Rational design and synthesis of novel anti-prostate cancer agents bearing a 3,5-bis-trifluoromethylphenyl moiety

Article abstract of DOI:10.1016/j.bmcl.2016.06.001

Prostate cancer is a major cause of male death worldwide and the identification of new and improved treatments is constantly required. Among the available options, different non-steroidal androgen receptor (AR) antagonists are approved also to treat castration-resistant forms. Most of these drugs show limited application due to the development of resistant mutants of their biological target. Following docking-based studies on a homology model for the AR open antagonist conformation, a series of novel 3,5-bis-trifluoromethylphenyl compounds was designed with the aim to improve the antiproliferative activity of anti-androgen drugs bicalutamide and enzalutamide. The new structural modifications might impede the receptor to adopt its closed agonist conformation also in the presence of adaptive mutations. Among the novel compounds synthesised, several displayed significantly improved in vitro activity in comparison with the parent structures, with IC₅₀values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145, 22Rv1). Selected hits demonstrated full AR antagonistic behaviour and promising candidates for further development were identified.

Full text of DOI:10.1016/j.bmcl.2016.06.001

Accepted Manuscript
Rational design and synthesis of novel anti-prostate cancer agents bearing a 3,5-
bis-trifluoromethylphenyl moiety
Salvatore Ferla, Marcella Bassetto, Fabrizio Pertusati, Sahar Kandil, Andrew D.
Westwell, Andrea Brancale, Christopher McGuigan
PII:
S0960-894X(16)30605-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.001
BMCL 23954
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2016
31 May 2016
1 June 2016
Please cite this article as: Ferla, S., Bassetto, M., Pertusati, F., Kandil, S., Westwell, A.D., Brancale, A., McGuigan,
C., Rational design and synthesis of novel anti-prostate cancer agents bearing a 3,5-bis-trifluoromethylphenyl
moiety, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.001
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Rational design and synthesis of novel anti-prostate cancer agents
bearing a 3,5-bis-trifluoromethylphenyl moiety.
Salvatore Ferlaa*, Marcella Bassettoa*1, Fabrizio Pertusatia*, Sahar Kandila, Andrew D. Westwella,
Andrea Brancalea, Christopher McGuigana
a School of Pharmacy and Pharmaceutical Sciences, Redwood Building, King Edward VII Avenue, CF10 3NB, Cardiff, Wales,
UK
Abstract
Prostate cancer is a major cause of male death worldwide and the identification of new and improved
treatments is constantly required. Among the available options, different non-steroidal androgen
receptor (AR) antagonists are approved also to treat castration-resistant forms. Most of these drugs
show limited application due to the development of resistant mutants of their biological target.
Following docking-based studies on a homology model for the AR open antagonist conformation, a
series of novel 3,5-bis-trifluoromethylphenyl compounds was designed with the aim to improve the
antiproliferative activity of anti-androgen drugs bicalutamide and enzalutamide. The new structural
modifications might impede the receptor to adopt its closed agonist conformation also in the presence
of adaptive mutations.
Among the novel compounds synthesised, several displayed significantly improved in vitro activity in
comparison with the parent structures, with IC50 values in the low micromolar range against four
different prostate cancer cell lines (LNCaP, VCaP, DU-145, 22Rv1). Selected hits demonstrated full
AR antagonistic behaviour and promising candidates for further development were identified.
Key words
Homology modelling; rational drug design; prostate cancer; androgen-receptor.
Prostate cancer (PC) represents a major cause of male death worldwide and it is the second most
common cancer for males, the fourth most common cancer overall.1 Currently, among the several
treatment options available, androgen deprivation is one of the main strategies to treat the disease at
various stages of its development.2 Flutamide (1) (in its active form as hydroxyflutamide (2)),
bicalutamide (3) and enzalutamide (4) are non-steroidal androgen receptor antagonists approved for the
treatment of PC (Figure 1). In many cases, these anti-androgens become ineffective after few years of
treatment due to the emergence of different mutations on the AR, which cause the anti-androgen
1
Corresponding author:E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
* These authors contributed equally to this work
This work is dedicated to the memory of Prof. Chris McGuigan, a great colleague and scientist,
invaluable source of inspiration and love for research.
1

