-methylacyl-CoAs are degraded by β-oxidation [1–4],
while the resulting R-2-methylacyl-CoAs are further metabolized to
their S-epimers by AMACR, thus ensuring complete metabolism. In
addition to its role in fatty acid metabolism, AMACR is also involved in
the irreversible pathway which converts R-ibuprofen to S-ibuprofen via
the corresponding acyl-CoA esters [1,2,10]. As only the S-enantiomer of
ibuprofen is a potent inhibitor of cyclooxygenases-1 and -2 [13], this
pathway results in the pharmacological activation of the inactive R-
ibuprofen. Several other ‘profens’ also undergo similar R- to S-conver-
sion in humans and other mammals [1,2,10]. AMACR has also been
claimed to be involved in the S- to R- conversion of mandelic acid in
mammalian cells [14], but this role was subsequently disproved [15].
There has been increased attention towards targeting AMACR as a
chemotherapeutic strategy to combat prostate cancer (PCa). AMACR
activity is increased by 4- to 10-fold in clinical prostate cancer tissue
samples, compared with the corresponding normal tissue [16,17].
Many spliced variants of AMACR have been reported [18–21], with the
most highly expressed spliced variant, AMACR 1A, demonstrated to
have ‘racemase’ activity [9,12]. Several other spliced variants of
AMACR are predicted not to possess racemase activity, as they lack key
catalytic residues or a C-terminal dimerization domain [1,2,18–21].
Knockdown of AMACR 1A significantly reduces the proliferation of
several androgen-dependent (AD) [17,22] and androgen-independent
(AI) [23] PCa cell lines at similar levels to that observed for the current
standard of care (androgen-deprivation) or androgen-receptor (AR)
antagonists (Casodex) [17]. Additionally, knock-down of AMACR has
shown to revert advanced PCa cells from AI to AD status by post-
transcriptional upregulation of the AR [23]. Many investigations, e.g.
Takahara et al. [23], demonstrate that AMACR is over-expressed at
significantly higher levels in AI PCa cells compared to AD PCa cells,
which has stimulated great interest in exploring AMACR as a PCa bio-
marker [1,24–27] and drug target [22,24,28–33]. Inhibitors of AMACR
could be exploited as mono-therapeutic agents or used in combination
with androgen-deprivation therapy to target CRPC [17,23].
reported to date; however, numerous structures of ligands bound to the
M. tuberculosis homologue (MtMCR) [8,11] reveal the binding mode of
several known substrates/inhibitors. There is 43% sequence identity
between MtMCR and AMACR 1A [22] (Supplementary Information, Fig.
S1), including residues comprising active site catalytic bases, residues
binding to the CoA moiety, and other active site residues. Therefore,
structural information can be used to help design rational inhibitors of
AMACR.
These MtMCR structures suggest that AMACR 1A contains a well-
defined binding site for the CoA moiety, which is lined by cationic ar-
ginine and lysine residues binding to the phosphate groups. The methyl
group is thought to bind in a well-defined pocket, and this hypothesis is
supported by the observation that the presence of an α-methyl group
[30] (or similar group [29]) enhances inhibition of AMACR, compared
to corresponding analogues that contain a hydrogen atom at the same
position. Similarly, biochemical studies show that 2-methylacyl-CoA
substrates are better substrates than those lacking the α-methyl group
[37]. The α-carbon of the 2-methylacyl-CoA substrate is positioned so
that the active site bases (His-122/Glu-241 and Asp-152; numbering
refers to human AMACR 1A) are located either side of the α-carbon in
order to perform the deprotonation/reprotonation reaction effectively.
Two overlapping side-chain binding sites, termed the R- and S-pockets,
accommodate substrate/inhibitor side-chains and this probably ac-
counts for the observed differences in potency between different epi-
mers of 2-methylacyl-CoA-like inhibitors [28]. This led to the design of
gem-disubstituted substrate-product analogues, which are thought to
inhibit MtMCR by simultaneous binding of side-chains to both the R-
and S-binding sites [31].
Previous work on AMACR have attempted to develop inhibitors by
increasing the acidity of the α-proton [28] or by mimicking the planar
enolate intermediate [24,29,30,36]. The first inhibitors with highly
acidic α-protons contained fluorine [28] but these were subsequently
shown to be substrates which eliminate HF and form the corresponding
unsaturated analogue [12,30,35]. We therefore decided to prepare a
focused set of 2-arylthiopropanoyl-CoA analogues (‘thiolactate in-
hibitors’), in the expectation that these would form stabilized enolates
which would bind tightly to AMACR. Previous work on 3-thia analo-
gues of straight-chain acyl-CoA oxidase substrates confirm a diminution
of the pKa of the α-proton by ca. 5 units [38–41] (∼16 for 3-thiaoc-
tanoyl-CoA vs. ∼ 21 for octanoyl-CoA [38]), supporting this approach.
