Novel improved synthesis of hsp70 inhibitor, pifithrin-μ in vitro synergy quantification of pifithrin-μ combined with pt drugs in prostate and colorectal cancer cells

Article abstract of DOI:10.3390/molecules21070949

We describe a novel improved approach to the synthesis of the important and well-known heat shock protein 70 inhibitor (HSP70), pifithrin-μ, with corresponding and previously unreported characterisation. The first example of a combination study comprising

Full text of DOI:10.3390/molecules21070949

molecules
Article
Novel Improved Synthesis of HSP70 Inhibitor,
Pifithrin-µ. In Vitro Synergy Quantification of
Pifithrin-µ Combined with Pt Drugs in Prostate
and Colorectal Cancer Cells
Aoife M. McKeon 1, Alan Egan 2, Jay Chandanshive 1, Helena McMahon 2 and Darren M. Griffith 1,*
1
Centre for Synthesis and Chemical Biology, Department of Pharmaceutical and Medicinal Chemistry,
Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2 D02 YN77, Ireland;
aoifemckeon@rcsi.ie (A.M.M.); jaychandanshive@rcsi.ie (J.C.)
Shannon ABC, South Campus, IT Tralee, Clash, Tralee, Co., Kerry V92 CX88, Ireland; ae12@hw.ac.uk (A.E.);
2
helena.mcmahon@staff.ittralee.ie (H.M.)
*
Correspondence: dgriffith@rcsi.ie; Tel.: +353-1-4022246
Academic Editor: Diego Muñoz-Torrero
Received: 2 June 2016; Accepted: 15 July 2016; Published: 21 July 2016
Abstract: We describe a novel improved approach to the synthesis of the important and well-known
heat shock protein 70 inhibitor (HSP70), pifithrin-
characterisation. The first example of a combination study comprising HSP70 inhibitor pifithrin-
and cisplatin or oxaliplatin is reported. We have determined, using the Chou-Talalay method,
(i) moderate synergistic and synergistic effects in co-treating PC-3 prostate cancer cells with pifithrin-
µ, with corresponding and previously unreported
µ
µ
and cisplatin and (ii) significant synergistic effects including strong synergism in cotreating HT29
colorectal cancer cells with oxaliplatin and pifithrin-µ.
Keywords: synthesis; pifithrin-µ; heat shock protein 70; cancer; platinum; combination study;
Chou-Talalay method
1. Introduction
Pifithrin-
µ, (2-phenylethynesulfonamide, PES, Scheme 1) is a potent and selective small molecule
and direct inhibitor of heat shock protein 70 [1,2]. Heat shock proteins (HSPs) , extensively studied
in the literature, are highly conserved proteins whose expression is increased by cells in response to
a variety of cellular stresses including elevated temperatures, hypoxia, and anti-cancer chemotherapy
for example [3]. There are at least eight highly homologous members of the human HSP70 family,
which can be loosely organised by subcellular localisation, tissue-specific expression and stress
induced expression.
HSP70-1 is a stress-inducible chaperone, which maintains protein homeostasis during normal cell
growth but during a stress response is overexpressed and binds to and stabilises its protein substrates
against denaturation or aggregation until adverse conditions improve [3]. It is an exciting anti-cancer
target as it is overexpressed in colorectal and prostate cancers, amongst others, and is associated with
cancer progression, chemotherapy resistance and poor prognosis. It is thought to provide cancer cells
with a survival advantage by conferring protection against apoptosis, influencing senescence and
inhibiting autophagy and HSP90 function [4].
Pifithrin- has been reported to interact with the C-terminal substrate (peptide) binding domain
µ
of HSP70 [1,5] and in doing so disrupts the association between HSP70 and (i) a number of its
cofactors such as HSP40 and (ii) client proteins including APAF-1 (apoptotic peptidase activating
factor 1, a key protein in the apoptosis regulatory network), p53 (a tumour suppressor protein)
Molecules 2016, 21, 949; doi:10.3390/molecules21070949
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and p62 (autophagy-related proteins) [
1
]. Significantly pifithrin-
µ
selectively kills cancer cells via
a caspase-independent mechanism involving increased protein aggregation, impairment of the
autophagy-lysosomal system and the proteasome pathway, as well as indirectly effecting the activity
of HSP90 [1,2].
