Studies toward the development of new silicon-containing building blocks for the direct 18F-labeling of peptides

Article abstract of DOI:10.1021/jm400857f

Silicon-containing prosthetic groups have been conjugated to peptides to allow for a single-step labeling with ¹⁸F radioisotope. The fairly lipophilic di-tert-butylphenylsilane building block contributes unfavorably to the pharmacokinetic profile of bombesin conjugates. In this article, theoretical and experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional theory calculations were used to predict the hydrolytic stability of di-tert-butylfluorosilanes 2-23 with the aim to improve the in vivo properties of ¹⁸F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile, hydrophilic linkers were introduced between the lipophilic di-tert-butylphenylsilane building block and the bombesin congeners. Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of ¹⁸F-labeled silicon-bombesin conjugates.

Full text of DOI:10.1021/jm400857f

Article
pubs.acs.org/jmc
Studies toward the Development of New Silicon-Containing Building
18
Blocks for the Direct F‑Labeling of Peptides
Lukas O. Dialer,† Svetlana V. Selivanova,† Carmen J. Muller,† Adrienne Muller,† Timo Stellfeld,‡
̈
̈
Keith Graham,‡ Ludger M. Dinkelborg,‡,∥ Stefanie D. Kramer,† Roger Schibli,† Markus Reiher,§
̈
and Simon M. Ametamey*,†
†Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of Chemistry and Applied Biosciences,
Swiss Federal Institute of Technology (ETH) Zurich, Wolfgang-Pauli Strasse 10, CH-8093, Zurich, Switzerland
‡Global Drug Discovery, Bayer Healthcare, Muellerstrasse 178, 13353 Berlin, Germany
§Laboratory of Physical Chemistry, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH)
Zurich, Wolfgang-Pauli Strasse 10, CH-8093, Zurich, Switzerland
S
* Supporting Information
ABSTRACT: Silicon-containing prosthetic groups have been
conjugated to peptides to allow for a single-step labeling with
18F radioisotope. The fairly lipophilic di-tert-butylphenylsilane
building block contributes unfavorably to the pharmacokinetic
profile of bombesin conjugates. In this article, theoretical and
experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional
theory calculations were used to predict the hydrolytic stability of di-tert-butylfluorosilanes 2−23 with the aim to improve the in
vivo properties of 18F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile,
hydrophilic linkers were introduced between the lipophilic di-tert-butylphenylsilane building block and the bombesin congeners.
Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography
and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of
18F-labeled silicon−bombesin conjugates.
high silicon−fluorine (Si−F) bond energy (565 vs 485 kJ mol−1
INTRODUCTION
■
for carbon−fluorine (C−F) bond), silicon has a high affinity for
Radiolabeled biomolecules such as proteins and peptides have
fluorine and is easily fluorinated, allowing for a direct one-step
been applied for positron emission tomography (PET) imaging
labeling under mild conditions.16,19 However, the Si−F bond is
due to their fast and specific targeting properties.1−3 Often, metal
prone to dissociation in the presence of water. To shield the Si−F
radioisotopes are used to radiolabel biomolecules using chelators
which allow for highly efficient labeling under mild conditions.4
bond from hydrolysis, bulky lipophilic di-tert-butyl groups were
employed for the design of the currently used di-tert-
However, fluorine-18 (18F) generally possesses better nuclear
butylphenylsilyl building block. Di-tert-butylphenylsilyl, in turn,
characteristics for PET due to the low positron (β+) energy
confers its high lipophilicity to the final peptide conjugate. In vivo
(0.64 MeV), including an appropriate physical half-life
studies in mice using bombesin derivatives labeled with 18F via di-
tert-butyphenylsilyl building block confirmed that high lipo-
philicity of the final conjugate negatively affected its systemic
distribution and revealed only low and unspecific uptake in
gastrin-releasing peptide receptor (GRPr) positive xeno-
grafts.20 Bombesin and its derivatives exhibit high affinity and
selectivity for the GRPr, which is overexpressed in various
human tumors including prostate, breast, pancreatic, and
small cell lung cancers.21−23 A reduction of the overall
lipophilicity may lead to an improved pharmacokinetic profile
of the radiolabeled peptide by shifting hepatobiliary to renal
clearance.
(109.7 min).5,6 Procedures for the direct labeling with 18F
normally require harsh (strong bases, high temperatures)
reaction conditions which are not compatible with sensitive
biomolecules.5 Usually, site-specific labeling of peptides or
proteins with 18F is achieved using suitable 18F-labeled
intermediates or prosthetic groups.7−10 This approach includes
multistep reaction procedures and is, therefore, time-
consuming. A more efficient, one-step procedure for radio-
fluorination under mild conditions would be beneficial to
accommodate for the short half-life of 18F and the lability of
peptides. Great advancements have been made in recent years in
18F labeling using organoboron, aluminum chelate, and
4-trimethylammonium-3-cyano-benzoyl moiety bearing biocon-
jugates.11−13
In the present study, we report the synthesis and evaluation of
silicon containing compounds with enhanced hydrophilic
Application of prosthetic groups containing silicon for site-
specific 18F labeling of peptides and other biomolecules was
investigated in our laboratory and by others.14−18 Because of a
Received: April 5, 2013
© XXXX American Chemical Society
A
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properties. Stability of the Si−F bond toward hydrolysis was
predicted using density functional theory (DFT) calculations
and tested experimentally. Modification of the linker between the
di-tert-butylphenylsilyl building block and bombesin analogues
was also investigated. Two bombesin analogues were radio-
fluorinated using a one-step labeling protocol and tested for
their binding affinity in vitro and their performance in vivo in
mice.
Table 1. Silicon-Based Building Blocks 1−23 with Their cLogP Values, Experimentally Evaluated Hydrolytic t1/2, and Calculated
(Si−F) Values
Δ
B
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Table 1. continued
a
Δ(Si−F) values of DFT structure optimizations of isomers, for which the starting structure guaranteed convergence to a stable conformer free of
intramolecular hydrogen bonds.
a
Scheme 1. Synthesis of Silylethanol 25, Triazole Amino Acid 27, Silylacetamide 29, and Fluorosilylacetamide 8
a
Reagents and conditions: (a) N2CHCO2Et, Rh2(OAc)4, DCM, 35 °C; (b) LAH, THF, 0 °C to reflux, 64% (two steps); (c) PPh3, CBr4, DCM, 0 °C
t
to rt; (d) NaN3, DMF, 57% (two steps); (e) propargyl glycine, Cu(II) acetate, (+)-sodium L-ascorbate, BuOH/water, 22%; (f) PCC, DCM; (g)
NaO2Cl, sulfamic acid, acetone/water, 0 °C, 26% (two steps); (h) BnNH2, DIPEA, T3P, THF, 0 °C to rt, 35%; (i-1) KF, K222, THF or toluene, rt,
(i-2) KF, K222, AcOH, THF, rt or reflux, (i-3) KF, K222, K2CO3, (i-4) TBAF, THF, with or without AcOH, (i-5) TBABF, KHF2, THF, (i-6) CuF2,
CCl4, (i-7) CuCl2, CuI, KF, Et2O, (j) Rh2(OAc)4, DCM, 5%.
intramolecular hydrogen bonds are, we performed additional
calculations using the shared-electron-numbers (SENs) method24
and found hydrogen bond energies of 21.4, 16.0, and 13.3 kJ/mol
for silanes 6, 7, and 8, respectively. DFT calculations for com-
pounds 6, 7, and 8 were newly performed in such a way that these
intramolecular hydrogen bonds were broken to have a different
stable conformer. The new Δ(Si−F) values were 0.27, 0.19, and
0.23 Å for silanes 6, 7 and 8, respectively, and are assigned with
the footnote (a) in Table 1.
Chemistry. The syntheses of silanes 8, 25, 27, and 29 were
accomplished as shown in Scheme 1. Di-tert-butylchlorosilane
(24) was reacted with ethyl diazoacetate in the presence of a
rhodium(II) catalyst via a rhodium carbene complex. The
obtained silylethylester was further reduced with LAH to yield
silylethanol 25 in 64% yield over two steps. Primary alcohol 25
was brominated using triphenylphosphine and carbon tetra-
bromide. The obtained bromide was converted to azide 26 in
57% yield. Coupling with L-propargylic glycine via copper-catalyzed
RESULTS
■
DFT Calculations. The difference in Si−F bond lengths
(Δ(Si−F)) of the silane and its solvated hydrolysis intermediate
was obtained using DFT calculations (see the Experimental
Section for the computational methodology). The Δ(Si−F) values
for model silane compounds 2−23 are depicted in Table 1.
