Phosphotyrosine prodrugs: design, synthesis and anti-STAT3 activity of ISS-610 aryloxy triester phosphoramidate prodrugs

Article abstract of DOI:10.1039/c8md00244d

Unmasked phohate groups of phosphotyrosine-containing molecules carry two negative charges at physiological pH, which compromise their (passive) cellular uptake. Also, these phosphate groups are often cleaved off by phosphatases. Together, these ultimatel

Full text of DOI:10.1039/c8md00244d

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RESEARCH ARTICLE
Phosphotyrosine prodrugs: design, synthesis and
anti-STAT3 activity of ISS-610 aryloxy triester
phosphoramidate prodrugs†‡
Cite this: DOI: 10.1039/
c8md00244d
*
Ageo Miccoli, Binar A. Dhiani and Youcef Mehellou
Unmasked phohate groups of phosphotyrosine-containing molecules carry two negative charges at physi-
ological pH, which compromise their (passive) cellular uptake. Also, these phosphate groups are often
cleaved off by phosphatases. Together, these ultimately limit the pharmacological efficacy of the
phosphotyrosine-containing compounds. To address these drawbacks, we herein present the application
of the aryloxy triester phosphoramidate prodrug technology, a monophosphate prodrug technology, to the
phosphotyrosine-containing compound ISS-610-Met, an analogue of the anticancer STAT3 dimerization
inhibitor ISS-610. Our data shows that the generated ISS-610-Met prodrugs exhibited enhanced pharma-
cological activity and inhibition of STAT3 downstream signaling compared to the parent compound ISS-
610-Met and the known STAT3 dimerization inhibitor ISS-610. These encouraging results provide a com-
pelling proof of concept for the potential of the aryloxy triester phosphoramidate prodrug technology in
the discovery of novel therapeutics that contain phosphotyrosine and its phospho mimics.
Received 10th May 2018,
Accepted 6th November 2018
DOI: 10.1039/c8md00244d
rsc.li/medchemcomm
and this led to a series of compounds with interesting biologi-
cal activities.5 However, none of these compounds have
1. Introduction
Phosphotyrosine mediates the formation of protein com-
plexes by binding to various phosphotyrosine-binding do-
mains such as Src homology 2 (SH2) domains,1 which are
present in many of proteins that include kinases, phospha-
tases and transcription factors. Given the vital roles SH2 do-
mains play in mediating many cell signaling events that are
implicated in the pathogenesis of human diseases,2–4 the
targeting of these phosphotyrosine-binding domains by small
molecules has been noted for a long time as a promising ap-
proach in drug discovery.5 However, the development of these
molecules has largely been hindered by the requirement of a
phosphotyrosine motif to mimic the endogenous moiety and
achieve binding to the target proteins with high affinity. This
is because the incorporation of phosphate groups makes
these phosphotyrosine-containing molecules unstable in vivo
due to dephosphorylation by alkyl phosphatases. Additionally,
the charged phosphotyrosine species at physiological pH limit
their (passive) cellular entry. To address these shortages,
phosphate bioisosteres, e.g. carboxylic acids, have been used
progressed to late clinical development.
In order to improve the drug-like properties of
phosphotyrosine-containing molecules and their phospho-
mimics, we hypothesized that this could be achieved by ap-
plying monophosphate prodrug technologies to these com-
pounds. Although there are many prodrug strategies reported
to be effective in delivering monophosphorylated molecules
into cells, we decided to use the aryloxy triester
phosphoramidate prodrug technology6 as this has already de-
livered two FDA-approved drugs and over 10 clinical candi-
dates.7 In this prodrug technology, the monophosphate
group is masked by an aryl motif and an amino acid ester
(Fig. 1). The hydrolysis of these masking groups is now well
understood to proceed in vivo via the involvement of two dis-
tinct enzymes; carboxypeptidase Y8 and a phosphoramidase-
Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University,
Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK.
E-mail: MehellouY1@cardiff.ac.uk
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00244d
‡ Binar A. Dhiani would like to dedicate this article to her father, who passed
away during the write up of this manuscript.
Fig.
1 Metabolism of the aryloxy phosphoramidate prodrug
technology to release the monophosphate species.
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type enzyme (Hint-1)9 (Fig. 1) [see Mehellou Y. et al.6 for a de-
tailed account on the aryloxy triester prodrug technology].
To determine the suitability of the aryloxy triester
phosphoramidate prodrug technology, we chose the STAT3
dimerization inhibitor ISS-610, a peptidomimetic that con-
tains a phosphotyrosine motif (Fig. 2). ISS-610 was first
reported in 2004 as a binder of a transcription factor known
as the signal transducer and activator of transcription 3
(STAT3).10 This molecule contains a phosphotyrosine moiety
by design, which allows it to bind the SH2 domain of STAT3
resulting in the inhibition of its dimerization and down-
stream signaling. The interest in the discovery of small mole-
cules that inhibit STAT3 is driven by the fact that STAT3 is
constitutively active in numerous cancers and contributes to
tumor cell proliferation and survival.11 At the molecular level,
the activation of STAT3 involves phosphorylation by upstream
kinases, which leads to its homo- and heterodimers with
other STAT isoforms. This dimerization is critical for its
downstream signaling and thus the inhibition of this dimer-
ization process emerged as a favorable approach in the dis-
covery of anticancer STAT3 inhibitors.
ISS-610 was found to be able to inhibit the binding of
STAT3 to the target DNA with moderate efficacy, IC50 = 42
μM.10 Although cellular data also supported the ability of ISS-
610 to inhibit STAT3 dimerization and subsequent down-
stream signaling, the concentrations required to achieve
these in cells were relatively high (∼1 mM).10 We suspected
that this was due to poor cellular uptake of ISS-610 that re-
sults from its relatively large size, charged nature at physio-
logical pH and dephosphorylation of the phosphotyrosine
moiety. These limitations made ISS-610 an ideal candidate
for the application of the powerful monophosphate prodrug
technology, the aryloxy triester phosphoramidate technology.
