Structure-based virtual screening, synthesis and biological evaluation of potential FAK-FAT domain inhibitors for treatment of metastatic cancer

Article abstract of DOI:10.3390/molecules25153488

Focal adhesion kinase (FAK) is a tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers. In cancer cells, FAK promotes the progression and metastasis of tumours. In this study, we used structure-based virtual screening to filter a library of more than 210K compounds against the focal adhesion targeting FAK-focal adhesion targeting (FAT) domain to identify 25 virtual hit compounds which were screened in the invasive breast cancer line (MDA-MB-231). Most notably, compound I showed low micromolar antiproliferative activity, as well as antimigratory activity. Moreover, examination in a model of triple negative breast cancer (TNBC), revealed that, despite not effecting FAK phosphorylation, compound I significantly impairs proliferation whilst impairing focal adhesion growth and turnover leading to reduced migration. Further optimisation and synthesis of analogues of the lead compound I using a four-step synthetic procedure was performed, and analogues were assessed for their antiproliferative activity against three breast cancer (MDA-MB-231, T47D, BT474) cell lines and one pancreatic cancer (MIAPaCa2) cell line. Compound 5f was identified as a promising lead compound with IC₅₀ values in the range of 4.59–5.28 μM in MDA-MB-231, T47D, BT474, and MIAPaCa2. Molecular modelling and pharmacokinetic studies provided more insight into the therapeutic features of this new series.

Full text of DOI:10.3390/molecules25153488

molecules
Article
Structure-Based Virtual Screening, Synthesis and
Biological Evaluation of Potential FAK-FAT Domain
Inhibitors for Treatment of Metastatic Cancer
Sahar B. Kandil *, Samuel R. Jones, Sonia Smith, Stephen E. Hiscox and Andrew D. Westwell
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Wales CF10 3NB, UK;
jonessr15@cardiff.ac.uk (S.R.J.); sonia.m.smith@hotmail.co.uk (S.S.); hiscoxse1@cardiff.ac.uk (S.E.H.);
westwella@cardiff.ac.uk (A.D.W.)
* Correspondence: kandils1@cardiff.ac.uk; Tel.: +44-(0)2920-875800
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 June 2020; Accepted: 29 July 2020; Published: 31 July 2020
Abstract: Focal adhesion kinase (FAK) is a tyrosine kinase that is overexpressed and activated in
several advanced-stage solid cancers. In cancer cells, FAK promotes the progression and metastasis
of tumours. In this study, we used structure-based virtual screening to filter a library of more
than 210K compounds against the focal adhesion targeting FAK-focal adhesion targeting (FAT)
domain to identify 25 virtual hit compounds which were screened in the invasive breast cancer
line (MDA-MB-231). Most notably, compound
as well as antimigratory activity. Moreover, examination in a model of triple negative breast cancer
(TNBC), revealed that, despite not effecting FAK phosphorylation, compound significantly impairs
proliferation whilst impairing focal adhesion growth and turnover leading to reduced migration.
Further optimisation and synthesis of analogues of the lead compound using a four-step synthetic
I showed low micromolar antiproliferative activity,
I
I
procedure was performed, and analogues were assessed for their antiproliferative activity against
three breast cancer (MDA-MB-231, T47D, BT474) cell lines and one pancreatic cancer (MIAPaCa2)
cell line. Compound 5f was identified as a promising lead compound with IC50 values in the
range of 4.59–5.28 µM in MDA-MB-231, T47D, BT474, and MIAPaCa2. Molecular modelling and
pharmacokinetic studies provided more insight into the therapeutic features of this new series.
Keywords: focal adhesion kinase (FAK); breast cancer (BC); pancreatic cancer; triple negative breast
cancer (TNBC); chloropyramine (C4); rational drug design; virtual screening
1. Introduction
Focal adhesion kinase (FAK) was first described more than 20 years ago and represents
a promising target for the treatment of several advanced-stage solid cancers, as well as mesothelioma
and haematological malignancies. FAK is a multifunctional non-receptor tyrosine kinase whose
overexpression and activation has been linked to tumour progression, survival, migration, invasion,
epithelial-to-mesenchymal transition (EMT), and drug resistance through effects on the cancer cells as
well as stromal cells of the tumour microenvironment [
tissues or benign tumours [ ]. The amplification of FAK in a number of solid cancers (e.g., colorectal,
ovarian, breast, prostate, and sarcoma) has been previously documented [ ]. FAK plays a pivotal role
1,2]. FAK also exists at low levels in normal
2
3–8
in protecting pancreatic ductal adenocarcinomas, through the development of an immunosuppressive
and fibrotic microenvironment, and promoting immune evasion [9,10].
Molecules 2020, 25, 3488; doi:10.3390/molecules25153488
www.mdpi.com/journal/molecules

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FAK exerts its effects on cancer cells, as well as stromal cells of the tumour microenvironment,
through both kinase-dependent and kinase-independent effects. In humans, FAK consists of the
amino N-terminal region containing the FERM (4.1 protein-ezrin-radixin-moesin) domain, the central
kinase domain, and the C-terminal focal adhesion targeting (FAT) domain (Figure 1). FAK possesses
both kinase-dependent and kinase-independent (scaffolding) functions which control cell movement,
invasion, survival, gene expression, and cancer stem cell self-renewal and is therefore an attractive
target for anticancer therapy [11
many tumour-related processes [
cells can be subdivided into two main strategies [14].
–13]. It is established that FAK phosphorylation and activation drives
2]. The therapeutic approach of FAK function inhibition in cancer
Figure 1.
followed by the central kinase domain. The focal adhesion targeting domain (FAT) is the C-terminal
part of the protein, showing key tyrosine phosphorylation site Y925. ( ) Chemical structure of the
(A) Schematic representation of focal adhesion kinase (FAK); FERM is the N-terminal domain
B
kinase-dependent FAK inhibitors TAE-226, PF-271 (VS-6062), Defactinib (VS-6063), and GSK2256098;
and the kinase-independent chloropyramine (C4), Y 15, Y 11, and Roslin-2 compounds.
The first strategy has been to develop ATP-competitive kinase inhibitors which are designed
to bind residues surrounding the ATP-binding pocket of FAK around the tyrosine 397 (Y397)
autophosphorylation site. As this pocket is similar in many different kinases, there is the potential
for off-target effects stemming from a lack of kinase selectivity. For example, compound TAE-226
(Figure 1) which is a small ATP-competitive molecule, exhibits nanomolar activity, prevents cell
invasion, reduces cell proliferation, increases apoptosis, and enhances docetaxel-mediated growth
inhibition both in vitro and in vivo [15–18]. Nevertheless, the development of this molecule was later
abandoned due to off-target effects. GlaxoSmithKline developed compound GSK2256098 (Figure 1)
that displayed minor clinical responses in mesothelioma patients. When tested in combination with
trametinib (MEK inhibitor), GSK2256098 did not demonstrate any improved efficacy compared to that
observed with GSK2256098 monotherapy [19
underwent Phase I clinical trial and was found to be tolerated with few adverse effects. However,
PF-271 was discontinued because of its nonlinear pharmacokinetics profile [ ]. Furthermore, Defactinib
,20]. Additionally, compound PF-271 (VS-6062) (Figure 1)
1
(VS-6063, PF-04554878) (Figure 1) is a late generation FAK inhibitor in phase I/II clinical trials and
is tested as monotherapy as well as in combination with pembrolizumab or paclitaxel for patients
with different types of solid tumours [21,22]. These studies suggest that targeting the FAK kinase
activity may be promising especially when used in combination therapies. Notably, kinase selectivity
is a well-known problem of many ATP binding site competitive inhibitors. Additionally, the ability of