The target compounds were synthesized as epimeric mixtures at the
2-position, as formation of the enolate and loss of stereochemical con-
figuration was anticipated to be facile. In parallel, we also investigated
the corresponding sulfone analogues, reasoning that the sulfone group
would promote formation of the enolate even more strongly, potentially
resulting in even more potent inhibition. Fenoprofenoyl-CoA 1 pro-
vided the starting side-chain structure, as this was previously shown to
be the best of the series of substrates with aromatic side-chains [10] and
is a known inhibitor [30].
Most previously reported AMACR inhibitors have been developed
using rational design, based on substrate structures and the enzyme
catalytic mechanism [24,28–30,33]. Inhibitors of the M. tuberculosis
homologue enzyme (MtMCR), for which X-ray crystal structures have
been solved [8,11,34], include the gem-disubstituted substrate-product
analogues 2,2-bis(4-(2-methylpropyl)lphenyl)propanoyl-CoA and 2,2-
bis(4-tert-butylphenyl)propanoyl-CoA, which inhibit MtMCR with Ki
values of 16.9
0.6 and 21
4 µM, respectively [32]. Carbamate
analogues of these inhibitors have recently been reported to be irre-
versible inhibitors of MtMCR [31]. However, until recently, there has
been no convenient assay to measure AMACR activity or evaluate the
potency of inhibitors, and this has hampered inhibitor development
[35,36]. We recently developed a continuous assay which is convenient
to use for inhibitor characterization, using a chromogenic substrate
which produces the intensely yellow 2,4-dinitrophenolate anion upon
incubation with AMACR [30,36].
2.2. Synthesis of inhibitors
Herein, we report the synthesis of
a
panel of novel 2-ar-
Synthesis of initially designed inhibitors used the reaction of 3- or 4-
phenoxybenzenethiol 1 with ethyl ( )-2-bromopropanoate to afford
the ethyl esters 2 (Scheme 1). The required phenoxybenzenethiols 1
have been synthesized from the corresponding phenoxyphenols 3 by
treatment with dimethylthiocarbamoyl chloride [42] to give 4. A
Newman-Kwart thermal rearrangement was subsequently used to pro-
vide the required carbamothioate 5. Only low levels of conversion were
observed at 260 °C [43], while extensive decomposition occurred at
300 °C. A compromise temperature of 280 °C gave the required product
in moderate yield whilst minimizing decomposition. Hydrolysis of 5
furnished the required phenoxybenzenethiols 1 but these rapidly oxi-
dized to the corresponding disulfides so they were treated with
ethyl 2-bromopropanoate without purification. The resulting esters 2
were hydrolyzed to the acids 6, which were activated with N,N′-
ylthiopropanoyl-CoA derivatives (‘thiolactate inhibitors’). These in-
hibitors were tested in vitro against human AMACR 1A in the first
systematic examination of rational inhibitor side-chain interactions and
their influence on inhibitor potency. We then examined selected ex-
amples of the carboxylic acid precursors of these inhibitors against a
panel of AD- and AI-PCa cell lines to evaluate their potential as treat-
ments for prostate cancer.
2. Results and discussion
2.1. Rational design of inhibitors
The X-ray crystal structure of mammalian AMACR has not been
2

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
Scheme 1. Synthesis of 2-arylthiopropanoyl-CoAs 7 and 2-arylsulfonylpropanoyl-CoAs 10. a: R1 = PhO, R2 = H; b: R1 = H, R2 = PhO. Reagents and conditions: i.
dimethylthiocarbamoyl chloride, DMF, ii. Δ; iii. NaOH, H2O, MeOH; iv. K2CO3, ethyl ( )-2-bromopropanoate, MeCN; iv. CDI, CH2Cl2; v. CoA-Li+3 , NaHCO3, H2O,
THF; vi. OXONE®, H2O. Note that no product was obtained for 10a.
carbonyl diimidazole and converted into their corresponding acyl-CoA
esters 7 [10,12,30,36,44].
diminution in inhibitory activity was unexpected, as 10b was expected
to undergo exchange of the α-proton even more readily than the thia
analogues and hence was predicted to bind to AMACR even more
tightly. This loss of inhibitory activity correlates with a reduction in
lipophilicity [46] (Table 1, Fig. 1, Supporting Information Fig. S4),
suggesting that potency is largely driven by side-chain interactions ra-
ther than formation of the enolate. Further experiments showed that all
three inhibitors gave rise to rapidly reversible inhibition (Supporting
Information).
Synthesis of the sulfone analogues was achieved by oxidation of the
ethyl ester intermediate 2 (Scheme 1). OXONE® treatment [45] of 2
resulted in formation of the sulfone ethyl esters 8 in ca. 80% yield,
which were subsequently hydrolyzed to the acids 9 with aqueous base
in high yield. Conversion of the 3-substituted-acid 9a to the corre-
sponding acyl-CoA 10a was attempted using the standard carbonyl
diimidazole method [10,12,30,36,44] but this did not result in the
desired product. This failure may be due to the low nucleophilicity of
the carboxylate anion of 9a, as further experiments showed low yields
of the intermediate acyl-imidazole. Alternatively, formation of the en-
olate may be facile under the conditions used to make the acyl-CoA and
this results in side-reactions or depletion of base resulting in low levels
of the required imidazole intermediate. Similarly, use of a mixed an-
hydride method [9,24] did not give 10a and starting material was re-
covered. Activation of the isomeric carboxylic acid 9b with carbonyl
diimidazole and coupling with reduced CoA [10,12,30,36,44] resulted
in formation of the corresponding acyl-CoA ester 10b in low yield.