C-terminal carboxylic group of the L-leucine amino acid, a
compound we termed ISS-610-Met. As a control, we also
synthesised the ISS-610-Met monophosphate (7) (Fig. 3). No-
tably, modifications of ISS-610 in its C-terminal region have
been extensively explored and led to the discovery of potent
STAT3 dimerization inhibitors.12
The synthesis of the ISS-610-Met started by the coupling
of the Boc-tyrosine (2) and L-leucine methyl ester via the use
of the peptide coupling reagent benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)
in the presence of triethylamine (TEA) to attain dipeptide 3
in a high yield (94%). Removal of the Boc-protecting group
with trifluoroacetic acid (TFA) afforded fragment 4 in almost
quantitative yield (99%) and this was subsequently coupled
to 4-cyanobenzoyl chloride, which had been prepared as
reported.13 Subsequently, the phosphorodiamidate of com-
pound 5 was synthesised by reacting intermediate 5 with N,N,
N′,N′-tetramethylphosphorodiamidic chloride in the presence
of 4-dimethylaminopyridine (DMAP) and 1,8-diazabicyclo-
ij5.4.0]undec-7-ene (DBU) to generate the desired product, 6,
in a moderate yield, 50%.12 Finally, the acidification diamidate
6 with TFA yielded acid ISS-610-Met (7) in high yield, 76%.12
Next, we synthesised ISS-610-Met aryloxy triester phosphoramidate
prodrugs (Fig. 4), the phosphorochloridates were initially
synthesised as previously reported.14,15 Briefly, 1-napthol (8)
in dry diethyl ether at −78 °C was treated with phosphorous
oxychloride (POCl3) and TEA to afford phosphorodichloridate
9, which was used in the next step without purification. Sub-
sequently, this was dissolved in anhydrous dichloromethane
(DCM) and reacted with the desired L-amino acid esters in
2. Results and discussion
2.1. Synthesis of ISS-610 and its prodrugs
In the design of ISS-610 phosphoramidate prodrugs, the car-
boxylic acid of ISS-610 had to be protected to avoid a nucleo-
philic attack from the free carboxylate group onto the phos-
phorous atom leading to the breakdown of the prodrug motif
akin to its in vivo metabolism. Thus, ISS-610 prodrugs
presented in this work carry a methyl group that blocks the
Fig.
3 Synthesis of ISS-610-Met (7). Reagents and conditions: a)
PyBop, L-leucine methyl ester hydrochloride, NEt3, THF, 94%; b) TFA,
DCM, 99%; c) 4-cyanobenzoyl chloride, NEt3, THF, 42%; d) N,N,N′,N′-
tetramethylphosphorodiamidic chloride, DMAP, DBU, THF, 50%; e)
TFA, H2O, 76%.
Fig. 2 Chemical structure of the phosphotyrosine containing STAT3
dimerization inhibitor ISS-610.
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Fig.
5
Postulated mechanism of aryloxytriester phosphoramidate
prodrugs.6
Fig.
4 Synthesis of ISS-610-Met aryloxy triester phosphoramidate
prodrugs. Reagents and conditions: a) POCl3, TEA, diethyl ether, −78
°C, 98%; b) for compound 10 (R = methyl): L-alanine methyl ester hy-
drochloride, TEA, DCM, −78 °C, 70%, for compound 11 (R = isopropyl):
L-alanine isopropyl ester hydrochloride, TEA, DCM, −78 °C, 72%, for
compound 12 (R = benzyl): L-alanine benzyl ester hydrochloride, TEA,
mation of the highly unstable anhydrous intermediate 17.
This subsequently gets opened up by a nucleophilic attack
from a water molecule on the phosphorous center generating
phosphoramidate 18. Finally, a phosphoramidase-type en-
zyme. e.g. Hint-1, cleaves the P–N bond to release the
unmasked monophosphate 19, ISS-610-Met in this case.
DCM, −78 °C, 97%; c) for compound 13 (R
=
methyl):
phosphorochloridate 10, TEA, DCM, 72%, for compound 14 (R = iso-
propyl): phosphorochloridate 11, TEA, DCM, 91%, for compound 15 (R
= benzyl): phosphorochloridate 12, TEA, DCM, 93%.
2.2. Serum stability and metabolism of ISS-610 prodrugs
As compounds 13–15 are prodrugs, we initially studied their
stability in human serum. For this prodrug 14 was incubated
with human serum at 37 °C for 12 h and the mixture was
monitored by 31P-NMR as we reported previously.14 At t = 0 h,
two singlets appeared on the 31P-NMR, δP = −1.43 and 0.93
ppm, which correspond to the two diastereoisomers of
prodrug 14 (Fig. 6). Upon the addition of the human serum
and monitoring the reaction for 12 h, no new phosphorous
peaks were detected indicating the stability of this prodrug in
human serum. Although the intensity of the prodrug peaks at
t = 12 h appeared lower than those at t = 0 h, this was due to
some of the prodrug crashing out of solution following the ad-
dition of the serum (Fig. 6). This is due to the relatively high
lipophilicity of prodrug 14 (cLog P = 6.06). This is a common
observation is studying these prodrugs when they have a rela-
tively high lipophilicity. To further confirm the stability of
prodrug 14, we run a mass spectrometry of the parent com-
pound by itself and then the sample following 12 h incuba-
tion in human serum. The results showed that no new mass
spectrometry peaks appeared following incubation with hu-
man serum for 12 h. This is a further confirmation of the sta-
bility of this prodrug in human serum for the 12 h studied.
Notably, this stability profile is in line with the human serum
stability profiles of the aryloxy triester phosphoramidate
prodrugs of nucleoside monophosphates.14,15
the presence of triethylamine to give the desired
phosphorochloridates in excellent yields (70–97%).
