The discovery of new and more potent chloropyramine (C4) analogues for the potential treatment of invasive breast cancer

Article abstract of DOI:10.1111/cbdd.13083

Breast cancer is the second most common cancer worldwide, accounting for 25% of all female cancers. Although the survival rate has increased significantly in the past few decades, patients who develop secondary site metastasis as well as those diagnosed with triple negative breast cancer still represent a real unmet medical challenge. Previous studies have shown that chloropyramine (C4) inhibits FAK-VEGFR3 signalling. More recently, C4 is reported to have SASH1 inducing properties. However, C4 exerts its antitumour and antiangiogenic effects at high micromolar concentrations (>100?μm) that would not be compatible with further drug development against invasive breast cancer driven by FAK signalling. In this study, molecular modelling guided structural modifications have been introduced to the chloropyramine C4 scaffold to improve its activity in breast cancer cell lines. Seventeen compounds were designed and synthesized, and their antiproliferative activity was evaluated against three human breast cancer lines (MDA-MB-231, BT474 and T47D). Compound 5c was identified to display an average activity of IC₅₀?=?23.5–31.3?μm, which represents a significant improvement of C4 activity in the same assay model. Molecular modelling and pharmacokinetic studies provided more promising insights into the mechanistic features of this new series.

Full text of DOI:10.1111/cbdd.13083

DR SAHAR KANDIL (Orcid ID : 0000-0003-1806-9623)
DR ANDREW WESTWELL (Orcid ID : 0000-0002-5166-9236)
Article type : Research Article
The discovery of new and more potent chloropyramine (C4)
analogues for the potential treatment of invasive breast cancer
Sahar Kandil*, Filippo Prencipe, Samuel Jones, Stephen Hiscox and Andrew D. Westwell
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB,
Wales, United Kingdom.
Keywords: Focal adhesion kinase (FAK), metastatic breast cancer (BC), chloropyramine (C4), lead optimisation,
reductive amination.
Graphical abstract
Highlights
• 17 New chloropyramine analogues were designed and synthesised.
• All new compounds were evaluated for their activity in three different breast cancer cell lines.
• For the most active compound 5c, in vitro pharmacokinetic studies were performed.
*
Corresponding author
E-mail address: Kandils1@cardiff.ac.uk (S. Kandil)
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13083
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ABSTRACT
Breast cancer (BC) is the second most common cancer worldwide, accounting for 25% of all female
cancers. Although the survival rate has increased significantly in the past few decades, patients who
develop secondary site metastasis as well as those diagnosed with triple negative breast cancer
(TNBC) still represent a real unmet medical challenge. Previous studies have shown that
chloropyramine (C4) inhibits FAK-VEGFR3 signaling. More recently, C4 is reported to have SASH1
inducing properties. However, C4 exerts its antitumour and antiangiogenic effects at high
micromolar concentrations (>100 µM) that would not be compatible with further drug development
against invasive breast cancer driven by FAK signaling. In this study, molecular modeling guided
structural modifications have been introduced to the chloropyramine C4 scaffold to improve its
activity in breast cancer cell lines. Seventeen compounds were designed and synthesised and their
anti-proliferative activity was evaluated against three human breast cancer lines (MDA-MB-231,
BT474 and T47D). Compound 5c was identified to display an average activity of IC50 = 23.5- 31.3 µM,
which represents a significant improvement of C4 activity in the same assay model. Molecular
modelling and pharmacokinetic studies provided more promising insights into the mechanistic
features of this new series.