compounds to function as agonists.2
In order to improve the antiproliferative properties of standard anti-androgens 3 and 4, and to possibly
impede the receptor to adopt its closed agonist conformation in the presence of adaptive mutations,
diverse new analogues were designed to increase the molecule occupational volume in proximity to the
receptor helix 12, responsible for the closure movement to the agonist conformation, with the aim to
impede this conformational change. Specifically, different hydrophobic aromatic substituents were
explored with docking studies, and in particular attention was focused on fluorinated groups, in order to
take advantage of the potential of fluorine to enhance the pharmacological properties and drug-like
characteristics of compounds.3 A 3,5-bis-trifluoromethyl group was identified as the best substituent for
aromatic ring B of the parent structures to achieve this result (Figure 1). The new analogues showed a
marked improvement in in vitro antiproliferative activity on a range of human PC cell lines (VCap,
LNCaP, DU-145 and 22Rv1), reaching sub-micromolar IC50 values. In addition, full AR antagonistic
effect was observed for selected compounds and preclinical candidates were identified.
Figure 1. Structure of AR antagonists in the market for the treatment of PC and their proposed modifications.
A homology model of the human WT-AR open conformation was built using a single template
approach in MOE2015.4 The crystal structure of the progesterone receptor in its open antagonist form
(PDB ID: 2OVM) was used as template (54% sequence identity with the human AR).5 Docking of (R)-
bicalutamide, enzalutamide and of the newly designed modifications was performed in the ligand
binding domain (LBD) of the AR model using Plants docking software.6 The predicted binding mode
found for the parent compounds revealed the presence of free occupational space in the model structure
in proximity to helix 12. As shown in Figure 2a, in silico studies suggest that the best substituent to fill
this space is a 3,5-bis-trifluoromethyl group on aromatic ring B of the standard compounds. Due to the
much bigger occupational volume in comparison with the 4-F substituent of bicalutamide, this
2

modification might impede the receptor closure even in the presence of adaptive mutations, such as the
W741L mutation, the most common for bicalutamide.7 As a further indication of this potential effect,
docking of the newly designed compounds on the crystal structure of the complex bicalutamide-
W741L AR (PDB ID 1Z95), which corresponds to the receptor closed conformation, reveals the
presence of steric clashes between the new bis-trifluoromethyl portion of the molecule and helix 12
(Figure 2b).
Figure 2. Predicted binding mode of 25a in the AR homology model (a) and in the bicalutamide-W741A AR closed
conformation crystal structure 1Z95 (b). The bis-CF3 aromatic substituent in ring B shows major clashes (red grid) with the
protein surface (grey grid) of the closed AR structure, indicating that it might impede the AR closure movement even in the
presence of adaptive mutations.
Along with the insertion of a 3,5-bis-trifluoromethyl group in aromatic ring B of the parent structures,
its replacement with a bulkier 3,5-di-tert-butyl substituent was also envisaged (compound 30f).
Moreover, ring A was explored as well for the repositioning and replacement of its original substituents
with a bis-CF3 function. All planned modifications were carried out according to an optimised three or
four-step synthetic pathway, adapted from literature procedures,8-14 summarised in Schemes 1 and
Scheme 2.
Racemic bicalutamide derivatives, along with racemic bicalutamide,10 were prepared by reacting
methacryloyl chloride (5) with substituted anilines 6-11 in dimethylacetamide (DMA) to obtain
phenylacrylamides 12-17, which were converted into the corresponding epoxides 18-23 in the presence
of a large excess of hydrogen peroxide and trifluoroacetic anhydride in dichloromethane. Opening of
the epoxide rings of 18-23 with substituted phenols or thiophenols gave a series of ethers 30-34 and
thioethers 24-29. Thioethers 24-27 and 29 were oxidized to the corresponding sulfones 35-39 with
mCPBA at 25 °C. For the synthesis of R-bicalutamide12 and R-derivatives 41-42, intermediate 40 was
prepared according to literature,11 and then reacted with the sodium salt of 3,5-bis-CF3-thiophenol in
tetrahydrofuran to give the desired (R)-thioether 41.12 Oxidation of 41 with mCPBA provided sulfone
42. Enzalutamide analogues were obtained by the preparation of isothiocyanates 43-46, treating the
corresponding anilines 6-9 with thiophosgene.13 Strecker reaction of substituted aniline 11 with acetone
and trimethylsilyl cyanide generated the desired cyanoamine 47, which was reacted with
isothiocyanates 43-46 in DMF followed by HCl and MeOH addition to give the desired thiohydantoins
48-51. Thiohydantoin 54 was prepared by heating intermediate 53, obtained according to literature
procedures,14 with isothiocyanate 52 in a mixture of DMSO and isopropylacetate (iPAC) at 85 °C for
14 hours. Standard enzalutamide was also prepared according to this procedure.14 All final compounds
3

