Synthesis, in Vitro Evaluation, and Radiolabeling of Fluorinated Puromycin Analogues: Potential Candidates for PET Imaging of Protein Synthesis

Article abstract of DOI:10.1021/acs.jmedchem.6b00968

There is currently no ideal radiotracer for imaging of protein synthesis rate (PSR) by positron emission tomography (PET). Existing fluorine-18-labeled amino acid-based radiotracers predominantly visualize amino acid transporter processes, and in many cases they are not incorporated into nascent proteins at all. Others are radiolabeled with the short-half-life positron emitter carbon-11, which is rather impractical for many PET centers. Based on the puromycin (6) structural manifold, a series of 10 novel derivatives of 6 was prepared via Williamson ether synthesis from a common intermediate. A bioluminescence assay was employed to study their inhibitory action on protein synthesis, which identified the fluoroethyl analogue 7b as a lead compound. The fluorine-18 analogue was prepared via nucleophilic substitution of the corresponding tosylate precursor in a modest radiochemical yield of 2 ± 0.6% with excellent radiochemical purity (>99%) and showed complete stability over 3 h at ambient temperature.

Full text of DOI:10.1021/acs.jmedchem.6b00968

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Article
Synthesis, in vitro Evaluation and Radiolabelling of Fluorinated Puromycin
Analogues: Potential Candidates for PET Imaging of Protein Synthesis.
Helen M. Betts, Selena Milicevic Sephton, Carmen Tong, Ramla
O. Awais, Philip J. Hill, Alan C. Perkins, and Franklin I. Aigbirhio
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00968 • Publication Date (Web): 03 Oct 2016
Downloaded from http://pubs.acs.org on October 4, 2016
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Synthesis, in vitro Evaluation and Radiolabelling of
Fluorinated Puromycin Analogues: Potential
Candidates for PET Imaging of Protein Synthesis
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Helen M. Betts‡†*, Selena Milicevic Sephton‡§*, Carmen Tong¶, Ramla O. Awais#, Philip J.
Hill,ǁ Alan C. Perkins#, Franklin I. Aigbirhio§.
† Nottingham University Hospitals NHS Trust, PET/CT Center, Nottingham City Hospital,
Hucknall Road, Nottingham, NG5 1PB, UK.
§ Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Center, University of
Cambridge, Cambridge, CB2 0QQ, UK.
¶ School of Life Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD,
UK.
# Radiological Sciences, School of Medicine, University of Nottingham, Queen’s Medical
Center, Derby Road, Nottingham, NG7 2UH, UK.
ǁ School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton
Bonington, LE12 5RD, UK.
KEYWORDS Positron emission tomography; protein synthesis; puromycin; fluorine-18;
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ABSTRACT
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There is currently no ideal radiotracer for imaging protein synthesis rate (PSR) by positron
emission tomography (PET). Existing fluorine-18 labelled amino acid-based radiotracers
predominantly visualize amino acid transporter processes, and in many cases they are not
incorporated into nascent proteins at all. Others are radiolabelled with the short half-life positron
emitter carbon-11 which is rather impractical for many PET centers. Based on the puromycin (6)
structural manifold, a series of 10 novel derivatives of 6 was prepared via Williamson ether
synthesis from a common intermediate. A bioluminescence assay was employed to study their
inhibitory action on protein synthesis which identified fluoroethyl analogue (7b) as a lead
compound. The fluorine-18 analogue was prepared via nucleophilic substitution of the
corresponding tosylate precursor in modest radiochemical yield 2±0.6% and excellent
radiochemical purity (>99%) and showed complete stability over 3 h at ambient temperature.
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INTRODUCTION
Protein translation is a fundamental process for function and survival of all organisms.
Enzymes, membrane receptors, structural proteins, growth factors amongst many more are
constantly produced within the cell under tight control.1 Disruption of cellular protein synthesis
indicates disease, characterized for instance by an increase of the rate of protein synthesis in
malignant growth, or by a reduction in rate in certain neurodegenerative disorders.2,3 The ability
to visualize protein synthesis rate (PSR) is therefore an important goal in diagnosis, treatment
and monitoring of these conditions. Positron emission tomography (PET) is an ideal technique
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with which to do this. Since PET requires sub-nanomolar concentrations of radiolabelled tracer
molecules, cellular processes such as PSR can be probed without causing a pharmacological
effect.4 Several amino acids labelled with positron emitters have been investigated to this end,
although their application in PSR imaging has several critical limitations. Carbon-11 labelled
methionine and leucine ([11C]1, [11C]MET and [11C]2, [11C]LEU, Figure 1) are used for imaging
PSR, but the short physical half-life of carbon-11 (20 min) makes their use practically
impossible at PET scanning centers without a cyclotron, severely limiting their accessibility.5,6
Furthermore, [11C]1 produces brain-penetrating radiolabelled metabolites.6 Fluorine-18 (t1/2 =
110 min) labelled analogues such as S-(3-[18F]fluoropropyl)homocysteine ([18F]3,
[18F]FPHCYS) and O-(2-[18F]fluoroethyl)tyrosine ([18F]4, [18F]FET), are not recognized by the
cellular protein synthesis mechanism, and therefore report amino acid active uptake, rather than
PSR.7,8 The tyrosine analogue 2-[18F]fluorotyrosine ([18F]5, [18F]FTYR), is incorporated into
nascent proteins, but in addition to its challenging electrophilic radiosynthesis via [18F]F2 gas
rather than [18F]F—, the resulting PET images are also dominated by the amino acid transporter
processes.8 (Figure 1). A new fluorine-18 labelled radiotracer capable of imaging PSR is
therefore required.
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O
O
S
11C
OH
H311C
OH
NH2
NH2
[
11C]1, [11C]MET
[
11C]2, [11C]LEU
7
8
9
S
18F
[
18F]3, R =
18F]4, R =
18F]5, R =
[
[
[
18F]FPHCYS
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O
R
OH
[
[
18F]FET
18F
O
NH2
18F]FTYR
18F
OH
Figure 1. Selected carbon-11 and fluorine-18 amino acid radiotracers.