Preparation of further 2-arylthiopropanoyl-CoA derivatives 7c-o
(Scheme 2) began with selective alkylation of a mercaptophenol 11
with ethyl ( )-2-bromopropanoate to provide the corresponding ethyl
The further series of 3- and 4-substituted 2-arylthiopropanoyl-CoAs
7c-o were then tested as inhibitors of AMACR activity (Table 1). As
expected, potency was generally correlated with drug (side-chain) li-
pophilicity, although there were exceptions (Fig. 1 and Supporting
Information Figs. S3 and S4). This is consistent with the proposed
model in which the inhibitor side-chain binds to the methionine-rich
surface (‘side-chain binding site’), as observed in the X-ray crystal
structures [8] of ligands bound to the M. tuberculosis homologue, MCR.
In the 3-substituted series, the most potent inhibition was observed
for the benzyl derivative 7c (IC50 = 22.3 nM) and this was far more
potent than expected based on calculated lipophilicity (Fig. S3). Addi-
tion of a 3-MeO group to the benzyl to give 7d decreased potency to the
level predicted by the lipophilicity. Extending the side-chain with a
further CH2- group in 7e did not make a significant difference in po-
tency, compared to 7d. Addition of a MeO- group to the terminal aro-
matic ring in 7f gave a small improvement in potency. However, the
(
)-2-(hydroxyphenylthio)propanoates 12. Alkylation of the phenolic
oxygen of 12 with benzyl or alkyl bromide derivatives provided a series
of substituted benzyloxy or alkoxy esters 2c-o. Hydrolyses of the esters
revealed the corresponding carboxylic acids 6c-o, which were con-
verted into their CoA esters 7c-o using N,N′-carbonyl diimidazole.
expected increase in potency upon addition of
a lipophilic tri-
fluoromethyl- group was not observed and 7g had a similar level of
potency as 7f (Fig. S3). Compound 7h, substituted with a 3-(2-me-
thylpropoxy) group, was less potent, as expected based on lipophilicity
calculations.
2.3. Evaluation of compounds in vitro
In contrast, substitutions at the 4-position gave potent inhibition for
almost all derivatives. Extension of the linker from 7b (no CH2- groups)
to 7i (one CH2- group) resulted in an increase in potency
(IC50 = 354 nM vs. 113 nM). Addition of a MeO-group to the terminal
Initially compounds 7a and 7b were tested using our deuterium-
exchange assay [9,10] (Supporting information, Fig. S2). Significant
exchange of 7a/7b α-1H for α-2H was observed in negative controls
containing heat-inactivated AMACR, showing that formation of the
enolate occurred rapidly. In the case of the 3-substituted isomer, 7a ca.
70% exchange was observed in the absence of active AMACR. Incuba-
tion in the presence of active AMACR resulted in even greater levels of
exchange for the 4-substituted isomer 7b, indicating that this com-
pound was a substrate. In the case of the 3-substituted isomer 7a,
limited further exchange was observed against the high non-enzymatic
background levels showing that it was also a substrate.
aromatic ring in 7j made
a small difference to the potency
(IC50 = 71 nM vs. 113 nM]. Analogue 7k had similar potency
[IC50 = 99 nM vs. 113 nM] to 7i, showing that increasing the linker by a
further CH2 group did not have a significant effect, as observed for the
corresponding regioisomers. Substitution of the terminal phenyl ring
with a MeO- group in 7l or with a CF3 group in 7m gave similar levels of
inhibitory potency, again showing that inhibitor potency was not in-
creased as much as expected upon introduction of the lipophilic tri-
fluoromethyl- group. 4-Substitution with an aliphatic pentoxyl chain
also resulted in potent inhibition, as shown by 7n with an IC50 value of
90 nM, consistent with predictions based on lipophilicity. Surprisingly,
substitution with the smaller 2-methylpropoxy- group in 7o gave five-
fold more potent inhibition than in the 3-substituted analogue 7h
(IC50 = 106 nM vs. 522 nM).
Inhibition of AMACR activity was subsequently evaluated using our
colorimetric assay [30,36]. Initial evaluation showed that 7a and 7b
were moderately potent inhibitors, with IC50 values of 247 nM and
354 nM, respectively. Fenoprofenoyl-CoA 1 showed an IC50 value of
340 nM (consistent with the literature report of 400 nM [30]). Thus,
both phenoxy-phenylthiopropanoyl-CoAs 7a and 7b were inhibitors of
comparable potency to fenoprofenoyl-CoA 1. In contrast, the 4-phe-
noxyphenylsulfone analogue 10b showed an IC50 value of ca. 46 µM,
showing a highly significant decrease in potency compared to 1. This
As expected, activity in the presence of inhibitors was reversed upon
rapid dilution, showing reversible inhibition for all compounds
3

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
Scheme 2. Synthesis of further 2-arylthiopropanoyl-CoAs 7c-o designed as inhibitors of AMACR. Reagents and conditions: i, ethyl ( )-2-bromopropanoate, CHCl3,
NEt3; ii, R-Br, DMF, K2CO3 or R-OH, THF, PPh3, DIAD; iii, NaOH, H2O, MeOH; iv, CDI, CH2Cl2; v CoA-Li+3 , NaHCO3, H2O, THF.
