Synthesis and anticancer properties of RGD peptides conjugated to nitric oxide releasing functional groups and abiraterone

Article abstract of DOI:10.1016/j.tet.2014.09.004

A series of analogues of the integrin binding aspartic acid-glycine-arginine (RGD) peptide sequence were synthesised conjugated to nitric oxide (NO) donating functional groups. Also the cytotoxicity of abiraterone, a prostate cancer drug, was explored when it was conjugated in three part constructs to RGD sequences and NO releasing heterocycles. In general the analogues showed integrin binding affinity comparable to RGD reference compounds, and all released NO by the Griess test assay. Two analogues exhibited significant cytotoxic effects against PC3 and MCF7 cell lines.

Full text of DOI:10.1016/j.tet.2014.09.004

Tetrahedron xxx (2014) 1e5
Contents lists available at ScienceDirect
Tetrahedron
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Synthesis and anticancer properties of RGD peptides conjugated to
nitric oxide releasing functional groups and abiraterone
,
,
Andrew Nortcliffe a, Ian N. Fleming b, Nigel P. Botting a y, David O’Hagan a
*
a EaStCHEM School of Chemistry, Biomolecular Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
b Aberdeen Biomedical Imaging Centre, Lilian Sutton Building, Foresterhill, Aberdeen AB25 2ZD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2014
Received in revised form 28 August 2014
Accepted 2 September 2014
Available online xxx
A series of analogues of the integrin binding aspartic acid-glycine-arginine (RGD) peptide sequence were
synthesised conjugated to nitric oxide (NO) donating functional groups. Also the cytotoxicity of abir-
aterone, a prostate cancer drug, was explored when it was conjugated in three part constructs to RGD
sequences and NO releasing heterocycles. In general the analogues showed integrin binding affinity
comparable to RGD reference compounds, and all released NO by the Griess test assay. Two analogues
exhibited significant cytotoxic effects against PC3 and MCF7 cell lines.
Keywords:
Ó 2014 Published by Elsevier Ltd.
Anticancer
RGD peptide
Nitric oxide
Abiraterone
1. Introduction
activities and has been implicated in the control of blood pres-
sure,14 neurotransmission,15 inhibition of platelet aggregation and1
The arginine-glycine-aspartic acid (RGD) sequence has been
immune system regulation.16 It is also implicated in cytostasis and
cytotoxicity in cancer.17 Previous research has shown that the
combination of a cytotoxic drug and an NO-release functional
group can increase the overall cytotoxic activity of the com-
pound.18e22 In our previous work we observed that the addition of
an NO-release functional group to Sulindac, a NSAID, generated
a cytotoxic effect in PC3 prostate cancer cells.18 It was identified by
Stewart et al. that NO modulates the hypoxic response in these cells
resulting in ‘NO bioactivity’ contributing to increased cytotoxicity.23
One of the key challenges in NO research is the delivery of NO to
a specific organ or tissue. Based on previous research into the use of
the RGD motif in our laboratory13 we sought to investigate whether
RGD peptides conjugated to NO release functionalities would be an
effective delivery agent for NO. It has previously been shown that
modifications to the N-terminus of the linear RGD sequence are
compatible for combining integrin binding affinity with conjuga-
tion to other molecular units.10,24,25 In addition, we sought to
combine the RGD/NO conjugate with a compound with a known
biological affect against prostate cancer cells. Abiraterone, a CYP17
inhibitor used for the treatment of late stage, metastatic prostate
cancer.26,27 While abiraterone has shown promising results for in-
creasing the life-expectancy of terminal patients,27 3e50% of pa-
tients exhibit a primary resistance to abiraterone after beginning
treatment and all patients ultimately exhibit resistance.28 In addi-
tion, abiraterone is currently undergoing clinical trials for the
treatment of advanced and metastatic breast cancer.29 We sought
recognised as a significant motif in molecular recognition for cell
adhesion.1 avb3-Integrin is
a heterodimeric receptor highly
expressed in malignant tumour cells and angiogenic tumour en-
dothelial cells. avb3-integrin is part of a family of 24 distinct
integrin heterodimers made up of 18a and 8b subunits (reviewed in
Raymond et al.).2 The integrin family act as cell surface receptors for
a range of soluble and insoluble extracellular ligands, including
collagens, laminins, fibronectin and vitronectin.3 The avb3-integrin
was identified to be a receptor for the RGD sequence,4 In addition to
avb3, the RGD sequence is recognised by a3b1, a5b1, a8b1, avb1, avb5,
avb6 and aIIbb3-integrins.3 The bioactivity of RGD-integrin in-
teractions has expanded to involve systems including cell migra-
tion,5 growth,6 differentiation4 and apoptosis,7 in addition to
adhesion.8 In cancer, changes in cell adhesion have roles in me-
tastasis, tumour angiogenesis and the evasion of apoptosis.9 As
a result of this the RGD sequence has been exploited as a targeting
mechanism for the conjugation of therapeutic agents,10,11 or as
a tool for molecular imaging.12,13 Nitric oxide (NO) is widely
thought of as a vasodilator14 but has a variety of biological activities.