The aim of this project was to develop a novel radiotracer which: (1) enables measurement of
PSR in vivo; (2) is not an amino acid and thus does not participate in the amino acid active
transport mechanism; and (3) is radiolabelled with fluorine-18, via [18F]fluoride.
Puromycin (6, PURO, Figure 2) is an aminonucleoside antibiotic that inhibits protein synthesis
in both bacteria and eukaryotes by mimicking aminoacyl-tRNA (8) at the ribosome.9,10
Compound 6 and some structural analogues, enter the ribosomal site A and form an amide bond
with the C-terminus of a nascent protein, thus anchoring at sites of active protein growth.9
Studies have demonstrated that 6 can be used directly to assess ribosome mediated peptide bond
formation since it does not require soluble translation factors for function, it does not induce
EF-Tu-GTPase activity and it can enter the ribosome independently.11–14 Furthermore,
incorporation of 6 into newly synthesized protein has been established as a direct readout of
translation rate, by comparison with classical [35S]MET studies.15 Fluorescent analogues of 6
have been successfully used to monitor protein synthesis (PS) in tissue samples (ex vivo)16 and
scandium-44, gallium-68 and carbon-11 radiolabelled 6 derivatives have been described for
PET.11,17,18 However, the scandium-44 and gallium-68 complexes are structurally dominated by
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the metal chelate, and carbon-11 radiolabelled 6 is limited by the short physical half-life of the
radionuclide. We hypothesized that a fluorinated analogue of 6 would be a preferable
radiotracer for PET imaging, as well as incorporating a more widely available radioisotope. The
literature precedent demonstrates that derivatives of 6 modified at the phenyl ring retain the
inhibitory activity of the parent,13,16,19which prompted us to investigate the effect of introduction
of a fluorine atom into the scaffold of 6 by varying functional groups at the phenol as point of
modification (7b-k, Figure 2). We also considered potential routes for facile introduction of
fluorine-18 in this position using a nucleophilic approach. Herein, we report the synthesis of
several derivatives of 6, their ability to inhibit PS, as well as the radiolabelling of the most
prominent derivative as a potential PET radiotracer.
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N
NMe2
O
N
HO
O
N
O
P
NH2
N
N
t
O
RNA
N
NH OH
NH2
O
O
N
O
N
O
OH
NH2
O
R
6, R = CH3, PURO
OR 7b-k, R = various functional groups
(this work; OR for b-k shown below)
8, Aminoacyl-tRNA
F
O
b
c
d
O
O
O
e
f
i
O
O
O
N
N
N
N
N
F
F
F
O
CN
j
N
F
F
N
g
h
O
O
F
F
k
N
N
N
N
O
Figure 2. The structures of 6, envisioned derivatives (7) and aminoacyl-tRNA (8).
RESULTS AND DISCUSSION
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Syntheses of investigated derivatives 7 (Figure 2 and Table 2) were envisioned following the
method previously reported for phenol 12 (Scheme 1) by the Aigbirhio group.18 Starting with
the commercially available Boc-protected tyrosine 9, EDC/HOBt amide coupling with TBS-
protected puromycin aminonucleoside 11 afforded phenol 12 which served as a common
intermediate to access TBS and Boc-protected derivatives of 6 (13a-f). These derivatives were
prepared from 12 by employing the classical Williamson ether synthesis20 with several
bromides, or electrophiles containing tosylate, mesylate or iodide as leaving groups (direct
method). Alternatively, acid 9 was esterified to afford methyl ester 10 which was successfully
converted to ethers 15g-h under analogous Williamson conditions in excellent yields (>95%).
Removal of the methyl ester group was performed using trimethyltin hydroxide in order to
avoid possible racemization at the chiral center18 and acids 16g-h were coupled with 11 to
afford amides 13g-h (Scheme 1, indirect method). Using the indirect method ether 15g was
prepared in excellent yield of 95% but the additional reaction step in the synthesis of 13g
negligibly lowered overall yield to 57% from 62% for the indirect and direct methods
respectively.
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O
OR1
O
OMe
O
OH
4
5
NHBoc
NHBoc
NHBoc
6
R2Br
Me3SnOH
7
8
9
K2CO3, DMF
R1 = Me
OH
OR2
OR2
9, R1 = H
15g-h
16g-h
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MeI, DMF
10, R1 = Me
N
NMe2
O
N
TBSO
11, THF
N
N
H2N
OTBS
R1 = H
1118
TBSO
TBSO
O
N
N
N
NMe2
NMe2
O
O
N
N
N
NH OTBS
N
N
O
NH OTBS
NHBoc
R2X
NHBoc
K2CO3, DMF
12
13a-h
for a-f
X = Br, OTs, OMs, I
OH
OR2
O
13f, OR2
=
R3
N3
14i-k, OR2
=
O
N
R3
N
N
Scheme 1. Syntheses of TBS and Boc protected derivatives 7 and 14, using direct and indirect
Williamson ether methods (please refer to Supporting Information for respective yields).
Conversion to products with some electrophiles using the Williamson method was sluggish,
likely because of the structural complexity of phenol 12. In support of this theory, the reactivity
of 10 and 12 with fluoroethyl electrophiles was compared (Table 1). Using mesylate as a
leaving group in reaction with 12, conversion of 16% was observed by NMR after 25 h (entry
1) due to the significant amount of by-products formed. Reaction of 12 with 2-fluoroethyl
tosylate gave only trace amount of product (entry 2) whereas heating the reaction mixture to 70
°C increased the yield of 13b to a modest 11% after 2 h and further to 44% when mixture was
heated over 17 h (entries 3 and 4). On the other hand, reaction of 10 with 2-fluoroethyl tosylate
proceeded with 37% NMR conversion (entry 6) at ambient temperature. Further improvement
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to 42% of 15b (entry 7) was achieved by longer reaction time at ambient temperature as well as
the addition of 18-crown-6, thus avoiding the possibility of decomposition or racemization upon
extended heating. The yield could be improved further to 64% by heating to reflux in MeCN
(entry 8), however racemization was observed (by chiral HPLC) and therefore these reaction
conditions were not pursued. To our surprise, reaction of 10 with 2-fluoroethyl mesylate
showed only slightly improved conversion (vs reaction with 12) of 20% by NMR (entry 5), for
which reason we deterred from employing mesylate as the leaving group.