(Supporting information). Kinetic analysis using varying substrate
concentrations in the presence or absence of 22.5 nM 7c was used to
determine the mode of inhibition. This analysis showed that mixed
competitive inhibition was the most likely kinetic mode of action, with
activity was observed at 100 µM. ‘Unusual’ fatty acids and derivatives
are generally taken up into cells with subsequent import into peroxi-
somes as their acyl-CoA esters for metabolism and detoxification [1,4];
it was therefore anticipated that 6 would be taken up into cells (as has
been shown for other AMACR inhibitor precursors [24]) and co-localize
with AMACR in peroxisomes and mitochondria [47,48]. Conversion of
the carboxylic acid into the acyl-CoA ester to produce the drug was
expected to take place. It appears that 6 are not converted efficiently
into 7, and that this is responsible for the observed lack of activity. It is
also possible that inhibitors 7 are removed by rapid metabolism, but
previous work on 3-thia acyl-CoAs have suggested this is not the case
[38,41,49].
a Ki value of 14.6
2.0 nM (mean
S.E.M.) (Supporting informa-
tion).
2.4. Cellular evaluation of compounds
Inhibitors of AMACR have been evaluated as their carboxylic acid
precursors at the cellular level because of the poor cell permeability, the
hydrolytic instability of the acyl-CoAs, and the known facile acyl-CoA
formation from carboxylate inhibitor precursors in cells [24,28].
Therefore, the carboxylic acid precursors (6c,h-j,n,o) were tested
against AD (LnCaP) and AI (Du145 and PC3) prostate cancer cell lines.
The selected compounds were chosen as the corresponding acyl-CoA
esters represented a range of 3- and 4-substituted derivatives (7c,h-
j,n,o) with the inhibitors giving a range of activities, including the most
potent (7c). Fenoprofen was included as a positive control.
3. Conclusions
Development of the 2-arylthiapropanoyl-CoA (‘thiolactate’) series of
inhibitors demonstrates that highly potent inhibition of AMACR can be
achieved in vitro. The structure-activity relationships can be largely
predicted based on lipophilicity calculations, although there are ex-
amples where inhibitors of both higher and lower potency than ex-
pected have been produced, which results from side-chain interactions
with the enzyme [8]. Although the inhibitor precursors failed to sig-
nificantly inhibit growth of prostate cancer cells, the study demon-
strates the potential for development of rationally-designed drugs
These compounds were initially screened at 500 µM, 200 µM and
100 µM against the three cell lines. The 2-arylthiopropanoic acids 6
provided strong anti-proliferative activity (33–88%; Supporting
Information, Table S1) against all three prostate cancer cell lines at
500 µM, and moderate activity at 200 µM. However, no significant
4

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
Table 1
IC50 values for inhibitor acyl-CoAs 1, 7a-o, 10b, as measured by the colorimetric assay [36], and the calculated lipophilicity (calc. LogP). IC50 values are expressed as
geometric means for three independent repeats with lower and upper geometric Standard Error of the Mean values in parentheses (see Experimental for details).
aOnly one IC50 determination was performed for this compound due to the low synthetic yield of the acyl-CoA. bLogP values calculated using http://www.
molinspiration.com/cgi-bin/properties.
Fenoprofenoyl-CoA1
Compound
7a - n
10b
R1
R2
IC50 (nM)
Calc. logPb
1
–
–
340 (282, 408)
247 (217, 281)
354 (324, 379)
22.3 (20.1, 24.7)
420 (399, 442)
437 (423, 452)
294 (267, 322)
313 (301, 325)
520 (492, 549)
113 (103, 124)
133 (120, 147)
99 (93, 106)
−3.77
−3.95
−3.93
−4.05
−4.00
−3.85
−3.81
−2.97
−4.42
−4.03
−4.00
−3.82
−3.79
−2.95
−3.68
−4.41
−4.91
7a
7b
7c
7d
7e
7f
PhO
H
H
PhO
BnO
H
3-MeOBnO
Ph(CH2)2O
H
H
3-MeOPh(CH2)2O
H
7g
7h
7i
3-F3CPh(CH2)2O
H
Me2CHCH2O
H
H
H
H
H
H
H
H
H
BnO
7j
3-MeOBnO
Ph(CH2)2O
3-MeOPh(CH2)2O
3-F3CPh(CH2)2O
Me(CH2)4O
Me2CHCH2O
PhO
7k
7l
71 (69, 73)
7m
7n
7o
10b
51 (46, 56)
90 (80, 100)
105 (95, 115)
a
4.6 × 104
-4.0
synthesized as previously described [10].