In addition to this has been shown to display a variety of biological
* Corresponding author. Tel.: þ44 (0)1334 463800; fax: þ44 (0)1334 467171;
e-mail address: do1@st-andrews.ac.uk (D. O’Hagan).
y
Nigel P. Botting passed away on 4th June 2011.
http://dx.doi.org/10.1016/j.tet.2014.09.004
0040-4020/Ó 2014 Published by Elsevier Ltd.
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2
A. Nortcliffe et al. / Tetrahedron xxx (2014) 1e5
to examine whether RGD conjugates in combination with NO do-
nors could elicit a cytotoxic effect from localised NO release, and if
this effect could be combined with abiraterone to combine the bi-
ological activity of each of the components. We have developed
a series of RGD peptides conjugated to nitric oxide donors, and to
the CYP17 inhibitor abiraterone.30 The resulting RGD peptide con-
jugates were assessed for NO release, avb3 integrin binding affinity
and for cytotoxicity against prostate (PC3) and breast (MCF7) can-
cer cell lines.
2. Results and discussion
2.1. Nitric oxide donor synthesis
2.2. RGD synthesis
A series of NO-donating carboxylic acids 1e3 were prepared
using a nitrate ester, furoxan and sydnonimine as the NO-donating
functional groups. 4-Nitrooxybutyric acid was prepared in three
steps from 4-bromobutyric acid 4 (Scheme 1). Esterification under
acid conditions provided bromobutyrate 5,31 and subsequent nu-
cleophilic substitution using silver nitrate furnished the nitrate
ester 6 (Scheme 1).32 Saponification of ester 6 using aqueous LiOH
at 5 ꢀC provided the desired carboxylic acid 1 with no observed
nitrate ester hydrolysis or elimination (Scheme 1).32
Initially we sought to develop an NOeRGD conjugate based on
a cyclo-RGD motif. Attempts at preparing a cyclo-RGD containing an
NO-amino acid, or bioconjugation to a commercial cyclo-RGD were
unsuccessful. As such our attention turned to linear RGD sequences
prepared by solution phase peptide synthesis, based on successful
reports by Boisbrun et al.,24 and by Welsh and Smith.35
Aspartic acid di-tert butyl ester 16 was synthesised in two steps
from commercially available Cbz-aspartic acid 17 (Scheme 3).36
Protection of the carboxylic acids was accomplished using tert-
butyl acetate and BF3(OEt)236 to give di-tert butyl ester 18. Transfer
hydrogenolysis of the Cbz group using Pd/C and ammonium for-
mate provided amine 16 (Scheme 3).36
Scheme 1. Reagents and conditions: (a.) AcCl, CH3OH, 0 ꢀC to rt, 18 h, quant.; (b.)
AgNO3, CH3CN, 90 ꢀC, 18 h, 97%; (c.) LiOH, CH3OH, 5 ꢀC, 18 h, 77%.