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Table 1. Williamson ether synthesis with 10 and 12 and fluoroethyl electrophiles.
NHBoc
NHBoc
LG
F
K2CO3, DMF
Additive
OH
10 or 12
Entry Phenol
O
F
Temp., Time
15b or 13b
LG
Temp. Time Yield (S):(R)
(°C)
23
(h)
25
19
2
(%)
16a
trace
11
1
2
3
4
5
6
7
8
12
12
12
12
10
10
10
10
OMs
OTs
OTs
OTs
OMs
OTs
OTs
OTs
—
—
23
70
—
70
17
25
16
48
21
44
—
23
20a
37a
42b
—
23
—
23
99:1
reflux
64b,c 89:11
a NMR conversion; b 18-c-6 used as additive; c MeCN was used as solvent
Alkyne 13f was further reacted with aryl azides under Cu(I)-mediated [2 + 3] cycloaddition
conditions to give “click” chemistry analogues 14a-c (Scheme 1).16,21
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Structural characterization of derivative 7f has been previously reported16 and we selected 7f
as a reference to establish conditions for deprotection of both the TBS and Boc groups without
epimerization (Scheme 2). Alkyne 13f was therefore treated with TBAF·THF resulting in
deprotection of both primary and secondary TBS groups in 17f. TFA was employed for the
removal of the Boc protecting group and 7f was afforded in 34% yield. NMR analysis of 7f was
in complete agreement with that previously reported (see Supporting Information).16 An
alternative two step deprotection was explored in which the Boc group was removed by TFA
quantitatively first, and 18f was subsequently treated with TBAF·THF to yield 7f in 57% yield.
Interestingly, the primary TBS group in 13f was cleaved during the Boc removal with TFA;
however it was the secondary TBS which required application of TBAF.
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TBSO
HO
N
N
NMe2
NMe2
O
O
N
N
N
N
N
N
O
NH OTBS
O
NH OH
NHBoc
TBAF, THF
79%
NHBoc
13f
17f
O
O
TFA, CH2Cl2
quant.
TFA, CH2Cl2
34%
TBSO
HO
N
N
NMe2
NMe2
O
O
N
N
N
N
N
N
O
O
NH OTBS
O
NH OH
NH2
TBAF, THF
57%
NH2
18f
7f
O
Scheme 2. Two step deprotection of 13f via two routes, yielding desired 7f in structural
agreement with the literature report.16
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With the deprotection route established, the syntheses of other derivatives of 6 were completed
in a one pot two step method. Intermediates 17 and 18 were not isolated; instead the second step
was performed with the crude material to afford 7b-k (Scheme 3a). Treatment of 13a and 13k
with TBAF in a single step yielded Boc-protected analogues 17a and 17k (Scheme 3b). Using
the synthetic method depicted in Schemes 1 and 2, derivative 7f was obtained in 20% yield over
4 steps from commercially available starting materials, which was an improvement to the
previously reported preparation of 7f in a total of six steps, with 9% overall yield.16 Yields
obtained for the syntheses of various derivatives of 6 are shown in Table 2.
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TBSO
O
HO
N
N
NMe2
NMe2
O
O
N
N
N
N
N
N
NH OTBS
O
NH OH
NH2
a) TBAF, THF
b) TFA
NHBoc
See Table 2
OR1 13b-h and 14i-k
a.
OR1 7b-k, R2 = H
b.
TBSO
HO
N
N
NMe2
NMe2
O
O
N
N
N
N
N
N
O
NH OTBS
O
NH OH
NHBoc
TBAF, THF
NHBoc
See Table 2
OR1 13a, 14k
OR1 17a, 17k
Scheme 3. (a) Two step, one pot deprotection of TBS and Boc groups in 13/14 to afford
derivatives 7. (b) TBAF deprotection of TBS groups to obtain derivatives 17.
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Table 2. Structures of derivatives 7 and 17, deprotection yields and inhibitory affinity data.
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Entry
1
Derivative
OR1
Yield GLuc
(%) (%)a
—
100
6
OCH3
34b
52
14
38
27
48
33
24
15
37
47
F
2
7b
7c
7d
7e
7f
O
195
184
22
3
O
F
F
O
O
4
N
N
NMe2
HO
O
O
O
O
O
O
O
5
O
N
N
O
NH OH
NH2
87
6
F
99
7
7g
7h
7i
OR1
F
136
165
187
150
8
N
N
9
N
N
N
N
F
CN
N
10
11
7j
N
F
F
N
7k
N
N
OR1 = OH, R2 = TBS
12
13
14
15
16
12
—
—
99
96
112
192
156
184
N
N
NMe2
R2O
OR1 = OCH3, R2 = TBS
OR1 = OCH3, R2 = H
13a
17a
17f
17k
O
N
N
O
NH OR2
NHBoc
OR1 = O
, R2 = H
OR1
78c 177
O
=
N
OR1
N
N
N
R2 = H
F
a
% GLuc production (vs. 6 inhibition) at 50 µM
b
c
Material was accessed via two different methods;
Material was prepared via [2 + 3] cycloaddition of azide directly with 17f;
Please refer to Supporting information for details
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With the target molecules (6, 7b-k, 12, 13a, 17a, 17f, 17k) in hand, their PS inhibitory
activity was assessed using a bioluminescent protein reporter system in bacteria. This technique
has been previously established for the assessment of compounds that inhibit PS.22 In this assay,
Gram positive bacteria Staphylococcus aureus 8325.4 were engineered to produce Gaussia
Luciferase (GLuc), a 19 kD photoprotein. On addition of the GLuc substrate coelenterazine, a
bioluminescent signal at 475 nm is produced proportional to the concentration of the GLuc
protein, thus providing a sensitive method with which to detect differences in PS in bacterial
culture.23 The test compound or 6 dihydrochloride was independently applied to S. aureus
culture, and the quantity of GLuc protein synthesized was measured by bioluminescence after
2.5 h. Cell number was estimated by optical density (OD) to normalize the response. The
efficiency of the novel derivatives for PS inhibition compared with 6·2HCl (set at 100%) is
displayed in Table 2.