1. INTRODUCTION
Current therapeutic strategies for breast cancer (BC) are mainly directed towards primary tumour
site histopathology i.e. expression of estrogen (ER), progesterone (PR) and human epidermal growth
factor 2 (HER2) receptors. Due to improved therapeutic strategies, the survival rate for ER-positive
breast cancer in particular has increased significantly in the past few decades. However, the sub-
group of aggressive and invasive breast cancers, including triple-negative disease (around 15% of
cases), is characterised by rapid relapse following chemotherapy and progression to metastatic
disease, with a poor overall prognosis.1
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase and scaffold protein, which is localised
to focal adhesions, and forms points of contacts between cells and the extracellular matrix. FAK is
highly expressed in cancer, acts as a multifunctional mediator of cell signaling, and its involvement in
cancer cell proliferation, invasion and metastasis is well documented.2-7 It is overexpressed in many
types of cancer such as thyroid, brain, liver, prostate, breast, colon, head and neck. Moreover, this
overexpression is associated with an invasive phenotype in such tumours.7 Numerous approaches
are being developed to inhibit FAK catalytic function (mainly targeting the Y397 tyrosine
phosphorylation site), which have successfully led to the development of clinical stage FAK kinase
inhibitors such as defactinib, currently in Phase I/II trials in ovarian, non-small cell lung,
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mesothelioma and pancreatic cancer (Verastem, Inc.)8 Alternatively, destabilisation of the FAK
scaffolding function is believed to be an advantageous and potentially more specific strategy to
develop FAK targeting therapeutics rather than focusing on the kinase function only.7-11 In silico
screening of the National Cancer Institute’s small molecule library for compounds targeting the FAK–
VEGFR3 interactions led to the identification of the clinically approved antihistamine drug
chloropyramine (C4) which belongs to the first generation of H1-antihistamines.12,13 Further in vitro
and in vivo studies have shown that treatment with C4 showed reduction in tumour burden and
neovascularization in mouse models of breast cancer.12 This activity has been demonstrated, both
alone and synergistically with other chemotherapeutic agents (e.g. doxorubicin and gemcitabine),
sensitising breast cancer cells to chemotherapy.12,13 Moreover, C4 is under development by
CureFAKtor Pharmaceuticals LLC, and received orphan drug designation from the FDA in
combination with gemcitabine for the treatment of pancreatic cancer. 14 More recently, Burgess et al
have independently used in silico connectivity mapping and in vitro modelling to demonstrate that
chloropyramine C4, increased SASH1 protein levels in breast cancer cells.15 Expression of the SASH1
protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell
death. Although C4 is an attractive candidate, it exerts its antitumour and antiangiogenic effects at
high micromolar concentrations (>100 µM).13 In order to get more insights into the structure activity
relationships (SAR) and improve the antitumour potency of C4, we introduced new chemical motifs
into the scaffold of C4. These modifications were tested in a preliminary in silico study that
suggested an improved interaction with the FAK-FAT domain. The newly designed compounds were
synthesised and screened in vitro against three human breast cancer cell lines (MDA-MB-231, BT474
and T47D). Our findings show that compound 5c, which displayed the best anti-proliferative potency
amongst all tested analogues, is a novel compound that warrants further investigation as a
candidate for breast cancer therapy.
2. METHODS AND MATERIALS
2.1 Lead optimisation and design of novel chloropyramine (C4) analogues
Based on the previous promising findings with chloropyramine (C4),12,13 we sought to improve upon
the chemical structure of C4 to understand better its structure activity relationship (SAR) and to
improve its anti-proliferative potency (C4 IC50 >100 µM). To investigate the impact of various
substituents of C4 on the antitumour efficacy, a series of novel derivatives were designed and
prepared, as detailed in Figure 1, and Schemes 1 and 2. Structural modifications involved replacing
the pyridine moiety with other aromatic systems containing a carboxylate moiety to enhance the
interaction with Lys 1032 within the FAK-FAT domain, introducing an extended carbon atom moiety
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(region A, Figure 1) or introducing a carbonyl group into N,N-dimethylamino ethyl side chain (region
C, Figure 1). The p-chlorobenzyl group (region B, Figure 1) was retained unmodified in most
compounds since it is involved in key interactions via a π-π stacking with the aromatic side chain of
Tyr 925. The salicylate aromatic moiety replacing the pyridine ring was reported to mimic the
phosphorylated tyrosine (pTyr).16,17 Molecular modeling studies of the newly designed compounds
showed improved binding interaction modes within the FAK-FAT pocket compared to the parent C4
compound due to a better orientation effect on the side chain (region C, Figure 1). All the prepared
compounds were evaluated against three breast cancer lines i.e. MDA-MB-231 (TNBC), BT474 (ER+,
HER+) and T47D (ER+, HER-).