To date only one synthesis of pifithrin-µ is reported in the literature, as Scheme 1; reaction
of trimethyl(phenylethynyl)silane in dichloromethane (DCM) with a titanium sulfamoyl chloride
complex, ClSO2NH2-TiCl4, in a reported 35% yield. No specific data associated with the
characterisation of pifithrin-µ was provided [6].
Scheme 1. Previously reported synthetic strategy for synthesis of pifithrin-µ [6].
Over the past 30 years, platinum (Pt) compounds have played a very important and well
documented role in treating cancer and currently Pt-based drugs are employed in nearly 50% of
anti-cancer therapies or regimens [7,8]. Pt drugs; cisplatin, carboplatin and oxaliplatin, Figure 1, are in
worldwide clinical use, whereas nedaplatin, lobaplatin and heptaplatin are solely approved for use
in Japan, China and South Korea, respectively. The cytotoxicity of Pt drugs is attributed to multiple
mechanisms [9] but, primarily, their ability to enter cells, hydrolyse (loss of chlorido or carboxylato
ligands) and covalently bind DNA, forming DNA adducts. These events can lead to DNA damage
responses, senescence and ultimately programmed cell death, apoptosis [7–9].
Figure 1. Structures of cisplatin, carboplatin and oxaliplatin.
Though Pt drugs are associated with high rates of clinical responses, many cancers including
colorectal and prostate cancers, for example, are intrinsically resistant to Pt-based therapies. In addition,
many cancers acquire chemoresistance, which further compounds therapeutic failure and tumour
recurrence over the longer term [9].
Several mechanisms that account for the Pt resistant phenotype of tumour cells have been
described in a recent review by Galluzzi et al., and they can be classified according to their position
relative to Pt binding DNA, the accepted primary target; (i) pre-target resistance; (ii) on-target resistance;
(iii) post-target resistance; and (iv) off-target resistance. Off-target resistance affects pathways that
positively regulate pro-survival signals that nullify or diminish cisplatin cytotoxicity although they are
typically not directly activated by cisplatin. The up-regulation of HSPs for example has been associated
with off-target resistance [9].
Though metal complexes have a crucial role to play in anti-cancer treatment, it is apparent
that most effective clinical regimens involve the use of combination therapy and that strategies to
circumvent resistance should target at least two distinct mechanisms [
7,9
]. Consequently, we are
interested in investigating if the HSP70 inhibitor pifithrin-µ can synergistically enhance the activity of
Pt drugs.
Herein, we describe (i) a novel improved synthesis of HSP70 inhibitor pifithrin-
(ii) an in vitro cytotoxicity combination study of a Pt drug and pifithrin- against prostate and colorectal
cancer cells using the state-of-the-art method for determining synergy, the Chou-Talalay method.
µ and
µ

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2. Results and Discussion
2.1. Syntheses of Pifithrin-µ, PES
To date, only one synthesis of pifithrin-
µ
is reported in the literature, as Scheme 1; reaction of
trimethyl(phenylethynyl)silane in DCM with a titanium sulfamoyl chloride complex, ClSO2NH2-TiCl4,
in a reported 35% yield. No specific data associated with the characterisation of pifithrin-
provided [6].
µ was
We report a facile, reproducible and improved synthesis of pifithrin-µ Reaction of commercially
available phenylacteylene with the organosilicon base LiHMDS (Li salt of hexamethyldisilazane) in
anhydrous THF in the presence of HMPA (hexamethylphosphoramide), gives the corresponding
acetylide anion, which subsequently reacts in situ with freshly prepared sulfamoyl chloride to give
1
pifithrin-
µ
in 68% yield and excellent purity, Scheme 2. Pifithrin-
µ
was characterised by H- and
13C-NMR spectroscopy, mass spectrometry and elemental analysis. In the H-NMR spectrum of
1
pifithrin-µ (DMSO-d6) three resonances, a doublet at 7.61 integrating for two, a triplet integrating for
one at 7.56 and a triplet integrating for two at 7.48 ppm, correspond to the five protons of the aromatic
ring. The resonance observed at 8.24 ppm is attributed to the two protons of the sulfonamide NH2.