10b
-4.5
4.2. General experimental procedures
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
Syntheses were carried out at ambient temperature, unless other-
wise specified. Solutions in organic solvents were dried with MgSO4.
Thin layer chromatography was performed on Merck silica aluminium
plates 60 (F254) and visualized with UV light, potassium permanganate
or phosphomolybdic acid. Column chromatography was performed
using Fisher silica gel (particle size 35–70 µm). Purification of acyl-CoA
esters was performed by solid-phase extraction using Oasis HLB 6 cc
(200 mg) extraction cartridges. Columns were conditioned with acet-
onitrile and water. After loading the columns were eluted with water,
10%, 25% and 50% of MeCN in water (v/v). NMR spectra were re-
corded at 22 °C at 400.04 or 500.13 MHz (1H) and 100.59 or
125.76 MHz (13C) on Bruker Avance III NMR spectrometers in D2O or
CDCl3. 1H NMR spectra recorded for known compounds matched data
reported in the literature unless otherwise stated. Mass spectra were
recorded on an ESI-TOF instrument. High resolution mass spectra were
recorded in ES mode. Acyl-CoA esters were characterized by 1H NMR
and HRMS. Compounds 1a [50], 1b [43], and 12b [45] were synthe-
sized by known literature methods. Compound 5b was synthesized by
the literature method [43] except that the temperature was increased to
280 °C.
7h
7e
7d
7b
7a
Fenoprofenoyl-CoA 1
7g
7f
7k
7j
7i
7o
7n
7l
7m
7c
-5.5
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5
LogP
Fig. 1. Lipophilicity of 2-arylthiopropanoyl-CoA inhibitors 7a-o, sulfone 10b
and fenoprofenoyl-CoA 1. Log10 IC50 values are reported
1 SD. Calculated
LogP used in this Figure are presented in Table 1. Compounds in green are more
potent than expected, and compounds in red are less potent than expected
based on calculated lipophilicity.
targeting AMACR.
Aqueous solutions for biological experiments were prepared in
Nanopure water of 18.2 MΩ.cm−1 quality and were pH-adjusted with
aq. HCl or NaOH. The pH of aqueous solutions was measured using a
Corning 240 pH meter and Corning general purpose combination
electrode. The pH meter was calibrated using Fisher Chemicals stan-
dard buffer solutions (pH 4.0 - phthalate, 7.0 - phosphate, and 10.0 -
borate) at either pH 7.0 and 10.0 or 7.0 and 4.0. Calibration and
measurements were carried out at ambient room temperature. Stock
concentrations of acyl-CoA esters for assays were determined using 1H
NMR.
4. Materials and methods
4.1. Sources of materials
Reagents were purchased from the Sigma-Aldrich Chemical Co.,
Fisher Scientific or Fluorochem. Reduced coenzyme A, tri-lithium salt
was purchased from Calbiochem. Deuterated solvents were purchased
from Goss Scientific. Anhydrous and general grade solvents were pur-
chased from the Sigma-Aldrich Chemical Co. and used without further
purification. Water for aqueous solutions was obtained from
a
Nanopure Diamond system (18.2 MΩ.cm−1). Human recombinant
AMACR 1A was expressed and purified and colorimetric substrate
synthesized as previously described [36]. Fenoprofenoyl-CoA 1 was
5

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
4.3. Synthesis of compounds
8a (123 mg, 97%) as a colourless oil (> 95% pure by 1H NMR), which
was used in the next step without further purification. IR νmax/cm−1
1738 (C]O), 1326, 1139 (S]O); δ (500.13 MHz, CDCl3) 7.58 (1H, dt,
J = 7.5, 1.1 Hz), 7.51 (1H, t, J = 8.1 Hz), 7.46 (1H, t, J = 2.0 Hz), 7.38
(2H, tt, J = 7.5, 2.0 Hz), 7.28 (1H, ddd, J = 8.2, 2.3, 1.0), 7.18 (1H, tt,
J = 7.4, 0.9 Hz), 7.03 (2H, d, J = 8.0 Hz), 4.10 (2H, q, J = 7.3 Hz),
4.02 (1H, q, J = 7.3 Hz), 1.55 (3H, d, J = 7.3 Hz) and 1.17 (3H, t,
J = 7.3 Hz); δ (125.76 MHz; CDCl3) 165.96, 158.04, 155.65, 138.37,
130.34, 130.10, 124.52, 123.86, 123.42, 119.48, 118.53, 65.31, 62.21,
13.78 and 11.63; ESI-MS m/z 333.0799 [M - H]- (C17H17O5S requires
333.0797).