The coupling of the phosphorochloridates, 10–12, to the
ISS-610-Met core structure, 5, was pursued in dry tetrahydro-
furan (THF) using triethylamine as a base. This gave the de-
sired ISS-610-Met phosphoramidate prodrugs, 13–15, in good
yields (72–93%). Notably, the final prodrugs, 13–15, were gen-
erated as a mixture of two diastereoisomers due to the chiral
phosphorous center and as indicated by the phosphorous
31P-NMR of these compounds (see ESI†). This is typical for
the synthesis of aryloxy triester phosphoramidate prodrugs
and many of these prodrugs are synthesised and studied as a
mixture of diastereoisomers6 with some of them even under-
going (late) clinical trials in this form, e.g. NUC-1031.15
In the design of the aryloxy triester phosphoramidate
prodrugs, the most common aryl motif used is the phenyl
followed by naphthyl. In this work, we chose the naphthyl as
the aryl motif of the prodrug moiety as it is a better leaving
group than phenol and hence in the metabolism of the
prodrugs the –P–O– bond would favor cleavage to release the
1-naphthol group (option 1, Fig. 5) instead of cleaving the
bond to release the phenol of the phosphotyrosine (option 2,
Fig. 5). Indeed, following entry to cells, the ester group of the
aryloxy triester phosphoramidate prodrugs is cleaved off by
esterases, e.g. carboxypeptidase Y, to yield an unmasked car-
boxylate group (16). This then performs a nucleophilic attack
on the phosphorous center. This leads to the cleavage of one
of the –O–P– phosphorous bonds as labelled options 1 and 2
in Fig. 5. Thus, in this case, having a naphthyl group as a
leaving group would favor option 1 and hence lead to the for-
2.3. Effect of ISS-610 and ISS-610-Met prodrugs on cancer
cells
The activation of the JAK/STAT3 signalling cascade leads to
STAT3 phosphorylation, dimerization and translocation to
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Fig. 6 Human serum stability of prodrug 14. A: 31P-NMR scan of prodrug 14 in 0.15 mL DMSO and 0.15 mL D2O. δP = −1.43 and 0.93. B: Final 31P-
NMR scan of the sample from A after being incubated with 0.3 mL of human serum at 37 °C for 12 h. C: LCMS trace of prodrug 14 alone. The peak
corresponding to the mass of the parent compound, 779.2750 g mol−1 (+Na), is highlighted in light yellow. D: LCMC trace the human serum alone.
E: LCMS trace of the organic fraction of the sample of prodrug 14 after being incubated with 0.3 mL of human serum at 37 °C for 12 h. The peak
corresponding to the mass of the parent compound, 779.2750 g mol−1 (+Na), is highlighted in light yellow.
the nucleus where it mediates the transcription of anti-
apoptotic genes such as Survivin and Bcl-xl.16 Hence, in de-
termining the potency of our prodrugs' inhibition of STAT3
dimerization, we probed for the expression levels of Survivin
and Bcl-xl. For this, we chose the breast cancer cell line MDA-
MB-468, which has the JAK/STAT3 signalling cascade consti-
tutively active and is widely used in studying the biological
activity of STAT3 inhibitors.17–19 Thus, the cells were treated
with the parent compound ISS-610 (1) or its C-methylated de-
rivative ISS-610-Met (7) as positive controls, or prodrugs
13–15 at the indicated concentrations for 24 h. Following
lysis, the cell lysates underwent Western blotting for total
Survivin and Bcl-xl as a readout of the compound's ability to
inhibit STAT3 dimerization and downstream signalling. As
shown in Fig. 7, the parent compounds, ISS-610 and ISS-610-
Met, did not have any significant downregulation of the
Fig. 7 Inhibition of the STAT3-dependent expression of the anti-apoptotic proteins Survivin and Bcl-xl by ISS-610 prodrugs. Cultured MDA-MB-
468 cells were treated with DMSO (control), ISS-610, prodrugs 13–15 at the indicated concentrations for 24 h. The cell lysates subsequently
underwent Western blotting for total STAT3, total Survivin and total Bcl-xl. Total β-actin was included as a loading control.
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expression of the anti-apoptotic proteins Survivin and Bcl-xl,
which is in line with the poor cellular activity of the IS-610
peptidomimetic.10 However, prodrugs 13–15 showed more
profound downregulation of the expression of Survivin and
Bcl-xl. This was most notable with compounds 13 and 14,
which exhibited significant reduction of Survivin even at 30
μM. In terms of the structure–activity relationship of aryloxy
triester phosphoramidate prodrugs, the ones with benzyl es-
ter tend to exhibit more potent biological activity than the
ones with methyl and isopropyl esters.6 However, in this
work, the prodrugs with methyl and isopropyl esters, 13 and
14 respectively, exerted more potent activity than their
prodrug counterpart with the benzyl ester, compound 15.
This is most likely due to the relatively high lipophilicity of
compound 15 as compared to compounds 13 and 14, which
could limit their solubility in the cell media. Indeed, in
treating the cells with prodrugs 13–15, only prodrug 15 upon
addition to the cells resulted in a cloudy media, which rap-
idly cleared up after gentle shaking. Nevertheless, the ob-
served downregulation of Survivin and Bcl-xl with prodrugs
13 and 14 relative to the lack of any effect on these proteins
by the parent compounds ISS-610 and ISS-610-Met was nota-
ble and indicates the potential of this prodrug approach in
increasing the potency of phosphotyrosine-containing STAT3
dimerization inhibitors.
purchased from Alta Biosciences (Birmingham, UK) and it
had a 99.2% purity. All reactions were carried out under an ar-
gon atmosphere. Reactions were monitored with analytical
TLC on silica gel 60-F254 precoated aluminum plates and vi-
sualized under UV (254 nm) and/or with 31P NMR spectra.