Acids 2 and 3 were prepared from succinic anhydride 7. Ring
opening with tert-butanol furnished ester 8 (Scheme 2).33 Selective
reduction of the carboxylic acid with BH3$Me2S provided the de-
sired precursor 9.33 Reaction of 9 with bis(phenylsulfonyl)furoxan
10 and DBU provided the ether 11 in 95% yield.34 Deprotection with
TFA generating the desired carboxylic acid 2. For the preparation of
acid 3, the 4-nitrophenylcarbamate 12 previously prepared in our
group for the introduction of sydnonimines was used.18,34 As such,
reaction of alcohol 9 with activated ester 12, furnished the desired
sydnonimine 13 in 70% yield. Once again, deprotection with TFA
provided the desired carboxylic acid 3.
Scheme 3. Reagents and conditions: (a.) tert-butyl acetate, BF3(OEt2), rt, 18 h, 60%; (b.)
(i.) NH4CO2H, 10% Pd/C, THF/MeOH (1:1), rt, 18 h, (ii.) satd HCl in EtOAc, 1 h, ꢁ20 ꢀC,
80%.
From here, the NH2-Arg(Pbf)-Gly-Asp(OtBu)-OtBu 19 sequence
was prepared using the procedure reported by Welsh and Smith
(Scheme 4)35 using 1-n-propane-phosphonic cyclic anhydride
(T3PÒ) for the peptide coupling reactions, and piperidine in DMF
for Fmoc deprotection (Scheme 4). Fmoc deprotection of Fmoc-
Arg(Pbf)-Gly-Asp(OtBu)-OtBu 22 under the conditions reported by
Welsh et al. (piperidine/CH2Cl2), resulted in significant epimerisa-
tion of the peptide. Using DMF rather than dichloromethane as the
solvent, this problem was avoided.
Scheme 4. Reagents and conditions: (a.) Fmoc-Gly-OH, T3PÒ, (iPr)2EtN, CH2Cl2, 0 ꢀC,
16 h, 95%; (b.) piperidine, CH2Cl2, 0 ꢀC to rt, 2 h, 87%; (c.) Fmoc-Arg(Pbf)-OH, T3PÒ,
(iPr)2EtN, CH2Cl2, 0 ꢀC, 16 h, 90%; (d.) piperidine, DMF, 0 ꢀC to rt, 0.5 h, 85%.
Scheme 2. Reagents and conditions: (a.) tert-Butanol, Et3N, N-hydroxysuccinimide,
DMAP, toluene, reflux, 18 h, 77%; (b.) BH3$Me2S, rt, 18 h, quant.; (c.) 9, DBU, CH2Cl2, rt,
2 h, 95%; (d.) TFA, CH2Cl2, rt, 16 h, 2¼quant., 3¼quant.; (e.) 9, CH3CN, reflux, 18 h, 70%.
2.3. RGDeNO synthesis
In addition to acids 1e3, two amino acids 14 and 15 were used.
These were prepared from Boc-Tyr-OMe and bis(phenylsulfonyl)
furoxan 10 and 4-nitrophenylcarbamate 11 as previously
reported.34
The condensation of NH2-Arg(Pbf)-Gly-Asp(OtBu)-OtBu 19 with
the prepared NO-donating carboxylic acids 1e3 using HATU/dii-
sopropylethylamine in DMF furnished the desired peptides 23e25
in 65e80% yield (Scheme 5). Global deprotection of the Pbf-
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A. Nortcliffe et al. / Tetrahedron xxx (2014) 1e5
3
2.4. RGDeNO-abiraterone
In addition to NOeRGD conjugates, we sought to combine NO
release with a chemotherapeutic agent. Abiraterone, a CYP17 in-
hibitor used for the treatment of castration resistant prostate can-
cer was used.30 To this effect, amino acid 34 was prepared as
previously reported.34 Coupling of Fmoc amino acid 34 to NH2-
Arg(Pbf)-Gly-Asp(OtBu)-OtBu 19 with HATU/diisopropylethylamine
provided the desired tetrapeptide 35 in 76% yield (Scheme 7).
Deprotection with piperidine in DMF provided the free N-terminus
amine 36 in 90% yield (Scheme 7).
Scheme 5. Reagents and conditions: (a.) 19, HATU, DMF, (iPr)2EtN, 0 ꢀC to rt, 18 h,
23¼65%, 24¼76%, 25¼80%; (b.) TFA:TIPS:H2O, rt, 2 h, 26¼85%, 27¼90%, 28¼88%.