Fluorosilanes 6, 14, and 20 show Δ(Si−F) values below 0.19 Å and
are predicted to be hydrolytically stable according to our previous
discussion in Hohne et al.19 Fluorosilanes 2−5, 7−13, 15−19,
̈
and 21−23 all exhibit a Δ(Si−F) value ≥0.19 Å and were therefore
considered to be hydrolytically unstable.
When re-evaluating model compounds 6, 7, and 8, we
observed that in the microsolvated intermediate structure the
−NH− group in one of the silicon substituents formed a hydro-
gen bond to the OH− moiety and/or to the water molecule,
respectively. In the former case, the Si−F bond elongation was
reduced resulting in a smaller Δ(Si−F) value, while the opposite
occurred in the latter case. To investigate how strong these
C
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azide−alkyne cycloaddition (CuAAC) afforded triazole 27 in
22% yield. Silylethanol 25 was treated with PCC to form the
intermediate aldehyde, which was subsequently oxidized with
sodium chlorite and sulfamic acid to carboxylic acid 28 in 26%
yield. Coupling of 28 to benzyl amine with 2,4,6-tripropyl-
1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide (T3P) afforded
silylamide 29 in 35% yield. Fluorosilyl amide 8 was synthesized
by reacting fluorosilane 30 with diazoacetamide 31 using a
rhodium(II) catalyst, whereas direct fluorination of silane 29
using various reagents and conditions was not successful
(Scheme 1, i-(1−7)). NMR studies showed complete decom-
position of 8 after seven days in CDCl3 (probably promoted by
the moisture in the NMR tube and/or the presence of DCl/HCl
traces in CDCl3). Decomposition products were identified
by NMR analysis to be di-tert-butylfluorosilanol (32) and
N-benzylacetamide (33) as illustrated in Figure 1.
resin-bound peptide with 2-(4-(di-tert-butylsilyl)phenyl)acetic
acid required a coupling reagent system (HBTU (O-(benzo-
triazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophos-
phate) and HOBT (1-hydroxybenzotriazole)/DIPEA (N,N-
diisopropylethylamine)). Nonradioactive reference compound
39 (Figure 2) was obtained by direct fluorination of precursor 38
with KF in the presence of K222 and glacial acetic acid. Higher
yields for peptides 41 and 43 were obtained when DMTMM-BF4
(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
tetrafluoroborate) and NMM (N-methylmorpholine) were used
as coupling reagents. Fluorosilane 43 contained impurities of
silanol 44 (44/43 = 3:1). The IC50 values determined for
peptides 39 and 41 were 8.3 1.4 and 23 13 nM, respectively.
Radiolabeling, Hydrolytic Stability of the Si−18F Bond,
and log D7.4 Measurement. The direct 18F-fluorination
protocol developed previously in our laboratory20 was applied
for the radiolabeling of hydrosilanes 25, 27, and 29 as depicted in
Scheme 3. 18F-Incorporation of >95% was achieved for all these
hydrosilanes. The measured hydrolytic half-lives of [18F]2 and
[18F]18 in the presence of water were 8 and 16 h, respectively.
For both compounds, hydrolysis was much faster in PBS or in
0.9% NaCl than in water. 18F-Labeling of 29 gave N-
benzylacetamide (33) and di-tert-butyl[18F]fluorosilanol ([18F]
32) instead of [18F]8.
A similar labeling approach was used for the radiosynthesis of
peptides [18F]39 and [18F]43. For both peptides, the best 18F-
incorporation yield was obtained when 10 μL of acetic acid was
used as an additive. Then 32 GBq 18F− provided 350 MBq of
[18F]39 with a specific radioactivity of 35 GBq/μmol at the end
of synthesis (EOS). Then 190 MBq of [18F]43 with a specific
radioactivity of 70 GBq/μmol (EOS) was produced starting from
30 GBq 18F−. Both peptides were stable in PBS over 2 h. The
logarithmic distribution coefficient (log D7.4) value as a measure
of lipophilicity was determined by the shake flask method and
amounted to 0.3 0.1 (n = 3) for [18F]39. The lipophilicity of
[18F]43 was not determined.
Small Animal PET. PET images of human prostate
adenocarcinoma (PC-3) xenograft-bearing mice after injection
of [18F]39 and [18F]43, respectively, are shown in Figure 3. The
highest radioactivity concentrations for both radiolabeled
peptides were observed in the abdominal region in all tested
mice. The tumors were clearly visualized with both [18F]39
and [18F]43 (Figures 3A,C,D), consistent with the ex vivo
biodistribution data (see next Ex Vivo Biodistribution). Co-
injection of [18F]39 with 50 μg of bombesin resulted in a
reduction of radioactivity uptake in the tumor (Figure 3B).
Figure 1. Proposed mechanism for the hydrolysis of [18F]8.
The synthetic pathway leading to silane 36 and fluorosilane 37
is shown in Scheme 2. Primary amine 34 was protected with the
BOC-group using di-tert-butyl dicarbonate to afford compound
35 in quantitative yield. Bromoaryl 35 was treated with
n-butyllithium (n-BuLi), and the resulting lithiate was trapped
by di-tert-butylchlorosilane to give the intermediate silylaryl
compound. Deprotection of the primary amine under acidic
conditions and further reaction with di-O-acetyl-tartaric acid
anhydride yielded silane 36 in 21% yield. Direct fluorination of
silane 36 with potassium fluoride (KF) in the presence of
Kryptofix 222 (K222) and acetic acid (AcOH) gave fluorosilane
37 in 36% yield.
Peptide Synthesis and in Vitro Receptor Binding
Assay. Peptide synthesis was carried out using Rink amide
resin following standard Fmoc strategy.25 The conjugation of the
a
Scheme 2. Synthesis of Silane 36 and Fluorosilane 37
a
Reagents and conditions: (a) Boc2O, NEt3, CH3OH, 0 °C to rt, quant; (b) n-BuLi, THF, tBu2SiHCl (16), −78 °C to rt; (c) 1.25 M HCl/CH3OH,
rt; (d) di-O-acetyl-tartaric acid anhydride, NEt3, CoCl2(cat), CH3CN, 21% (three steps); (e) KF, K222, AcOH, THF, reflux, 36%.
D
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Figure 2. Structures of silicon containing bombesin analogues.
a
Scheme 3. 18F-Radiolabeling of Model Compounds 25, 27, and 29 and of Peptides 38 and 42
a
R = Ala(SO3H)-Ala(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2.
Ex Vivo Biodistribution. Tables 2 and 3 summarize the ex
vivo biodistribution data of [18F]39 and [18F]43 in PC-3
xenograft-bearing mice, which were sacrificed after PET imaging.
The tumor uptake of [18F]39 was 1.8 0.7%ID/g (n =3)at117min
after injection and was reduced by coinjection of nonradioactive
bombesin (50 μg per mouse) to 1.24 0.09%ID/g (n = 3).
Tumor to blood ratio was 2.0 at baseline and was reduced to 1.1
under blocking conditions. The gallbladder uptake of [18F]39
E
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a
b
c
Maximum intensity projections. Arrows point at PC-3 tumor xenografts. Image data were normalized to SUV (A,B, SUVmax = 2; C,D, SUVmax = 4).
a
Figure 3. PET images (MIP) with [18F]39 (60−105 min pi) under baseline (A) and blocking (B) conditions and with [18F]43 (C, 62−
92 min pi; D, 140−170 min pi) under baseline conditions
bc
,
Table 2. Ex Vivo Biodistribution of [18F]39 in PC-3
Tumor-Bearing Mice in Comparison to Previously
Reported Data of [18F]45
after injection were 5 and 8, respectively. Because of the lower
tumor to blood ratio and the higher radioactivity values in blood,
liver, and kidneys, we did not further investigate the specificity of
[18F]43. As expected for both peptides, a high accumulation of
radioactivity was measured in the pancreas, adrenal glands, and
intestines due to the high physiological expression of GRPr in
these organs.