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24
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Comparable PS inhibitory activity of 7f to that of 6 was observed, as expected based on the
published data (Table 2, entries 6 and 1).16 Only three other analogues showed comparable or
better PS inhibitory effects at 50 µM concentration: 7b, 7e and 7g (entries 2, 5 and 7). At this
concentration, non-fluorinated 7e proved the most potent PS inhibitor. Functionalization of 6
with either aromatic substituents (entries 3, 4 and 8) or [2 + 3] cycloaddition products, the
“click” analogues (entries 9-11), resulted in reduction in inhibitory potency compared with 6.
Similarly, masking of the hydroxyl functional groups (entries 12 and 13), in addition to amine
group (entries 14-16) confirmed importance of free hydroxyl and amine functionalities to
maintain inhibitory activity. Figure 3 shows the concentration dependence of PS inhibitory
activity of the novel derivatives. At all concentrations tested, 7b was the most potent of the
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fluorinated derivatives. At the lowest concentration tested (5 µM), 7b reduced protein
production by 36% compared with 6, and proved to be a superior PS inhibitor to 7e (25%
reduction compared with 6).
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40000
5 µM
50 µM
5 µΜ 50 µΜ
500 µM
500 µΜ
35000
30000
25000
20000
15000
10000
5000
0
6
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
12
13a 17a
17f
17k
Figure 3. Effect of derivatives of 6 on protein synthesis, as indicated by inhibition of luciferase
synthesis. RLU/OD = relative light units/optical density.
With evidence to support the use of 7b (FEPURO, Table 2) as the lead fluorinated compound in
the series, we next sought to develop the respective radiolabelling precursor (Scheme 4). An
obvious selection of phenol 12 (Scheme 1) as a precursor for radiosynthesis with 2-
[18F]fluoroethyl tosylate24 was quickly discarded for following reasons: the presence of TBS
protecting groups in 12 would facilitate side reactions due to the affinity of fluoride for silicon;
and reaction of 12 with 2-fluoroethyl tosylate (Scheme 1 and Supporting Information)
proceeded with modest yield therefore predicting similarly poor conversion with 2-
[18F]fluoroethyl tosylate. Silicon-free protecting groups on the hydroxyl groups were necessary
for successful radiolabelling, and we chose to investigate a direct fluorination approach. The
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radiolabelling precursor 24 (Scheme 4) was therefore envisioned as most appropriate.
Alongside synthesis of the radiolabelling precursor, we revisited the route to access 7b. Methyl
ester 10 was reacted with 2-fluoroethyl tosylate to afford 15b in 42% (Table 1), and in analogy,
reaction of 10 with 1,2-dibromoethane gave 19, in 89% yield, using crown ether 18-c-6 as
additive. Heating this reaction to 80 °C did not induce racemization of the bromo-analogue (see
the Supporting Information). Hydrolysis of the methyl esters was accomplished with LiOH, and
using DCC the N-succinimidyl ester was successfully installed to yield 20 and 21. Amide
coupling of activated esters 20 and 21 with commercially available puromycin aminonucleoside
22 furnished 77% of 17b and 96% of 23. For 17b, the Boc group was removed under acidic
conditions leading to 7b in total of five steps and 7% overall yield. The hydroxyl groups in
bromide 23 were acetylated catalysed by DMAP, and subsequently the bromide functionality
was converted to a tosylate leaving group using silver tosylate, providing 24 in 46% yield from
23 (Scheme 4).
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O
4
5
6
7
8
9
O
OMe
O
O
OMe
O
O
N
NHBoc
NHBoc
NHBoc
O
LG
a) LiOH, THF/H2O
X
18-c-6, K2CO3
DMF
LG = OTs or Br
b) DCC, NHS
EtOAc
X
OH
O
10
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12
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17
18
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X
10
15b, X = F, 42%
19, X = Br, 89%
20, X = F, 63%
21, X = Br, 34%
N
NMe2
O
N
HO
N
N
THF
H2N
OH
22
N
N
NMe2
HO
NMe2
O
O
N
N
AcO
N
N
N
N
O
NH OAc
O
NH OH
NHR
a) Ac2O, Pyr, DMAP
NHBoc
24
b) AgOTs, MeCN
46% with 23
17b, X = F, R = Boc
77%
TFA
34%
O
O
7b, X = F, R = H
OTs
X
23, X = Br, R = Boc, 97%
Scheme 4. Synthesis of radiolabelling precursor 24 and revisited synthesis of 7b.
Next, conditions for radiolabelling of 24 were explored (Scheme 5) with small (288-480
MBq) amounts of [18F]F— in manually conducted experiments. Aqueous [18F]F— was
azeotropically dried with acetonitrile in the presence of base. Variables investigated included
time (5-30 min), reaction temperature (80-130 °C), reaction solvent (DMSO, MeCN, DMF),
base (KHCO3, K2CO3), phase transfer agent (18-c-6, K222, TBAHCO3) and precursor quantity
(5-10 mg). Full details can be found in Table 3. The optimal conversion of 22% was achieved
using DMSO at 120 °C over 15 min as analysed by HPLC (entry 8). Heating the reaction to 130
°C resulted in only a slight increase in conversion to 24%, however five additional,
radiolabelled species were observed by HPLC that eluted closely with [18F]25 (entry 9). Use of
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the mild base KHCO3 with K222 also favoured [18F]25 formation. Reducing the amount of 24
5
6
used in the reaction resulted in a significant reduction in conversion to 12% (entry 11).
7
8
9
10
11
12
13
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17
18
19
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Table 3. Optimisation of radiolabelling for preparation of [18F]25 from 10 mg of 24 (0.3 mL
reaction volume).