In The 13C-NMR spectrum signals at 132.2 (2
ˆ
C), 131.2 (1
are associated with the six aromatic carbons and signals at 87.5 and 84.3 ppm are assigned to the two
alkyne carbons. ESI-MS in the negative mode assisted in identifying pifithrin- with a mass peak at
180.2 a.m.u. Elemental analysis correlated with required analysis for pifithrin-µ.
ˆ
C), 129.2 (2
ˆ
C) and 117.9 (1
ˆ
C) ppm
µ
Scheme 2. Novel A synthetic strategy for synthesis of pifithrin-µ.
2.2. In Vitro Cytotoxicity
The in vitro anti-cancer chemotherapeutic potential of cisplatin and pifithrin-µ were determined
against three prostate cancer cell lines; LNCaP (androgen sensitive) and PC-3, and DU145 (androgen
insensitive) with a view to selecting one relatively Pt resistant cell line from each cancer type for the
combination study described below. IC50 is defined as the concentration of compound that inhibits
cell proliferation by 50% relative to the untreated cells. All three cell lines were found to have very
similar sensitivity to treatment with both cisplatin and pifithrin-
and pifithrin- (17–22 M), Table 1. The values for cisplatin correspond with previously reported IC50
values in the literature [10].
µ for 72 h treatment; cisplatin (4–6 µM)
µ
µ
Table 1. IC50
(µM) values calculated for cisplatin, oxaliplatin or pifithrin-
µ
against prostate and
colorectal cell lines on 72 h treatment (n = 3).
Test Compounds
PC-3
DU145 LNCaP HT29 LoVo
HCT116
Pifithrin-µ
Cisplatin
Oxaliplatin
17.4
6.01
-
18.8
4.02
-
21.6
5.3
-
40
-
9
5
-
0.5
26
-
0.3
The in vitro anti-cancer chemotherapeutic potential of oxaliplatin, the Pt drug of choice for the
treatment of stage IV colorectal cancer, and pifithrin- were determined against three colorectal cancer
cell lines; HT29, LoVo and HCT116. HCT116 and LoVo were found to be most sensitive to oxaliplatin
treatment with IC50 value of 0.3 and 0.5 M, respectively, relative to the HT29 cells (9 M). The values
for oxaliplatin correspond with previously reported IC50 values in the literature [11,12].
µ
µ
µ

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HT29 and HCT116 had somewhat similar sensitivity when treated with pifithrin-
respectively), which reasonably correspond with the IC50 values determined for the prostate cancer
cell lines (17–22 M), Table 1. It is noteworthy that the LoVo cells are particularly sensitive to treatment
µ (40 and 26 µM
µ
with pifithrin-µ with an IC50 of 5 µM.
Significantly little or no data on pifithrin-
been reported in the literature, however, pifithrin-
µ
against any prostate or colorectal cancer cell lines has
has been shown to have IC50 values ranging from
µ
2.5 to 12.7 µM against a number of leukemic cell lines [13].
2.3. In Vitro Combination Study
Unsubstantiated claims of synergy however are prevalent in the literature. Determination of
a greater combined effect than each drug alone does not necessarily indicate synergism for example.
Such an observation can be a result of an additive effect or even a slight antagonism. Synergy should
not be confused with enhancement, potentiation, or augmentation [14].
An in vitro cytotoxicity combination study (n = 3) was therefore undertaken using the
Chou-Talalay method, the state of the art method for determining a synergistic, additive or antagonistic
effect on co-treatment of cells with two or more drugs [14]. This method for drug combination is based
on the median-effect equation derived from the mass-action law principle, which is considered the
unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation
and Scatchard equation. These equations provide the theoretical basis for the resulting combination
index (CI) equation, a quantitative definition for synergism where CI < 1, antagonism where CI > 1 and
additive effect where CI = 1. Based on these algorithms, Compusyn, a computer software developed
for single drug and drug combinations, generates CI indexes [14,15].
PC-3 cells and HT29 cells were investigated as representative examples of prostate and colorectal
cancer cells respectively given their relative resistance to Pt treatment in the in vitro cytotoxicity study
described, Table 1.
Both cell lines were treated with fixed ratios of pifithrin-µ and a Pt drug (cisplatin or oxaliplatin)
as outlined in Tables 2 and 3 where selection of concentrations was principally informed by the relevant
IC50 values. Combination indexes were determined using Compusyn and plotted in Figures 2a–c
and 3a–c.