Molecules 2020, 25, 3488
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PYK2 (a FAK-related kinase) to take over certain FAK functions after FAK deletion has to be taken into
account [14].
A second strategy towards targeting FAK is based on the inhibition of the key FAK
kinase-independent (scaffolding) functions which are not blocked by FAK kinase inhibitors and
are possibly enhanced by it. The scaffolding function effects help to explain why FAK kinase inhibition
can lead to unpredictable therapeutic outcomes [23].
Moreover, several reports suggest that although FAK kinase activity is necessary for cell motility,
it may be not essential for cell proliferation and survival. Additionally, to avoid selectivity problems
typically found with competitive inhibitors of the ATP binding site, inhibiting the scaffolding function
of FAK might be a useful alternative approach [14]. Targeting the FAT domain will potentially allow
greater selectivity compared to the majority of other FAK inhibitors in clinical trials that target the
kinase domain only. In this strategy, small molecules were identified via molecular modelling studies
that may disrupt different scaffolding protein–protein interaction functions (PPI). These molecules
include Y11, Y15, and Roslin (R2) plus chloropyramine hydrochloride (C4), a licenced H1 histamine
receptor antagonist (Figure 1) [24
targeting (FAT) domain interactions, inhibits cell proliferation in vitro, and reduces tumour growth
in vivo within xenograft mouse models [27 28]. FAK inhibitors can also be used as chemotherapy
–26]. It is proposed that C4 blocks FAK C-terminal focal adhesion
,
sensitizers since they enhance the antitumour activity when used in combination with other cytotoxic
chemotherapeutics; however, questions remain about target selectivity [17,21].
Increased FAK mRNA levels are found in several human malignancies. Large databases such
as the Cancer Genome Atlas show that FAK mRNA levels are increased in invasive breast cancers
(26%) [29], and that these increased levels are correlated with poor overall patient survival [30,31].
It is established that FAK has a crucial role and is considered to be an indicator of development,
progression, and invasive potential of human breast cancer (BC). FAK shows upregulated expression,
phosphorylation, and processing in BC tissue compared to the adjacent non-tumour tissue of the same
patient. Upregulation of FAK was found to be increased in parallel with the advancement of cancer
stages where FAK is upregulated both at protein and mRNA levels. Overexpression of FAK was
frequently found in ER-positive and PR-positive BC, but not Her2/neu-negative breast cancer cases [32].
Moreover, the mRNA expression levels of the FAK encoding gene (PTK2), FAK phosphorylation as
well as the focal adhesion points (FAs) are higher in triple negative breast cancer (TNBC), an aggressive
BC type characterised by high metastatic potential. FAK inhibition prevented the oestrogen induced
migration of invasive MDA-MB 231 cells [33]. Furthermore, FAK inhibition in a pancreatic cancer
cell line (MiaPaCa2) resulted in a significant decrease in the number of colonies in a dose-dependent
manner [34]. Additionally, treatment with FAK inhibitors resulted in decreased primary tumour size
and fewer tumour-associated macrophages (TAMs), which are key contributors to tumour progression
and inflammation, in a mouse model of pancreatic ductal adenocarcinoma [35]. Herein, we used
a structure-based approach to design and synthesise potential FAK-FAT domain inhibitors.
2. Results and Discussion
2.1. Virtual Screening (Molecular Modelling)
The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localisation
of FAK to focal adhesions and subsequent activation. Phosphorylation of tyrosine 925 (Y925) in the FAT
domain has been shown to promote tumour metastasis and invasion in vivo
hydrochloride) (Figure 1) is a clinically approved histamine receptor H1 antagonist and was identified
in a previous study to inhibit FAK-FAT domain mediated signalling [27 28]. However, C4 displays
its anticancer activity at a remarkably high micromolar concentration (>100 M). Our previous lead
[36]. C4 (chloropyramine
,
µ
optimisation work on the chemical structure of chloropyramine (C4) led to the identification of a more
active analogue against three human breast cancer lines (MDA-MB-231, BT474, and T47D) with an
average antiproliferative activity (IC50) of approximately 23 µM [37].

Molecules 2020, 25, 3488
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In this study, we aimed to use a structure-based drug design and virtual screening approach to
identify a small molecule that can bind the FAK Tyr 925 phosphorylation site according to the workflow
depicted in Figure 2A. Therefore, the crystal structure of the focal adhesion targeting (FAT) domain
was obtained from the Protein Data Bank (PDB code 1K05) and prepared for docking [38]. The pocket
used for the high throughout virtual screening was focused on the area surrounding the three amino
acids: Asp 1036, Ser 910, and Lys 1032 which also encompasses the Tyr 925 residue (Figure 3).
Figure 2.
FAK-FAT domain. (
screening experiment.
(
A
) Schematic representation of the virtual screening of SPECS virtual library against
B
) Chemical structures of the top 25 preliminary FAK-FAT hits in the virtual
Figure 3.
(A) The predicted binding mode of compound I (cyan sticks) inside the pocket of the FAK-FAT
domain (grey) pocket, showing interaction with Asp 1036 (pink), Ser 910 (yellow), Pro 911 (blue)
,
Lys 1032 (green), and Tyr 925 (purple); hydrogen bonds are indicated by the cyan dashed lines.
(B) The detailed interactions between compound I and the FAK-FAT domain.

Molecules 2020, 25, 3488
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Approximately 210,000 small molecules from the SPECS virtual library of compounds were
imported, rendered, and minimised using a MMFF94x force field and run in a virtual screening
simulations programme using Glide SP within Maestro software (Glide, version 9.5, Schrödinger;
http://www.schrodinger.com). These compounds underwent four stages of filtration including
prefiltration of the non-drug-like compounds in accordance with Lipinski’s rules and excluding
chemically unstable and potentially toxic groups. This was followed by high throughput virtual
screening (HTVS), standard precision virtual screening (SPVS) and extra precision virtual screening
(XPVS). Each compound was positioned in the chosen pocket, which was made up of the area
surrounding the three amino acids Asp 1036, Ser 910, and Lys 1032 within a 12 A◦ radius and scored
for electrostatic and van der Waals interactions as implemented in the Schrodinger virtual screening
package (version 9.5). This process resulted in the identification of twenty-five virtual hits (Figure 2B).
The predicted binding modes to the FAK-FAT domain of selected hits are shown in Figure 2A.
These 25 hits underwent preliminary in vitro profiling in the metastatic triple negative breast cancer
(TNBC) cell line (MDA-MB-231) for antiproliferative and antimigratory properties. This revealed that
compound I in particular exhibited low micromolar antiproliferative and antimigratory properties
exceeding those of chloropyramine (C4) and thus was considered as a hit compound.
According to the surface representation of the FAK-FAT domain, the predicted docking mode of
compound
I
binds in a comparatively polar cavity surrounded by residues Ser 910, Thr 929, Pro 911
,
Pro 913, Lys 1032, and Asp 1036, as shown in Figure 3. Key interactions of compound
I
due to the
presence of the free amine (NH2) group include a salt bridge with the side chain carboxyl group
(COOH) of Asp 1036, two H-bonds with the backbone carbonyl (C=O) group of Pro 911, and the
side chain hydroxy group (OH) of Ser 910. Additionally, hydrophobic interactions were noticed
between the p-tert-butyl phenyl and p-chlorophenyl moieties and the surrounding hydrophobic pocket
formed of residues Tyr 925, Val 928, Val 932, Pro 913, and Leu 1035 (Figure 3). Compound I possesses
a chiral centre and both isomers are accommodated at the binding site and bind similarly with minimal
difference in binding potential between enantiomers (Figure 4).
Figure 4. The predicted binding mode of the two enantiomers of compound
I showing similar
interactions with the FAK-FAT domain; hydrogen bond is indicated by the dashed lines.
It is worth mentioning that the previous study published by Kurenova et al. [27] used the nuclear
magnetic resonance analysis (NMR) of the FAT/VEGFR-3 peptide complex to localise the chemical
shift of residue histidine 1025 on the FAT domain, to hypothesise that a small molecule binding to this
site could disrupt the FAK-VEGFR-3 interaction (Figure 5A). On the other hand, our virtual screening
study focused on the pocket near the catalytic tyrosine 925, which provides the chemical and geometric
features appropriate for small molecule binding as indicated by the red box shown in Figure 5B.
The grid generation module of the Schrodinger software was used to specify the pocket used to rank
the potential ligands, based on the interactions with the target pocket (Figure 5B).

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Figure 5. Comparison between the site selection (red dashed box) for the high throughout virtual
screening of the drug-like FAK-FAT inhibitor; ( ) previously identified chloropyramine (C4) centred
around His 1025. ( ) Our virtual screening hit compound centred around the Asp 1036, Ser 910,
and Lys 1032 pocket.
A
B
I
Interestingly, there appears to be some chemical structure similarity between compound
I and C4
(Figure 5). However, the significantly enhanced activity of compound over C4 appears to be a result
I
of the improved interaction between the amino ethyl side chain of compound I and the COOH group
of Asp 1036 (Figure 3), compared to C4 (Figure 6).
Figure 6. The predicted binding mode of chloropyramine (C4) showing detailed interactions with the
pocket of the FAK-FAT domain; hydrogen bond is indicated by the dashed lines.
2.2. Characterisation of the Cellular Effects of Compound I
2.2.1. Compound I Inhibits TNBC Cell Proliferation and Migration
The effect of compound
I on cell behaviour was explored in an in vitro model of TNBC,
MDA-MB-231, using C4 and the FAK kinase inhibitor PF271 as control compounds. Initial Western
blotting of FAK activity revealed no significant changes in the activity of either FAKY397or FAKY925
,
as well as no changes in the stability of total FAK protein following treatment with compound I
(
Figure S1; Supplementary Information). This was expected, given that this compound limits
protein–protein scaffolding interaction rather than kinase activity and subsequent phosphorylation
of key FAK residues. However, despite its inability to impair FAK phosphorylation, compound
could significantly reduce proliferation rates of MDA-MB-231 cells (Figure 7A). More relevant to FAK
I

Molecules 2020, 25, 3488
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function, compound
I
also elicited a significant reduction in serum-stimulated migration in Boyden
chamber assays (Figure 7B,C).
Figure 7.
compound
(
A
I
) Relative 7-day proliferation of MDA-MB-231 cells in vitro in the presence of 1
or PF271 compared to vehicle-only control. ) Representative images of Boyden
or PF271, post 18 h serum-stimulated migration.
) Quantified results of anti-migratory effects of compound and PF271 from 18 h Boyden chamber
µM
(
B
chamber migration assays incubated with compound
I
(
C
I
migration assay. Negative (-ve) control represents non-serum stimulated cell migration. Data represent
the average number of cells/membrane across duplicate wells and 3 replicates. Error bars show SEM:
*p < 0.05; ***p < 0.001.
2.2.2. Compound
To validate that this change in migration was resulting from altered FAK functionality, we evaluated
the changes in subcellular distribution of active FAK in response to compound . Given its ability to bind
I Causes Changes in Cellular Morphology and Cellular Localisation of Active FAK
I
and impair protein–protein interactions of the FAT-domain of FAK, we hypothesised that treatment
could impair co-localisation and activity of FAK with focal adhesions. As such, we co-incubated
MDA-MB-231 cells with a marker of active FAK (FAKY861) with the well-established focal adhesion
marker vinculin (Figure 8A). Both compound
dynamics, with treatments leading to increased localisation of FAK to the cell periphery versus
vehicle-only controls. However, compound treated cells had significantly more active FAK displayed
I and PF271 caused a significant alteration in FAK
I
throughout the cytoplasm versus PF271 treated cells. These changes were also reflected in the dynamics
of the focal adhesions, with a significant increase in focal adhesions being noted in treated cells
(Figure 8B) implying impaired turnover of these regions. Interestingly, compound
the size of focal adhesions, unlike PF271 (Figure 8C). Given the decrease in co-localisation with vinculin
versus PF271, we hypothesised that compound impairs migration through partial sequestering of
I had no effect on
I
active FAK to the cytoplasm, limiting its recruitment and activation of subsequent FAK-activated
factors necessary for the growth and turnover of nascent focal adhesions.