Scheme 7. Reagents and conditions: (a.) 19, HATU, DMF, (iPr)2EtN, 0 ꢀC, 18 h, 76%; (b.)
piperidine, DMF, 0 ꢀC to rt, 0.5 h, 90%.
To append abiraterone 37 to amine 36, it was converted to the
tert-butyl hemisuccinate ester 38 using tert-butyl hemisuccinate 8
and EDCI.HCl/DMAP (Scheme 8). Deprotection of the tert-butyl
ester furnished the desired succinate ester 39 (Scheme 8).
guanidyl and tert-butyl ester groups was accomplished using
a standard cocktail of TFA, triisopropylsilane and water (95:2.5:2.5)
(Scheme 5). Purification of the crude peptides by trituration with
cold diethyl ether, followed by repeat lyophilisation from tert-
butanol:water (1:3, 1:9, 0:10) allowed the full removal of organic
residues and isolation of the desired peptides 26e28 as the tri-
fluoroacetate salt.
In a similar manner, amino acids 14 and 15 were coupled to
amine 19 using HATU. Global deprotection of the Pbf-guanidyl, tert-
butyl esters and tert-butoxycarbonyl group furnished the peptides
31 and 32 (Scheme 6).
Scheme 8. Reagents and conditions: (a.) 8, EDCI$HCl, DMAP, CH2Cl2, rt, 16 h, 95%; (b.)
TFA, CH2Cl2, rt, 16 h, quant.
Condensation of tetrapeptide amine 36 and abiraterone ester 39
using HATU provided the abiraterone conjugate 40 (Scheme 9).
Global deprotection with TFA:TIPS:H2O and subsequent trituration
and lyophilisation provided the peptide 41 (Scheme 9).
Scheme 6. Reagents and conditions: (a.) 19, HATU, DMF, (iPr)2EtN, 0 ꢀC, 18 h, 29¼70%,
30¼69%; (b.) TFA:TIPS:H2O, rt, 2 h, 31¼92%, 32¼90%.
Scheme 9. Reagents and conditions: (a.) 36, HATU, DMF, (iPr)2EtN, 0 ꢀC to rt, 18 h, 83%;
(b.) TFA:TIPS:H2O, rt, 2 h, 75%.
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4
A. Nortcliffe et al. / Tetrahedron xxx (2014) 1e5
Table 2
3. Biological evaluation
Inhibition of biotinylated c[RGDfK]-(PEG)2 binding to immobilized avb3. integrin.
Data are the averages of triplicate experiments. Q¼normalised activity as ratio
The NOeRGD peptides prepared were assessed for NO release,
avb3 integrin affinity, and cytotoxicity against prostate (PC3) and
breast (MCF7) cancer cell lines.
IC50(peptide)/IC50(RGD)
Compound
IC50
/m
M
Q
RGD
GRGDSPK
8.5ꢂ3.1
6.4ꢂ1.3
1
0.75
0.66
0.56
0.73
0.70
0.68
3.1. NO/NOL2 release assay
26
27
28
31
32
41
5.6ꢂ0.5
4.7ꢂ0.7
6.2ꢂ0.2
The level of NO release from the NOeRGD peptides was de-
termined by the Griess test.37 The formation of nitrite (NOꢁ2 ) is
a reliable proxy measure for nitric oxide, in the presence of thiol.38
To this effect, the Griess test for nitrite was used to determine NO
production from the NOeRGD peptides compared to the RGD tri-
peptide. Solutions of RGD peptides 26e28, 31e32 and 41 (1 mmol,
6.0ꢂ0.3
5.8ꢂ1.6
107.6ꢂ45.7
12.6
terminus as shown with peptide 41 results in a decreased affinity
towards the avb3 integrin when a linear chain is used.