Table 2. Combined drug concentrations investigated against PC-3 prostate cancer cells.
Cisplatin Concentration
Pifithrin-µ 15 µM
Pifithrin-µ 10 µM
Pifithrin-µ 5 µM
20 µM Cisplatin
10 µM Cisplatin
5 µM Cisplatin
2.5 µM Cisplatin
1 µM Cisplatin
20, 15
10, 15
5, 15
2.5, 15
1, 15
20, 10
10, 10
5, 10
2.5, 10
1, 10
20, 5
10, 5
5, 5
2.5, 5
1, 5
Table 3. Combined drug concentrations investigated against HT29 colorectal cancer cells.
Oxaliplatin Concentration
Pifithrin-µ 20 µM
Pifithrin-µ 10 µM
Pifithrin-µ 5 µM
40 µM Oxaliplatin
20 µM Oxaliplatin
10 µM Oxaliplatin
5 µM Oxaliplatin
2.5 µM Oxaliplatin
40, 20
20, 20
10, 20
5, 20
40, 10
20, 10
10, 10
5, 10
40, 5
20, 5
10, 5
5, 5
2.5, 20
2.5, 10
2.5, 5
Of the 15 different combined concentrations of pifithrin-
µ
and cisplatin against PC-3 cells tested,
seven combinations have CI values > 1 and eight have CI values < 1, Figure 2a–c. CIs can be interpreted
in finer detail and categorised as Table 4 and as follows [15]. One combination, 10 M pifithrin-
and 5 M cisplatin, with a CI of 1.16 exhibits slight antagonism, (CI category of 1.1–1.2), and three
µ
µ
µ

Molecules 2016, 21, 949
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additional combinations exhibit moderate antagonism (CI category of 1.20–1.45). Three combinations
(pifithrin- 15 M and cisplatin 1, 2.5 and 5 M) have CI indexes near to 1 and can be considered to
µ
µ
µ
exhibit a nearly additive effect (CI category of 0.9–1.1).
(a)
(d)
(b)
(e)
(c)
(f)
Figure 2. CI values for pifithrin-
5, 10, and 20
synergism where CI < 1, antagonism where CI > 1 and additive effect where CI = 1. The average
of three independent experiments relative to control, 0.2% DMSO treated cells. Fa effect for pifithrin-
(PES) at 15 M ( ); 10 M ( ) and 5 M ( ) in combination with 1, 2.5, 5, 10, and 20 M cisplatin.
Fa (fraction affected) represents the respective proliferation inhibition, where 0% inhibition fa = 0%
µ
(PES) at 15
µM (a); 10
µM (b
) and 5
µM (c) in combination with 1, 2.5,
µ
M cisplatin against HT29 cells. CI (Combination index) is a quantitative definition for
SEM
˘
ď
µ
µ
d
µ
e
µ
f
µ
and 100% inhibition fa = 1. The average
ď0.2% DMSO treated cells.
˘
SEM of three independent experiments relative to control,
Two combinations, 5
µM pifithrin-
µ
and 10
µM cisplatin and 10
µM pifithrin-
µ
and 10
µM
cisplatin fall in the moderate synergistic category (0.7–0.85) and four further combinations fall within
the synergistic category (0.3–0.7). 5
of 0.55.
µM pifithrin-µ and 20 µM cisplatin exhibit the lowest CI index

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There are no previous reports in the literature of pifithrin-
µ
in combination with cisplatin against
prostate cancer cells. Interestingly, the HSP70 inhibitor pifithrin-µ has been previously shown to
increase the antitumor effects of hyperthermia against LNCaP, PC-3, and DU-145 prostate cancer
cells [16].
Of the fifteen different combined concentrations of pifithrin-
tested, Figure 3a–c, one combination, 5 M pifithrin- and 10 M oxaliplatin, resulted in a CI value of
c. 1, and is therefore considered to exhibit a nearly additive effect, Table 4.
µ and oxaliplatin against HT29 cells
µ
µ
µ
(a)
(d)
(b)
(e)
(c)
(f)
Figure 3. CI values for pifithrin-
µ
(PES) at 20
µM (a); 10
µM (b
) and 5
µM (c) in combination with 2.5, 5,
10, 20 and 40
synergism where CI < 1, antagonism where CI > 1 and additive effect where CI = 1. The average ˘ SEM
of three independent experiments relative to control, 0.2% DMSO treated cells. Fa effect for pifithrin-
(PES) at 20 M ( ); 10 M ( ) and 5 M ( ) in combination with 2.5, 5, 10, 20 and 40 M oxaliplatin.