Molecules 2020, 25, 3488
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Figure 8. Exploration of subcellular dynamic changes in active FAK and FA-marker vinculin following
compound or PF271 treatment. ( ) Images are representative of MDA-MB-231 cells following
I
A
1 h of serum stimulated migration in the presence of compound I, PF271, or a vehicle-only control.
The generated images were subsequently used to quantify changes in the average number of focal
adhesions/cell (
B) and the average size of focal adhesions (C). All error bars represent SEM; n = 3.
**p < 0.01; ***p < 0.001.
2.3. Chemistry
The preliminary activity results suggest that compound
development, and that the aminoethyl group and the diaryl moiety are promising structural features.
This prompted us to synthesise a series of compound analogues to optimise the structure–activity
I
is an interesting starting point for further
I
profile. We investigated the impact of introducing various substituents to the diarylethylamine scaffold
while keeping a bulky hydrophobic substituent at the para position of one of the phenyl groups.
A four-step synthetic pathway was devised for the synthesis of compound
The hydrochloride salts of the diarylethylamine derivatives (5a ) were prepared according to Scheme 1.
The first step involved the reaction of the corresponding Grignard reagent (1a ) and the respective
aromatic aldehyde (2a ) to prepare the diarylmethanol intermediates (3a ). Pure products were
obtained in good yields (47–77%). The conversion of the sterically congested alcohol group in
3a into the corresponding nitrile group was achieved via two steps. Firstly, the generation of
I analogues.
–
i
–c
–
h
–i
–
i
the chloride derivatives was achieved using thionyl chloride (SOCl2). Secondly, the reaction of the
chloride derivatives with titanium tetrachloride (TiCl4) and trimethylsilylcyanide (TMSCN) successfully
provided the nitrile analogues (4a
lithium aluminium hydride (LiAlH4), followed by treatment with 2M hydrogen chloride in anhydrous
diethyl ether to yield the target hydrochloride salts 5a (Scheme 1). Column chromatography and/or
–i). The reduction of the nitrile group into amine was achieved using
–
i
recrystallisation were used to purify all compounds. Salt forms of the final compounds were prepared
to give crystalline products and avoid the purification problems of the free amines. The confirmation
of the structures of all the synthesised compounds was achieved using analytical and spectroscopic
data (1H, 13C, 19F NMR, and mass spectrometry). The acquired data were in full accordance with the
depicted structures. (Figures S2–S4; Supplementary Information).

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Scheme 1. Synthesis of diphenylethylamine derivatives (5a–i). Reagents and conditions: (i) THF, rt,
24 h; (ii) SOCl2, CH2Cl2, rt, 3 h; (iii) TMSCN, TiCl4, CH2Cl2, rt, 24 h; (iv) LiAlH4, Et2O, rt, 24 h, then
2 M HCl in anh. Et2O.
2.4. Cell Viability Assay
The antiproliferative activity of compound I as well as its newly synthesised analogues (5a–i)
was evaluated in vitro against the three human breast cancer cell lines MDA-MB-231 (TNBC),
T47D (ER+, HER−), and BT474 (ER+, HER+) as well as a human pancreatic cancer cell line MIAPaCa2.
Chloropyramine (C4) was used as a positive control, and the results are shown in Table 1. The seeding
of the corresponding cell lines started one day before incubation, which lasted for 72 h with the
different concentrations of the tested compounds. CellTiter Glo reagent (25 µL) was added to the
cell plate. The luminescence was analysed after 10 min using a PerkinElmer Envision instrument.
The test compounds were used at ten different triplicate concentrations in half log increments up
to 100
chloropyramine (C4). Notably, C4 did not show any significant anti-proliferative activity up to a
concentration of 100 M across the three human breast (hBC) cell lines and one pancreatic cancer
(hPC) cell line. Marginally, the best activity was associated with compound 5f, which showed low
micromolar activity in the three hBC cell lines (MDA-MB-231, IC50 = 5.06 M), (BT474, IC50 = 4.59 M),
and (T47D, IC50 = 4.70 µM) and the pancreatic cancer line (MIAPaCa2, IC50 = 5.28 µM) (Figure 9).
µM. The results showed that all compounds displayed better anti-proliferative activity than
µ
µ
µ
Table 1. In vitro anti-proliferative activity (IC50 in
µM) of compounds (I, 5a–i) compared to
chloropyramine (C4) in human breast (MDA-MB-231, T47D, and BT474) and pancreatic (MIAPaCa2)
cancer cell lines. IC50 values represent the mean of independent triplicate experiments.
T47D
BT474
MDA-MB-231
ER− PR− HER−
ID
R1
R2
MIAPaCa2
ER+ PR+/− HER−
ER+ PR+/− HER+
I
4-tBu
4-tBut
4-tBut
4-tBut
4-tBut
4-tBut
4-tBut
4-OCF3
4-Cl
4-OCF3
4-SCF3
4-F
6.37
5.63
6.71
4.94
2.67
5.00
12.46
8.94
14.78
14.97
4.70
15.53
13.19
44.50
>100
5.66
5.51
5a
5b
5c
5d
5e
5f
5g
5h
5i
11.78
12.76
14.79
14.12
5.06
16.95
12.75
46.25
99.02
11.44
13.38
14.74
14.38
4.59
15.26
13.23
43.93
>100
15.72
15.12
15.80
16.97
5.28
45.32
39.89
49.04
>100
2-F
3-F
4-tBut
4-Cl
4-OCF3 4-OCF3
4-OMe
-
4-OMe
-
C4

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Figure 9. Antiproliferative activity of compounds I and 5f across three human breast cancer cell lines
(MDA-MB-231, BT474, and T47D) and one pancreatic cancer (Mia PaCa-2) cell line.
Figure 10 illustrates the predicted docking mode of compound 5f which offers the advantage
of lacking the chiral centre present in compound I. Compound 5f sits in the pocket surrounded by
residues Ser 910, Thr 925, Thr 929, Pro 911, Pro 913, Lys 1032, and Asp 1036, as shown in Figure 5.
Key interactions of compound 5f due to the presence of the free amine (NH2) group include a salt
bridge with the side chain carboxyl group (COOH) of Asp 1036, two H-bonds with the backbone
carbonyl (C=O) group of Pro 911, and the side chain hydroxy group (OH) of Ser 910. In addition,
hydrophobic interactions were noticed between the p-tert butyl phenyl moiety and the surrounding
hydrophobic pocket formed of residues Tyr 925, Val 928, Val 932, Pro 913, and Leu 1035 (Figure 10).
Figure 10. (A) The predicted binding mode of compound 5f inside the pocket of the FAK-FAT domain
showing hydrogen bonds as blue dashed lines. (B) The detailed interactions between compound 5f
and FAK-FAT domain.
2.5. Pharmacokinetic Studies
2.5.1. In Vitro Aqueous Solubility
Furthermore, in vitro pharmacokinetic (PK) studies of the most active compounds were carried
out. The in vitro aqueous solubility ◦was evaluated using five different concentrations (1, 3, 10, 30,
and 100 µM), after incubation at 37 C for 2 h. Both the estimated precipitation range (lower and
upper bound) and a mid-range value were measured. Nicardipine and pyrene were used as control
compounds. Compound and 5c showed high aqueous solubility with an estimated precipitation
I