4.3.1. Ethyl ( )-2-(4-phenoxyphenylthio)propanoate (2b)
Anhydrous K2CO3 (367 mg, 2.65 mmol) was stirred with 4-phe-
noxybenzenethiol 1b (534 mg, 2.64 mmol) in dry MeCN (4.9 mL) at
−45 °C for 15 min. MeCN (1.8 mL) was added, followed by ethyl
(
)-2-bromopropanoate (0.35 mL, 2.64 mmol) in MeCN (1.2 mL). The
mixture was warmed to 20 °C and stirred for 20 h. Filtration (Celite®),
evaporation and chromatography (PE/EtOAc 30:1) afforded 2b
(630 mg, 79%) as a colourless oil. IR νmax/cm−1 1731 (C]O); 1H NMR
(400.04 MHz, CDCl3): δ 7.44 (2H, d, J = 8.8 Hz), 7.37–7.31 (2H, m),
7.13 (1H, tt, J = 7.3, 1.1 Hz), 7.04–6.98 (2H, m), 6.93 (2H, d,
J = 8.8 Hz), 4.18–4.07 (2H, m), 3.68 (1H, q, J = 7.1 Hz), 1.45 (3H, d,
J = 7.1 Hz) and 1.20 (3H, t, J = 7.1 Hz); δ (100.59 MHz, CDCl3)
172.47, 157.97, 156.30, 135.91, 129.76, 126.09, 123.75, 119.31,
118.61, 60.98, 45.69, 17.13 and 13.99; ESI–MS m/z 325.0991
[M + Na]+ (C17H18NaO3S requires 325.0869). Compound 2b was
hydrolyzed to the corresponding acid using General Method C and
converted into the acyl-CoA ester by General Method D. Spectroscopic
data are available in the Supporting Information.
4.3.6. Ethyl ( )-2-(4-phenoxyphenylsulfonyl)propanoate (8b)
Compound 2b (143 mg. 0.47 mmol) was treated with OXONE®, as
for 8a, to give 8b (129 mg, 82%) as a colourless oil. IR νmax/cm−1
1738 (C]O), 1324, 1144 (S]O); 1H NMR (400.04 MHz, CDCl3): δ 7.81
(2H, d, J = 9.0 Hz), 7.45–7.37 (2H, m), 7.23 (1H, tt, J = 7.4, 1.1 Hz),
7.10–7.02 (4H, m), 4.14 (2H, q, J = 7.2 Hz), 4.02 (1H, q, J = 7.1 Hz,
CH), 1.55 (3H, d, J = 7.1 Hz), 1.20 (3H, t, J = 7.2 Hz); δ (100.59 MHz,
CDCl3) 166.33, 162.94, 154.63, 131.66, 130.20, 130.02, 125.22,
120.45, 117.13, 65.44, 62.13, 13.82 and 11.90; ESI-MS m/z 333.0807
[M - H]- (C17H17O5S requires 333.0797).
4.3.2. O-(3-Phenoxyphenyl) N,N-dimethylthiocarbamate (4a)
NaH (60% w/w dispersion in oil, 1.20 g, 30.0 mmol) was added
slowly 3–phenoxyphenol 3a (1.86 g, 10.0 mmol) in dry DMF (27 mL) at
10 °C. N,N-dimethylthiocarbamoyl chloride (5.78 g, 46.8 mmol) was
added after evolution of H2 had ceased. The mixture was stirred at 70 °C
for 21 h and then cooled to ambient temperature. Water (100 mL) was
added and the mixture was extracted thrice with CHCl3. The organic
layers were combined, washed with aq. KOH (0.89 M, 50 mL) and brine.
Drying, evaporation and chromatography (PE/EtOAc 5:1) afforded 4a
(1.86 g, 68%) as a colourless oil. IR νmax/cm−1 1139 (C]S); 1H NMR
(400.04 MHz, CDCl3): δ 7.38–7.30 (3H, m), 7.15–7.05 (3H, m), 6.93
(1H, ddd, J = 8.3, 2.4, 1.0 Hz), 6.86 (1H, ddd, J = 8.1, 2.2, 1.0 Hz),
6.78 (1H, t, J = 2.2 Hz), 3.41 (3H, s), 3.26 (3H, s); δ (100.59 MHz,
CDCl3) 185.78, 157.41, 156.11, 154.50, 129.43, 129.31, 123.26,
118.76, 117.08, 115.53, 113.15, 42.76, 38.27; ESI-MS m/z 274.0900
[M + H]+ (C15H16NO2S requires 274.0902).
4.3.7. Ethyl ( )-2-(3-hydroxyphenylthio)propanoate (12a)
3-Hydroxythiophenol 11a (3.00 g, 23.8 mmol) and ethyl 2-bromo-
propanoate (3.1 mL, 24 mmol) were stirred at reflux in CHCl3 (80 mL)
with NEt3 (5.0 mL, 36 mmol) for 1 h. The cooled mixture was washed
with water and brine. Drying, evaporation and chromatography gave
12a (4.65 g, 86%) as a yellow oil. 1H NMR (400.04 MHz, CDCl3): δ 7.16
(1H, t, J = 8.0 Hz), 7.00 (1H, ddd, J = 7.8, 1.8, 1.0 Hz), 6.95 (1H, ddd,
J = 2.5, 1.7, 0.4 Hz), 6.75 (1H, ddd, J = 8.2, 2.5, 1.0 Hz), 5.46 (1H, br
s), 4.20–4.08 (2H, m), 3.81 (1H, q, J = 7.1 Hz), 1.50 (3H, d,
J = 7.1 Hz), 1.20 (3H, t, J = 7.1 Hz). 13C NMR (100.59 MHz, CDCl3) δ
173.07, 155.89, 134.67, 129.90, 124.70, 119.23, 115.08, 61.42, 45.16,
17.36, 13.99. ESI-MS m/z 249.0577 [M + Na]+ (C11H14NaO3S re-
quires 249.0556).