were fully characterised by 1H NMR, 13C NMR, 19F NMR and HRMS. Purity >95% was confirmed by
HPLC (see Supplementary Material).
Scheme 1 Synthetic pathways for the synthesis of racemic and R-bicalutamide analogues adapted from reported procedures.8-12
Reagents and conditions: (a) 5 (8 equiv.), DMA, RT, 3 h (77-95%); (b) H2O2 (4 equiv.)/(CF3CO)2O (5 equiv.), DCM, RT, 24 h
(60-86%); (c) NaH (1.2 equiv.), phenol or thiophenol (1.2 equiv.), THF, RT, 24h (29-89%); (d) mCPBA (1.4 equiv.), DCM, RT,
4-6 h (40-89%); (e) 3,5-bis-CF3-thiophenol (1.2 equiv.), 60% NaH (1.2 equiv.), THF, RT, 24 h (75%); (f) mCPBA (1.4 equiv.),
DCM, RT, 6 h (68%).
Scheme 2. Synthetic strategy toward enzalutamide derivatives. Adapted from literature procedures.13, 14 Reagents and conditions:
(a) CSCl2 (1.5 equiv.), NaHCO3, H2O, DCM, RT, 24 h (98-100%); (b) TMSCN (5.1 equiv.), acetone, 80 °C, 12 h (80-97%); (c)
DMF, RT 48 h, followed by HCl, MeOH, reflux, 6 h (10-51%). d) DMSO, iPAc, 85 °C, 14 h (67%).
All newly synthesised compounds were evaluated for their antiproliferative effect in an in vitro 2D
monolayer assay using four human prostate cancer cell lines (Table 1, absolute IC50 in µM). LNCaP,
4

VCaP and 22Rv1 cell lines exhibit some androgen sensitivity, whereas DU-145 is hormone-insensitive
(Supporting Information, section S.3).
The activity of reference compounds 3 and 4 is consistent with previously reported data.15-17 As recent
research studies carried out in our group have shown the lack of significant difference in
antiproliferative activity between racemic and R-bicalutamide analogues,18 the major part of our new
bicalutamide-derived structures have been prepared as racemic mixtures. Most of the new derivatives
performed better than standard bicalutamide, significantly improving its antiproliferative activity up to
60-fold (overall antiproliferative activity in the four cell lines reported as geometric mean). Active
inhibitors showed sigmoidal concentration-effect curves with total cell kill at high concentrations
(Supplementary data).
Scaffold 1
Scaffold
Scaffold 2
Compound
Ar (B ring)
X
R
(A Absolute IC50 (µM)
ring)
22Rv1
DU-
145
LNCaP
45.27
45.20
2.34
VCaP
68.37
51.61
2.73
Geom.mean
52.42
47.05
2.43
1
1
1
1
1
1
4-F-Ph
SO2
SO2
S
4-CN, 3- 49.58
CF3
49.20
R,S-
Bicalutamide
4-F-Ph
4-CN, 3- 46.25
CF3
45.41
2.85
2.78
9.09
3.38
3
(R-
Bicalutamide)
24a
3,5- CF3-Ph
3,5- CF3-Ph
3,5-CF3-Ph
3,5- CF3-Ph
4-CN, 3- 1.90
CF3
S
4- NO2, 1.50
3-CF3
1.50
1.53
1.76
25a
26a
27a
S
4-CN, 2- 4.65
CF3
7.76
6.65
6.84
S
4-NO2,
2-CF3
2.29
3.11
3.52
3.03
1
1
1
1
1
1
3,5-CF3-Ph
3-CF3-Ph
S
S
S
S
S
O
4-CF3
9.75
9.67
4.37
6.42
2.94
16.39
10.03
8.66
9.61
6.41
4.70
9.61
8.75
3.57
7.76
2.91
3.01
9.88
9.86
3.84
9.29
2.90
2.47
11.10
9.56
4.78
8.17
3.55
3.10
28a
29b
29c
29d
29e
30a
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
2-CF3-Ph
3-OCF3-Ph
2-OCF3-Ph
3,5-CF3-Ph
4-CN, 3- 2.64
CF3
1
1
1
1
3,5- tBu-Ph
3,5- CF3-Ph
3,5-CF3-Ph
3,5-CF3-Ph
O
S
4-CN, 3- 2.27
CF3
3.15
2.90
8.05
11.64
2.67
0.99
3.56
23.98
2.98
1.66
4.77
23.73
2.69
1.68
5.11
14.18
30f
31a
32a
33a
4-NO2,
3-CF3
1.09
O
O
4-CN, 2- 4.07
CF3
4-NO2,
2-CF3
4-CF3
16.10
1
1
3,5-CF3-Ph
3,5-CF3-Ph
O
8.49
9.23
5.58
3.35
2.86
4.15
6.21
5.75
4.19
34a
35a
SO2
4-CN, 3- 3.11
CF3
5