Column chromatography was performed on silica gel (35–70
μM). NMR data were recorded on a Bruker AV300, AVIII300,
AV400, AVIII400, or DRX500 spectrometer in the deuterated
solvents indicated, and the spectra were calibrated on resid-
ual solvent peaks. Chemical shifts (δ) are quoted in ppm, and
J values are quoted in Hz. In reporting spectral data, the fol-
lowing abbreviations were used: s (singlet), d (doublet), t (trip-
let), q (quartet), dd (doublet of doublets), and m (multiplet).
HPLC was carried out on a DIONEX summit P580 quaternary
low-pressure gradient pump with a built-in vacuum degasser
using a Summit UVD 170 s UV/vis multichannel detector. Sol-
vents were used as HPLC grade. Chromeleon software was
used to visualize and process the obtained chromatograms.
Analytical separations used a flow rate of 1 mL min−1
,
semipreparative used a flow rate of 3 mL min−1, and prepara-
tive used a flow rate of 20 mL min−1. All tested compounds
had a purity of ≥95% unless otherwise stated (see ESI†).
(S)-Methyl 2-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-
hydroxyphenyl)propanamido)-4-methylpentanoate (3)
To a stirring, colorless solution of Boc-L-tyrosine 2 (125 mg,
0.444 mmol), in dry THF (5 mL), was added PyBOP (277 mg,
0.532 mmol), L-leucine methyl ester hydrochloride (162 mg,
0.889 mmol) and triethylamine (0.19 mL, 1.332 mmol). The
white suspended solution was stirred at room temperature
for eight hours under an argon atmosphere. After reaction
completion, the orange solution was concentrated under re-
duced pressure and diluted with water (50 mL), where the
product was extracted with ethyl acetate (3 × 50 mL). The
combined organic layers were concentrated to 50 mL and
washed with water (1 × 50 mL) and brine (1 × 50 mL), dried
with MgSO4, filtered and concentrated under reduced pres-
sure to afford a colorless oil. Flash column chromatography
(2 : 1 ethyl acetate : hexane) was utilized to purify the product
and yield dipeptide 3 as a white crystalline solid (170 mg,
3. Conclusion
STAT3 dimerization is an essential process for its down-
stream signalling and cancer cell survival. The inhibition of
this process using phosphotyrosine-containing small mole-
cules that bind the STAT3 SH2 domain is an attractive ap-
proach in treating cancer. As small molecules that contain
phosphotyrosine and its phosphomimics often have poor
cellular uptake, we herein described – as a proof of concept –
the application of the aryloxy triester phosphoramidate ap-
proach to ISS-610-Met, an analogue of phosphotyrosine-
containing STAT3 dimerization inhibitor ISS-610. Our data
showed that these prodrugs are stable in human serum at 37
°C for 12 h. In cells, these prodrugs were relatively more
potent in inhibiting STAT3 downstream signalling than their
parent compounds ISS-610 and ISS-610-Met. Collectively, this
work highlights the novel applicability of the aryloxy triester
phosphoramidate prodrug approach in discovering new ther-
apeutics that contain phosphotyrosine or its phosphomimics,
an endeavour that has proved to be a stern challenge so far.
1
94%). H NMR (500 MHz, CDCl3) δ = 7.03 (d, J = 8.2 Hz, 2H),
6.73 (d, J = 8.5 Hz, 2H), 6.48 (s, 1H), 6.34 (d, J = 8.2 Hz, 1H),
5.11 (s, 1H), 4.60–4.50 (m, 1H), 4.28 (s, 1H), 3.69 (s, 3H), 2.97
(d, J = 6.8 Hz, 2H), 1.62–1.52 (m, 2H), 1.50–1.45 (m, 1H), 1.43
(s, 9H), 0.90 (t, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ
= 172.9, 171.7, 155.4, 130.4, 127.5, 115.6, 80.5, 55.9, 52.3,
50.8, 41.4, 37.4, 28.2, 24.6, 27.7, 21.8. MS (ES) 431.2 [M +
Na]+, 331.2 [M-Boc + Na]+.
4. Experimental
General information
(S)-Methyl 2-((S)-2-amino-3-(4-hydroxyphenyl)propanamido)-4-
methylpentanoate (4)
Dichloromethane, diethyl ether, methanol, and toluene were
dried in-house using a Pure Solv-MD solvent purification sys-
tem. All the other solvents were used as received from com-
mercial suppliers. All of the other reagents used in the synthe-
sis were purchased from Sigma-Aldrich except L-alanine
isopropyl ester, which was synthesised in house. ISS-610 was
To a stirring solution of compound 3 (350 mg, 0.857 mmol)
in DCM (8 mL), TFA (2.5 mL) was carefully added. The or-
ange solution was left to stir at room temperature for two
hours. Upon reaction completion, the orange solution was
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concentrated under reduced pressure, diluted with water (50
mL) and neutralized to pH 8 with 10% NaOH. The product
was extracted from the orange solution with ethyl acetate (4 ×
50 mL). The combined organic layers were dried with MgSO4,
filtered and concentrated under reduced pressure to yield
peptide 4 as a cream/white solid (260 mg, 99%). 1H NMR
(500 MHz, MeOD) δ = 7.14 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4
Hz, 2H), 4.52 (t, J = 7.3 Hz, 1H), 4.13–3.99 (m, 1H), 3.73 (s,
3H), 3.24–2.87 (m, 2H), 1.75–1.58 (m, 3H), 0.97 (dd, J = 15.8,
6.1 Hz, 6H). 13C NMR (126 MHz, MeOD) δ = 172.5, 168.7,
156.8, 130.3, 124.6, 115.4, 54.4, 50.9, 40.2, 36.5, 24.5, 21.8,
20.4. MS (ES) m/z 317.1 [M + Na]+, 295.1 [M + H]+.
2H), 2.69 (dd, J = 15.7, 10.1 Hz, 12H), 1.68–1.56 (m, 2H), 1.53
(ddd, J = 12.0, 9.0, 4.5 Hz, 1H), 0.87 (dd, J = 6.1, 4.7 Hz, 6H).