18F–, Base, Additive
24
[
18F]25
Solvent, Temp., 15 min
Entry
Base
Additive Solvent Temp. HPLC conversion
(°C)
80
(%)
5
1
2
K2CO3
K2CO3
K2CO3
K222
K222
K222
—
DMF
DMSO
MeCN
DMSO
DMSO
DMF
80
13
3
80
4
4
TBAHCO3
KHCO3
KHCO3
KHCO3
KHCO3
KHCO3
KHCO3
KHCO3
80
12a
18
8
5
K222
K222
K222
K222
K222
18-c-6
K222
80
6
80
7
DMSO
DMSO
DMSO
DMSO
DMSO
120
120
130
120
120
20b
22
24a
0
8
9
10
11
12a,c
a Formation of by-products observed; b 10 min reaction time; c 7.5 mg of 24 used
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N
N
NMe2
NMe2
O
O
N
N
AcO
O
HO
O
N
N
N
N
NH OAc
NHBoc
NH OH
NHBoc
NaOH
9
[
18F]25
[
18F]17b
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O
[
18F]F–, K222, KHCO3
O
18F
18F
HCl
24
N
NMe2
O
N
HO
O
N
N
NH OH
NH2
[
18F]7b
18F]FEPURO
[
2%
O
18F
Scheme 5. Radiosynthesis of [18F]7b via nucleophilic substitution and deprotection. Overlaid
radio (red) and UV (blue) HPLC traces of formulated [18F]7b, and 7b. Axes have been slightly
offset for clarity.
After formation of [18F]25, the removal of protecting groups was investigated. Attempted total
deprotection to form [18F]7b directly from [18F]25 by acidification of the DMSO reaction
mixture with HCl was unsuccessful, resulting in no reaction at ambient temperature, or
decomposition upon heating. For this reason a two-step method was established. In the first
step, quantitative removal of both acetyl groups was accomplished in a short reaction time of 5
min using NaOH (aq.). An SPE purification was next performed to remove the DMSO from the
mixture. Application of the aqueous base in the first step facilitated transfer to the SPE cartridge
by solubilizing unreacted precursor 24. The Boc-group was then removed in the second step.
Successfully employed reaction conditions to form 7b from 17b with TFA (Scheme 4) were
investigated, and although [18F]17b was completely consumed, an unidentified by-product was
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additionally formed resulting in only 47% of [18F]7b by HPLC. Use of TFA was not pursued for
this reason, as well as its incompatibility with the polymer components of automated
radiochemistry modules. Instead, hydrochloric acid (3M) was successfully employed for Boc-
deprotection to form [18F]7b without by-products at ambient temperature over 5 min. [18F]7b
was purified by semi-preparative HPLC and formulated in phosphate buffered saline (PBS)
containing 10% ethanol. Structural identity of [18F]7b was confirmed by HPLC co-injection
with reference sample 7b (Scheme 5).
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Reactions performed using 0.9-1.8 GBq of aqueous [18F]fluoride yielded formulated [18F]7b in
non-decay corrected yield of 2±0.6% (n = 3) in 140 min, with specific activity of 5 GBq/µmol
and radiochemical purity of >99% after purification (Scheme 5). Conducting the radiosynthesis
manually limited any further increase in the amount of starting activity and therefore specific
activity.
Radiochemical stability of a formulated solution of [18F]7b was tested by analytical HPLC over
a three hour period, and showed no reduction in radiochemical purity.
CONCLUSIONS
We have developed a series of novel derivatives of 6, with potential for imaging PSR by PET.
The synthesis of a common intermediate was accomplished via EDC/HOBt mediated amide
coupling from commercially available materials, and derivatisation of the phenol functionality
was achieved employing the classical Williamson synthesis in good yields. Using a luciferase
PS assay 7b was identified as the lead compound having higher PSR inhibitory potency than
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reference 6. The radiolabelling precursor 24 was prepared in seven steps with 14% overall yield
and successfully employed in the radiosynthesis of [18F]7b via nucleophilic substitution.
Radiolabelled [18F]7b was prepared in excellent radiochemical purity. Implementation of a
modular radiosynthesis of [18F]7b as well as in vitro and in vivo evaluation of [18F]7b are
currently underway in our laboratories and will be reported in a due course.
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EXPERIMENTAL SECTION
General. All reactions requiring anhydrous conditions were conducted in oven-dried glass
apparatus under an atmosphere of inert gas. All reagents were purchased from Sigma-Aldrich or
Alfa Aesar and used without further purification, unless otherwise stated. Preparative
chromatographic separations were performed on Aldrich Science silica gel 60 (35-75 μm) and
reactions followed by TLC analysis using Sigma-Aldrich silica gel 60 plates (2-25 μm) with
fluorescent indicator (254 nm) and visualized with UV or potassium permanganate. 1H and 13C
NMR spectra were recorded in Fourier transform mode at the field strength specified on a
Bruker Avance 400 MHz or a Bruker Avance 300 MHz spectrometer. Spectra were obtained in
the specified deuterated solvents in 5 mm diameter tubes. Chemical shift in ppm is quoted
relative to residual solvent signals calibrated as follows: CDCl3 δH (CHCl3) = 7.26 ppm, δC =
77.2 ppm; (CD3)2SO δH (CD3SOCHD2) = 2.50 ppm, δC = 39.5 ppm; MeOD-d4 δH (CD2HOD) =
3.31 ppm, δC = 49.0 ppm; (CD3)2NC(O)D δH ((CD3)2NC(O)H) = 7.92 ppm, δC = 165.5 ppm.
Multiplicities in the 1H NMR spectra are described as: s = singlet, d = doublet, t = triplet, q =
quartet, quint. = quintet, m = multiplet, b = broad; coupling constants are reported in Hz.