Fa (fraction affected) represents the respective proliferation inhibition, where 0% inhibition fa = 0%
µM oxaliplatin against HT29 cells. CI (Combination index) is a quantitative definition for
ď
µ
µ
d
µ
e
µ
f
µ
and 100% inhibition fa = 1. The average
ď0.2% DMSO treated cells.
˘
SEM of three independent experiments relative to control,

Molecules 2016, 21, 949
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Table 4. CI ranges and their descriptions using the Chou-Talalay method [14].
Combination Index (CI)
Description
<0.1
Very strong synergism
Strong synergism
Synergism
Moderate synergism
Slight synergism
Nearly additive
Slight antagonism
Moderate antagonism
Antagonism
Strong antagonism
Very strong antagonism
0.1–0.3
0.3–0.7
0.7–0.85
0.85–0.9
0.9–1.10
1.1–1.2
1.2–1.45
1.45–3.3
3.3–10
>10
The remaining fourteen combined concentrations tested resulted in the determination of CI values
below 1 and in turn exhibit synergistic effects. 20 M pifithrin- and 40 M oxaliplatin resulted in
a CI index of 0.77 which falls within the moderate synergism category of 0.7–0.85. In contrast 10
pifithrin- and 10 M oxaliplatin with a CI value of 0.24 falls within the strong synergism category of
0.1–0.3. The remaining 12 combinations are found within 0.3–0.7 synergism category, Table 4.
µ
µ
µ
µM
µ
µ
Given the importance of synergism being associated with high inhibition of cell proliferation
fractions affected (fa, where 0% inhibition fa = 0% and 100% inhibition fa = 1) for all combinations
investigated are plotted in Figures 2d–f and 3d–f for ease of comparison with corresponding CI values.
It is apparent that the majority of combinations associated with a synergistic value have a relatively
high fa value.
Pifithrin-µ in combination with both oxaliplatin and cisplatin was determined to synergistically
enhance the in vitro cytotoxic activity of the Pt drugs at particular concentrations and ratios and
with corresponding high fa values. It is clear in this study that synergism was far more prevalent in
the combination study undertaken for oxaliplatin and pifithrin-µ against the HT29 colorectal cancer
cell lines as opposed to pifithrin- and cisplatin against the PC-3 cells. Similarly it is particularly
µ
noteworthy that the fa for the majority of combinations of pifithrin-µ and oxaliplatin against the HT29
cells were greater than 75%, Figure 3d–f.
The primary aim of identifying drug combinations is to achieve a synergistic therapeutic effect,
dose and toxicity reduction, and to diminish or defer the onset of drug resistance [14]. Therefore HSP70
inhibitors, such as Pifithrin-
anticancer agents.
µ, merit further investigation in combination with Pt drugs and alternative
3. Materials and Methods
3.1. Materials and Instrumentation
Phenylacetylene, LiHMDS, HMPA, solvents and deuterated solvents were all purchased from
Sigma Aldrich and used without further purification. Cisplatin [17], oxaliplatin [18] and sulfamoyl
chloride [19] were synthesised via previously reported methods. IR spectra were recorded as KBr discs
(400–4000 cm´1) on a Mattson Genesis II CSI FTIR spectrometer (Bruker, Billercia, MA, USA) and the
1
spectra analysed using OPUS software (Version 5.0, Bruker). H- and 13C-NMR spectra were recorded
on a Bruker Avance 400 NMR spectrometer and the spectra analysed using MestReNova software
(version 6.0.2-5475, Mestrelab, Santiago de Compostela, Spain). The residual undeuterated DMSO
signal at 2.505 ppm was used as an internal reference. Liquid chromatography-mass spectrometry
experiments were performed on a Quattro Micro quadrupole electrospray mass spectrometer
(Micromass, Waters Corp., Milford, MA, USA): 10 µL of the samples were injected in 300 µL of
acetonitrile:water (60:40, v/v). The mass spectrometry data were acquired both in positive and negative
ion modes. Analytical RP-HPLC was performed on a Perkin Elmer Series 200 apparatus (Perkin Elmer

Molecules 2016, 21, 949
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Inc., Waltham, MA, USA) using analytical column Intersil ODS-2 (150 Å, 5
µm); (A) Gradient: 0 to
40 min/0% to 100%; (B) flow rate = 1 mL/min´1; (C) UV detection was performed at 212 nm and
(D) the solvent system consisted of 60% Acetonitrile/40% Water. Retention times (tR) from analytical
RP-HPLC are reported in minutes. Elemental analysis (C, H, N) were performed by the RCSI Analytical