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range of 100 (lower bound) and >100
µM (upper bound). Compounds 5a and 5h showed moderate
solubility of 30 µM (lower bound) and 100 µM (upper bound), and compounds 5b and 5f showed
relatively reduced solubility comparable to pyrene (Table 2).
Table 2. In vitro aqueous solubility of diarylethylamine salt analogues (5a–5c, 5f, and 5h) compared to
nicardipine and pyrene (as control compounds) in buffered aqueous solution.
Estimated Precipitation Range (µM)
ID
Lower Bound
Upper Bound
Calculated Mid-Range
Compound I
100
30
3
100
1
30
10
3
>100
100
20
>100
6.5
100
30
10
>100
65
11.5
>100
3.75
65
5a
5b
5c
5f
5h
nicardipine
pyrene
10.5
6.5
2.5.2. Microsomal Metabolic Stability
The metabolic stability of compounds
I, 5a, 5c, 5f–5h was tested in human liver microsomes.
All compounds were incubated for 45 min with pooled liver microsomes and the intrinsic clearance
(CLint), which is the theoretical unrestricted maximum clearance of unbound drug without blood or
plasma protein binding limitations. Half-life (t1/2) values were measured at five time points. Both CLint
as well as t1/2 values were compared to dextromethorphan and verapamil as control compounds
(Table 3). Compound 5f displayed a microsomal half-life (t1/2) = 148 min, which is higher than that of
the drug controls—dextromethorphan (t1/2 = 49.2 min) and verapamil (t1/2 = 7.4 min)—and represents
a reasonable starting point for the development of an anticancer therapy.
Table 3. In vitro metabolic stability of compounds 5a, 5c, 5f
–h compared to control compounds
(dextromethorphan and verapamil) in human liver microsomes; CLint = intrinsic clearance,
SE CLint = standard error, t1/2 = half-life, and n = number of time points used to calculate CLint
.
Metabolic Stability (Human)
Compound Remaining (% of 0 min)
ID
CLint
t1/2 min
SE CLint
n
(µL/min/mg protein)
0 min
5 min
15 min 30 min 45 min Control
Compound I
9.10
8.91
8.26
9.34
13.9
2.35
28.2
187
3.35
0.532
1.15
0.699
2.14
2.31
3.73
2.82
152
156
168
148
99.9
589
49.2
7.40
5
5
5
5
5
5
5
3
100
100
100
100
100
100
-
89.4
95.8
94.7
97.2
99.0
102
-
92.3
92.3
95.0
92.2
96.2
108
-
77.9
86.0
86.1
88.0
86.8
100
-
81.3
81.4
82.5
80.7
72.8
95.8
-
95.7
91.0
98.0
88.0
86.6
97.8
-
5a
5c
5f
5g
5h
dextromethorphan
verapamil
-
-
-
-
-
-
2.5.3. Cardiotoxicity Assay
The hERG channel inhibition assay is a sensitive measurement to identify compounds exhibiting
cardiotoxicity related to hERG channel inhibition. Compounds 5a 5f, and 5i were tested for hERG
channel inhibition in the patch-clamp assay. The IC50 was calculated at 6 different concentration points
(0, 0.008, 0.04, 0.2, 1, 5, and 25 M). The results show that there was no in vitro cardiotoxicity observed
with compound 5f (IC50 = >25 M) which reflects a satisfactory degree of safety for this compound
(Table 4) [39]. (http://cyprotex.com).
I,
,
µ
µ

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Table 4. In vitro cardiotoxicity expressed in terms of IC50 of hERG channel inhibition
of diarylethylamine compounds (5a 5f 5i) compared to control compounds (quinidine and
,
,
dimethylsulfoxide). SE IC50: standard error; n: number of data points used to calculate IC50
.
hERG Channel Inhibition
ID
IC50 (µM)
SE IC50 (µM)
n
Compound I
5.47
3.59
>25
6.01
1.39
>25
0.616
0.833
-
1.22
0.366
-
15
11
12
11
12
15
5a
5f
5i
quinidine
dimethylsulfoxide
3. Materials and Methods
3.1. Molecular Modelling
The crystal structure of the focal adhesion targeting (FAT) domain was downloaded from the
Protein Data Bank (PDB code 1K05) [38] and prepared for docking using the MOE (Molecular
Operating Environment) protein preparation tools. The library of commercially available compounds
was downloaded from the SPECS website [40] (www.specs.net) in sdf format and prepared using
the conformational Search tool in MOE. The virtual screening simulations were performed using
the Glide SP within Maestro software using the default settings (Glide, version 9.5, Schrödinger;
http://www.schrodinger.com). The pocket used for the high throughout virtual screening was focused
on the area surrounding the three amino acids: Asp 1036, Ser 910, and Lys 1032 which also encompasses
the Tyr 925 residue within a 12A◦ radius. The virtual screening output database was saved as a mol2
file, and the visual inspection of the docking modes was performed in MOE.
3.2. Cell Proliferation Assay
To assess proliferation, cells were seeded at 4.2
×
105 cells/mL in 24-well plates. Following 24 h,
the media were replaced with fresh RPMI + 5% FCS, in the presence or absence of treatments and
a selection of wells counted (day 0 counts). Cells were subsequently counted each day up to 7 days to
assess proliferation. Briefly, this entailed the removal of old media and the addition of trypsin/EDTA
in order to lift cells before passing gently through a 25 G needle to achieve a single-cell suspension.
The resulting solution was then added to isoton in a counting cup and the cell number was determined
using a Coulter Multisizer III (Beckman Coulter Life Sciences, Indianopolis, IN, USA) Each well was
counted twice with all conditions performed in triplicate.
3.3. Boyden Chamber Migration
Migration was assessed utilising 24-well, transmembrane permeable support plates
(
Corning Lifesciences, Corning, New York, NY, USA) with 6.5 mm microporous membrane (8
pore size) inserts, each coated with 10 g/mL fibronectin in sterile PBS. Inserts were placed into wells
containing RPMI + 5% FCS ( treatments), before cells in serum-free RPMI were seeded into the top
µm
µ
±
portion of each insert at a density of 50,000 cells/mL. Cells were then incubated at 37 ◦C and 5% CO2
for 18 h. After this migratory period was completed, cells were fixed to the underside of the inserts in
3.7% PFA in PBS and stained with 0.5% crystal violet solution. Resulting inserts were then imaged
using a standard light microscope.
3.4. Immunofluorescence
MDA-MB-231 TNBC cells were seeded at 700,000 cells/mL onto fibronectin coated coverslips and
allowed to proliferate until 50% confluent, at which point they were treated with serum-free RPMI for
24 h. Following this, cells were incubated with RPMI+ 5% FCS for 1 h, before being washed briefly in