Numbers in parentheses following carbon atom chemical shifts refer to the number of attached
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hydrogen atoms as revealed by the DEPT/HSQC spectral editing technique. Mass spectra were
recorded on a Waters Micromass LCT instrument, a Bruker MicroTOF mass spectrometer using
electrospray Ionisation (ESI), or by the EPSRC Mass Spectrometry Service at the University of
Swansea. Purity of compounds was ≥95% as determined by analytical LC on a Waters Acquity-
H UPLC or Agilent Series 1200 system with UV detection (λ = 254 nm). The general
UPLC/HPLC methods were as follows. Waters general method: 0-4 min, 5-95% aq. MeCN at
0.6 mL/min (column: BEH C18, 1.7 µm, 2.1x50 mm. Agilent general method: 0- 1 min 5% B;
1-15 min 5-95% B; 15-18 min 95% B; 18-20 min 95-5% B; 20-25 min 5% B. A flow rate of 1
mL/min was used. Unless otherwise stated, solvent A = H2O and B = MeCN. A Phenomenex
Luna C18(2) column (150 mm x 4.6 mm) was used. All radioactive manipulations were
performed in designated lead shielded hot cells to reduce operator dose. [18F]Fluoride was
purchased from PETNET Nottingham, and was supplied as [18F]fluoride in H2[18O]O. Seppak
tC18 light cartridges were purchased from Waters. Radiochemical incorporation was
determined by the percentage of the desired product from integration of the analytical HPLC
radiotrace. Where radiochemical yields are given, they are calculated from HPLC purified
material as a percentage of starting aqueous [18F]fluoride, and are not corrected for decay. Semi-
preparative HPLC was performed using a Knauer Smartline pump, equipped with Knauer Azura
UV detector (254 nm) and diode radiodetector (Carroll Ramsey, USA), and a custom built
apparatus for HPLC loop load and product collection. Data were collected using SingleStream
software (Dr. R. Fortt). A Phenomenex Synergi Hydro-RP column (250 x 10 mm) 4µ 80A with
guard was used, with an isocratic method. The eluant was composed of 30% MeCN in 70%
ammonium acetate (aq. 50 mM), with a flow rate of 2 mL/min.
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(S)-2-Amino-N-{(2S,3S,4R,5R)-5-[6-(dimethylamino)-9H-purin-9-yl]-4-hydroxy-2-
5
6
(hydroxymethyl)tetrahydrofuran-3-yl}-3-[4-(2-fluoroethoxy)phenyl]propanamide
(7b).
7
8
9
17b (40.1 mg, 0.07 mmol) was dissolved in TFA (0.5 mL). The reaction stirred at room
temperature for 10 min, after which the volatiles were removed under a stream of N2. The
resulting solid was rinsed with diethyl ether (2x1 mL) and dried under a stream of N2. The solid
was redissolved in EtOAc (15 mL), and extracted into H2O (15 mL). The aqueous layer was
retained, and saturated aq. NaHCO3 (10 mL) was added. The product was extracted into EtOAc
(2x10 mL), and washed with H2O (10 mL). The organic layer was dried (MgSO4), and
evaporated under reduced pressure. The product was purified by column chromatography on
silica (eluting with 5% MeOH/CH2Cl2 containing 1% NEt3) yielding a white solid (11.4 mg,
0.02 mmol, 34%): 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.23 (s, 1H), 8.08 (bs, 1H),
7.16 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H,) 6.13 (bd, J = 2.9 Hz, 1H), 5.98 (d, J = 2.7
Hz, 1H), 5.15 (bt, J = 5.2 Hz, 1H), 4.80-4.64 (dm, JF-C-H = 47.8 Hz, 2H), 4.51-4.42 (m, 2H),
4.25-4.13 (dm, JF-C-C-H = 30.2 Hz, 2H), 3.96-3.90 (bm, 1H), 3.72-3.65 (b m, 1H) 3.54- 3.26 (b
m, 8H), 2.92 (dd, J = 13.5, 5.0 Hz, 1H), 2.57 (dd, J = 13.5, 8.5 Hz, 1H) ppm; 13C NMR (100
MHz, DMSO-d6) δ 174.3 (0), 156.6 (0), 154.2 (0), 151.7 (1), 149.5 (0), 137.8 (1), 130.7 (0),
130.2 (1, 2C), 119.5 (0), 114.1 (1, 2C), 89.3 (1), 83.5 (1), 82.1 (2, d, J = 166.6 Hz), 73.1 (1),
66.9 (2, d, J = 19.0 Hz), 60.9 (2), 56.0 (1), 50.0 (1), 39.7 (2, obscured by solvent, visible by
DEPT-135), 37.7 (b, 3, 2C) ppm; 19F NMR (376 MHz, DMSO-d6) δ -224.7 (tt, JH-C-F = 47.8, JH-
C-C-F = 30.2 Hz) ppm; HRMS m/z [M + H]+ 504.2372 (calcd. 504.2365 for C23H31FN7O5+); HPLC
(Solvent A = ammonium acetate, 50 mM, solvent B = MeOH) Rt = 12.2 min.
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(S)-2-amino-N-{(2S,3S,4R,5R)-5-[6-(dimethylamino)-9H-purin-9-yl]-4-hydroxy-2-
(hydroxymethyl)tetrahydrofuran-3-yl}-3-{4-[2-([18F]fluoro)ethoxy]phenyl}propanamide
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([18F]7b). The optimized procedure was as follows: aqueous [18F]fluoride (0.9-1.8 GBq) was
added to a mixture of K222 (5.0 mg) and KHCO3 (50 µL, 0.1 M) in MeCN (0.5 mL) in a v-vial
equipped with stirring bar. The mixture was dried azeotropically at 110 °C under a stream of N2
(without stirring). Two further portions of MeCN (2x0.5 mL) were added to ensure complete
drying (total drying time ca. 30 min). Precursor 24 (10.0 mg), was dissolved in DMSO (0.3 mL,
anhydrous) and added to the dried [18F]fluoride. The reaction was heated to 120 °C for 15 min
with stirring. After cooling to room temperature, NaOH (1 M, 3 mL) was added and stirred for
5 min, followed by DMSO (0.7 mL). The reaction mixture was loaded to a pre-conditioned
(MeCN 2 mL; H2O 5 mL) tC18 Seppak light cartridge, and the cartridge was washed with H2O
(4 mL). The cartridge was briefly dried for 2 min after washing. The cartridge was eluted with
MeCN (0.5 mL), concentrated under a stream of N2 at 80 °C for 5 min, cooled, and HCl (3 M,
300 µL) added. The reaction stirred for 5 min at room temperature. After neutralization with
NaOH (1M) and dilution with mobile phase (1 mL), the mixture was loaded to a semi-
preparative HPLC system for purification. The radioactive peak corresponding to the product
(Rt = 12 min) was collected. The HPLC cut was diluted with H2O (15 mL), loaded to a primed
tC18 light cartridge (EtOH 2mL; H2O 5 mL), and washed with H2O (2 mL). The desired product
[18F]7b was eluted with EtOH (300 µL) and diluted in phosphate buffered saline (2.7 mL).