4.3.8. General method A: Synthesis of compounds 2c, d, h, i, j, n and o
Compounds 12a or 12b, in DMF (15 mL), was stirred at 100 °C with
K2CO3 (1.22 g, 8.84 mmol) and the appropriate alkyl bromide [benzyl
bromide (0.32 mL; 2c and 2i), 3-methoxybenzyl bromide (0.32 mL or
0.37 mL; 2d and 2j), 1-bromo-2-methylpropane (0.29 mL; 2 h and 2o)
or 1-bromopentane (0.24 mL; 2n)] (2.66 mmol), for 1 h. The reaction
mixture was cooled and the DMF was evaporated. The residue, in
CH2Cl2, was washed twice with water and once with brine. Drying and
evaporation gave the required compounds. Spectroscopic data for these
compounds are given in the Supporting information.
4.3.3. S-3-Phenoxyphenyl-N,N-dimethylcarbamothioate (5a)
Compound 4a (1.76 g, 6.43 mmol) was heated at 280 °C under Ar
for 100 min. Chromatography (PE/EtOAc 19:1 → 3:1) gave 5a (664 mg,
38%) as a brown oil. IR νmax/cm−1 1671 (C]O); δ (400.04 MHz,
CDCl3) 7.39–7.30 (3H, m), 7.29–7.24 (1H, m), 7.20 (1H, dd, J = 2.2,
1.7 Hz), 7.13 (1H, tt, J = 8.4, 1.1 Hz), 7.09–7.01 (3H, m) and 3.03 (6H,
br s); δ (100.59 MHz; CDCl3) 166.01, 157.09, 156.46, 130.10, 129.98,
129.63, 129.59, 125.37, 123.32, 119.14, 118.91, 36.60; ESI-MS m/z
274.0879 [M + H]+ (C15H16NO2S requires 274.0902).
4.3.4. Ethyl ( )-2-(3-phenoxyphenylthio)propanoate (2a).
4.3.9. General method B: Synthesis of compounds 2e-g, k-m
Compound 5a was prepared from 3–phenoxybenzenethiol 1a
(94 mg, 0.47 mmol), using the procedure as for compound 5b, to afford
a colourless oil (94 mg, 67%). IR νmax/cm−1 1732 (C]O); 1H NMR
(400.04 MHz, CDCl3): δ 7.38–7.30 (2H, m), 7.25 (1H, t, J = 8.0 Hz),
7.20–7.07 (3H, m), 7.04–6.97 (2H, m), 6.94–6.89 (1H, m), 4.15–4.03
(2H, m), 3.78 (1H, q, J = 7.2 Hz), 1.48 (3H, d, J = 7.2 Hz) and 1.17
(3H, t, J = 7.2 Hz); δ (100.59 MHz; CDCl3) 172.38, 157.44, 156.59,
135.02, 129.88, 129.75, 126.95, 123.54, 122.24, 119.01, 118.04,
61.17, 44.94, 17.29 and 13.97; ESI-MS m/z 325.0868 [M + Na]+
(C17H18NaO3S requires 325.0874).
DIAD (0.65 mL, 3.31 mmol) was added dropwise to 12a or 12b
(500 mg, 2.21 mmol), PPh3 (869 mg, 3.31 mmol) and appropriate al-
cohol [2-phenylethanol (0.27 mL, 2.21 mmol, 2e; or 0.17 mL,
1.46 mmol, 2k), 2-(3-methoxyphenyl)ethanol (0.31 mL, 2.21 mmol, 2f;
0.20 mL, 1.46 mmol, 2l), or 2-(3-trifluoromethylphenyl)ethanol
(0.33 mL, 2.21 mmol, 2g and 2m] in THF (10 mL), and the mixture was
stirred for 18 h. Drying, evaporation and chromatography (PE/EtOAc
20:1) gave the esters 2e-g, k-m. Spectroscopic data for these com-
pounds are given in the Supporting information.
4.3.10. General method C: Hydrolysis of the ethyl ester to give compounds
6a-o
4.3.5. Ethyl ( )-2-(3-phenoxyphenylsulfonyl)propanoate (8a)
A solution of OXONE® (470 mg, excess) in water (4.7 mL) was added
to 2a (115 mg, 0.38 mmol) in MeOH/THF (1:1, 4.7 mL) and the mixture
was stirred for 4 h. The mixture was filtered (Celite®). Water was added
to the evaporation residue, which was extracted thrice with EtOAc.