Molecules 2020, 25, 3488
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sterile PBS, and fixed in 3.7% PFA for 15 min. The resulting fixed cells were subsequently permeabilised
with 0.2% Triton-X100 in PBS for 8 min. This was proceeded by a 40 min block in 10% normal goat serum
(in 1% BSA in PBS) prior to 30 min primary antibody incubation. Both FAKY861 and vinculin antibodies
were diluted to working concentration (1:100 and 1:200, respectively) in 1% BSA in PBS. Specific
fluorophore-conjugated secondary antibodies were then applied (Alexafluor-488 and Alexfluor-594
diluted 1:1000 in 1% BSA in PBS), and resulting coverslips were briefly washed in PBS and mounted to
glass slides using hard-set Vectashield mounting media plus DAPI (40, 6-diamidino-2-phenylindole).
Slides were viewed using a 63X oil immersion lens on a Leica DM IRE2 microscope (Leica Microsystems,
Wetzlar, Germany).
3.5. General Procedure for the Preparation of Diaryl Alcohols (3a–i)
The diaryl methanol derivatives (3a
corresponding substituted phenyl magnesium bromide (Grignard reagent, 1a
(5.7mmol) to a solution of the respective aldehyde (5.7mmol) (2a–d
mixture was stirred for 24 h from 0 ◦C to room temperature. The mixture was concentrated under
vacuum then quenched by adding saturated aqueous NH4Cl (30 mL) and extracted with ethyl acetate
–
i
) were prepared by the dropwise addition of the
) 0.5M solution in THF
) in THF (20 mL) at 0 ◦C. The reaction
–c
(3
×
30 mL). The combined organic layers were washed with brine (20 mL) and water (30 mL), dried
over anhydrous magnesium sulphate, filtered, and concentrated under vacuum. The crude residue
was purified by column chromatography eluting with hexane-ethyl acetate gradually increasing from
100:0 to 90:10 v/v. Pure products were obtained in good yields (47%–77%).
(4-tert-butylphenyl)(4-(trifluoromethoxy)phenyl)methanol (3a), 1H NMR (CDCl3)
δ 7.45 (d, J = 8.5 Hz, 2H,
ArH), 7.41 (d, J = 8.5 Hz, 2H, ArH), 7.31 (d, J = 8.5 Hz, 2H, ArH), 7.21 (dd, J = 1, 9 Hz, 2H, ArH),
5.86 (d, J = 3 Hz, 1H, CH), 2.57 (d, J = 3.5 Hz, 1H, OH), 1.34 (s, 9H, 3
×
CH3). 19F (CDCl3) δ −57.84
.
,
13C NMR (CDCl3) δ 150.99 (ArC), 148.40 (ArC), 142.44 (ArC), 140.45 (ArC), 127.86 (ArCH), 126.32 (ArCH)
125.65 (ArCH), 120.90 (ArCH), 120.47 (1JC-F = 255.5Hz, OCF3), 75.40 (CH), 34.58 (C(CH3)3), 31.33 (C(CH3)3),
MS [ESI, m/z]: calcd for C18H19F3O2 [M + H-OH] 308.1388; found 308.1384.
(4-tert-butylphenyl)(4-((trifluoromethyl)thio)phenyl)methanol (3b), 1H NMR (CDCl3)
δ 7.67 (d, J = 8.5 Hz,
2H, ArH), 7.48 (d, J = 8.5 Hz, 2H, ArH), 7.44 (d, J = 8.5 Hz, 2H, ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 5.79
(d, J = 3 Hz, 1H, CH), 2.95 (d, J = 3.5 Hz, 1H, OH), 1.39 (s, 9H, (CH3)3). 19F (CDCl3) δ −42.65, 13C NMR
(CDCl3) δ
151.09 (ArC), 146.88 (ArC), 140.20 (ArC), 136.35 (ArCH), 129.67 (q, 1JC-F = 306.3 Hz, SCF3),
127.52 (ArCH), 126.49 (ArCH), 125.68 (ArCH), 75.49 (CH), 31.59 (C(CH3)3), 31.34 (CH3)3. MS [ESI, m/z]:
calcd C18H19F3OS [M − H] 339.1031; found 339.1033.
(4-tert-Butylphenyl)(4-fluorophenyl) methanol (3c), 1H NMR (CDCl3)
δ 7.44–7.36 (m, 4H, ArH), 7.34–7.28
(m, 2H, ArH), 7.05 (t, J = 8.5Hz, 2H ArH), 5.82 (s, 1H, CH), 2.50–2.25 (m, 1H, OH), 1.33 (m, 9H, (CH3)3.
19F (CDCl3) δ −115.24, 13C NMR (CDCl3)
δ
162.15 (d, 1JC-F = 243.8 Hz, ArC), 150.75 (ArC), 140.78 (ArC),
139.67 (ArC), 128.19 (d, 3JC-F = 8.0 Hz, ArCH), 126.28 (ArCH), 125.53 (ArCH), 115.22 (d, 2JC-F = 21.1
Hz, ArCH), 70.87 (CH), 31.50 C(CH3)3, 25.10 (CH3)3. MS [ESI, m/z]: calcd C17H19FO [M − H] 257.1342;
found 257.1338 and [M−H2O + H] 241.1393; found 241.1390.
1
(4-tert-butylphenyl)(2-fluorophenyl)methanol (3d), H NMR (CDCl3)
δ 7.57 (t, J = 7.5Hz, 1H, ArH),
7.39–7.35 (m, 4H, ArH), 7.29–7.26 (m, 2H, ArH), 7.19 (t, J = 7.5Hz, 1H, ArH), 6.16 (d, J = 4Hz, 1H,
CH), 2.26 (d, J = 4.5Hz, 1H, OH), 1.33 (s, 9H, (CH3)3). 19F (CDCl3) δ −118.50, 13C NMR (CDCl3)
δ
159.91 (d, 1JC-F = 244.6 Hz, ArC), 150.71 (ArC), 139.79 (ArC), 131.04 (d, 2JC-F = 12.1Hz, ArCH), 129.00
(d, 2JC-F = 8.8 Hz, ArCH), 127.64 (d, 2JC-F = 3.8 Hz, ArCH), 126.14 (ArC), 125.46 (ArC), 124.28 (d, 1JC-F
= 3.8 Hz, ArCH), 70.01 (CH), 34.53 (C(CH3)3), 31.33 (CH3)3. MS [ESI, m/z]: calcd C17H19FO [M
257.1342; found 257.1341 and [M−H2O + H] 241.1; found 241.1.
−
H]

Molecules 2020, 25, 3488
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1
(4-tert-Butylphenyl)(3-fluorophenyl) methanol (3e), H NMR (CDCl3)
δ
7.40 (d, J = 8.5Hz, 2H, ArH),
7.35–7.29 (m, 3H, ArH), 7.21–7.14 (m, 2H, ArH), 7.97 (td, J = 2.5, 8.5Hz, 1H, ArH), 5.84 (d, J = 3Hz, 1H,
CH), 2.21 (d, J = 3.5Hz, 1H, OH), 1.34 (s, 9H, (CH3)3). 19F (CDCl3) δ −112.92, 13C NMR (CDCl3)
δ 162.94
(d, 1JC-F = 244.5 Hz, ArC), 150.95 (ArC), 146.45 (d, 2JC-F = 6.5 ArC), 140.40 (ArC), 129.87 (d, 3JC-F = 8.1Hz,
ArCH), 126.35 (ArCH),), 125.60 (ArCH), 122.00 (d, 4JC-F = 3.1 Hz, ArCH), 114.23 (d, 2
ArCH), 113.36 (d, 2JC-F = 21.8 Hz, ArCH), 75.52 (d, 4JC-F = 1.6 Hz CH), 34.56 (C(CH3)3), 31.32 (CH3)3.
MS [ESI, m/z]: calcd C17H19FO [M H] 257.1342; found 257.1341 and [M H2O + H] 241.1; found 241.1.
JC-F = 20.9Hz,
−
−
Bis(4-tert-butylphenyl)methanol (3f), 1H NMR (CDCl3)
7.40–7.38 (m, 4H, ArH), 7.36–7.33 (m, 4H, ArH),
5.84 (d, J = 3Hz, OH), 2.16 (d, J = 3.5 Hz, CH), 1.34 (s, 2(CH3)3). 13C NMR (CDCl3)
150.40 (ArC),
δ
δ
141.00 (ArC), 126.25 (ArCH), 125.39 (ArCH), 79.95 (CH), 34.52 C(CH3)3, 31.36 (CH3)3. MS [ESI, m/z]:
calcd for C21H28O [M − H] 295.2062; found 295.2061 and [M−H2O + H] 279.2113; found 279.2115.
(4-Chlorophenyl)(4-trifluoromethoxyphenyl)methanol (3g), 1H NMR (CDCl3)
δ 7.38 (d, J = 8 Hz, 2H, ArH),
7.35 (d, J = 9 Hz, 2H, ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.21 (dd, J = 1, 8.5 Hz, 2H, ArH), 5.80 (d, J
= 3 Hz, 1H, CH), 2.57 (d, J = 3 Hz, 1H, OH). 19F (CDCl3) δ −57.85, 13C NMR (CDCl3)
δ 148.69 (ArC),
141.99 (ArC), 141.77 (ArC), 133.69 (ArC), 128.81 (ArCH), 128.81 (ArCH), 127.91 (ArCH), 127.86 (ArCH),
121.07 (ArCH), 120.44 (1JC-F = 255.5Hz, OCF3), 74.90 (CH). MS [ESI, m/z]: calcd for C14H10ClF3O2
[M + H-H2O] 285.0294; found 285.0298.
1
Bis(4-trifluoromethoxyphenyl)methanol (3h), H NMR (CDCl3)
δ 7.41 (dtd, J = 8.5, 3, 1 Hz, 4H, ArH),
7.23 (dd, J = 9, 1 Hz, 4H, ArH), 5.86 (d, J = 3 Hz, 1H, CH), 2.50 (d, J = 3.5 Hz, 1H, OH). 19F (CDCl3)
δ 148.70 (ArC), 141.87 (ArC), 127.93 (ArCH), 121.11 (ArCH), 120.45
(q, 1JC-F = 255.6 Hz, OCF3), 74.82 (CH). MS [ESI, m/z]: calcd C15H10F6O3 [M
δ −57.91, 13C NMR (CDCl3)
−
H] 351.0456; found
351.0456.
3.6. General Procedure for Formation of Diaryl Nitrile (4a–i)
A solution of thionyl chloride (10.80 mmol) and diaryl alcohol (3a–i, 7.20 mmol) in dichloromethane
(2 mL) was stirred at room temperature for 2 h. The reaction was concentrated under reduced pressure
to give the diaryl chloride. To a solution of the diaryl chloride (7.20 mmol) in dichloromethane
(33.12 mL), trimethylsilyl cyanide (7.20 mmol) and titanium tetrachloride (7.20 mL, 1 M solution in
dichloromethane) were added. After stirring under argon at room temperature for 2 h, the reaction
was quenched with methanol (13.90 mL) and water (41.62 mL) and diluted with dichloromethane
(104 mL). The organic layer was washed with saturated, aqueous sodium bicarbonate (68.25 mL) and
water (68.25 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to give the
crude product.
2-(4-tert-butylphenyl)-2-(4-trifluoromethoxyphenyl)acetonitrile (4a), 1H NMR (CDCl3)
δ 7.48–7.41 (m, 4H,
ArH), 7.31 (d, J = 8 Hz, 2H, ArH), 7.26 (d, J = 8 Hz, 2H, ArH), 5.18 (s, 1H, CH), 1.36 (s, 9H, 3 × CH3).
19F (CDCl3) δ −57.87. 13C NMR (CDCl3)
δ 151.68 (ArC), 148.98 (ArC), 134.79 (ArC), 132.26 (ArC), 129.28
(ArCH), 127.39 (ArCH), 126.34 (ArCH), 121.58 (ArCH), 120.41 (1JC-F = 256.4Hz, OCF3), 119.46 (CN),
41.58 (CH), 34.63 (C(CH3)3), 31.24 (C(CH3)3). MS [ESI, m/z]: calcd for C19H18F3NO [M + Na], 356.1233;
found, 356.1236.
2-(4-tert-butylphenyl)-2-(4-(trifluoromethyl)thiophenyl)acetonitrile (4b), 1H NMR (CDCl3)
2H, ArH), 7.47–7.42 (m, 4H, ArH), 7.30–7.27 (m, 2H, ArH), 5.17 (s, 1H, CH), 1.34 (s, 9H, (CH3)3).
19F (CDCl3) -42.45, 13C NMR (CDCl3)
151.81 (ArC), 139.04 (ArC), 136.89 (ArCH), 131.88 (ArC),
129.40 (q, 1JC-F = 306 Hz, SCF3), 128.83 (ArCH), 127.43 (ArCH), 126.37 (ArCH), 124.61 (q, 3
C-F = 2.3 Hz
δ 7.69 (d, J = 8 Hz,
δ
δ
J
,
ArC), 119.13 (CN), 41.93 (CH), 34.64 (C(CH3)3), 31.23 (CH3)3. MS [ESI, m/z]: calcd C19H18F3NS [M + H]
350.1190; found 350.1185, [M + H − HCN] 323.1081; found 323.1082.