a
b
[18F]39
[18F]45
tissue and ratio baseline [%ID/g] blockade [%ID/g] baseline [%ID/g]
tumor
1.8 0.7
1.24 0.09
1.17 0.08
3.4 0.5
0.7 0.6
5.6 0.4
2.1 0.7
0.40 0.05
0.42 0.04
4.08 0.67
n/d
blood
0.91 0.14
pancreas
prostate
liver
10
5
DISCUSSION
■
0.37 0.16
4.4 1.2
1.7 0.8
21 12
Previous studies by both our group and the Schirrmacher group
have documented the importance of the tert-butyl substituents
for designing hydrolytically stable silicon building blocks.14,19
Therefore, in our approach to design new building blocks with
reduced lipophilicity, we have decided to retain two tert-butyl
substituents and to replace the aromatic part of the new building
blocks with less lipophilic moieties. Previous DFT calculations by
4.40 1.04
1.73 0.24
kidney
intestine
lung
16
9
16
n/d
4
0.65 0.12
194 12
0.57 0.25
2.0
1.4 0.4
236 78
0.57 0.24
1.1
gallbladder
spleen
146 126
0.46 0.06
0.95
tumor/blood
a
Hohne et al. showed that compounds with Δ(Si−F) ≥ 0.19 Å tend
̈
Biodistribution at 117 min pi under baseline (8.3−15.3 MBq, n = 3)
b
to be unstable in aqueous solutions, while compounds with
and blocking conditions (4.8−10.5 MBq, n = 3). Biodistribution at
Δ
(Si−F) < 0.19 Å are considered to be hydrolytically stable.19
In the present study, DFT calculations showed that model
120 min pi under baseline conditions.20
compounds 2−23 exhibit Δ(Si−F) values ≥0.19 Å, except com-
pounds 6, 14, and 20, which all have Δ(Si−F) values below 0.19 Å
(Table 1). No correlation exists between the lipophilicity of the
building blocks and their Δ(Si−F) values. To verify the theoretical
calculations, precursors 25, 27, and 29 of model compounds 2, 8,
and 18, respectively, were labeled with 18F− (Scheme 3) and
was 194 12%ID/g, indicating hepatobiliary clearance of [18F]
39. [18F]43 showed tumor uptake of 3.5%ID/g (104 min post-
injection (pi)) and 2.4%ID/g (182 min pi), respectively. The
tumor to blood ratio (0.9) was higher at 182 min pi than at
104 min pi, and tumor to muscle ratios at 104 min and at 182 min
Table 3. Ex Vivo Biodistribution of [18F]43 in PC-3 Tumor-Bearing Mice
[18F]43
tissue and ratios
baseline (104 min pi) [%ID/g]
baseline (182 min pi) [%ID/g]
tumor
3.47
5.37
46
2.44
2.58
21
blood
pancreas
prostate
liver
0.44
25
0.29
13
kidney
6
4.1
intestine
lung
12.6
4.2
9.5
1.94
128
20
gallbladder
urine
36
25
spleen
2.15
1.37
1.24
0.69
5.01
1.09
0.42
0.65
5.03
1.32
0.85
0.51
0.29
2.07
0.92
0.8
heart
fat
muscle
adrenal gland
bone
stomach
tumor/blood
tumor/muscle
0.95
8.41
F
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subjected to hydrolytic stability studies. The predicted hydrolytic
instability of the Si−18F bond of [18F]2 and [18F]18 in aqueous
solutions could be confirmed experimentally. On the basis of
these results, we did not further synthesize reference compounds
2 and 18 and we conclude that relatively fast hydrolysis of the
Si−F bond of these compounds takes place due to a decreased
steric hindrance around the silicon atom. This may also explain
the greater than 0.19 Å Δ(Si−F) values for 3−5, 7−13, 15, 17−19,
and 21−23. Replacement of the phenyl ring by a similarly bulky
triazole ring increased the Δ(Si−F) value to 0.24 Å as calculated
for 16. This might be due to the basic triazole nitrogen atoms,
which enhance the nucleophilic character of surrounding water
molecules and thus facilitate hydrolysis of the Si−F bond.
Because of the hydrolytic instability of triazole 16, its “click”
precursor acetylene 14 with a Δ(Si−F) value of 0.17 Å was not
further investigated although 14 is predicted to be hydrolytically
stable. Methoxymethoxysilanes 20 and 21 differ only in their
terminal group and thus it is reasonable to assume that the
remote functionalities might have influenced their Δ(Si−F) values.
Compound 20 with an advantageous Δ(Si−F) value of 0.18 Å was
not further evaluated because it is synthetically not easily
accessible.
The hydrolytic stability of acetamide 6 was verified using
model compound 8. Unexpectedly, 18F-labeling of precursor
compound 29 which was anticipated to afford [18F]8 did not lead
to the desired radiolabeled compound but instead to di-tert-
butyl[18F]fluorosilanol ([18F]32) and N-benzylacetamide (33).
Mechanistically, we assume fluorosilane [18F]8 undergoes hydro-
lysis corresponding to a mechanism described for the solvolysis
of β-ketosilanes.26 According to this mechanism, the silicon−
methylene (Si−CH2) bond of [18F]8 is cleaved instead of the
Si−F bond (Figure 1). In the future, we plan to focus DFT
calculations not only on the stability of Si−F bond but also to
evaluate the stability of all silicon−carbon bonds.
In the DFT calculation of microsolvated intermediate model
structures, from which we extracted one Si−F bond length
needed for the Δ(Si−F) measure, we assumed that a hydroxide
anion (OH−) binds to the silicon atom, while a water molecule
builds up a hydrogen bond to the fluorine atom of the com-
pounds.19 Hence, the measured elongation of the Si−F bond
depends on both the binding of OH− and the hydrogen bond of
the water molecule. However, we use a small microsolvated
model system for the intermediate in which strong intra-
molecular hydrogen bonds can be expected to lead to artifacts in
the interpretation. In contrast, intramolecular hydrogen bonds
are not likely to occur in aqueous solution as other water
molecules can saturate these contacts, thus making their
formation very difficult if not impossible. Therefore, we propose
that Δ(Si−F) values of conformers without intramolecular hydro-
gen bonds are more suitable to predict the hydrolytic stability of
Si−F bonds. Accordingly, we changed the intermediate
conformations of model compounds 6, 7, and 8 in order to
make sure that no intramolecular hydrogen bonds occurred in
our microsolvated structures. The new Δ(Si−F) values differed a
lot from the Δ(Si−F) values of the conformers with intramolecular
hydrogen bonding showing the influence of neglecting intra-
molecular hydrogen bonds in the calculations. However, the
Schirrmacher and his co-workers for silicon-based carbohydrate-
conjugated octreotate derivatives.27 As a proof of concept, we
selected the previously investigated peptide 4520 and replaced
the arginine linker by two L-cysteic acids (Ala(SO3H)) to give 39
as shown in Figure 2. In addition, incorporation of hydrophilic
tartaric acid between arginine or L-cysteic acid and the silicon
building block afforded peptides 41 and 43. The synthesis of
silicon building block 36 is shown in Scheme 2. The binding
affinities (IC50) of 39 and 41 determined in competition assays
with [125I]-Tyr4-bombesin were 8.3
1.4 and 23
13 nM,
respectively, and found to be comparable to previously reported
18F-labeled bombesin analogues 45 (IC50 = 22.9 nM) and FB-
[Lys3]-bombesin (IC50 = 5.3 nM).20,28 The binding affinity of 43
was not determined as several attempts to produce pure 43 failed,
mainly due to difficulties in separating 43 from its silanol
counterpart 44. However, we anticipate that the IC50 value of 43
would be in the same range as peptide 39 because 39 and 43
differ only in the linker, which does not participate in the binding
to the GRPr. The radiosyntheses of peptides [18F]39 and [18F]43
were accomplished via a one-step reaction using hydride as a
leaving group (Scheme 3). Both [18F]39 and [18F]43 were
produced in sufficient radiochemical yields, specific radioactivity,
and good radiochemical purity for animal experiments. The log
D
7.4 value for [18F]39 was 0.3 0.1, which is 1 unit lower than the
log D7.4 value of [18F]45 (log D7.4 = 1.3 0.1).20
PET studies showed that [18F]39 accumulated in the tumor
but to a greater extent in the abdominal region and the urinary
bladder. Bone uptake was not observed and thus defluorination
did not occur. The tumor was not visualized under blocking
conditions and the GRPr expressing pancreas was slightly
blocked in ex vivo biodistribution. Compared to the ex vivo
biodistribution data of [18F]45, the more hydrophilic [18F]39
showed a 4.5-fold higher tumor accumulation and a 2-fold higher
tumor to blood ratio at 120 min pi but still moderate specificity.
Preliminary ex vivo biodistribution studies of [18F]43 revealed a
1.4−1.9-fold higher accumulation than for [18F]39. However,
substantially higher liver uptake was also observed and the tumor
to blood ratio was 3.1−2.1-fold lower than with [18F]39.
Nevertheless, our hypothesis that increased hydrophilicity of the
conjugate will facilitate tumor uptake compared to [18F]45 was
confirmed.