13C NMR (126 MHz, CDCl3) δ = 172.8, 170.8, 165.5, 150.3,
137.7, 132.5, 132.2, 130.7, 127.9, 120.2, 118.0, 115.1, 54.8, 52.3,
51.1, 41.1, 37.1, 36.7, 24.8, 22.7, 21.8. 31P NMR (202 MHz,
CDCl3) δ = 15.79. MS (ES) m/z 594.3 [M + Na]+.
Methyl ((S)-2-(4-cyanobenzamido)-3-(4-
(phosphonooxy)phenyl)propanoyl)-L-leucinate (7)
A stirring solution of diamidate 6 (60 mg, 0.102 mmol) in 9 : 1
TFA (9 mL) : H2O (1 mL) was stirred overnight at ambient tem-
perature. Upon reaction completion, the solvent was removed
to afford a cream solid. The solid was further dried under vac-
uum until constant weight, yielding acid 7 as a white solid (40
(S)-Methyl 2-((S)-2-(4-cyanobenzamido)-3-(4-hydroxyphenyl)
propanamido)-4-methylpentanoate (5)
1
mg, 76%). The compound was used without purification. H
NMR (500 MHz, MeOD) δ = 7.74 (d, J = 8.1 Hz, 2H), 7.70 (d, J =
8.1 Hz, 2H), 7.15 (d, J = 7.2 Hz, 2H), 7.05 (d, J = 7.6 Hz, 2H),
4.39 (dd, J = 9.4, 5.6 Hz, 1H), 3.86–3.75 (m, 1H), 3.60 (s, 3H),
3.01 (ddd, J = 23.9, 14.3, 7.5 Hz, 2H), 1.62 (dd, J = 12.5, 6.6 Hz,
1H), 1.58–1.50 (m, 2H), 0.83 (dd, J = 22.1, 6.5 Hz, 6H). 13C
NMR (126 MHz, MeOD) δ = 173.0, 172.4, 167.1, 138.2, 132.1,
132.0, 131.9, 129.7, 128.0, 120.0, 117.6, 114.7, 55.2, 51.4, 50.9,
40.1, 36.5, 24.5, 21.9, 20.4. 31P NMR (202 MHz, MeOD) δ =
−4.34. MS (ES) m/z 516.3 [M − H]−. HRMS C24H27N3O8NaP
calcd. 516.1541 [M − H]−, found 516.1541.
Compound 4 (118 mg, 0.382 mmol) was dissolved in THF (4
mL) and triethylamine (0.11 mL, 0.764 mmol) and stirred un-
der an argon atmosphere at room temperature for 30 min. To
the colorless solution, 4-cyanobenzyl acid chloride13 (70 mg,
0.421 mmol) in THF (2 mL) was added dropwise and left to
stir overnight at room temperature. The cream solution, with
a suspension of white precipitate, was concentrated under re-
duced pressure and then diluted in water (20 mL), where the
product was extracted with ethyl acetate (3 × 20mL). The com-
bined orange organic layers were washed with water (20 mL)
and brine (20 mL), dried with MgSO4, filtered and concen-
trated under reduced pressure to afford an orange oil. Col-
umn chromatography (2 : 1 ethyl acetate : hexane) was used to
yield dipeptide 5 as white crystals (70 mg, 42%). 1H NMR (300
MHz, CDCl3) δ = 7.84 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz,
2H), 7.13 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 7.5 Hz, 1H), 6.75 (d, J
= 8.5 Hz, 2H), 6.07 (d, J = 7.9 Hz, 1H), 5.49 (s, 1H), 4.87–4.76
(m, 1H), 4.59–4.49 (m, 1H), 3.75 (s, 3H), 3.12 (ddd, J = 21.6,
13.9, 6.7 Hz, 2H), 1.58–1.39 (m, 3H), 0.89 (d, J = 5.6 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ = 172.8, 171.2, 165.5, 155.3,
137.4, 132.4, 130.5, 127.9, 122.3, 117.9, 115.6, 55.0, 52.5, 51.2,
41.2, 37.8, 24.8, 22.6, 21.9. MS (ES) m/z 460.2 [M + Na]+.
Naphthalen-1-yl phosphorodichloridate (9)
In a flame dried flask, POCl3 (0.56 mL, 6.588 mmol) was
added to a solution of 1-naphthol (864 mg, 6 mmol) in dry
diethyl ether (6 mL). The colorless solution was stirred at
room temperature for 30 minutes under an atmosphere of ar-
gon. The solution was then cooled to −78 °C, where triethyl-
amine (0.84 mL, 6.588 mmol) was added dropwise and then
allowed to stir for 30 minutes, before being left to stir for one
hour at room temperature. The white precipitate was filtered
out of the solution and washed with diethyl ether. The yellow
filtrate was concentrated under reduced pressure to afford
phosphorochloridate 9 as a yellow oil (1.54 g, 98%), which
was used without purification.