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2-(4-tert-butylphenyl)-2-(4-fluorophenyl)acetonitrile (4c), 1H NMR (CDCl3)
δ
7.46 (d, J = 8.5Hz, 2H, ArH),
7.39 (dd, J = 5, 8Hz, 2H, ArH), 7.36 (d, 8.5Hz, 2H ArH), 7.10 (t, J = 9Hz, 2H ArH), 5.17 (s, 1H CH),
1.38–1.37 (m, 9H, (CH3)3). 19F (CDCl3)
δ
-113.86. 13C NMR (CDCl3)
δ
162.45 (d, 1JC-F = 246.3 Hz, ArCH),
3
151.492 (ArC), 132.82 (ArC), 132.14 (ArC), 129.55 (d, JC-F = 7.5 Hz, ArCH), 127.40 (ArCH), 126.29
2
(ArCH), 116.06 (CN), 116.15 (d, JC-F = 21.3 Hz, ArCH), 41.46 (CH), 34.63 (C(CH3)3), 31.31 (CH3)3.
MS [ESI, m/z]: calcd C18H18FN [M + NH4] 285.1767; found 285.1766.
2-(4-tert-Butylphenyl)-2-(2-fluorophenyl)acetonitrile (4d), 1H NMR (CDCl3)
δ 7.38 (t, J = 7.5Hz, 1H, ArH),
7.30 (d, J = 7Hz, 2H ArH), 7.26–7.22 (m, 3H, ArH), 7.10 (t, J = 7.5Hz, 1H, ArH), 7.01 (t, J = 9Hz
,
1H, ArH), 5.34 (s, 1H CH), 1.22 (s, 9H, (CH3)3). 19F (CDCl3)
δ
-117.28. 13C NMR (CDCl3)
δ
159.70
(d, 1JC-F = 246.3 Hz, ArC), 151.43 (ArC), 131.65 (ArC), 130.26 (d, JC-F = 7.5 Hz, ArCH), 129.31
(d, 4JC-F = 2.8 Hz, ArCH), 127.17 (ArCH), 126.15 (ArCH), 124.92 (d, JC-F = 3.8 Hz, ArCH), 123.66
3
3
(d, 2 C-F = 14.1 Hz, ArC), 119.05 (CN), 116.25 (d, 2JC-F = 21.4 Hz, ArCH), 35.66 (d, 3JC-F = 3.8 Hz, CH),
J
34.59 C(CH3)3, 31.25 (CH3)3. MS [ESI, m/z]: calcd C18H18FN [M + NH4] 285.1767; found 285.1765.
2-(4-tert-butylphenyl)-2-(3-fluorophenyl)acetonitrile (4e), 1H NMR (CDCl3)
7.43 (d, J = 8.5Hz, 1H, ArH),
7.41–7.35 (m, 1H, ArH), 7.29 (d, J = 8, 2H, ArH), 7.20 (d, J = 7.5Hz, 1H, ArH), 7.11- 7.02 (m, 2H, ArH),
5.13 (s, 1H CH), 1.34 (s, 9H, (CH3)3). 19F (CDCl3) -111.36. 13C NMR (CDCl3) 162.99 (d, 1JC-F = 246.4
Hz, ArC), 151.65 (ArC), 138.37 (d, 3JC-F = 7.3 Hz, ArC), 132.15 (ArC), 130.73 (d, 3
C-F = 8.3 Hz, ArCH),
δ
δ
δ
J
127.38 (ArCH), 126.28 (ArCH), 123.41 (d, 4JC-F = 2.8 Hz, ArCH), 119.31 (CN), 115.28 (d, 2JC-F = 21.0 Hz
,
ArCH), 114.99 (d, 2JC-F = 22.8 Hz, ArC), 41.89 (d, 4JC-F = 1.9 Hz, CH), 34.62 C(CH3)3, 31.24 (CH3)3.
MS [ESI, m/z]: calcd C18H18FN [M + NH4] 268.1501; found 268.1494.
2,2-Bis(4-tert-butylphenyl)acetonitrile (4f), 1H NMR (CDCl3)
ArH), 5.11 (s, 1H, CH), 1.33 (s, 9H, 2(CH3)3). 13C NMR (CDCl3)
δ
7.42–7.40 (m, 4H, ArH), 7.31–7.29 (m, 4H,
151.20 (ArC), 133.03 (ArC), 127.39
δ
(ArCH), 126.08 (ArCH), 120.04 (CN), 41.82 (CH), 34.57 C(CH3)3, 31.27 (CH3)3. MS [ESI, m/z]: calcd
C22H27N [M + NH4] 323.2487; found 323.2487.
1
2-(4-Chlorophenyl)-2-(4-trifluoromethoxyphenyl)acetonitrile (4g), H NMR (CDCl3)
δ 7.42–7.37 (m, 4H,
ArH), 7.31 (d, J = 8 Hz, 2H, ArH), 7.26 (dd, J = 1, 9 Hz, 2H, ArH), 5.16 (s, 1H, CH). 19F (CDCl3)
δ
-57.90.13C NMR (CDCl3)
δ 149.19 (ArC), 134.73 (ArC), 134.04 (ArC), 133.78 (ArC), 129.59 (ArCH),
129.23 (ArCH), 129.06 (ArCH), 121.75 (ArCH), 120.35 (1JC-F = 256.5Hz, OCF3), 118.83 (CN), 41.37 (CH).
MS [ESI, m/z]: calcd for C15H9ClF3NO [M] 311.0325; found 311.0327.
2,2-Bis(4-trifluoromethoxyphenyl)acetonitrile (4h), 1H NMR (CDCl3) δ 7.41 (d, J = 8.5 Hz, 4H, ArH), 7.28
(d, J = 9 Hz, 4H, ArH), 5.21 (s, 1H, CH). 19F (CDCl3)
(ArC), 129.27 (ArCH), 121.76 (ArCH), 120.36 (q, JC-F = 256.4 Hz, OCF3), 118.83 (CN), 41.30 (CH).
MS [ESI, m/z]: calcd C16H9F6NO2 [M] 361.0537; found 361.0531, [M + H] 362.0616; found 362.0595,
[M + H-HCN] 335.0507; found 335.0530.
δ δ 149.26 (ArC), 133.93
-57.97, 13C NMR (CDCl3)
1
2,2-Bis(4-methoxyphenyl)acetonitrile (4i), 1H NMR (CDCl3)
4H, ArH), 5.08 (s, 1H, CH), 3.83 (s, 6H, 2xCH3). 13C NMR (CDCl3)
δ
7.27 (d, J = 9 Hz, 4H, ArH), 6.91 (d, J = 8.5 Hz
,
δ
159.40 (ArC), 128.82 (ArCH), 128.30
(ArC), 120.13 (CN), 114.50 (ArCH), 55.36 (2xCH3), 41.07 (CH). MS [ESI, m/z]: calcd for C16H15NO2
[M − H] 252.1019; found 252.1021.
3.7. General Procedure for the Reduction of Nitrile to Amine Hydrochloride (5a–i)
To a suspension of lithium aluminum hydride (2.35 mmol) in anhydrous diethylether (20 mL) at
0 ◦C, a solution of nitrile (4a
–
i
, 0.78 mmol) in diethylether (10 mL) was added dropwise. After stirring
under nitrogen for 24 h, the reaction was quenched with water (10 mL). The reaction mixture was
extracted with ethyl acetate (3 50mL). The combined organic layer was dried over MgSO4, filtered,
×
and concentrated under reduced pressure to give the crude product. The crude mixture was treated
with a 2 M anhydrous HCl solution in diethyl ether (5–10 mL), and the resulting amine hydrochloride
salts were washed with diethyl ether. If the amine hydrochloride salts did not form a precipitate,