Service, Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland,
123 St. Stephen’s Green, Dublin 2, Ireland.
3.2. Syntheses of Pifithrin-µ
˝
Phenylacetylene (0.54 mL, 4.9 mmol) was added to anhydrous THF (15 mL) and cooled to
´
78 C
using a mixture of liquid nitrogen and acetone. Once cooled, a 1 M solution of LiHMDS (5.4 mL,
5.4 mmol) was added dropwise and the mixture was left to stir for 10 min. HMPA (0.94 mL, 5.4 mmol)
was then added before an additional 10 min of stirring. Freshly prepared sulfamoyl chloride (620 mg,
5.4 mmol) dissolved in anhydrous THF (5 mL) was subsequently added and the reaction mixture was
left to stir for 1 h, maintaining the temperature at
(15 mL) was added and the mixture washed with aqueous NH4Cl (15 mL). The aqueous layer was
extracted with EtOAc (3 15 mL), the organic layers combined, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The resulting crude oil was subject to column chromatography
eluting with 30% EtOAc/hexane to yield pifithrin- as a white solid. Yield 0.603 g (68%). δH (400 MHz,
DMSO-d6) 8.24 (2H, br s, NH2), 7.61 (2H, d, 3J 8 Hz, aromatic H), 7.56 (1H, t, 3J 8 Hz, aromatic H), 7.48
´
78 ˝C. After stirring and returning to RT, EtOAc
ˆ
µ
(2H, t, 3J 8 Hz, aromatic H). δC (100 MHz, DMSO-d6) 132.2 (aromatic C
129.2 (aromatic C 2), 117.9 (aromatic C
ˆ
2), 131.2 (aromatic C
ˆ
1),
1
ˆ
ˆ
1), 87.5 (alkyne C), 84.3 (alkyne C). (C8H7NO2S¨ {2 H2O
requires C, 50.52; H, 4.24; N, 7.36%. Found: C, 50.86; H, 4.67; N, 6.99%); HPLC: C18 column, isocratic
60% acetonitrile/40% water as an eluent, retention time: 4.19 min. Purity > 99%. MS (ESI-) m/z: 180.2.
3.3. In Vitro Cell Culture
Three prostate cancer cell lines; PC-3, LNCaP and DU145 were generously provided by
Prof. Bill Watson, School Of Medicine, Conway Institute, University College Dublin and 3 colorectal
cancer cell lines; HT29, LoVo, and HCT116 were kindly gifted by Prof. Jochen Prehn at the Department
of Physiology and Medical Physics and the Centre for Systems Medicine at the Royal College of
Surgeons in Ireland.
LNCaP cells were cultured in RPMI 1640 medium supplemented with 10% Heat-inactivated
Foetal Bovine Serum (HI-FBS), PC-3 cells in Ham’s F12 Kaighn’s modified medium supplemented
with 10% HI-FBS and DU145 cells in Modified Eagle Medium (MEM) supplemented with 10% HI-FBS.
HT29 and LoVo cell lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium)
supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% FBS. The HCT-116 cells
were cultured in RPMI 1640 supplemented with 1% penicillin-streptomycin, 1% L-glutamine and
10% FBS. Culture reagents and media were purchased from Biosera and used within 6 months of the
purchase date.
˝
In all cases cells were kept in an incubator set at 37 C with 5% CO2. Together the CO2 environment
and the hydrogen carbonate from the medium generate a physiology pH of 7.4.
3.4. In Vitro Cytotoxicity Assays
One hundred microliters of stock solutions of cisplatin and oxaliplatin were made freshly in
medium and diluted out with medium to required concentrations. A 20 mM DMSO pifithrin-µ stock
solution was diluted out with medium to required concentrations with final DMSO concentration
of
ď
0.2%. 0.2% DMSO in medium was used as a control for experiments pertaining to pifithrin-µ.