Methyl ((S)-3-(4-((bisIJdimethylamino)phosphoryl)oxy)phenyl)-
2-(4-cyanobenzamido)propanoyl)-L-leucinate (6)
Methyl (chloroIJnaphthalen-1-yloxy)phosphoryl)-L-alaninate (10)
To a stirring solution of compound 5 (100 mg, 0.229 mmol),
under a nitrogen atmosphere in anhydrous THF (20 mL), was
added N,N,N′,N′-tetramethylphosphorodiamidic chloride (0.07
mL, 0.457 mmol), DMAP (56 mg, 0.457 mmol) and DBU (0.05
mL, 0.344 mmol). The orange solution was stirred at room
temperature overnight. Upon reaction completion the mixture
was quenched with methanol (10 mL) and stirred for 10 mi-
nutes. The solvent was then removed under reduced pressure
and the resultant orange oil was subjected to silica flash col-
umn chromatography (3 : 97 methanol : DCM) to isolate
diamidate 6 as a white solid (65 mg, 50%). 1H NMR (500 MHz,
CDCl3) δ = 7.79 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 7.6 Hz, 1H), 7.62
(d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 7.8 Hz,
2H), 7.02 (d, J = 7.9 Hz, 1H), 4.89 (q, J = 6.9 Hz, 1H), 4.55 (td, J
= 8.5, 5.3 Hz, 1H), 3.73 (s, 3H), 3.11 (ddd, J = 35.6, 14.1, 6.7 Hz,
To a stirring solution of compound 9 (513 mg, 1.974 mmol)
and dry DCM (5 mL), in a flame dried flask, was added
L-alanine methyl ester hydrochloride (250 mg, 1.792 mmol)
and stirred for 15 minutes at room temperature, under an ar-
gon atmosphere. Triethylamine (0.5 mL, 3.585 mmol) was
added dropwise to the yellow solution at −78 °C to form a
suspended white precipitate. The reaction mixture was stirred
for 30 minutes before being left to stir and warm to room
temperature for 2.5 hours. Upon reaction completion, the
grey/white solution was concentrated under reduced pressure
and filtered with ether. The yellow filtrate was concentrated
under reduced pressure to afford a yellow oil, which was puri-
fied with column chromatography (6:4 ethyl acetate: hexane)
to yield phosphoramidate 10 as a yellow oil (411 mg, 70%).
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1H NMR (300 MHz, CDCl3) δ = 8.10–8.02 (m, 1H), 7.88 (d, J =
7.5 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.64–7.50 (m, 3H), 7.44
(t, J = 7.9 Hz, 1H), 4.45–4.24 (m, 2H), 3.79 (d, J = 14.2 Hz,
3H), 1.55 (dd, J =9.2, 5.3 Hz, 3H). 31P NMR (121 MHz, CDCl3)
δ = 8.2, 7.9.
amine (0.07 mL, 0.504 mmol) at room temperature for 15 mi-
nutes under an argon atmosphere. Phosphorochloridate 10
(113 mg, 0.344 mmol) was added dropwise in DCM (2 mL).
The yellow solution was stirred at room temperature for 5
hours; where it was then concentrated under reduced pres-
sure to form a dense yellow oil. The oil was diluted in water
(20 mL) and the product was extracted with ethyl acetate (3 ×
20 mL), where the yellow combined organic layers were
washed with water (20 mL) and brine (20 mL), dried with
MgSO4, filtered and concentrated to form a white solid. Col-
umn chromatography (2 : 1 ethyl acetate : hexane) was used to
Isopropyl (chloroIJnaphthalen-1-yloxy)phosphoryl)-L-alaninate
(11)
To a stirring solution of compound 9 (513 mg, 1.97 mmol)
and dry DCM (5 mL), in a flame dried flask, was added
L-alanine isopropyl ester hydrochloride (300 mg, 1.794 mmol)
and stirred for 15 minutes at room temperature, under an ar-
gon atmosphere. Triethylamine (0.5 mL, 3.581 mmol) was
added dropwise to the yellow solution at −78 °C to form a
suspended white precipitate. The reaction mixture was stirred
for 30 minutes before being left to stir and warm to room
temperature for 2.5 hours. Upon reaction completion, the
grey/white solution was concentrated under reduced pressure
and filtered with ether. The yellow filtrate was concentrated
under reduced pressure to afford a yellow oil, which was puri-
fied with column chromatography (6:4 ethyl acetate: hexane)
1
isolate prodrug 13 as white crystals (120 mg, 72%). H NMR
(400 MHz, CDCl3) δ = 8.00 (dd, J = 12.5, 6.6 Hz, 1H), 7.83 (dd,
J = 10.6, 7.8 Hz, 1H), 7.72 (dd, J = 14.6, 7.8 Hz, 2H), 7.65 (dd,
J = 8.0, 4.7 Hz, 1H), 7.57–7.43 (m, 5H), 7.41–7.30 (m, 1H),
7.22–7.04 (m, 4H), 6.94 (d, J = 7.9 Hz, 1H), 4.97–4.82 (m, 2H),
4.55 (dd, J = 13.1, 8.1 Hz, 1H), 4.50–4.40 (m, 1H), 4.25–4.09
(m, 1H), 3.67 (d, J = 6.4 Hz, 3H), 3.57 (d, J = 16.3 Hz, 3H),
3.23–3.01 (m, 2H), 1.67–1.43 (m, 3H), 1.36 (dd, J = 22.2, 7.0
Hz, 3H), 0.82 (d, J = 5.8 Hz, 6H). 13C NMR (101 MHz, CDCl3)
δ = 173.6, 172.9, 170.9, 165.4, 149.7, 146.5, 137.4, 134.8,
133.6, 133.5, 132.2, 130.7, 127.9, 126.7, 126.5, 125.56, 125.4,
125.0, 121.4, 120.4, 118.0, 114.9, 54.8, 52.4, 51.0, 50.5, 41.2,
37.4, 37.0, 24.8, 22.6, 22.0, 20.8. 31P NMR (121 MHz, CDCl3) δ
1
to yield compound 11 as a yellow oil (504 mg, 72%). H NMR
(300 MHz, CDCl3) δ = 8.07 (dd, J = 6.9, 4.7 Hz, 1H), 7.87 (t, J =
3.0 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 7.62–7.51 (m, 3H), 7.44
(t, J = 8.0 Hz, 1H), 5.09 (tt, J = 17.1, 6.3 Hz, 1H), 4.47–4.16 (m,
2H), 1.54 (t, J = 6.5 Hz, 3H), 1.32–1.24 (m, 6H). 31P NMR (121
MHz, CDCl3) δ = 8.3, 7.9.