Molecules 2020, 25, 3488
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the diethyl ether solution was concentrated under reduced pressure and rinsed with diethyl ether to
give the hydrochloride salts.
2-(4-tert-butylphenyl)-2-(4-trifluoromethoxyphenyl)ethan-1-aminium hydrochloride (5a), 1H NMR (CDCl3)
δ
8.12 (bs, 3H, +NH3), 7.35 (d, J = 8 Hz, 2H, ArH), 7.31 (d, J = 8.5 Hz, 2H, ArH), 7.17 (d, J = 8.5, 2H,
ArH), 7.14 (d, J = 8.5, 2H, ArH), 4.44 (t, J = 8 Hz, 1H, CH), 3.57 -3.42 (m, 2H, CH2), 1.29 (s, 9H, 3xCH3).
19F (CDCl3) δ −57.81. 13C NMR (CDCl3)
δ 150.89 (ArC), 148.50 (ArC), 138.30 (ArC), 135.90 (ArC),
129.53 (ArCH), 127.40 (ArCH), 126.19 (ArCH), 121.49 (ArCH), 120.39 (1JC-F = 255.6Hz, OCF3), 47.57
(CH), 43.78 (CH2), 34.49 (C(CH3)3), 31.23 (C(CH3)3). MS [ESI, m/z]: calcd for C19H23ClF3NO [M + H]
338.1727; found 338.1726.
2-(4-tert-butylphenyl)-2-(4-(trifluoromethyl)thiophenyl)ethan-1-aminium chloride (5b), 19F (CDCl3) δ −42.45,
1H NMR (CDCl3)
δ
8.05 (bs, 3H, +NH3), 7.43–7.32 (m, 4H, ArH), 7.27–7.10 (m, 4H, ArH), 4.49
(t, J = 8Hz, 1H, CH), 3.60–3.30 (m, 2H, CH2), 1.31 (s, 9H, (CH3)3). 13C NMR (CDCl3)
δ 151.49 (ArC),
1
139.63 (ArC), 136.98 (ArCH), 130.78 (ArC), 128.24 (q, JC-F = 296 Hz, SCF3), 128.01 (ArCH), 127.41
(ArCH), 126.38 (ArCH), 42.15 (CH), 34.43 C(CH3)3, 31.30 C(CH3)3. MS [ESI, m/z]: calcd C19H23ClF3NS
[M + H]+354.1498; found 354.1501.
2-(4-tert-butylphenyl)-2-(4-fluorophenyl)ethan-1-aminium chloride (5c), 19F (CDCl3) δ −114.15, 1H NMR
(CDCl3)
δ
8.07 (bs, 3H, +NH3), 7.36–7.31 (m, 4H, ArH), 7.03–6.94 (m, 4H, ArH), 4.42 (t, J = 8Hz, 1H,
CH), 3.51–3.50 (m, 2H, CH2), 1.29 (s, 9H, (CH3)3). 13C NMR (CDCl3)
δ
161.99 (d, JC-F = 245.0 Hz,
1
3
ArCH), 150.48 (ArC), 136.73 (ArC), 135.58 (ArC), 129.78 (d, JC-F = 7.9 Hz, ArCH), 127.40 (ArCH),
126.03 (ArCH), 125.57 (d, 2JC-F = 20.0 Hz, ArCH), 34.45 (CH), 34.46 (CH2), 31.25 C(CH3)3, 29.71 (CH3).
MS [ESI, m/z]: calcd C18H23ClFN [M-Cl]+ 272.1809; found 272.1808.
2-(4-tert-butylphenyl)-2-(2-fluorophenyl)ethan-1-aminium chloride (5d), 1H NMR (CDCl3)
+NH3), 7.35–7.29 (m, 4H, ArH), 7.22–7.19 (m, 4H, ArH), 3.94 (t, J = 8Hz, 1H, CH), 3.62–3.57 (m, 1H,
CH2), 3.52–3.48 (m, 1H, 1.32, CH2), 1.29 (s, 9H, (CH3)3). 19F (CDCl3) -115.13. 13C NMR (CDCl3)
149.70 (d, 1JC-F = 256.1 Hz, ArC), 143.94 (ArC), 135.51 (ArC), 130.18 (d, 3JC-F = 8.5 Hz, ArCH), 129.28
δ 8.10 (bs, 3H,
δ
δ
4
3
(d, JC-F = 3.9 Hz, ArCH), 127.54 (ArCH), 126.00 (ArCH), 122.95 (d, JC-F = 6.1 Hz, ArCH), 118.30
(d, 2 C-F = 12.5 Hz, ArC), 116.32 (d, 2JC-F = 22.5 Hz, ArCH), 42.82 (CH2), 31.25 (d, 3JC-F = 3.4 Hz, CH),
J
30.56 (C(CH3)), 30.03 (CH3). C18H23ClFN [M-Cl]+, 272.1809; found 272.1806.
2-(4-tert-butylphenyl)-2-(3-fluorophenyl)ethan-1-aminium chloride (5e), 1H NMR (CDCl3)
δ 8.15–7.81 (m, 3H,
+NH3), 7.38–7.32 (m, 4H, ArH), 7.26–7.20 (m, 2H, ArH), 7.19–7.16 (m, 2H, ArH), 4.48–4.40 (m, 1H,
CH), 3.60–3.40 (m, 2H, CH2), 1.31–1.27 (m, 9H, (CH3)3). 19F (CDCl3)
δ δ
-111.70. 13C NMR (CDCl3)
163.06 (d, 1JC-F = 245.3 Hz, ArC), 143.94 (ArC), 135.51 (ArC), 130.18 (d, 3JC-F = 8.5 Hz, ArCH), 129.28
(d, 4JC-F = 3.9 Hz, ArCH), 127.54 (ArCH), 126.00 (ArCH), 122.95 (d, JC-F = 6.1 Hz, ArCH), 118.30
3
(d, 2JC-F = 12.5 Hz, ArC), 116.32 (d, 2JC-F = 22.5 Hz, ArCH), 43.71 (CH2), 47.79 (CH), 34.49 (C(CH3)3),
31.24 ((CH3)3). C18H23ClFN [M-Cl]+, 272.1809; found 272.1808.
1
2,2-Bis(4-tert-butylphenyl)ethan-1-aminium chloride (5f), H NMR (CDCl3)
δ 8.08 (bs, 2H, NH2), 7.33
(d, J = 8Hz, 4H, ArH), 7.21 (d, J = 8Hz, 4H, ArH), 3.94 (t, J = 8Hz, 1H, CH), 3.52 (bs, 2H, CH2), 1.29
(s, 9H, 2(CH3)3). 13C NMR (CDCl3)
150.41 (ArC), 136.55 (ArC), 127.57 (ArCH), 126.05 (ArCH), 47.65
δ
(CH), 44.08 (CH2), 34.46 C(CH3)3, 31.30 (CH3)3. MS [ESI, m/z]: calcd C22H32ClN [M + H] 310.2529;
found 310.2529.
1
2-(4-chlorophenyl)-2-(4-trifluoromethoxyphenyl)ethan-1-aminium chloride (5g), H NMR (CDCl3)
δ 8.13
(bs, 3H, +NH3), 7.32 (d, J = 8 Hz, 2H, ArH), 7.27 (d, J = 8 Hz, 2H, ArH), 7.23–7.16 (m, 4H, ArH), 4.43
(t, J = 8 Hz, 1H, CH), 3.44 (bs, 2H, CH2). 19F (CDCl3) -57.85, 13C NMR (CDCl3)
148.69 (ArC), 137.72
δ
δ
(ArC), 137.15 (ArC), 133.88 (ArC), 129.44 (ArCH), 129.40 (ArCH), 129.31 (ArCH), 121.64 (ArCH), 120.37
(1JC-F = 212.5Hz, OCF3), 47.48 (CH), 43.31 (CH2). MS [ESI, m/z]: calcd for C15H14Cl2F3NO [M + H]
316.0711; found 316.0712.

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2,2-Bis(4-trifluoromethoxyphenyl)ethan-1-aminium chloride (5h), 19F (CDCl3) δ −57.84, 1H NMR (CDCl3)
δ
8.15 (bs, 3H, +NH3), 7.42–7.33 (m, 4H, ArH), 7.12–7.08 (m, 4H, ArH), 4.26–4.20 (m, 1H, CH), 3.35–3.28
(m, 2H, CH2). 13C NMR (CDCl3) 148.27 (ArC), 142.90 (ArC), 128.88 (ArCH), 121.34 (q, 1JC-F=252.3
δ
Hz, OCF3), 120.96 (ArCH), 52.25 (CH), 50.71 (CH2). MS [ESI, m/z]: calcd C16H14ClF6NO2 [M + H]+
366.0923; found 366.0924.
1
+
2,2-Bis(4-methoxyphenyl)ethan-1-aminium chloride (5i), H NMR (CDCl3)
δ 8.04 (bs, 3H, NH3), 7.17
(d, J = 7.5 Hz, 4H, ArH), 6.85 (d, J = 7.5 Hz, 4H, ArH), 4.38 (t, J = 8.5 Hz, 1H, CH), 3.76 (s, 6H, 2OCH3),
3.48 (bs, 2H, CH2). 13C NMR (CDCl3)
δ 158.59 (ArC), 133.15 (ArC), 128.95 (ArCH), 114.31 (ArCH),
55.25 (2xCH3), 53.27 (CH), 45.23 (CH2). MS [ESI, m/z]: calcd for C16H20ClNO2 [M + H] + 258.1489;
found 258.1486.
3.8. In Vitro Aqueous Solubility, Metabolic Stability and Cardiotoxicity Studies
All in vitro biological evaluations were performed by Cyprotex, according to their internal
procedures. The detailed protocols procedure can be found online.
3.8.1. Turbidimetric Aqueous Solubility
The test compound was diluted in buffer to give a range of concentrations (1, 3, 10, 30, and
100 µ
M; final DMSO concentration 1%) and incubated at 37 ◦C for 2 hr. Absorbance was measured at a
wavelength of 620 nm and the solubility was estimated from the concentration of the test compound
that produces an increase in absorbance above the vehicle control (i.e., 1% DMSO in buffer).
Experimental Procedure
The test compound (10 mM in DMSO) was serially diluted to give solutions of 0.1, 0.3, 1, and
3 mM in DMSO. Each test compound concentration was then further diluted 1 to 100 in buffer (typically
0.01 M phosphate buffered saline pH 7.4) so that the final DMSO concentration was 1%, and the
final test compound concentrations were 1, 3, 10, 30, and 100 µM. The experiment was performed at
37 ◦C and each concentration was incubated in 7 replicate wells. The plates were incubated for 2 h
at 37 ◦C before the absorbance was measured at 620 nm. Nicardipine and pyrene were included as
control compounds. The solubility of nicardipine is pH dependent whereas the solubility of pyrene is
pH independent.
Data Analysis
The solubility was estimated from the concentration of the test compound that produced an
increase in absorbance above the vehicle control (i.e., 1% DMSO in buffer).
An estimated precipitation range (lower and upper bound) and a calculated mid-range value
were returned, along with any relevant comments, in the form of an Excel spreadsheet.
3.8.2. Microsomal Metabolic Stability
The test compound (3 µM) was incubated with pooled liver microsomes at 5 time points over the
course of a 45 min experiment, and the test compound was analysed by LC-MS/MS. Intrinsic clearance
values (CLint) with standard error and t1⁄2 values were measured.
A total of 50 µL of the 10 mM test compound in DMSO per species per assay condition.
Experimental Procedure
Microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4, and the test
compound (final substrate concentration 3
at 37 ◦C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final
incubation volume was 50 L. A minus cofactor control incubation was included for each compound
tested where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). Two control
µM; final DMSO concentration 0.25%) were pre-incubated
µ