2.2 Chemical synthesis
The first series of compounds containing an extended carbon unit in the C4 skeleton (5a-d, region A
modification) were synthesised as shown in Scheme 1. The first step involved reductive amination of
various substituted aromatic aldehydes (1a-d) with N, N-dimethyl-ethylenediamine 2 which provided
intermediates (3a-d) in good yields.18 It is worth mentioning that the reaction conditions of this step
were optimized in terms of solvent and temperature choice for the different aldehydes used (see
experimental section). These intermediates were alkylated with p-chlorobenzyl chloride 4 to give the
target analogues (5a-d) in good to moderate yield (57-69 %). Interestingly, in the case of 4-((2-
(dimethylamino)ethylamino)methyl) benzonitrile 3a, a quaternary ammonium side product 6 was
obtained alongside the expected product 5a (in 1:3 ratio, respectively). Unexpectedly, in the case of 2-
cyanobenzaldehyde 7, the cyclised isoindolenone derivative 8 was isolated instead of the desired
Schiff’s base, Scheme 2 (A). A plausible mechanism is suggested where the initial step is the
nucleophilic addition of the amine 2 to the formyl group of 7 affording the adduct 7a, which
subsequently cyclises to 7b by intramolecular nucleophilic addition of the hydroxyl group to the cyano
group of 7a, followed by conversion to the isoindolenone derivative 8, Scheme 2 (B).19
The second series of compounds (13a-m), featuring an additional carbonyl group in the N,N-
dimethylamino ethyl side chain moiety of C4, were prepared according to Scheme 3 (A). The first step
involved a reductive amination reaction20 between the corresponding aniline derivative (9a-l) and p-
halobenzaldehyde (10a or 10b) to give the key intermediates (11a-l). The final compounds were
obtained by reacting N,N-dimethylaminoacetyl chloride hydrochloride 12 with intermediates (11a-l) in
tetrahydrofuran at room temperature. The reductive amination of 5-amino-2-hydroxybenzoic acid with
p-chlorobenzaldehyde (10a) was not feasible due to the instability of the intermediate product (11m).
For this reason, we managed to get the corresponding target product 13m via the hydrolysis of 13c
using NaOH solution in a very good yield (92 %), Scheme 3 (A). Interestingly, double reductive
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amination occurred in the case of compound 9m to give 14 which was hydrolysed to 15 using NaOH.
The structures of all the synthesised compounds were confirmed using analytical and spectroscopic
data (NMR, mass spectrometry), which were in full accordance with their depicted structure. All final
compounds were purified and tested as free bases.
3. RESULTS AND DISCUSSION
3.1 Cell Viability Assay
The anti-proliferative activity of all the synthesised compounds 5a-d, 6, 8, 13a-m, 14 and 15 was
evaluated as free bases against three human breast cancer cell lines; MDA-MB-231 (TNBC), BT474
(ER+, HER+) and T47D (ER+, HER-). Chloropyramine C4 was used as a control drug. The corresponding
cell lines were seeded the day before incubation, then incubated for 72 h with the different
concentrations of compound tested. CellTiter Glo reagent (25 μL) was added to the cell plate. The
luminescence was recorded after 10 mins on a PerkinElmer Envision instrument. All the twenty-one
compounds were tested at 10 concentrations in half log increments up to 100 μM in triplicate. The
results indicated that three compounds (5c, 6 and 15) have better anti-proliferative activity than
chloropyramine C4, which did not show any anti-proliferative activity up to 100 μM concentration
across the three BC cell lines. The most active compound 5c showed micromolar activity across the
three BC cell lines; (MDA-MB-231, IC50 = 23.5 μM), (BT474, IC50 = 73.2 μM) and (T47D, IC50 = 31.3
μM), Figure 2A. Interestingly, the quaternary ammonium compound 6 and the double reductive
amination product 15, displayed better anti-proliferative activity than C4, Figure 2B and C,
respectively. All the other tested analogues showed no observed improvement in the
antiproliferative activity over the parent compound C4 (all compounds were tested at maximum
concentration of 100 µM).