=
−2.5, −2.6. MS (ES) m/z 751.4 [M
+
Na]+. HRMS
C38H41N4O9NaP calcd. 751.2509 [M + Na]+, found 751.2513.
Methyl ((2S)-2-(4-cyanobenzamido)-3-(4-(((((S)-1-isopropoxy-1-
oxopropan-2-yl)amino)IJnaphthalen-1-
yloxy)phosphoryl)oxy)phenyl)propanoyl)-L-leucinate (14)
Benzyl (chloroIJnaphthalen-1-yloxy)phosphoryl)-L-
alaninatealaninate (12)
To a stirring solution of compound 9 (513 mg, 1.972 mmol)
and dry DCM (5 mL), in a flame dried flask, was added
L-alanine benzyl ester hydrochloride (386 mg, 1.794 mmol)
and stirred for 15 minutes at room temperature, under an ar-
gon atmosphere. Triethylamine (0.5 mL, 3.583 mmol) was
added dropwise to the yellow solution at −78 °C to form a
suspended white precipitate. The reaction mixture was stirred
for 30 minutes before being left to stir and warm to room
temperature for 2.5 hours. Upon reaction completion, the
grey/white solution was concentrated under reduced pressure
and filtered with ether. The yellow filtrate was concentrated
under reduced pressure to afford a yellow oil, which was puri-
fied with column chromatography (6:4 ethyl acetate: hexane)
In a flame dried flask, a solution of compound 5 (70 mg,
0.160 mmol) in DCM (5 mL) was stirred with triethylamine
(0.05 mL, 0.352 mmol) at room temperature for 15 minutes
under an argon atmosphere. Compound 11 (252 mg, 0.708
mmol) was added dropwise in DCM (3 mL). The yellow solu-
tion was stirred at room temperature for 5 hours; where it
was then concentrated under reduced pressure to form a
dense yellow oil. The oil was diluted in water (20 mL) and
the product was extracted with ethyl acetate (3 × 20 mL),
where the yellow oil was washed with water (20 mL) and
brine (20 mL), dried with MgSO4, filtered and concentrated
to form a white solid. Column chromatography (2 : 1 ethyl ac-
etate : hexane) was used to isolate prodrug 14 as white crys-
1
to yield compound 12 as a yellow oil (700 mg, 97%). H NMR
1
tals (110 mg, 91%). H NMR (400 MHz, CDCl3) δ = 8.01 (dd, J
(300 MHz, CDCl3) δ = 8.09–7.98 (m, 1H), 7.87 (dd, J = 7.8, 3.5
Hz, 1H), 7.73 (d, J = 8.3 Hz, 1H), 7.61–7.52 (m, 3H), 7.44 (d, J
= 7.9 Hz, 1H), 7.40–7.30 (m, 5H), 5.18 (dd, J = 16.4, 11.9 Hz,
2H), 4.44–4.23 (m, 2H), 1.56 (t, J = 6.6 Hz, 3H). 31P NMR (121
MHz, CDCl3) δ = 8.1, 7.8.
= 8.0, 5.3 Hz, 1H), 7.87–7.80 (m, 1H), 7.74 (dd, J = 9.8, 8.5 Hz,
2H), 7.65 (d, J = 8.3 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.53 (t, J
= 3.1 Hz, 1H), 7.52–7.44 (m, 3H), 7.41–7.35 (m, 2H), 7.23–7.12
(m, 4H), 6.64 (dd, J = 46.4, 8.1 Hz, 1H), 5.04–4.78 (m, 2H),
4.62–4.50 (m, 1H), 4.23 (dd, J = 12.3, 9.8 Hz, 1H), 4.17–4.07
(m, 1H), 3.68 (d, J = 9.2 Hz, 3H), 3.13 (t, J = 5.8 Hz, 2H), 1.65–
1.43 (m, 3H), 1.38–1.31 (m, 3H), 1.25 (dd, J = 17.7, 7.3 Hz,
1H), 1.20–1.06 (m, 6H), 0.83 (d, J = 2.8 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ = 172.8, 170.5, 165.3, 149.9, 146.6, 137.5,
134.8, 133.6, 133.2, 132.3, 130.8, 127.8, 126.6, 126.4, 125.5,
125.0, 121.4, 120.5, 118.0, 115.2, 115.0, 69.4, 54.8, 52.4, 51.0,
Methyl ((2S)-2-(4-cyanobenzamido)-3-(4-(((((S)-1-methoxy-1-
oxopropan-2-yl)amino)IJnaphthalen-1-
yloxy)phosphoryl)oxy)phenyl)propanoyl)-L-leucinate (13)
In a flame dried flask, a solution of compound 5 (100 mg,
0.229 mmol) in dry DCM (3 mL) was stirred with dry triethyl-
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50.6, 41.3, 37.5, 37.1, 24.8, 22.6, 21.8, 21.6, 21.0. 31P NMR
(121 MHz, CDCl3) δ = −2.5, −2.5. MS (ES) m/z 757.3 [M + H]+,
779.3 [M + Na]+. HRMS C40H45N4O9NaP calcd. 779.2822 [M +
Na]+, found 779.2825.