Product [18F]7b was isolated in non-decay corrected yield of 2±0.6% (n = 3), with specific
activity of 5 GBq/µmol and radiochemical purity of >99%.
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Methyl-(S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(2-fluoroethoxy)phenyl]propanoate
(BocTyr(EtF)OMe, 15b). Boc(L)TyrOMe (1.01 g, 3.4 mmol, 1eq) was dissolved in DMF (6
mL) and K2CO3 (3.23 g, 23.4 mmol, 6.9 eq) was added. 18-Crown-6 (0.10 g, 0.4 mmol, 0.1 eq)
and 2-fluoroethyl tosylate (0.82 g, 3.8 mmol, 1.1 eq) were added and the reaction stirred at
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room temperature for 48 h. H2O (25 mL) was added to the reaction, and the product extracted
into EtOAc (2x30 mL). The combined organics were washed with brine (2x30 mL), dried
(MgSO4), and evaporated under reduced pressure. The product was purified by silica
chromatography (eluting with 20% EtOAc/hexane) yielding a colorless oil, which solidified on
standing (0.49 g, 1.4 mmol, 42%): 1H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 8.6 Hz, 2H), 6.88
(d, J = 8.6 Hz, 2H), 4.97 (d, J = 7.9 Hz, 1H), 4.77 (dt, J = 47.4, 4.2 Hz, 2H), 4.57 (m, 1H), 4.22
(dt, J = 27.7, 4.2 Hz, 2H), 3.73 (s, 3H), 3.09-2.96 (m, 2H), 1.44 (s, 9H) ppm; HRMS m/z [M +
Na]+ 364.1542 (calcd. 364.1531 for C17H24FNNaO5+); HPLC Rt = 13.3 min. The spectral data
were in complete agreement with the literature reported values.25
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Tert-butyl [(S)-1-({(2S,3S,4R,5R)-5-[6-(dimethylamino)-9H-purin-9-yl]-4-hydroxy-2-
(hydroxymethyl)tetrahydrofuran-3-yl}amino)-3-[4-(2-fluoroethoxy)phenyl]-1-oxopropan-
2-yl]carbamate (17b). Puromycin aminonucleoside (0.14 g, 0.5 mmol, 1 eq) was dissolved in
THF (8 mL) and BocTyr(EtF)OSu 20 (0.20 g, 0.5 mmol, 1 eq) was added. The reaction stirred
at room temperature for 20 h. The reaction was diluted with H2O and product extracted with
EtOAc (3x20 mL). The organic extracts were washed with H2O (20 mL) and brine (20 mL),
dried (MgSO4), and evaporated to dryness. The product was purified by column
chromatography on silica (eluting with 5% MeOH/CH2Cl2) yielding a white solid (0.22 g, 3.6
mmol, 77%): 1H NMR (400 MHz, DMSO-d6) δ 8.44 (s, 1H), 8.24 (s, 1H), 8.00 (d, J = 7.6 Hz,
1H), 7.20 (d, J = 8.5 Hz, 2H), 6.90-6.84 (m, 3H), 6.06 (d, J = 4.7 Hz, 1H), 5.99 (d, J = 2.8 Hz,
1H), 5.16 (bm, 1H), 4.72 (m, JH-C-F = 47.8 Hz, 2H), 4.53-4.43 (m, 2H), 4.24-4.13 (m, JH-C-C-F
=
30.2 Hz, 3H), 3.96-3.91 (bm, 1H), 3.68 (bd, J = 11.6 Hz, 1H), 3.61-3.34 (bm, 7H), 2.91 (dd, J
= 13.6, 4.6 Hz, 1H), 2.70 (dd, J = 13.3, 9.9 Hz, 1H), 1.30 (s, 9H) ppm;13C NMR (100 MHz,
DMSO-d6) δ 172.0 (0), 156.6 (0), 155.1 (0), 154.2 (0), 151.8 (1), 149.6 (0), 137.8 (1), 130.2 (1,
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2C), 119.5 (0), 114.0 (1, 2C), 89.2 (1), 83.3 (1), 82.1 (2) (d, J = 166.5 Hz), 78.0 (0), 73.0 (1),
66.9 (2, d, J = 19.0 Hz), 60.8 (2), 55.8 (1), 50.2 (1), 37.8 (3, 2C), 36.8 (2), 28.0 (3, 3C) ppm.
One carbon resonance was missing (quaternary aromatic carbon); 19F NMR (376 MHz, DMSO-
d6) δ -224.7 (tt, JH-C-F = 47.8, JH-C-C-F = 30.2 Hz) ppm; HRMS [M + H]+ m/z 604.2887 (calcd.
604.2890 for C28H39FN7O7+); HPLC Rt = 10.9 min.
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(S)-2-[(Tert-butoxycarbonyl)amino]-3-[4-(2-fluoroethoxy)phenyl]propanoic acid
(BocTyr(EtF)OH, en route to 20). BocTyr(EtF)OMe 15b (0.46 g, 1.4 mmol) was dissolved in
THF (8 mL) and cooled to 0 °C in an ice bath. LiOH (aq. 2M, 2 mL) was added and the reaction
stirred for 2 h. The THF was evaporated and the mixture acidified with KHSO4 (5%, 30 mL).