CONCLUSION
■
In the present study, several silicon-based building blocks with
enhanced hydrophilic properties were investigated. None of the
synthesized compounds preserved their stability in aqueous
solution. An overall enhanced hydrophilicity of the silicon-
containing peptide through modification of the linker leads to an
improved pharmacokinetic profile and to an enhanced tumor
uptake. Greater improvements may be achieved if a hydrolyti-
cally stable silicon-based building block with significantly
reduced lipophilicity would become available.
EXPERIMENTAL SECTION
■
DFT Calculations. The DFT calculations to predict the hydrolytic
stability of the Si−F bond in fluorosilanes 2−23 (Table 1) were carried
out according to the previously published method.19 Briefly, a SN2
mechanism for the hydrolysis of the Si−F bond occurring under
inversion via a pentacoordinate intermediate is assumed. The difference
of the calculated Si−F bond lengths of the fluorosilane and its
corresponding fluorosilanol pentacoordinate intermediate provides the
Δ(Si−F) value. The all-electron Kohn−Sham DFT calculations were
performed using the quantum-chemical program package Turbomole.29
Δ
(Si−F) values of 6 and 8 are not similar and we lack a reasonable
explanation for this discrepancy.
One possibility to compensate for the high lipophilicity of the
di-tert-butylsilylphenyl building block is to introduce hydrophilic
linkers between the peptide sequence and the silicon containing
building block. This strategy has successfully been applied by
G
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The pure functional TPSS in combination with the resolution-of-the-
identity (“RI”) density fitting technique, and the def-TZVP basis set for
all atoms apart from Si were applied.30,31 For Si, we used a def-TZVPP
basis set30 with Dunning-type polarization functions as implemented in
Turbomole 6.4. To estimate intramolecular hydrogen bond energies, we
employed the SENs approach by Reiher et al.24 For this, single-point
BP86/RI/TZVP calculations, as described in ref 24 were carried out on
top of the TPSS-optimized structures.
temperature overnight and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (9:1 → 4:1,
1
nBuOH/AcOH) to obtain 27 (47 mg, 22%) as white solid. H NMR
(400 MHz, C2D6OS) δ 8.01 (s, 1H, CHN), 4.45−4.40 (m, 2H, CH2N),
3.86 (s, 1H, CHCH2), 3.23−3.02 (m, 2H, CH2CH), 1.31−1.24 (m, 2H,
SiCH2), 1.03 (s, 18H, CH3). 29Si NMR (CDCl3, 99 MHz) δ 10.1. HRMS
(ESI) calcd for [C15H31N4O2Si]+, 327.2211; found, 327.2200.
2-(Ditert-butylsilyl)acetic Acid (28). To a solution of 25 (750 mg,
4 mmol) in DCM (52 mL), PCC (1.32 g, 6 mmol) was added and the
mixture was stirred at room temperature for 3 h. The reaction mixture
was diluted with diethyl ether and filtered through Celite (diethyl ether).
The residue was concentrated in vacuo, redissolved in acetone (40 mL),
and cooled to 0 °C. A solution of sodium chlorite (1.36 g, 12 mmol) and
sulfamic acid (1.09 g, 11.2 mmol) in water (40 mL) was added dropwise,
and the mixture was stirred at 0 °C. After 30 min, the reaction was
diluted with water and extracted with EtOAc (4×). The organic phases
were combined, dried over MgSO4, and concentrated in vacuo. The
residue was purified by flash column chromatography on Reprospher
Acidosil-S, 50 μm (hexane), to yield 28 (209 mg, 26% over two steps)
Chemistry. All reactions were carried out under an atmosphere of
argon in oven-dried glassware with magnetic stirring, unless otherwise
indicated. The reagents and solvents were purchased from Sigma-
Aldrich Chemie GmbH, Fluka Chemie AG, Archimica GmBH, Chemie
Brunschwig AG, Acros Organics, ABCR GmbH & Co., or VWR
International AG and were used as supplied unless stated otherwise.
Flash chromatography was performed with Fluka silica gel 60 (0.040−
0.063 μm grade). Analytical TLC was performed with commercial
aluminum sheets coated with 0.25 mm silica gel (E. Merck, Kieselgel
60 F254). Compounds were visualized by UV light at 254 nm and by
dipping the plates in an aqueous potassium permanganate solution or in
an ethanolic vanillin/sulfuric acid solution followed by heating. 1H, 13C,
19F, and 29Si NMR data were acquired on a Bruker AV400 (400 MHz) or
AV500 (500 MHz) spectrometer. Chemical shifts are reported in delta
(δ) units, in parts per million (ppm) downfield from tetramethylsilane
(1H NMR and 29Si NMR), from trichlorofluoromethane (19F NMR)
and relative to the center line of a triplet at 77.0 ppm for chloroform-d
(13C NMR). Splitting patterns are designated as: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad peaks. All coupling constants
(J) are given in Hz. IR data were recorded on a Perkin-Elmer, Spectrum
100, FT-IR spectrometer. Absorbance frequencies are reported in
reciprocal centimeters (cm−1). HRMS were performed by the MS
service at the Laboratory of Organic Chemistry, ETH Zurich, and are
given in m/z.
1
as colorless solid. Rf 0.24 (1:9, EtOAc/hexane). H NMR (CDCl3,
400 MHz) δ 3.59 (t, J = 2.9 Hz, 1H, SiH), 1.99 (d, J = 3.0 Hz, 2H, CH2),
1.06 (s, 18H, CH3). 13C NMR (CDCl3, 100 MHz) δ 180.4, 28.3, 19.2,
18.9. 29Si NMR (CDCl3, 99 MHz) δ 11.3. IR (neat): 2932, 2890, 2859,
2118, 1690, 1469, 1422, 1390, 1367, 1289, 1146, 1109, 1013 cm−1.
HRMS (ESI) calcd for [C10H21O2Si]−, 201.1316; found, 201.1318.
N-Benzyl-2-(di-tert-butylsilyl)acetamide (29). To a solution of 28
(51 mg, 0.25 mmol) in THF (2.5 mL), DIPEA (0.11 mL, 0.63 mmol)
and benzylamine (0.03 mL, 0.25 mmol) were added and the mixture was
cooled to 0 °C. T3P as a 50% solution in THF (1.2 mL, 2 mmol) was
added dropwise, and the mixture was stirred at 0 °C for 30 min, warmed
to room temperature, and stirred overnight. The reaction was diluted
with EtOAc and washed with water (1×) and brine (1×). After drying
over MgSO4, the organic phase was concentrated in vacuo. The residue
was purified by flash column chromatography on silica gel (9:1 → 4:1,
hexane/EtOAc) to afford 29 (26 mg, 35%) as white crystals. Rf 0.27 (4:1,
hexane/EtOAc). 1H NMR (CDCl3, 400 MHz) δ 7.24−7.36 (m, 5H, Ar-
H), 5.61 (s, 1H, NH), 4.41 (d, J = 5.7 Hz, 2H, NHCH2), 3.59 (t, J = 3.3
2-(Di-tert-butylsilyl)ethanol (25). To a mixture of di-tert-butylchlor-
osilane (24, 5.3 mL, 26 mmol) and rhodium(II) acetate dimer (0.11 g,
0.25 mmol) in dry DCM (14 mL) a solution of ethyl diazoacetate
(4.8 mL, 39 mmol) was added slowly over 7 h at 35 °C. The mixture was
filtered through Celite (DCM) and concentrated in vacuo. The residue
was added dropwise to a mixture of LAH (5.6 g, 147 mmol) in THF
(74 mL) at 0 °C. After heating at reflux for 2.5 h, the reaction was
quenched with 1 M HCl, diluted with ethylacetate (EtOAc), and filtered.
The organic phase was washed with 1 M HCl (1×), water (1×), and
brine (1×) and dried over MgSO4. The residue was purified by flash
column chromatography on silica gel (19:1 → 4:1, hexane/EtOAc) to
yield 25 (3.14 g, 64% over two steps) as colorless liquid. Rf 0.43 (7:3,
Hz, 1H, SiH), 1.90 (d, J = 3.5 Hz, 2H, SiCH2), 1.05 (s, 18H, CH3). 13
C
NMR (CDCl3, 100 MHz) δ 138.5, 128.6, 127.9, 127.4, 44.0, 28.5, 21.0,
19.0. 29Si NMR (CDCl3, 99 MHz) δ 11.4. IR (neat): 3291, 2929, 2886,
2857, 2111, 1630, 1542, 1497, 1469, 1455, 1364, 1291, 1261, 1155,
1131, 1012 cm−1. HRMS (ESI) calcd for [C17H30NOSi]+, 292.2091;
found, 292.2094.