[final], 400
thione (GT) buffer containing superoxide dismutase (SOD) ([GT]
6 mmol, final [GT] 600
M, pH 7.6, [SOD] 4557 U mLꢁ1). RGD was
mM) in DMSO:H2O (1:9) were incubated with a gluta-
m
3.3. Cytotoxicity results
used as a control. All of the prepared NOeRGD peptides demon-
strated GT dependent NO release in the Griess assay (Table 1). The
RGD sequence alone did not show any NO-release in GT or pH 7.6
buffer. The highest levels of NO release were observed from the
furoxan RGD peptides 27 and 32. The levels of NO release were
Cell cytotoxicity assays were performed in 96-well plates as
described in Smith et al.40 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) cell proliferation reagent was used
to estimate the cell number in each well. Preliminary cytotoxicity
results indicate that peptides 27 and 41 show concentration de-
pendent cytotoxic activity against PC3 (prostate) and MCF7 (breast)
cancer cell lines (Table 3). The other peptides tested did not show
an observable cytotoxic effect. The differences in cytotoxicity
maybe a consequence of the different mechanism of NO release in
the cell following internalisation of the peptide after integrin
41.4ꢂ1.1
Sydnonimine RGD peptide 31 had a similar level of NO release
(40.8ꢂ2.7 mol, 10.2%). Nitrate ester 26 and abiraterone conjugate
41 had comparable levels of NO release (32.2ꢂ2.5 mol and
mmol and 52.5ꢂ5.1
mmol (10.4 and 13.1%), respectively.
m
m
31.6ꢂ4.8
mmol). NO release was comparable to previously reported
compounds.18,34
Table 1
NO release from RGD peptides. Data are the averages of triplicate experiments
Compound
NOꢁ2 production
mol) (GT buffer)
NOꢁ2 production as %
(GST buffer)
NOꢁ2 production
mol) (pH 7.6 buffer)
NOꢁ2 production as %
(pH 7.6 buffer)
(
m
(m
26
27
28
31
32
41
RGD
32.2ꢂ2.5
41.4ꢂ1.1
29.6ꢂ6.6
40.8ꢂ2.7
52.5ꢂ5.4
31.6ꢂ4.8
2.6ꢂ0.6
8.1
10.4
7.4
10.2
13.1
7.9
2.1ꢂ0.4
1.6ꢂ1.1
3.1ꢂ2.3
4.0ꢂ1.6
1.0ꢂ0.7
2.4ꢂ1.1
2.2ꢂ0.7
<1.0
<1.0
<1.0
1.0
<1.0
<1.0
<1.0
<1.0
3.2. avb3 integrin affinity
binding.38,41,42 Abiraterone containing peptide 41 showed de-
creased cytotoxicity compared with abiraterone acetate (IC50
The RGD peptides 26e28, 31e32 and 41 were submitted to
biochemical assays to measure their affinity for avb3 receptor.
Competitive affinities were calculated by analyzing the interactions
between immobilized avb3 and the commercially available c
[RGDfK(Biotin-PEG-PEG)] peptide in the presence of the RGD
peptides.39 Commercially available RGD and GRGDSPK were used
as reference sequences. IC50 values were calculated for each pep-
tide, and were normalised with respect to the IC50 of RGD to allow
comparisons between experiments. The results are summarised in
22.5ꢂ0.4 and 12.6ꢂ1.8
mM in PC3 and MCF7 cells, respectively). As
shown in Tables 1 and 2, each of the peptides displayed NO release
and integrin binding in the respective Griess and avb3 affinity as-
says, although only 27 and 41 had some cytotoxic activity. It has
been previously reported that it is not possible to detect selective
cytotoxicity of RGD linked paclitaxel in cell culture models,43 as
a result, the observed activity of abiraterone conjugate 41 may not
reflect the true cytotoxic potential of the compound due to the
limitations of the cell culture assay. Non-toxic RGD/NO conjugates
may find use in other areas of NO research.
Table 2. The highest affinity (4.7 mM) was observed for furoxan RGD
peptide 27. This was nearly twofold higher affinity than the RGD
control, and nearly 1.5-fold higher affinity than GRGDSPK. RGD
peptides 26, 28, 31 and 32 all displayed greater affinity for avb3
compared to RGD, and they had a comparable activity to GRGDSPK.