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compounds were included with each species. All incubations were performed singularly for each
test compound.
Each compound was incubated for 0, 5, 15, 30, and 45 min. The control (minus NADPH) was
incubated for 45 min only. The reactions were stopped by transferring 20 µL of incubate to 60 µL
methanol at the appropriate time points. The termination plates were centrifuged at 2500 rpm for
20 min at 4 ◦C to precipitate the protein.
Quantitative Analysis
Following protein precipitation, the sample supernatants were combined in cassettes of up to
4 compounds, the internal standard was added, and samples were analysed using Cyprotex generic
LC-MS/MS conditions.
Data Analysis
From a plot of ln peak area ratio (compound peak area/internal standard peak area) against time,
the gradient of the line was determined. Subsequently, half-life and intrinsic clearance were calculated
using the equations below:
elimination rate constant (k) = (- gradient)
(1)
half-life (t1⁄2)(min) = 0.693/k
(2)
(3)
(4)
intrinsic clearance (CLint)(µL/min/mg protein) = V × 0.693/t1⁄2
V = Incubation volume (µL)/Microsomal protein (mg)
relevant control compounds were assessed, ensuring intrinsic clearance values fall within the
specified limits.
3.8.3. In Vitro Cardiotoxicity; hERG Channel Inhibition (IC50 Determination)
Mammalian cells expressing the hERG potassium channel were dispensed into 384-well planar
arrays and hERG tail-currents measured by whole-cell voltage-clamping. A range of concentrations
of the test compound was then added to the cells and a second recording of the hERG current was
made. The percent change in hERG current was calculated and used to calculate an IC50 value (test
compound concentration which produces 50% inhibition). IC50 is delivered, standard error (SE IC50),
and n (number of data points used to calculate IC50) for the test compound. The experiments were
performed on an Ion Works TM automated patch clamp instrument (Molecular Devices LLC (San Jose
,
California, United States)), which simultaneously performs electrophysiology measurements for
48 single cells in a specialised 384-well plate (PatchPlateTM(manufacturer, city, state if USA or Canada,
country)). All cell suspensions, buffers, and test compound solutions were at room temperature during
the experiment.
The cells used were Chinese hamster ovary (CHO) cells stably transfected with hERG (cell-line
obtained from Cytomyx, UK). A single-cell suspension was prepared in extracellular solution
(Dulbecco’s phosphate buffered saline with calcium and magnesium pH 7.2) and aliquots added
automatically to each well of a Patch Plate TM. The cells were then positioned over a small hole at the
bottom of each well by applying a vacuum beneath the plate to form an electrical seal. The vacuum
was applied through a single compartment common to all wells which was filled with intracellular
solution (buffered to pH 7.2 with HEPES). The resistance of each seal was measured via a common
ground-electrode in the intracellular compartment and individual electrodes placed into each of the
upper wells.
Electrical access to the cell was then achieved by circulating a perforating agent, amphotericin B,
underneath the PatchPlateTM. The pre-compound hERG current was then measured. An electrode
was positioned in the extracellular compartment and a holding potential of 80 mV applied for 15 s.
−

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The hERG channels were then activated by applying a depolarising step to +40 mV for 5 s and then
clamped at −50 mV for 4 s to elicit the hERG tail current, before returning to −80 mV for 0.3 s.
Compound dilutions were prepared by diluting a DMSO solution (default 10 mM) of the test
compound using a factor 5 dilution scheme into DMSO, followed by dilution into extracellular buffer
such that the final concentrations tested were typically 0.008, 0.04, 0.2, 1, 5, and 25 µM (final DMSO
concentration 0.25%). The Ion Works TM instrument automatically adds test compound dilutions to the
upper wells of the PatchPlateTM. The test compound was left in contact with the cells for 300 s before
recording currents using the same voltage-step protocol as in the pre-compound scan. Quinidine,
an established hERG inhibitor, was included as a positive control and vehicle control (0.25% DMSO) as
negative control.
Each concentration was tested in 4 replicate wells on the PatchPlateTM (maximum of
24 data points). Filters were applied to ensure only acceptable cells were used to assess hERG inhibition.
The cell must maintain a seal resistance of greater than 50 MOhm and a pre-compound current of at
least 0.1 nA and ensure cell stability between pre-and post-compound measurements.
For each replicate, the hERG response was calculated using the following equation:
% hERG response = [Post-compound current (nA)/Pre-compound current (nA)] × 100
(5)
The % hERG response was plotted against concentration for the test compound and, where
concentration-dependent inhibition was observed, the data were fitted to the following equation and
an IC50 value calculated:
y = [ymax − ymin/1 + (IC50/x)s]+ y min
(6)
where y = hERG response; ymax = mean vehicle control response; x = concentration; IC50 = concentration
required to inhibit current by 50%; s = Hill slope.
4. Conclusions
So far, the main approach for FAK inhibition has been focused on FAK catalytic function,
which mainly targets the Y397 tyrosine phosphorylation site by interfering with the binding of ATP.
This has resulted in the development of several FAK kinase inhibitors such as defactinib in the
clinical stage. However, this approach lacks specificity with varying degrees of cross-reactivity with
other tyrosine kinases and associated unwanted side effects. Alternatively, the inhibition of the FAK
scaffolding function and the downstream signalling is believed to be a favourable strategy, and likely
offers more specific FAK inhibitors rather than focusing solely on the kinase function. In this study,
we used a structure based virtual screening approach to identify a small molecule hit compound
I.
Subsequently, a series of compound analogues was synthesised and screened for antiproliferative
I
activity in three breast cancer (MDA-MB-231, T47D, BT474) cell lines and one pancreatic cancer
(MIAPaCa2) cell line. Compounds I and 5f displayed low micromolar antiproliferative activity across
the four cell lines. Moreover, compounds I and 5f have promising metabolic stability in the liver
microsome, and compound 5f lacks cardiotoxicity in the hERG inhibition assay. With no FAK inhibitors
currently on the market, compound 5f represents a promising drug-like small molecule that justifies
further development as a lead compound for breast and pancreatic cancer therapy.
Supplementary Materials: Supplementary data associated with this article can be found in the online version.
These data include Figure S1; Western blot analysis of compound I effects on activation and total stability of FAK.
Figures S2–S4; the spectroscopic data of compound 5f (1H, 13C, 19F NMR, and mass spectrometry).
Author Contributions: Conceptualization, S.B.K., S.E.H. and A.D.W.; methodology, S.B.K. and S.R.J.; investigation,
S.B.K., S.R.J. and S.S.; funding and resources, S.E.H. and A.D.W.; writing–original draft preparation, S.B.K. and
A.D.W.; writing–review and editing, S.B.K., S.R.J. and A.D.W.; supervision, S.E.H. and A.D.W. All authors have
read and agreed to the published version of the manuscript.
Funding: Life Sciences Research Network of Wales (Welsh Government).

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Acknowledgments: We would like to thank Life Sciences Research Network of Wales (Welsh Government) for
postdoctoral support to SK. Antiproliferative assays were run as an outsourced service through WuxiApptech Co.
Ltd. All in vitro solubility and stability evaluations were performed by Cyprotex, and the detailed protocols can
be found at http://www.cyprotex.com [39]. We would also like to thank the EPSRC National Mass Spectrometry
centre (Swansea, UK) for accurate mass spectrometry provision.
Conflicts of Interest: The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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