N-Benzyl-2-(di-tert-butylfluorosilyl)acetamide (8). To a mixture of
di-tert-butylfluorosilane (30, 228 mg, 1.40 mmol; see ref 32 for its
preparation) in DCM (0.7 mL) was added rhodium(II) acetate dimer
(3.10 mg, 7 μmol) followed by dropwise addition of N-benzyl-2-
diazoacetamide (31, 123 mg, 0.702 mmol; see ref 33 for its preparation)
in DCM (1.2 mL). The reaction was stirred at room temperature for 15
min, filtered through Celite (DCM), and concentrated in vacuo. The
residue was purified by flash column chromatography (9:1 → 4:1,
hexane/EtOAc) to afford 8 (11 mg, 5%) as colorless solid. Rf 0.44 (7:3,
hexane/EtOAc). 1H NMR (CDCl3, 400 MHz) δ 7.36−7.25 (m, 5H, Ar-
H), 5.71 (s, 1H, NH), 4.42 (d, J = 5.4 Hz, 2H, NHCH2), 2.03 (d, J = 2.0
Hz, 2H, SiCH2), 1.07 (d, J = 1.0 Hz, 18H, CH3). 13C NMR (CDCl3, 100
MHz) δ 138.3, 128.7, 127.9, 127.6, 44.1, 27.0, 20.3, 20.1. 19F NMR
(CDCl3, 376 MHz) δ −181.2. HRMS (ESI) calcd for [C17H29FNOSi],+
310.1997; found, 310.1992.
The NMR tube containing the sample was stored at room tem-
perature for 7 d and was then analyzed again by 1H NMR and 19F NMR
spectroscopy. 1H NMR (CDCl3, 400 MHz) δ 7.36−7.28 (m, 5H), 5.70
(br s, 1H), 4.44 (d, J = 5.7 Hz, 2H), 2.03 (s, 3H), 1.06 (d, J = 1.0 Hz,
18H). 19F NMR (CDCl3, 376 MHz) δ −157.7. The observed chemical
shifts point to decomposition products 32 and 33. For verification,
compounds 32 and 33 were separately synthesized.
1
hexane/EtOAc). H NMR (400 MHz, CDCl3) δ 3.82−3.77 (m, 2H,
CH2OH), 3.28 (t, J = 2.7 Hz, 1H, SiH), 1.52 (s, 1H, OH), 1.09−1.01 (m,
2H, SiCH2), 1.00 (s, 18H, CH3). 13C NMR (100 MHz, CDCl3) δ 61.6,
28.6, 18.6, 14.8. 29Si NMR (CDCl3, 99 MHz) δ 8.4.
(2-Azidoethyl)di-tert-butylsilane (26). Triphenylphosphine
(306 mg, 1.17 mmol) was slowly added to a solution of 25 (200 mg,
1.06 mmol) in DCM (5.3 μL) at 0 °C. After 5 min of stirring, carbon
tetrabromide (387 mg, 1.17 mmol) was added slowly. The reaction was
stirred for 10 min at 0 °C and 20 min at room temperature. The mixture
was concentrated in vacuo, and the residue was purified by flash column
chromatography on silica gel (hexane) to afford crude (2-bromoethyl)-
di-tert-butylsilane. Crude (2-bromoethyl)di-tert-butylsilane and sodium
azide (105 mg, 1.61 mmol) were dissolved in DMF (3.4 mL) and stirred
at room temperature for 6 h. The reaction was diluted with EtOAc and
washed with brine (2×), and the organic phase was dried over MgSO4
and concentrated. The residue was purified by flash column
chromatography on silica gel (hexane) to obtain 26 (130 mg, 57%
1
over two steps) as colorless liquid. Rf 0.35 (hexane). H NMR (400
MHz, CDCl3) δ 3.41−3.37 (m, 2H, CH2N3), 3.31 (t, J = 2.5 Hz, 1H,
SiH), 1.08−1.05 (m, 2H, SiCH2), 1.03 (s, 18H, CH3). IR (neat) 2931,
2887, 2858, 2087, 1468, 1244 cm−1.
Di-tert-butylfluorosilanol (32). 32 was synthesized in analogy to a
published procedure.34 Briefly, dichloro-di-tert-butylsilane was reacted
with trifluorostibine to obtain di-tert-butyldifluorosilane. Further
2-Amino-3-(1-(2-(Di-tert-butylsilyl)ethyl)-1H-1,2,3-triazol-4-yl)-
propanoic Acid (27). Azide 26 (140 mg, 0.66 mmol), propargyl glycine
(75 mg, 0.66 mmol), copper(II) acetate (12 mg, 0.07 mmol), and
(+)-sodium L-ascorbate (130 mg, 0.66 mmol) were dissolved in tBuOH
(3.3 mL) and water (3.3 mL). The reaction was stirred at room
1
treatment with KOH afforded 32. H NMR (CDCl3, 400 MHz) δ
1.06 (d, J = 1.0 Hz, 18H, CH3). 19F NMR (CDCl3, 376 MHz) δ −157.6.
H
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N-Benzylacetamide (33). To a solution of benzylamine (0.5 mL,
4.58 mmol) in DCM (17.0 mL) was added triethylamine (1.28 mL,
9.16 mmol) and 4-(dimethylamino)pyridine (0.056 g, 0.458 mmol) at
0 °C. Acetic anhydride (0.432 mL, 4.58 mmol) was added dropwise, and
the mixture was stirred for 10 min at 0 °C and 30 min at room
temperature. The reaction was washed with saturated aqueous NaHCO3
(1×), 1 M HCl (1×), water (1×), and brine (1×). The organic phase
was concentrated in vacuo to obtain 33 (670 mg, 97%) as white solid. 1H
NMR (CDCl3, 400 MHz) δ 7.38−7.28 (m, 5H, Ar-H), 6.05 (br s, 1H,
NH), 4.44 (d, J = 5.8, 2H, CH2), 2.03 (s, 3H, CH3).
tert-Butyl 4-Bromophenethylcarbamate (35). To a solution of
4-bromophenylethylamine (34, 6.94 g, 34 mmol) in CH3OH (100 mL)
was added NEt3 (19 mL, 136 mmol) and di-tert-butyl dicarbonate
(15.3 g, 68 mmol) at 0 °C. The mixture was stirred at room temperature for
14 h. The reaction was concentrated, diluted with EtOAc, and washed with
water (2×) and brine (1×). After drying over MgSO4, concentration of the
organic phase in vacuo afforded a residue, which was further purified by
flash column chromatography on silica gel (19:1 → 17:5, hexane/EtOAc)
to afford 35 (10.2 g, quantitative) as white solid. Rf 0.34 (4:1, hexane/
EtOAc). 1H NMR (400 MHz, CDCl3) δ = 7.43−7.40 (m, 2H), 7.06 (d, J =
8.4 Hz, 2H), 4.54 (s, 1H), 3.36−3.32 (m, 2H), 2.75 (t, J = 7.0 Hz, 2H), 1.43
(s, 9H). 13C NMR (100 MHz, CDCl3) δ = 155.8, 138.0, 131.6, 130.5, 120.2,
79.3, 41.6, 35.7, 28.4. IR (neat) 3444, 3355, 2977, 2932, 2871, 1694, 1488,
1365, 1248, 1164 cm−1. HRMS (ESI) calcd for [C13H18BrNNaO2]+,
322.0413; found, 322.0414.
C(O)CH3), 1.04 (s, 18H, C(CH3)3). 13C NMR (CDCl3, 100 MHz) δ
170.0, 166.6, 139.8, 134.4, 134.3, 131.8, 131.7, 128.7, 128.6, 128.1, 72.2,
40.6, 35.5, 33.7, 29.7, 28.9, 27.3, 22.3, 20.5, 20.4, 20.3, 20.2,13.9. 19F
NMR (CDCl3, 376 MHz) δ −188.9. IR (neat) 3362, 2935, 2893, 2860,
1751, 1659, 1539, 1372, 1209, 1109, 1060 cm−1. HRMS (ESI) calcd for
[C24H37FNO7Si]+, 498.2318; found, 498.2305.
Peptide Synthesis. Fmoc deprotection (general procedure): The
resin-bound Fmoc peptide was treated with 20% piperidine in DMF
(v/v) for 5 min. This step was repeated with a reaction time of 20 min.
The resin was washed with DMF (2×), DCM (2×), and DMF (2×).