Drying, evaporation and chromatography (PE/EtOAc 20:1 → 3:1) gave
Aq. NaOH (2.5 M, 2.0 mL, 5.00 mmol) was stirred with of the ethyl
ester 2a-o in MeOH (15 mL) for 1.5 h. MeOH was evaporated and the
mixture was acidified with aq. HCl (1.0 M) to pH ∼ 3 and extracted
twice with CH2Cl2. The combined organic layers were washed with
brine. Drying, evaporation and chromatography (PE/EtOAc 2:1) gave
6

M. Yevglevskis, et al.
BioorganicChemistry92(2019)103263
the carboxylic acids 6a-6o. Spectroscopic data for these compounds are
Biochimie 98C (2014) 36–44.
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given in the Supporting information.
4.3.11. General method D: Formation of acyl-CoA esters 7a-o
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Carboxylic acids 6a-o in dry CH2Cl2 (3.0 mL) was treated with N,N′-
carbonyl diimidazole (32 mg, 0.2 mmol) in one portion and the mixture
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brine. Drying, filtration and evaporation gave the crude imidazolide.
This material was dissolved in THF (3.0 mL), then CoA-Li3 (31 mg,
0.04 mmol) in aq. NaHCO3 (0.1 M, 3.0 mL) was added and the mixture
was stirred for 18 h. The solution was acidified to pH ∼ 3 with aq. HCl
(1 M) and the solvents were partly removed under reduced pressure.
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4.3.12. Evaluation of AMACR inhibition by test compounds in vitro
Colorimetric assays were performed as previously described
[30,36], except that the concentration of DMSO in the final assay was
10% (v/v). Control experiments showed that no reduction in activity
was observed at this concentration. Assays were conducted in full- or
half-volume 96-well plates, in a final volume of 200 or 100 µL, re-
spectively. Control experiments have previously shown identical results
using full and half-volume plates [30,36]. Reaction rates were de-
termined by plotting A354 with time in Excel, and data was analysed
using SigmaPlot 13 as previously described [30,36]. Log10 IC50 values
were calculated from individual dose-response curves with inhibition of
binding plotted against Log10 drug concentration (in M). Mean Log10
IC50 values were then calculated from 3 independent repeats together
with corresponding Log10 Standard Error of the Mean (S.E.M.) values.
Data were then converted to non-logarithmic values to produce the
geometric mean with corresponding upper and lower limits of the
geometric S.E.M. values (Table 1). Ki values were determined as de-
scribed using 8 concentrations of substrate (150, 100, 66.6, 29.6, 19.8,
13.1, 8.8 and 5.9 µM in assay), in the presence of 0 or 22.5 nM 7c, and
analysed with SigmaPlot as previously described [36]. Data are
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humans, Org. Biomol. Chem. 12 (2014) 6737–6744.
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growth modifier in prostate cancer, Cancer Res. 63 (2003) 7365–7376.
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means
S.E.M., for 3 dependent repeats.
Funding sources
[19] J.N. Mubiru, A.J. Valente, D.A. Troyer, A variant of α-methylacyl-CoA racemase
gene created by a deletion in exon 5 and its expression in prostate cancer, Prostate
65 (2005) 117–123.
This work was funded by Prostate Cancer UK (S10-03 and PG14-
[20] G.L. Shen-Ong, Y. Feng, D.A. Troyer, Expression profiling identifies a novel α-me-
thylacyl-CoA racemase exon with fumarate hydratase homology, Cancer Res. 63
(2003) 3296–3301.
009),
a
University of Bath Overseas Research Studentship,
a
a
Biochemical Society Summer Vacation studentship (2016) and
Shandong-Bath undergraduate exchange studentship (2016).
[21] B. Ouyang, Y.-K. Leung, V. Wang, E. Chung, L. Levin, B. Bracken, L. Cheng, S.-M. Hi,
α-Methylacyl-CoA racemase spliced variants and their expression in normal and
malignant prostate tissues, Urology 77 (2011) e1–e7.
Notes
[22] B.A.P. Wilson, H. Wang, B.A. Nacev, R.C. Mease, J.O. Liu, M.G. Pomper,
W.B. Isaacs, High-throughput screen identifies novel inhibitors of cancer biomarker
α-methylacyl-coenzyme A racemase (AMACR/P504S), Mol. Cancer Ther. 10 (2011)
825–838.
The authors are members of the Cancer Research @ Bath (CR@B)
network.
[23] K. Takahara, H. Azuma, T. Sakamoto, S. Kiyama, T. Inamoto, N. Ibuki, T. Nishida,
H. Nomi, T. Ubai, N. Segawa, Y. Katsuoka, Conversion of prostate cancer from
hormone independency to dependency due to AMACR inhibition: Involvement of
increased AR expression and decreased IGF1 expression, Anticancer Res. 29 (2009)
2497–2505.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103263.
[24] A. Morgenroth, E.A. Urusova, C. Dinger, E. Al-Momani, T. Kull, G. Glatting,
H. Frauendorf, O. Jahn, F.M. Mottaghy, S.N. Reske, B.D. Zlatopolskiy, New mole-
cular markers for prostate tumor imaging: A study on 2-methylene substituted fatty
acids as new AMACR inhibitors, Chem. Eur. J. 17 (2011) 10144–10150.
[25] Z. Jiang, B.A. Woda, K.L. Rock, Y.D. Xu, L. Savas, A. Khan, G. Pihan, F. Cai,
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