3.2 Molecular modelling
In silico docking was performed on all the designed analogues using the downloaded crystal
structure of FAK (PDB ID: 1K05).21 The chemical structures of the compounds were constructed,
rendered and minimised with the MMFF94x force field in MOE. Docking simulations were performed
using Glide SP in Maestro (Glide, version 9.5, Schrödinger, LLC, New York, NY. http://www.
schrodinger.com). Based on the surface representation of the FAK-FAT domain, there appears to be
an improved interaction over the parent C4 upon the introduction of an extended carbon linker,
compounds (5a-d), or introduction of a carbonyl group as in compounds (13a-m) into the general
chloropyramine scaffold. Figure 3A shows the predicted docking mode of the most active compound
5c compared to that of the parent compound C4. The computational study showed an improved
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interaction between the N,N-dimethylamino ethyl side chain and Asp 1036 of 5c. Compound 5c sits
in a relatively polar cavity surrounded by residues Ser 910, Thr 929, Lys 1032, and Asp 1036. Key
interactions of 5c include two H-bonds between the N,N-dimethylamino ethyl group and both the
side chain carboxylate group of residue Asp 1036 and the backbone carbonyl group of Pro 911. In
addition, hydrophobic interactions were observed between the p-chlorobenzyl group moiety and
the surrounding hydrophobic pocket formed of residues Tyr 925, Val 928, Val 932, and Leu 1035
(Figure 3A). The relatively higher activity of compound 5c appears to be a result of the better
directing effect of the N,N-dimethylamino side chain conferred by the extra carbon moiety. On the
other hand, according to our model, the introduction of the carbonyl group into the N,N-
dimethylamino ethyl moiety of C4 (e.g. 13a) showed an improved effect on orienting the N,N-
dimethylamino ethyl group into the polar pocket around Asp 1036 as well as directing the other
aromatic group to the hydrophobic pocket, Figure 3B. However, none of the carbonyl containing
compounds (13a-m) were able to demonstrate any better anti-proliferative activity than C4,
probably because of the reduced basicity of the N,N-dimethylamino moiety upon the introduction of
the carbonyl group. Generally, our study of the two modified series of compounds suggests that
more substantial structural modifications of C4 are needed to significantly improve the anti-
proliferative activity in BC cell lines.
3.3 In vitro aqueous solubility and microsomal metabolic stability
Subsequently, in vitro pharmacokinetic studies of the most active compound 5c were performed.
The in vitro aqueous solubility was investigated using five concentrations of 5c i.e (1, 3, 10, 30 and
100 μM), which were incubated at 37 °C for 2 hr. The estimated precipitation range (lower and
upper bound) and a mid-range value were calculated for 5c. Nicardipine and pyrene are included as
control compounds. The results show that compound 5c has a very good aqueous solubility with
estimated precipitation range 100 µM (lower bound) and >100 µM (upper bound), Table 1. This
relatively high aqueous solubility could prove to be a possible solution to the sedation side effect of
chloropyramine because of its ability to cross the blood brain barrier (BBB).15 The calculated logP
values (C4 = 3.99, 5c= 4.42) are similar but tPSA values (C4 = 18.84, 5c= 32.78) suggest that 5c is less
likely to cross the BBB.
Furthermore, the metabolic stability of 5c was tested in human liver microsomes. Compound 5c was
incubated for 45 min with pooled liver microsomes and the intrinsic clearance (CL ) and half-life
int
(t1/2) values were calculated based on 5 time points. The values of intrinsic clearance (CLint) as well as
half-life values of 5c compared to control compounds, dextromethorphan and verapamil, are shown
in Table 2. Compound 5c has microsomal half-life (t1/2) of 59.4 minutes, which exceeds that of the
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registered drug controls dextromethorphan (t1/2 = 48.2 mins) and verapamil (t1/2 = 8.0 mins), and
represents a good starting point for optimisation towards a daily administrable product.
Estimated Precipitation Range (µM)
compound
Lower bound
Upper bound
Calculated Mid-range
5c
100
10
3
>100
30
>100
20
nicardipine
pyrene
10
6.5
Table 1. In vitro aqueous solubility of 5c versus control compounds (nicardipine and pyrene) in buffered
aqueous solution
CLint (uL/min/mg protein)
t1/2 (min)
59.4
5c
23.3
28.8
dextromethorphan
verapamil
48.2
173.0
8.0
Table 2. In vitro metabolic stability of 5c versus control compounds (dextromethorphan and verapamil) in
human liver microsomes; CLint: theoretical unrestricted maximum clearance of unbound drug by an eliminating
organ, in absence of blood or plasma protein binding limitations, t1/2 is the half-life.