HBTU/HOBT coupling (general procedure): A solution of Fmoc-Xaa-
OH (Xaa = amino acid, 4 equiv), HBTU (4 equiv), HOBT (4 equiv), and
DIPEA (4 equiv) in DMF was added to the resin-bound, free amine peptide
and shaken for 90 min at room temperature. This step was repeated with a
reaction time of 60 min. The resin was washed with DMF (2×), DCM (2×),
and DMF (2×). The peptides were typically prepared starting with 147 mg
(100 μmol) of the resin. The amounts of reagents and building blocks in all
subsequent reactions were calculated based on this amount.
Synthesis of 2-(4-(Di-tert-butylfluorosilyl)phenyl)acetyl-Ala-
(SO3H)-Ala(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2
(Precursor Compound 38). The resin-bound, side chain protected
peptide was prepared according to the general procedures described
above. 2-(4-(Di-tert-butylsilyl)phenyl)acetic acid (100 mg, 359 μmol)
and HBTU (136.2 mg, 359 μmol) were dissolved in DMF (5 mL), and
DIPEA (63 μL, 359 μmol) was added. The resin-bound peptide (179 μmol)
was suspended in this solution, and the suspension was shaken for 24 h at
room temperature. The resin was then filtered, washed with DMF (5 ×
5 mL) and DCM (5 × 5 mL), and dried in vacuo. Subsequent treatment of
the resin with 2 mL of TFA/water/triiso-propylsilane/phenol (85:5:5:5)
afforded the crude, fully deprotected peptide, which was precipitated and
washed with cold methyl tert-butyl ether. The crude peptide was dried in
vacuo, purified by preparative reversed phase (RP) HPLC, and lyophilized to
afford 38 (30 mg, 9.6%) as white solid. The product was analyzed by HPLC-
MS: m/z calcd, 1641.8; found, 1642.0 ([M + H]+).
Synthesis of 2-(4-(Di-tert-butylfluorosilyl)phenyl)acetyl-Ala-
(SO3H)-Ala(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2
(Reference Compound 39). 38 (9.3 mg, 5.7 μmol) was dissolved in
THF (1 mL). The solution was added to a mixture of KF (2.6 mg,
45.3 μmol), K222 (17.1 mg, 45.3 μmol), and K2CO3 (3.1 mg, 22.7 μmol).
Glacial acetic acid (7.8 μL, 135.9 μmol) was added, and the resulting
suspension was heated at 70 °C for 30 min. The crude mixture was directly
subjected to preparative RP-HPLC, and the purified product was
lyophilized to obtain 39 (6 mg, 58%) as white solid. The product was
analyzed by HPLC-MS: m/z calcd, 1659.8; found, 1660.4 ([M + H]+).
Procedure for the Syntheses of 4-((4-(Di-tert-butylsilyl)phenethyl)-
amino)-2,3-dihydroxy-4-oxobutanoyl-Arg-Ava-Gln-Trp-Ala-Val-
NMeGly-His-Sta-Leu-NH2 and 4-((4-(Di-tert-butylsilyl)phenethyl)-
amino)-2,3-dihydroxy-4-oxobutanoyl-Ala(SO3H)-Ala(SO3H)-Ava-
Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 (Precursor Compounds 40
and 42). The resin-bound, side chain protected peptide was prepared
according to the general procedures described above. 36 (165.0 mg,
344 μmol), DMTMM-BF4 (120.4 mg, 367 μmol), and NMM (69.6 μL,
688 μmol) were dissolved in DMF (10 mL). The resin-bound peptide
(230 μmol) was suspended in this solution, and the suspension was
shaken for 30 min at ambient temperature. Thereafter the resin was
filtered and washed with DMF (3 × 10 mL) and then treated with
hydrazine monohydrate (1.1 mL) in DMF (5 mL) at room temperature
for 3 h to remove the acetyl groups, washed with DMF (3 × 5 mL) and
DCM (3 × 5 mL), and dried in vacuo. Subsequent treatment of the resin
with 2.5 mL TFA/water/triiso-propylsilane/phenol (85:5:5:5) afforded
the crude, fully deprotected peptide, which was precipitated and washed
with cold methyl tert-butyl ether. The crude peptide was dried in vacuo,
purified by preparative RP-HPLC, and lyophilized. The products were
analyzed by HPLC-MS: 40 (5.7 mg, 1.5%) as white solid; m/z calcd,
807.0; found, 807.2 ([(M + 2H)/2]+). 42 (11.5 mg, 1.8%) as white
solid: m/z calcd, 1759.8; found, 1760.7 ([M + H]+).
2,3-Diacetoxy-4-(4-(di-tert-butylsilyl)phenethylamino)-4-oxobu-
tanoic Acid (36). n-BuLi (6.18 mL, 9.89 mmol) was added dropwise to a
solution of 35 (990 mg, 3.3 mmol) in THF (11 mL) at −78 °C. The
colorless mixture was stirred at −78 °C and after 1.5 h was treated with
di-tert-butylchlorosilane (1.04 mL, 4.95 mmol). After warming to room
temperature, the reaction mixture was further stirred for 20 h and the
resulting yellow mixture was poured into 0.3 M aqueous NaHCO3
solution and extracted with EtOAc (3×). The combined organic phases
were washed with water (1×) and brine (1×), dried over MgSO4, and
concentrated in vacuo. A 1.25 M HCl/CH3OH solution (5 mL) was
added to the residue, and the mixture was stirred at room temperature
for 1 h. After concentration in vacuo, the resulting solid was mixed with
acetonitrile (CH3CN, 16.5 mL) and dissolved by adding NEt3 (0.15 mL,
1.07 mmol). Cobalt(II) chloride (21 mg, 0.17 mmol) and di-O-acetyl-
tartaric acid anhydride (1.07 g, 4.95 mmol) were added, and the blue
solution was stirred at room temperature for 5 h. The reaction mixture
was poured into 1 M HCl solution and extracted with EtOAc (2×). The
combined organic phases were poured into 0.3 M aqueous NaHCO3
and extracted with EtOAc (3×). After drying over MgSO4, the com-
bined organic phases were concentrated in vacuo. The residue was
purified by flash column chromatography on Reprospher Acidosil-S,
50 μm (9:1 → 4:1, hexane/EtOAc), to yield 36 (338 mg, 21% over three
steps) as colorless foam. Rf 0.26 (75:27:5:0.5, CHCl3/CH3OH/H2O/
AcOH). 1H NMR (CDCl3, 400 MHz) δ 7.48 (d, J = 7.5 Hz, 2H), 7.15
(d, J = 7.3 Hz, 2H), 7.07 (s, 1H), 5.76 (s, 1H), 5.54 (s, 1H), 3.83 (s, 1H),
3.50 (s, 2H), 2.78 (s, 2H), 2.06 (s, 6H), 1.02 (s, 18H). 13C NMR
(CDCl3, 100 MHz) δ 170.1, 167.1, 139.3, 136.1, 133.4, 128.6, 127.9,
72.7, 40.7, 35.4, 35.2, 33.7, 28.9, 22.3, 20.6, 20.5, 19.0, 13.9. 29Si NMR
(CDCl3, 99 MHz) δ 12.9 (JSi−H = 186 Hz). IR (neat) 3358, 2931, 2856,
2097, 1749, 1631, 1544, 1413, 1372, 1212, 1055, 803 cm−1. HRMS
(ESI) calcd for [C24H36NNa2O7Si]+, 524.2051; found, 524.2055.