Abiraterone conjugate 41, displayed an integrin binding affinity of
Table 3
Cytotoxicity results of RGDeNO conjugates. All data are an average of triplicates
Compound
PC3 IC50
/
mM
MCF7 IC50/mM
26
27
28
31
32
41
>100
>100
107.6 mM, an approximately 13-fold decrease in affinity compared
38.2ꢂ3.8
46.0ꢂ18.2
to RGD. The integrin binding studies suggest that the addition of
a single amino acid residue, or a heterocycle appended chain onto
the N-terminus of the peptide has little affect on the affinity of the
peptide to avb3. However, installation of a large group on the N-
>100
>100
>100
>100
99.4ꢂ0.7
>100
>100
58.3ꢂ10.7
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A. Nortcliffe et al. / Tetrahedron xxx (2014) 1e5
5
4. Summary
References and notes
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In summary, a series of RGD peptides have been prepared,
conjugated to various NO releasing functional groups. The synthesis
provided six conjugate analogues using nitrate esters, furoxans and
sydnonimines as NO-release functional groups, either as linear
appended groups or using an NO-releasing amino acid. The bi-
ological activity was evaluated by NO release assays (Griess test),
avb3 integrin binding assays and cytotoxicity assays against PC3 and
MCF7 cancer cell lines. Furoxan containing peptide 27 displayed
a twofold increase in avb3 binding compared to the RGD peptide
sequence, along with upto 10% NO-release after incubation with GT
for one hour. This peptide also displayed some cytotoxicity against
PC3 and MCF7 cancer cells, although the mechanism of this cyto-
toxicity is still under investigation. NOeRGD peptide conjugates
have the potential to be useful delivery agents of NO to cells, which
have high expression of avb3, such as the epithelial lining of car-
diovascular tissue, or in cancer tissue, which overexpresses integrin
receptors.
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5. Experimental
€
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18. Nortcliffe, A.; Ekstrom, A. G.; Black, J. R.; Ross, J. A.; Habib, F. K.; Botting, N. P.;
5.1. Nitrite/NO release assay (Griess test)
O’Hagan, D. Bioorg. Med. Chem. 2014, 22, 756.
19. Bertinaria, M.; Rolando, B.; Giorgis, M.; Montanaro, G.; Marini, E.; Collino, M.;
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Procedure adapted from Nortcliffe et al.34 The peptide (1 mmol)
was dissolved in DMSO:H2O (1:9); Buffer solution was added (pH
7.6, containing 12 mmol GST). The mixture was incubated at room
temperature for 1 h. Griess reagent A (1% sulfanilamide, 5% H3PO4
in deionised H2O) was added and incubated for 5 min. Griess re-
21. Montanaro, G.; Bertinaria, M.; Rolando, B.; Fruttero, R.; Lucas, C. D.; Dorward, D.
A.; Rossi, A. G.; Megson, I. L.; Gasco, A. Bioorg. Med. Chem. 2013, 21, 2107.
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Int. J. Cancer 2009, 124, 223.
agent
deionised H2O) was added and the mixture incubated for 10 min.
The final concentration of peptide was 400 M. UV absorbance at
B
((N-1-naphthyl)ethylenediamine dihydrochloride in
ꢀ
24. Boisbrun, M.; Vanderesse, R.; Engrand, P.; Olie, A.; Hupont, S.; Regnouf-de-
m
Vains, J.-B.; Frochot, C. Tetrahedron 2008, 64, 3494.
568 nm was measured using a Multiskan FC Microplate Photometer
and calibrated using a standard curve prepared from a standard
solution of NaNO2 to give the nitrite concentration. Measurements
were made in triplicate.
25. Hersel, U.; Dahmen, C.; Kessler, H. Biomaterials 2003, 24, 4385.
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Flaig, T. W.; Hutson, T. E.; Cheng, T.; Patterson, H.; Hainsworth, J. D.; Ryan, C. J.;
ꢀ
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5.2. Binding affinity tests
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Acknowledgements
We thank CRUK for a Studentship (A.N.) and Dr. Terry K. Smith
(St. Andrews University) for access to a Microplate reader.
€
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Supplementary data
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Full compound synthesis and characterisation data can be found
in the Supplementary data. Supplementary data related to this ar-
ticle can be found at http://dx.doi.org/10.1016/j.tet.2014.09.004.
Please cite this article in press as: Nortcliffe, A.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.09.004