Cell assay and immunoblotting
MDA-MB-468 cells were cultured in RPMI 1640 media (Gibco)
supplemented with 10% fetal bovine serum (FBS) (Gibco)
and 1% of PenStrep (Gibco). The cell was maintained at 37
°C with 5% CO2 incubator to reach 70–80% confluency to be
used in the experiment. The cells were treated with ISS-610,
ISS-610-Met, or prodrugs 13–15 in various concentrations
(0.1, 1, 10, 30 and 100 μM) for 24 hours. Protein lysates were
prepared using 1% NP40 lysis buffer containing 50 mM Tris–
HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Nonidet-40, 0.27
M sucrose, 1 mM sodium orthovanadate, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate supplemented with 1
mM benzamidine and 0.1 mM PMSF. Protein concentrations
were measured using Bradford assay and SDS samples were
prepared by boiling the protein sample at 90 °C for 5 minutes
in SDS sample loading buffer. 20 μg of each sample was
loaded per lane and subjected to separation in polyacryl-
amide gels and transfer on to nitrocellulose membrane.
Membranes were blocked in 10% skim milk in TBST [50 mM
Tris/HCl (pH 7.5), 0.15 M NaCl containing 0.25% Tween-20]
for 30 minutes at room temperature. The membranes were
then incubated with the primary antibodies STAT3 (Cell Sig-
nalling Technology, #4904), Survivin (Cell Signalling Technol-
ogy, #2808), Bcl-xl (Cell Signalling Technology, #2764) and
β-actin (Cell Signalling Technology, #3700), overnight at 4 °C
and secondary antibodies: anti-rabbit (Cell Signalling Tech-
nology, #7074) or anti-mouse (Cell Signalling Technology,
#7076), for 1 h at room temperature. Finally, protein detec-
tion using horseradish peroxidase-conjugated secondary anti-
bodies and the ECL® reagent (Amersham Bioscience) was
performed.
Methyl ((2S)-3-(4-(((((S)-1-(benzyloxy)-1-oxopropan-2-
yl)amino)IJnaphthalen-1-yloxy)phosphoryl)oxy)phenyl)-2-(4-
cyanobenzamido)propanoyl)-L-leucinate (15)
In a flame dried flask, a solution of compound 5 (70 mg,
0.160 mmol) in DCM (5 mL) was stirred with triethylamine
(0.05 mL, 0.352 mmol) at room temperature for 15 minutes
under an argon atmosphere. Compound 12 (350 mg, 0.867
mmol) was added dropwise in DCM (3 mL). The yellow solu-
tion was stirred at room temperature for 5 hours; where it
was then concentrated under reduced pressure to form a
dense yellow oil. The oil was diluted in water (20 mL) and
the product was extracted with ethyl acetate (3 × 20 mL),
where the yellow oil was washed with water (20 mL) and
brine (20 mL), dried with MgSO4, filtered and concentrated
to form a white solid. Column chromatography (2 : 1 ethyl ac-
etate : hexane) was used to isolate prodrug 15 as white crys-
1
tals (120 mg, 93%). H NMR (400 MHz, CDCl3) δ = 7.99 (t, J =
7.8 Hz, 1H), 7.83 (t, J = 8.3 Hz, 1H), 7.72 (dd, J = 11.7, 8.5 Hz,
2H), 7.67–7.59 (m, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.50–7.39 (m,
3H), 7.38–7.31 (m, 1H), 7.30–7.19 (m, 5H), 7.18–7.04 (m, 4H),
6.81 (dd, J = 59.9, 8.1 Hz, 1H), 5.00 (q, J = 12.2 Hz, 2H), 4.92–
4.80 (m, 2H), 4.56 (dd, J = 8.5, 4.4 Hz, 1H), 4.39 (dd, J = 12.4,
10.0 Hz, 1H), 4.30–4.12 (m, 1H), 3.66 (d, J = 7.0 Hz, 3H),
3.19–3.02 (m, 2H), 1.65–1.45 (m, 3H), 1.37 (dd, J = 22.5, 7.1
Hz, 3H), 1.29–1.23 (m, 1H), 0.83 (d, J = 5.6 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ = 172.9, 170.7, 165.3, 149.8, 146.5, 137.5,
135.1, 134.7, 133.4, 132.3, 130.8, 128.6, 128.5, 128.1, 127.8,
126.7, 126.6, 126.4, 125.5, 125.4, 125.0, 121.4, 120.4, 118.0,
115.1, 67.2, 54.9, 52.4, 51.0, 50.5, 41.2, 37.4, 36.9, 24.8, 22.6,
21.8, 20.9. 31P NMR (121 MHz, CDCl3) δ = −2.5, −2.6. MS (ES)
m/z 827.3 [M + Na]+. HRMS C44H45N4O9NaP calcd. 827.2822
[M + Na]+, found 827.2826.
Author contributions
A. M. synthesized and characterized the compounds
presented in this work. B. A. D. executed the biological char-
acterization of the compounds. A. M., B. A. D. and Y. M.
designed all of the experiments. Y. M. wrote the manuscript
and all of the authors provided feedback and approved the fi-
nal version of the manuscript.
Serum stability of prodrug 14
Procedure was adapted from Slusarczyk et al.15 Compound Abbreviations
14 (5 mg) was dissolved in DMSO (0.15 mL) and D2O (0.15
POCl3 Phosphorous(V) oxychloride
SH2 Src homology 2
STAT3 Signal transducer and activator of transcription 3
mL) in an NMR tube. A 31P-NMR of the sample was initially
undertaken as a control. Defrosted human serum (0.3 mL)
was added to the NMR tube, which initially formed a cloudy
solution. Additional DMSO (0.15 mL) was treated to aid solu-
bility. The tube was then incubated at 37 °C and the spectra
were recorded at 30 min after the addition and then at even
time intervals over 12 h. At the end of the 31P-NMR experi-
ment, the organics were extracted from the serum with ethyl
acetate (3 × 1 mL), dried with MgSO4, filtered and concen-
trated under reduced pressure to yield a white solid. A 31P-
NMR of this in DMSO (0.15 mL) and D2O (0.15 mL) was
taken and this demonstrated the retention of the prodrug
singlet pair. The extract was additionally used for LCMS.
TEA
TFA
Triethylamine
Trifluoroacetic acid
Conflicts of interest
The authors declare no conflicts of interest.
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