2,3-Diacetoxy-4-(4-(di-tert-butylfluorosilyl)phenethylamino)-4-
oxobutanoic Acid (37). To a solution of 36 (200 mg, 0.42 mmol) in
THF (4.2 mL), AcOH (72 μL, 1.25 mmol), K222 (235 mg, 0.63 mmol),
and potassium fluoride (37 mg, 0.625 mmol) were added and the
reaction mixture was heated under reflux for 6 h. Thereafter, the yellow
mixture was washed with saturated aqueous NH4Cl solution (1×), water
(1×), and brine (1×), and the organic phase was dried over MgSO4 and
concentrated in vacuo. Purification of the residue was accomplished by
flash column chromatography on Reprospher Acidosil-S, 50 μm (9:1 →
4:1, hexane/EtOAc), to yield 37 (74 mg, 36%) as white−yellow solid. Rf
Synthesis of 4-((4-(Di-tert-butylfluorosilyl)phenethyl)amino)-2,3-dihy-
droxy-4-oxobutanoyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2
and 4-((4-(Di-tert-butylfluorosilyl)phenethyl)amino)-2,3-dihydroxy-4-ox-
obutanoyl-Ala(SO3H)-Ala(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-
Leu-NH2 (Reference Compounds 41 and 43). The resin-bound, side
1
0.10 (90:10:1:0.5, CHCl3/CH3OH/H2O/AcOH). H NMR (CDCl3,
400 MHz) δ 7.54 (d, J = 7.8 Hz, 2H, Ar-H), 7.20 (d, J = 8.0 Hz, 2H, Ar-
H), 6.65 (s, 1H, NH), 5.79 (s, 1H, CH), 5.60 (s, 1H, CH), 3.50−3.47
(m, 2H, CH2N), 2.81 (t, J = 7.2 Hz, 2H, CH2-Ar), 2.08 (s, 6H,
I
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Journal of Medicinal Chemistry
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chain protected peptide was prepared according to the general
procedures described above. 37 (22.4 mg, 45 μmol), DMTMM-BF4
(15.7 mg, 48 μmol), and NMM (10 μL, 90 μmol) were dissolved in
DMF (2 mL). The resin-bound peptide (30 μmol) was suspended in
this solution, and the suspension was shaken for 12 h at ambient
temperature. The resin was then filtered and washed with DMF (3 ×
3 mL). The resin was treated with hydrazine monohydrate (0.1 mL) in
DMF (0.9 mL) at room temperature for 6 h to remove the acetyl groups,
washed with DMF (3 × 5 mL) and DCM (3 × 5 mL), and dried in
vacuo. Subsequent treatment of the resin with 2 mL of TFA/water/
triiso-propylsilane/phenol (85:5:5:5) afforded the crude, fully depro-
tected peptide, which was precipitated and washed with cold methyl
tert-butyl ether. The crude peptide was dried in vacuo, purified by
preparative RP-HPLC, and lyophilized. The products were analyzed by
HPLC-MS: 41 (2.4 mg, 3.1%) as white solid; m/z calcd, 816.0; found,
816.2 ([(M + 2H)/2]+). 43 (3.8 mg, 3.6%) as white solid; m/z calcd,
1776.8; found, 1777.7 ([M + H]+).
In Vitro Receptor Binding Assay. The binding affinity of the
nonradioactive bombesin peptides 39 and 41 for human GRPr was
determined in a displacement assay with PC-3 cells (DSMZ, German
Collection of Microorganisms and Cell Cultures). Cells were seeded in
48-well plates (8 × 104 cells/well) and grown in Ham’s F-12 nutrient
mix with GlutaMax (Invitrogen) for 1 day to subconfluence. Cells were
washed twice with PBS, followed by the addition of incubation buffer.
The test compound was dissolved in DMSO to produce 1 mM stock
solutions and further diluted in incubation buffer (50 mM 2-(4-(2-
hydroxyethyl)-1-piperazine)ethanesulfonic acid (HEPES), protease
inhibitor complete (1 tablet/50 mL; Roche Diagnostics GmbH),
5 mM MgCl2, and 0.1% BSA (pH 7.4) in Dulbecco Modified Eagle
Medium with GlutaMAX I (Invitrogen)) to 10−10−10−3 M. Test
compound solutions and [125I]-Tyr4-bombesin (PerkinElmer; specific
radioactivity, 81.4 GBq/μmol; conc, 22.73 nM; KD = 0.81 nM) were
added to all well plates (final volume, 960 μL; test compound con-
centration range, 10−5−10−12 M; [125I]-Tyr4-bombesin concentration,
0.237 nM). Nonspecific binding was estimated with Tyr4-bombesin
(concentration per well: 1.0 μM). After incubation at room temperature
for 1 h, cells were washed twice with cold PBS (containing 0.1% BSA)
and solubilized with 0.25% trypsin ethylenediaminetetraacetic acid
solution (0.3 mL/well, incubation for 15 min at 37 °C). Cells were
pipetted into Eppendorf cups, and wells were washed with PBS (1 mL)
and added to cell solutions. Radioactivity was measured in a γ-counter
(1480 Wizard, PerkinElmer). The IC50 values were calculated using
KELL Radlig software (Biosoft).
acetic acid (10 μL) in anhydrous DMSO (150 μL) was added to the dry
K[18F]F/K222 complex and heated at 110 °C for 20 min. An aliquot of
the crude reaction mixture was analyzed using an analytical UPLC to
show an 18F-incorporation of ≥95%.
N-Benzyl-2-(di-tert-butyl[18F]fluorosilyl)acetamide ([18F]8). A sol-
ution of 29 (2.0 mg) and glacial acetic acid (10 μL) in anhydrous DMSO
(150 μL) was added to the dry K[18F]F/K222 complex and heated at
110 °C for 20 min. An aliquot of the crude reaction mixture was analyzed
using an analytical UPLC to show an 18F-incorporation of ≥95%. TLC
analysis (9:1, hexane/EtOAc) of the crude reaction mixture showed that
the 18F-labeled product did not correspond to compound 8 but rather
to 32.
2-(4-(Di-tert-butyl[18F]fluorosilyl)phenyl)acetyl-Ala(SO3H)-Ala-
(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 ([18F]39). A
solution of 38 (2.0 mg) and glacial acetic acid (10 μL) in anhydrous
DMSO (150 μL) was added to the dry K[18F]F/K222 complex and
heated at 110 °C for 20 min. An aliquot of the crude reaction mixture
was analyzed using an analytical HPLC (ACE C18, 50 mm × 4.6 mm,
5 μm; gradient CH3CN/H2O + 0.1% TFA 5:95 → 95:5 in 20 min,
1.0 mL/min) before addition of H2O/CH3CN (9:1, 2 mL) into the
reaction vial. The diluted reaction mixture was injected into a
semipreparative HPLC (ACE C18, 250 mm × 10 mm, 5 μm; isocratic
CH3CN/H2O + 0.1% TFA 37:63, 4.0 mL/min), and the product peak
was collected. The product fraction was diluted with water (20 mL) and
immobilized on a C18 cartridge (Sep-Pak Light C18, Waters, or
Chromafix C18 (s), Machery-Nagel). After washing with water (20 mL),
[18F]39 was eluted with ethanol (1 mL). The solvent was evaporated at
90 °C. For in vivo applications, [18F]39 was reconstituted in 0.15 M PBS
containing ≤5% ethanol (v/v) and the solution was filtered sterile. The
overall synthesis time was 80 min. Reverse-phase HPLC revealed a
radiochemical purity ≥95%. The synthesis afforded 350 MBq of [18F]39
starting from 32.05 GBq of the dried K[18F]F/K222 complex. The
product could be obtained in specific radioactivity of 35 GBq/μmol and
radiochemical yield (RCY) of 1.8% (decay corrected) and was stable in
PBS over 2 h.
4-((4-(Di-tert-butylfluorosilyl)phenethyl)amino)-2,3-dihydroxy-4-
oxobutanoyl-Ala(SO3H)-Ala(SO3H)-Ava-Gln-Trp-Ala-Val-NMeGly-
His-Sta-Leu-NH2 ([18F]43). A solution of 42 (2.0 mg) and glacial acetic
acid (10 μL) in anhydrous DMSO (150 μL) was added to the dry
K[18F]F/K222 complex and heated at 110 °C for 20 min. An aliquot of
the crude reaction mixture was analyzed using an analytical HPLC (ACE
C18, 50 mm × 4.6 mm, 5 μm; gradient CH3CN/H2O + 0.1% TFA 5:95
→ 95:5 in 20 min, 1.0 mL/min) before addition of H2O/CH3CN (9:1, 2
mL) into the reaction vial. The diluted reaction mixture was injected
into a semipreparative HPLC (ACE C18, 250 mm × 10 mm, 5 μm;
isocratic CH3CN/H2O + 0.1% TFA 40:60, 4.0 mL/min), and the
product peak was collected. The product fraction was diluted with water
(20 mL) and immobilized on a C18 cartridge (Sep-Pak light C18,
Waters, or Chromafix C18 (s), Machery-Nagel). After washing with
water (20 mL), [18F]43 was eluted with ethanol (1 mL). The solvent
was evaporated at 90 °C. For in vivo applications, [18F]43 was
reconstituted in 0.15 M PBS containing ≤5% ethanol (v/v), and the
solution was filtered sterile. The overall synthesis time was 80 min.
Reverse-phase HPLC revealed a radiochemical purity ≥90%. The
synthesis afforded 190 MBq of [18F]43 starting from 29.68 GBq of the
dried K[18F]F/K222 complex. The product could be obtained in specific
radioactivity of 70 GBq/μmol and RCY of 1.1% (decay corrected) and
was stable in PBS over 2 h.