The product was extracted into EtOAc (2x30 mL), and the combined organic extracts were
dried (MgSO4), filtered and evaporated to dryness. The product was isolated as a white solid
(0.42 g, 1.3 mmol, 93%). No further purification was necessary. 1H NMR (400 MHz, CDCl3) δ
7.11 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 4.92 (d, J = 7.3 Hz, 1H), 4.74 (dt, J = 47.4,
4.2 Hz, 2H), 4.57 (m, 1H), 4.19 (dt, J = 27.8, 4.2 Hz, 1H), 3.17-3.00 (m, 2H), 1.42 (s, 9H) ppm;
13C NMR (100 MHz, CDCl3) δ 176.5 (0), 157.8 (0), 155.6 (0), 130.7 (1, 2C), 128.6 (0), 115.0
(1, 2C), 82.1 (2, d, J = 170.6 Hz), 80.5 (0), 67.3 (2, d, J = 20.5 Hz), 54.6 (1), 37.1 (2), 28.5 (3,
3C) ppm; 19F NMR (376 MHz, CDCl3) δ -224.9 (tt, JH-C-F = 47.4, JH-C-C-F = 27.8 Hz) ppm;
HRMS m/z [M - H]⁻ 326.1408 (calcd. 326.1409 for C16H21FNO5⁻); HPLC (Solvent A = H2O +
0.1% TFA; B = MeCN + 0.1% TFA) Rt = 11.7 min.
2,5-Dioxopyrrolidin-1-yl (S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(2-
fluoroethoxy)phenyl]propanoate (BocTyr(EtF)OSu, 20). BocTyr(EtF)OH (0.41 g, 1.3 mmol,
1 eq), was dissolved in EtOAc (10 mL) and N-hydroxysuccinimide (0.15 g, 1.3 mmol, 1 eq) was
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added. The mixture was cooled to 0 °C in an ice bath. Dicyclohexylcarbodiimide (0.26 g, 1.3
mmol, 1 eq) was added dropwise as a solution in EtOAc (10 mL). The reaction stirred at room
temperature for 16 h, after which the white precipitate was filtered off. The filtrate was
evaporated to dryness, then recrystallized from hot 2-propanol. The resulting solid was filtered
and dried in vacuo (0.36 g, 0.8 mmol, 68%): 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 8.6 Hz,
2H), 6.88 (d, J = 8.6 Hz, 2H), 4.95-4.83 (m, 1H), 4.74 (dt, J = 47.4, 4.2 Hz, 2H), 4.20 (dt, J =
27.7, 4.2 Hz, 2H), 3.28-3.07 (m, 2H), 2.86 (s, 4H), 1.42 (s, 9H) ppm; 13C NMR (100 MHz,
CDCl3) δ 168.8 (0), 167.9 (0), 157.9 (0), 154.8 (0), 131.0 (1, 2C), 127.4 (0), 115.0 (1, 2C), 82.1
(2, d, J = 170.6 Hz), 80.7 (0), 67.3 (2, d, J = 20.6 Hz) 52.9 (1), 37.5 (2), 28.4 (3, 3C), 25.8 (2,
2C) ppm; 19F NMR (376 MHz, CDCl3) δ -224.9 (tt, JH-C-F = 47.4, JH-C-C-F = 27.7 Hz) ppm;
HRMS [M + Na]+ m/z 447.1530 (calcd. 447.1538 for C20H25FN2NaO7+); HPLC:
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BocTyr(EtF)OSu appeared to decompose on HPLC analysis under all conditions tested, using
both normal and reverse phase columns.
In vitro assays. S. aureus 8325.4 (pUNKPxyl/tet::GLuc) was grown overnight in 5 mL tryptone
soya broth (TSB), with erythromycin (5 µg/mL) and lincomycin (25 µg/mL) at 37 °C with 250
rpm shaking. The overnight culture was diluted to an optical density (OD600) of 0.05 with fresh
TSB, and anhydrotetracycline (ATc, 40 ng/mL) was added to induce GLuc expression.
Compounds for assessment were prepared as stock solutions of 50 mM in DMSO, and diluted
to final concentrations of 5 µM, 50 µM and 500 µM, containing 1% DMSO. To a 96-well plate
(Corning, black, clear bottom) was added bacterial culture (180 µL) and test compound (20 µL),
control wells contained 1% DMSO; all experiments were performed in triplicate. Bacterial cell
number: Plates were incubated at 37 °C in a 96 well plate reader (Tecan), and optical density
(OD600) was recorded every 15 min for 2.5 h. Bioluminescence: After 2.5 h, and whilst in the
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Tecan plate reader, coelenterazine (20 µM, 50 µL) was added to each well and bioluminescence
immediately recorded. The bioluminescent signal was normalized with optical density to correct
for cell number and reported in relative light units per OD (RLU/OD). Error bars indicate
standard deviation from the mean.
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ASSOCIATED CONTENT
Supporting Information.
Full experimental details for the synthesis of analogues of 6, NMR spectral data, radio-HPLC
traces and stability study data are available in the Supporting Information. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* Email: helen.betts@nottingham.ac.uk. Tel: 0044 (0)115 9709172; sms96@cam.ac.uk Tel:
0044 (0)1223 748188.
Author Contributions
‡These authors contributed equally. All authors have given approval to the final version of the
manuscript.
Funding Sources
H.M.B. acknowledges the Royal Society of Chemistry Research Fund for partial funding of this
project, and the NIHR Clinical Research Network (East Midlands) for funding her post. We are
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grateful to the UK Medical Research Council (MRC) for funding (Grant number G9219778).
Carmen Tong was supported by the MRC/University of Nottingham Doctoral Training
Program. SMS acknowledges EPSRC Mass Spectrometry Facility for funding her attendance at
the Mass Spectrometry Summer School 2016.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Dr. W. Chan (University of Nottingham) is acknowledged for access to synthetic chemistry
facilities. The EPSRC Mass Spectrometry Facility at the University of Swansea is
acknowledged for performing HRMS analyses.
ABBREVIATIONS
DSC disuccinimidyl; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; [18F]FET, O-(2-
[18F]fluoroethyl) tyrosine; [18F]FPHCYS, S-(3-[18F]fluoropropyl)homocysteine; [18F]FTYR, 2-
[18F]fluorotyrosine; HOBt, hydroxybenzotriazole; [11C]LEU, carbon-11 leucine; [11C]MET,
carbon-11 methionine; PET, positron emission tomography; PURO, puromycin; SPE, solid
phase extraction; TBS, tert-butyldimethylsilyl.
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