4. Conclusion
In this study, two series of novel chloropyramine C4 analogues were designed and synthesised. The
anti-proliferative activity of compounds (5a-d) and (13a-m) was evaluated as free bases in vitro
across three human breast cancer cell lines; MDA-MB-231 (TNBC), BT474 (ER+, HER+) and T47D (ER+,
HER-). The results identified compound 5c as having the most significant anti-proliferative activity,
displaying micromolar activity across the three breast cancer cell lines; (MDA-MB-231, IC50 = 23.5
μM), (BT474, IC50 = 73.2 μM) and (T47D, IC50 = 31.3 μM). This activity represents a significant
improvement to the chloropyramine C4, which did not display any anti-proliferative activity up to
100 μM. Further pharmacokinetic study showed that 5c performed well in both the aqueous
solubility and microsomal metabolic stability analysis. Considering the above findings, it could be
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concluded that 5c represents a promising basis for further development of breast cancer
therapeutics targeting the FAK scaffolding domain.
Acknowledgement
We thank the Welsh Government, Life Sciences Research Network Wales scheme for postdoctoral
support to SK. Anti-proliferative assays were run as an outsourced service through WuxiApptech
AppTec Co., Ltd. All in vitro biological evaluations were performed by Cyprotex, and the detailed
protocols can be found at http://www.cyprotex.com. We also thank the EPSRC National Mass
Spectrometry centre (Swansea, U.K.) for provision of accurate mass spectrometry. We thank the
analytical services unit, school of Chemistry, Cardiff University for provision of accurate mass
spectrometry. We thank Miss. Emma Jones for the technical support.
Supplementary data
Details of experimental procedures and spectroscopic characterisations data of the compounds 5a-
d, 6, 8, 13a-m, 14 and 15 associated with this article can be found in the online version at
Figure 1. Chemical structure of chloropyramine and regions of proposed structural modification.
Scheme 1. Synthesis of compounds (5a-d). Reagents and conditions: a) i. 3a and 3d, (MeOH, rt, 4h), 3b (DCE,
reflux, 4h), 3c (AcOH, 6h), ii. NaBH(OAc)3, rt, 24h, b) THF, rt. 24h.
Scheme 2; A) Synthesis of isoindolene derivative 8. Reagents and conditions; a) MeOH, rt, 4h, NaBH(OAc)3, rt,
24h. B) proposed mechanism for the formation of the isoindolenone derivative 8.
Scheme 3. A) Synthesis of compounds (13a-m). Reagents and conditions: a) MeOH, rt, 4h, NaBH(OAc)3, 24h, b)
THF, rt. 24h. B) Synthesis of compounds 14 and 15. Reagents and conditions: a) DCE, reflux, 4h, NaBH(OAc)3,
r.t. 24h, b) NaOH, MeOH, reflux, 4hr.
Figure 2. Anti-proliferative activity of compounds 5c (A), 6 (B) and 15 (C) against three human breast cancer
cell lines; MDA-MB-231, BT474 and T47D.
Figure 3. A) Superposition of the predicted binding mode of compound 5c (grey) versus C4 (green) showing
hydrogen bond interactions (blue dashed line) with key amino acids; Pro 911 and Asp 1036. B) compound 13a
(grey) versus C4 (green); both are inside the pocket of FAK-FAT domain (golden yellow ribbon representation).
Figure 4. Mass spectrum of compound 5c. (end of Supporting Information).
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Figure 5. 1H NMR spectrum of compound 5c. (end of Supporting Information).
Figure 6. 13C NMR spectrum of compound 5c. (end of Supporting Information).
Conflict of Interest:
The authors declare that there are no financial/commercial conflicts of interest.
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Scheme 1. Synthesis of compounds (5a-d). Reagents and conditions: a) i. 3a and 3d, (MeOH, rt, 4h), 3b (DCE,
reflux, 4h), 3c (AcOH, 6h), ii. NaBH(OAc)3, rt, 24h, b) THF, rt. 24h.
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Scheme 2; A) Synthesis of isoindolene derivative 8. Reagents and conditions; a) MeOH, rt, 4h, NaBH(OAc)3, rt,
24h. B) proposed mechanism for the formation of the isoindolenone derivative 8.
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Scheme 3. A) Synthesis of compounds (13a-m). Reagents and conditions: a) MeOH, rt, 4h, NaBH(OAc)3, 24h, b)
THF, rt. 24h. B) Synthesis of compounds 14 and 15. Reagents and conditions: a) DCE, reflux, 4h, NaBH(OAc)3,
r.t. 24h, b) NaOH, MeOH, reflux, 4hr.
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