Radiolabeling. No-carrier-added aqueous [18F]fluoride ion was
produced on an IBA Cyclone 18/9 cyclotron by irradiation of 98%
enriched [18O]H2O (2.0 mL) using an 18-MeV proton beam via the
[18O(p,n)18F] nuclear reaction. [18F]Fluoride was trapped on an anion-
exchange resin cartridge (Sep-Pak QMA Light, Waters; preconditioning
with 0.5 M K2CO3 solution (5 mL), water (10 mL), and air (10 mL)).
The cartridge was eluted with a solution of K222 (5.0 mg) and
potassium carbonate (1.0 mg) in H2O (0.3 mL) and CH3CN (1.2 mL).
Solvents were removed by heating at 95 °C for 20 min, applying a gentle
stream of nitrogen. During this time, CH3CN (3 × 1 mL) was added and
evaporated to give the dry K[18F]F/K222 complex. After the radio-
labeling reaction, the identity of the 18F-labeled products was confirmed
by comparison with the HPLC retention time of their nonradioactive
reference compounds or by coinjection using analytical radio-HPLC
(gradient CH3CN/H2O + 0.1% TFA 5:95−95:5 in 20 min, 1.0 mL/
min). For the analysis of crude reaction mixture, an ultraperformance
liquid chromatography (UPLC, Waters) system with an Acquity UPLC
BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters) and an attached
coincidence detector (FlowStar LB513, Berthold) was used (gradient
CH3CN/H2O + 0.1% TFA 5:95 → 95:5 in 4 min, 0.6 mL/min).
2-(Di-tert-butyl[18F]fluorosilyl)ethanol ([18F]2). A solution of 25
(2.0 mg) and glacial acetic acid (10 μL) in anhydrous DMSO (150 μL)
was added to the dry K[18F]F/K222 complex and heated at 110 °C for
20 min. An aliquot of the crude reaction mixture was analyzed using an
analytical UPLC to show an 18F-incorporation of ≥95%.
Hydrolytic Stability of the Si−18F Bond. The reaction mixture
containing [18F]2 or [18F]18 was diluted in water (2.0 mL) and passed
through on a C18 cartridge (Sep-Pak light C18, Waters, or Chromafix
C18 (s), Machery-Nagel). After washing with water (5.0 mL), [18F]2 or
[18F]18 was eluted with ethanol (1.0 mL) and aliquots were diluted in
either water, 0.9% NaCl, or 0.15 M PBS. Analysis was performed at
various time points (30, 50, 75, 80, 100, 210 min) using an UPLC
(Waters) system with an Acquity UPLC BEH C18 column (2.1 mm ×
50 mm, 1.7 μm, Waters) and an attached coincidence detector
(FlowStar LB513, Berthold, gradient CH3CN/H2O + 0.1% TFA 5:95 →
95:5 in 4 min, 0.6 mL/min). The hydrolysis of [18F]2 or [18F]18 was
followed by formation of corresponding amounts of 18F. The half-lives
2-Amino-3-(1-(2-(di-tert-butyl[18F]fluorosilyl)ethyl)-1H-1,2,3-tria-
zol-4-yl)propanoic Acid ([18F]18). A solution of 27 (2.0 mg) and glacial
J
dx.doi.org/10.1021/jm400857f | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were derived from the linear function of time and the amount of intact
labeled compound.
Present Address
∥L.M.D.: Piramal Imaging, Tegelerstrasse 6-7, 13353 Berlin,
Germany.
log D7.4 Measurement. The determination of log D7.4 was carried
out in analogy to a published procedure.35 Briefly, [18F]39 was added to
a mixture of PBS (0.5 mL, pH = 7.4) and 1-octanol (0.5 mL) at room
temperature. The mixture was equilibrated for 15 min in an overhead
shaker and further centrifuged (3 min, 5000 rpm). Aliquots (50 μL) of
each of two phases were analyzed in a γ-counter. The partition
coefficient is expressed as the ratio between the radioactivity
concentrations (cpm/mL) of the 1-octanol and PBS phase. Values
represent the mean standard deviation of three determinations from
one experiment.
Animals. Animal studies complied with Swiss laws on animal
protection and husbandry and were approved by the Veterinary office of
the Canton Zurich. After an acclimatization period, tumor xenografts
were produced in 6-week-old male NMRI nude mice (Charles River) by
subcutaneous injection in the right shoulder region of 5 × 106 PC-3cells
in 100 μL of PBS (pH 7.4) under 2−3% isoflurane anesthesia. PET and
ex vivo biodistribution experiments were conducted when the
xenografts reached a volume of about 1 cm3.
Small Animal PET. Xenograft-bearing animals (33−35 g, n = 3)
were injected via tail vein with [18F]39 (8.3−15.3 MBq in 100−150 μL
of PBS containing ≤5% ethanol, 390−630 pmol). To determine specific
binding, an additional group of mice (32−34 g, n = 3) received 50 μg of
nonradioactive bombesin coinjected with [18F]39 (4.8−10.5 MBq in
110−150 μL of PBS containing ≤5% ethanol, 510−575 pmol). In a
preliminary study, xenograft-bearing animals (30 and 32 g) were
administered [18F]43 (5.1 MBq in 100 μL of PBS containing ≤5%
ethanol, 93 pmol, n = 1 or 3.6 MBq in 100 μL PBS containing ≤5%
ethanol, 96 pmol, and n = 1). Anesthesia was induced with 5% isoflurane
(Abbott) in O2/air 10 min before PET acquisition. Depth of anesthesia
and body temperature were controlled as described by Honer et al.36
PET scans were performed under 2−3% isoflurane anesthesia with a GE
VISTA eXplore PET/CT tomograph. Static scans in two bed positions
(15 min upper body followed by 15 min lower body) with [18F]39 were
carried out 60−105 min after injection. Dynamic scans with [18F]43
were acquired in one bed position (list mode) from 2 to 92 min or from
80 to 170 min after tracer injection. Data were reconstructed by two-
dimensional ordered-subset expectation maximization (2D OSEM);
dynamic scans were reconstructed into 5 min time frames. Region of
interest analysis was conducted with the PMOD 3.3 software (PMOD,
Switzerland). Standardized uptake values (SUV) were calculated as a
ratio of tissue radioactivity concentration (kBq/cm3) and injected
activity dose per gram body weight (kBq/g), both decay-corrected.
Ex Vivo Biodistribution. Animals used for PET imaging were
subsequently sacrificed for ex vivo biodistribution (see section above for
amount of radioactivity injected). Animals (n = 6) injected with [18F]39
were sacrificed at 117 min after injection. Animals, which were
administered with [18F]43, were sacrificed at 104 min (n = 1) or 182 min
(n = 1) after injection. Organs and tissues of interest were collected and
weighed, and the amount of radioactivity was determined in a γ-counter
to calculate percentage uptake (% injected dose per gram of tissue).
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Claudia Keller, Peter Friese, Katrin
Dinse, Dr. Thomas Nauser, and Mathias Nobst for their
experimental assistance and technical support. We also thank
Dr. Steven Donald for carrying out some of the early calculations
on selected compounds.
ABBREVIATIONS USED
■
AcOH, acetic acid; β+, positron; C−F, carbon−fluorine;
CH3CN, acetonitrile; CuAAC, copper-catalyzed azide−alkyne
cycloaddition; DFT, density functional theory; DIPEA, N,N-
diisopropylethylamine; DMTMM-BF4, 4-(4,6-dimethoxy-1,3,5-
triazin-2-yl)-4-methylmorpholinium tetrafluoroborate; EOS,
end of synthesis; EtOAc, ethylacetate; 18F, fluorine-18; GBq,
gigabecquerel; GRPr, gastrin-releasing peptide receptor; HBTU,
O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluoro-
phosphate; HOBT, 1-hydroxybenzotriazole; KF, potassium
fluoride; K222, Kryptofix 222; log D7.4, logarithmic distribution
coefficient; MBq, megabecquerel; n-BuLi, n-butyllithium; NMM,
N-methylmorpholine; OH−, hydroxide anion; pi, postinjection;
PC-3, human prostate adenocarcinoma; PET, positron emission
tomography; RP, reversed phase; Si−CH2, silicon−methylene;
Si−F, silicon−fluorine; SENs, shared electron numbers; SUV,
standardized uptake values; T3P, 2,4,6-tripropyl-1,3,5,2,4,6-
trioxatriphosphorinane 2,4,6-trioxide; UPLC, ultraperformance
liquid chromatography
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ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC chromatograms of the quality control of compounds 18F-
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Corresponding Author
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