1
1
1
3,5- CF3-Ph
3,5-CF3-Ph
3,5-CF3-Ph
SO2
SO2
SO2
4-NO2,
3-CF3
2.15
5.26
n.d.
2.10
n.d.
3.30
n.d.
2.98
n.d.
36a
37a
38a
4-CN, 2- n.d.
CF3
4-NO2,
2-CF3
2.41
3.05
3.09
7.11
3.57
1
1
1
1
1
3-CF3-Ph
SO2
SO2
SO2
SO2
S
3,5-CF3
3,5-CF3
3,5-CF3
3,5-CF3
10.27
9.45
11.48
11.02
14.11
12.91
2.66
9.50
8.51
9.34
8.02
0.79
9.98
8.55
9.99
9.95
0.84
10.28
9.33
39b
39c
39d
39e
41
2-CF3-Ph
3-OCF3-Ph
2-OCF3-Ph
3,5- CF3-Ph
12.84
8.99
11.42
9.81
4-CN, 3- 1.13
CF3
1.19
1
2
2
2
2
2
2
3,5- CF3-Ph
SO2
4-CN, 3- 2.50
CF3
6.36
32.27
n.d.
1.99
11.47
n.d.
4.11
3.38
28.10
n.d.
42
3-F,4-
-
-
-
-
-
-
4-CN, 3- 31.76
CF3
53.04
n.d.
4
CONHMe-Ph
3,5- CF3-Ph
(Enzalutamide)
48
4-CN, 3- n.d.
CF3
3,5- CF3-Ph
3,5- CF3-Ph
3,5- CF3-Ph
4-NO2,
3-CF3
20.20
18.12
n.d.
9.93
n.d.
28.44
n.d.
17.93
n.d.
49
50
51
54
4-CN, 2- n.d.
CF3
4-NO2,
2-CF3
6.97
10
4.67
11.29
18.59
19.77
8.82
29.78
3-F,4-
3,5-CF3
26.37
47.86
CONHMe-Ph
Table 1. Antiproliferative activity for bicalutamide/enzalutamide derivatives. All data are mean values from triplicate
experiments.
In the bicalutamide-derived series of analogues (compounds 24-42), all the newly synthesised
compounds performed better than the standards in the antiproliferative assay, with the only exception
being 37a, for which an antiproliferative effect could not be observed due to solubility issues. The
presence of a 3,5-bis-trifluoromethyl substituent in aromatic ring B seems to strongly affect the
antiproliferative activity in all four prostate cancer cell lines, with the best results obtained for 41,
which reaches sub-micromolar IC50 values in two different cell lines (LNCaP and VCaP, both
androgen-sensitive), 31a, 25a, 24a, 36a, 27a and 30a. Replacement of the bis-CF3 substituent of 30a
with a bulkier 3,5-di-tert-butyl group (compound 30f) leads to slightly lower IC50 values, which
indicate activity retention. Insertion of a 3,5-bis-CF3 substituent in ring A of parent bicalutamide
(compounds 29b-e, 39b-e), according to our modelling studies, would be associated with a better
occupation of the inner portion of the AR ligand binding domain; this modification, combined with
optimised substituents for aromatic ring B recently found in our group (unpublished data), is associated
with a significant activity improvement in comparison with standard bicalutamide for all the new
derivatives prepared.
Due to solubility issues, the effect of the modification of ring B of enzalutamide with a 3,5-bis-
trifluoromethyl group could be assessed only for 49 and 51: also in this case, this structural change is
associated with activity improvement in all four prostate-cancer cell lines, with a particularly
6