Cell growth was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt, (MTS test, Promega, Southampton, UK), a colorimetric assay
based on the ability of viable cells to reduce a soluble yellow tetrazolium salt to blue formazan. 1
ˆ
104
prostate cancer cells or 3 µL
ˆ
104 colorectal cancer cells were seeded per well onto 96-well plates in 100

Molecules 2016, 21, 949
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of the appropriate culture medium. Twenty-four hours after seeding, the medium was removed and
the cells were treated by adding 100 L of the test compound solutions at appropriate concentrations.
After 72 h of treatment, 20 L of the MTS reagent was added to each well and the plates incubated
µ
µ
˝
for 2 hr at 37 C. The absorbance was measured at 490 nm using a Wallac 1420 Victor 3 V plate
reader (Perkin-Elmer Life Sciences, Boston, MA, USA). The percentage of surviving cells relative to
untreated controls was then determined. The IC50 value defined as the drug concentration required
to inhibit cell growth by 50%, was estimated graphically from dose-response plots using GraphPad
Prisim, a scientific 2D graphing and statistics software (Prisim 5 for Windows, version 5.01, GraphPad
Software Inc., La Jolla, CA, USA).
3.5. In Vitro Cytotoxicity Combination Study
A drug combination study was carried out and synergistic potential investigated using the
Chou-Talalay method [14].
One hundred microliters of stock solutions of cisplatin and oxaliplatin were made freshly in
medium and diluted out with medium to required concentrations. A 20 mM DMSO pifithrin-µ stock
solution was diluted out with medium to required concentrations with final DMSO concentration
of
Based on the in vitro cytotoxicity evaluation PC-3 prostate cancer cells and HT29 colorectal cancer
cells were chosen for the combination study. PC-3 cells at a concentration 1
104 or HT29 cells at
a concentration of 3 L of the appropriate culture
104 were seeded per well onto 96-well plates in 100
medium. Twenty-four hours after seeding the medium was removed and the cells were treated by
adding 100 L of the combinations in medium solutions at selected concentrations. Specifically, PC-3
cells were treated with combinations of pifithrin- and cisplatin, whereas HT29 cells were treated with
combinations of pifithrin-µ and oxaliplatin, Tables 2 and 3.
After 72 h of treatment, 20 L of MTS reagent was added to each well and the plates incubated
ď0.2%. 0.2% DMSO in medium was used as a control for experiments pertaining to pifithrin-µ.
ˆ
ˆ
µ
µ
µ
µ
for 2 h at 37 ˝C. The absorbance was measured at 490 nm using a Wallac 1420 Victor 3V plate reader
(Perkin-Elmer Life Sciences). The percentage of surviving cells relative to untreated controls were
calculated and the fraction affected (fa) which represents the respective proliferation inhibition was
determined, where 0% inhibition fa = 0% and 100% inhibition fa = 1.
The CI indexes were computed and graphically represented using dose-response plots in
Compusyn [15].
4. Conclusions
We described a novel improved synthesis for the important and well-known HSP70 inhibitor,
pifithrin-µ, with corresponding and previously unreported characterisation.
The first example of a combination study comprising HSP70 inhibitor pifithrin-
oxaliplatin is reported. We have determined moderate synergistic and synergistic effects in co-treating
PC-3 prostate cancer cells with pifithrin- and cisplatin and significant synergistic effects including
µ and cisplatin or
µ
strong synergism in co-treating HT29 colorectal cancer cells with pifithrin-µ and oxaliplatin.
This study indicates that HSP70 inhibition and Pt-based drug regimens should be investigated
further as potential anticancer combination therapies. In addition the impact of co-treatment of
pifithrin-µ and Pt drugs on cell death pathways merit investigation.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
7/949/s1.
Acknowledgments: This publication has emanated from research supported in part by a research grant from
Science Foundation Ireland (SFI) under Grant Number 12/IP/1305 and in part by Institute of Technology, Tralee,
Postgraduate Research Scholarship Programme.
Author Contributions: D.M.G., J.C. and H.M. conceived and designed the experiments; A.M.M. and A.E.
performed the experiments. D.M.G. and A.M.M. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 949
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Sample Availability: Samples of pifithrin-µ are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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