significant effect found for 51. Insertion of this substituent on aromatic ring A of enzalutamide
(compound 54) is associated with activity retention.
Considering the antiproliferative results obtained across the four different cell lines, the new
compounds show significant activity also on androgen-insensitive DU-145 cells. Even if the IC50
values obtained for most of the new compounds are higher in this cell line in comparison with the other
three, the improved antiproliferative activity suggests the presence of a potential off-target effect,
besides the canonical anti-androgen mechanism. Nevertheless, the presence of an off-target can be
speculated also for parent bicalutamide, for which the results obtained in our assay further confirm the
previously reported lack of selectivity between LNCaP and DU-145 cell lines.15 Due to this last
consideration, and given the marked improvement in antiproliferative activity associated with the new
structures, the presence of a potential off-target was not considered a limitation at this stage, while
further investigations to assess the molecular mechanism underlying this effect will be the object of
future studies.
As representative of the new series of compounds, 25a and 36a were evaluated for their
antagonist/agonist effect on the AR using the GeneBLAzer® Betalactamase reporter technology for
Nuclear receptors (NRs), to assess whether the antiproliferative activity found is correlated with any
interference with the AR function.19 Both compounds were found to be full antagonists of the androgen
receptor in a single concentration antagonism experiment (10µM concentration, antagonistic effect >
80%), reducing the receptor activation induced by the standard agonist R1881.20,21 A 10-concentration
antagonism assay showed that 25a possess an antagonistic IC50 in the same range of (R)-bicalutamide
and enzalutamide (Table 2). The IC50 value found for (R)-bicalutamide is comparable with previously
reported values in similar assays.22,23
Compound Antagonistic effect (%)* IC50 (µM)** Agonistic effect (%)***
83
95
90
93
0.490
0.361
0.710
3.1
5
3 (R-Bic.)
4 (Enzal.)
25a
N.E.
7
1
36a
Table 2: AR antagonist/agonist assay results. * Compounds were considered full antagonist if at a single concentration of 10µM
the reduction of receptor activation by R1881 was greater than 80%; ** Compounds tested at 10 different concentrations; ***
Compound at 10 µM concentration in absence of R1881 was used.
Despite the fact that 36a shows a significantly improved antiproliferative effect, its action as AR
antagonist seems to be reduced in terms of inhibitory IC50 in comparison with standard bicalutamide,
thus indicating the presence of other aspects that contribute to its anti-prostate cancer properties, as
already observed from the antiproliferative data. As a further confirmation of this potential off-target
effect, additional in vitro studies on 36a and 41, our most active new analogue, were performed in
order to evaluate their general cytotoxicity in comparison with 3, along with their metabolic stability
(Table 3).
Compound Metabolic stabilitya
MTT assayb
Antiproliferative resultsc
t1/2 (min)
MEC (µM)* AC50 (µM)** IC50 (µM)
7

214
567
6.49
19.2
3.6
54.3
9.01
2.35
47.05
2.98
1.19
3 (R-bical)
36a
41
1.22
a
Table 3: Metabolic stability and cytotoxicity assay results for selected compounds. t1/2 calculated in human liver microsomes;
compounds metabolically stable with no loss of parent detected for the duration of the assay. b MTT assay; compounds
have been tested at 8 different concentrations. * MEC: minimum effective concentration that significantly crosses vehicle control
threshold; ** AC50: concentration at which 50% of maximum effect is observed. c Antiproliferative assay results are expressed in
terms of geometric mean of the four different cell lines.
The MTT cell viability assay was performed in a human hepatocarcinoma (HepG2) cell line, as a
means to measure the effect of 36a and 41 on cell proliferation and other events that eventually lead to
cell death.24 Data are reported as minimum effective concentration (MEC) related to a vehicle control
(DMSO) and AC50. The tested compounds showed a concentration-dependent decrease in formazan
production across the range studied (data not shown), indicating a decrease in cell viability associated
with some growth inhibitory and toxic effects. Compared with control (R)-bicalutamide 3, 36a and 41
are characterized by a higher cytotoxicity, which at least partially correlates with the results obtained in
the antiproliferative assay. These results might further indicate that the newly prepared analogues,
besides the canonical AR antagonistic activity, possess an extra effect, which positively contributes to
their antiproliferative action. The presence of the new bis-CF3 substituent in aromatic ring B seems to
increase the metabolic stability of the bicalutamide scaffold, as suggested by the t1/2 found for 36a,
which is 2.6 fold higher than the value found for 3. The presence of the thioether group, which likely
undergoes rapid oxidation to sulfoxide or sulfone, is the most probable cause of the short half-life of
41, the most active new derivative found in the antiproliferative assay, thus making the sulfide scaffold
an unsuitable candidate for further development.
Due to the promising results obtained in the antiproliferative assay, different in vitro properties were
evaluated for selected new derivatives (sulfone, ether or thiohydantoin analogues), in order to aid the
identification of pre-clinical candidates. In particular, metabolic stability (t1/2), plasma protein binding
and cardiotoxicity were considered (Table 4).
Compound Metabolic stabilitya PPBb
Cardiotoxicityc
t1/2 (min)
% bound % hERG inhib. At 25 µM
214
9624
94.7
78.3
99.9
99.9
n.d.
3 (R-bical)
1930
7.64
13.4
29.3
9.44
30a
39e
42
290
Metabolically stable
71.5
51
a
Table 4: Metabolic stability, plasma protein binding and cardiotoxicity assay results for selected compounds. t1/2 calculated in
b
human liver microsomes; compounds metabolically stable with no loss of parent detected for the duration of the assay.
c
Plasma protein binding assay; results are expressed in terms of mean % bound from duplicate experiments. Cardiotoxicity
expressed in terms of % hERG inhibition at 25 µM compound concentration.
Except for 51, the compounds analysed are characterised by an increased metabolic stability in
comparison with standard bicalutamide. The tested analogues share a high affinity for human serum
albumin, with values similar to the ones of R-bicalutamide found for all except 39e. Finally, none of
8

the compounds analysed showed any significant cardiotoxic effect (no IC50 could be evaluated). As a
combination of its in vitro properties and antiproliferative profile in the four prostate-cancer cell lines,
42 has been chosen as pre-clinical candidate for the treatment of prostate cancer and will be evaluated
in 22Rv1 prostate cancer mice models. In particular, 42 is one of the most active compounds identified
in the antiproliferative assay, with an IC50 of 1.99 µM in the LNCaP cell-lines, the most active among
the new sulfone derivatives. Being a sulfone this compound is also associated with the highest
metabolic stability observed, and its plasma protein binding is highly similar to parent R-bicalutamide.
In summary, a novel series of anti-prostate cancer compounds bearing a 3,5-bis-trifluoromethylphenyl
group have been identified through a rational approach. Most of the new structures showed a
significant improvement in antiproliferative activity in comparison with the parent compounds against
four different prostate cancer cell lines. Full AR antagonistic behaviour has been demonstrated for
selected new structures and a pre-clinical candidate has been identified and is now undergoing further
exploration. The biological data obtained so far suggests that the new compounds, besides their AR
antagonist activity, might possess an off-target effect, which positively contributes to their anti-prostate
cancer properties. The evaluation of the molecular basis of this extra effect will be the focus of future
investigations.
Acknowledgements
The authors would like to thank Oncotest (Freiburg, Germany) for provision of human prostate cancer
cell line testing as an outsourced service. The AR binding assays were carried out by Invitrogen
(location), and Cyprotex (U.K.) were responsible for performing the metabolic stability, HepG2
cytotoxicity and cardiotoxicity assays as an outsourced service. Mr Derek Angus is warmly
acknowledged for many valuable comments on the ADME section of the manuscript. The Welsh
Government is acknowledged for funding (A4B-Academic Expertise for Business grant), in
collaboration with Catylix, Inc (Burbank, CA, USA) as industrial advisers to the project).
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