Synthesis, dynamic NMR characterization and XRD studies of novel N,N′-substituted piperazines for bioorthogonal labeling

Article abstract of DOI:10.3762/bjoc.12.242

Novel, functionalized piperazine derivatives were successfully synthesized and fully characterized by ¹H/¹³C/¹⁹F NMR, MS, elemental analysis and lipophilicity. All piperazine compounds occur as conformers resulting from the partial amide double bond. Furthermore, a second conformational shape was observed for all nitro derivatives due to the limited change of the piperazine chair conformation. Therefore, two coalescence points were determined and their resulting activation energy barriers were calculated using ¹H NMR. To support this result, single crystals of 1-(4-nitrobenzoyl)piperazine (3a, monoclinic, space group C2/c, a = 24.587(2), b = 7.0726(6), c = 14.171(1) ?, β = 119.257(8)°, V = 2149.9(4) ?³, Z = 4, D_obs = 1.454 g/cm³) and the alkyne derivative 4-(but-3-yn-1-yl)-1-(4-fluorobenzoyl)piperazine (4b, monoclinic, space group P2₁/n, a = 10.5982(2), b = 8.4705(1), c = 14.8929(3) ?, β = 97.430(1)°, V = 1325.74(4) ?³, Z = 4, D_obs = 1.304 g/cm³) were obtained from a saturated ethyl acetate solution. The rotational conformation of these compounds was also verified by XRD. As proof of concept for future labeling purposes, both nitropiperazines were reacted with [¹⁸F]F^-. To test the applicability of these compounds as possible ¹⁸F-building blocks, two biomolecules were modified and chosen for conjugation either using the Huisgen-click reaction or the traceless Staudinger ligation.

Full text of DOI:10.3762/bjoc.12.242

Synthesis, dynamic NMR characterization and XRD studies of
novel N,N’-substituted piperazines for bioorthogonal labeling
Constantin Mamat*1,2, Marc Pretze3, Matthew Gott1 and Martin Köckerling4
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 2478–2489.
1Helmholtz-Zentrum Dresden-Rossendorf, Institut für
Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400,
D-01328 Dresden, Germany, 2Technische Universität Dresden,
Fachrichtung Chemie und Lebensmittelchemie, D-01062 Dresden,
Germany, 3Medizinische Fakultät Mannheim der Universität
Heidelberg, Institut für Klinische Radiologie und Nuklearmedizin,
Theodor-Kutzner-Ufer 1-3, D-68167 Mannheim, Germany and
4Universität Rostock, Institut für Chemie – Festkörperchemie,
Albert-Einstein-Straße 3a, D-18059 Rostock, Germany
doi:10.3762/bjoc.12.242
Received: 10 August 2016
Accepted: 31 October 2016
Published: 21 November 2016
Associate Editor: J. A. Murphy
© 2016 Mamat et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Constantin Mamat* - c.mamat@hzdr.de
* Corresponding author
Keywords:
building blocks; coalescence; dynamic NMR; labeling; Staudinger
ligation
Abstract
Novel, functionalized piperazine derivatives were successfully synthesized and fully characterized by 1H/13C/19F NMR, MS,
elemental analysis and lipophilicity. All piperazine compounds occur as conformers resulting from the partial amide double bond.
Furthermore, a second conformational shape was observed for all nitro derivatives due to the limited change of the piperazine chair
conformation. Therefore, two coalescence points were determined and their resulting activation energy barriers were calculated
using 1H NMR. To support this result, single crystals of 1-(4-nitrobenzoyl)piperazine (3a, monoclinic, space group C2/c,
a = 24.587(2), b = 7.0726(6), c = 14.171(1) Å, β = 119.257(8)°, V = 2149.9(4) Å3, Z = 4, Dobs = 1.454 g/cm3) and the alkyne deriv-
ative 4-(but-3-yn-1-yl)-1-(4-fluorobenzoyl)piperazine (4b, monoclinic, space group P21/n, a = 10.5982(2), b = 8.4705(1),
c = 14.8929(3) Å, β = 97.430(1)°, V = 1325.74(4) Å3, Z = 4, Dobs = 1.304 g/cm3) were obtained from a saturated ethyl acetate solu-
tion. The rotational conformation of these compounds was also verified by XRD. As proof of concept for future labeling purposes,
both nitropiperazines were reacted with [18F]F–. To test the applicability of these compounds as possible 18F-building blocks, two
biomolecules were modified and chosen for conjugation either using the Huisgen-click reaction or the traceless Staudinger ligation.
Introduction
The development of new building blocks for specific and bio- blocks can influence (bio-)chemical parameters such as the
orthogonal labeling of biologically active compounds is of high lipophilicity (log P) affecting the solubility and the biological
importance. Depending on their size and composition, building behavior of the resulting labeled biomolecule [1]. To diversify
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and regulate this behavior, compounds with a piperazine a proof of concept study was performed for the site-selective
skeleton were chosen as excellent candidates for bispecific labeling of a pharmacologically active peptide and a small
modification.
organic compound. These compounds provide the respective
reference compounds for later radiolabeling purposes. Finally, a
Simple alkylated and acylated secondary amines/amides are procedure to synthesize the building blocks containing
known from the literature [2] as well as their NMR properties fluorine-18 was evaluated.
[3]. The widely used solvent DMF is an intensely investigated
Results and Discussion
example [4]. Functionalized piperazines [5], especially benzoy-
lated derivatives, are our interest. In the past, mono or bisacy- Synthesis of the piperazine compounds
lated piperazines with benzamide [6], nicotinamide [7] and Low-cost starting materials were applied for the preparation of
isonicotinamide [8-11] residue were explored in terms of their all novel building blocks. First, piperazine (2) was reacted with
NMR and complexation behavior. However, unsymmetrically functionalized benzoyl chlorides 1a,b to yield the mono-
substituted piperazines are rarely investigated [12,13].
acylated amides 3a,b according to literature procedures [14].
Next, the necessary functional groups for the later click reac-
N,N’-Unsymmetrically functionalized piperazines are the basis tions to connect the resulting building blocks to biomolecules
of the development of our new building blocks. In this case, one were introduced. Thus, 3a,b were alkylated with 4-tosylbutyne
of the nitrogen atoms is used for the connection to the label to give compounds 4a,b in high yields of 84% and 82%, respec-
(e.g., fluorescence dye, radionuclide) and the second is used for tively. These compounds are applicable in the classical Cu-cata-
the introduction of a (bioorthogonal) functional group (e.g., lyzed Huisgen-click reaction with azide-functionalized, biologi-
azide, alkyne, phosphane, tetrazine) to later connect to the bio- cally active molecules. Additionally, 3a,b were reacted with
molecule via bioorthogonal ligation.
3-azidopropyl tosylate to yield compounds 5a,b in high yields
of 87% and 81%, respectively. These derivatives can be utilized
Our aim was the development of novel, N,N’-unsymmetrically for both variants of the Staudinger ligation in addition to the
functionalized piperazine derivatives using a high-yielding, classical and strain-promoted variants of the Huisgen-click
simple synthesis with either a bioorthogonal alkyne or azide reaction. For future radiolabeling procedures, nitro derivatives
functionality for future labeling purposes. Furthermore, charac- 4a and 5a serve as starting material (precursor) whereas fluo-
terization of these piperazine derivatives was performed with rine compounds 4b and 5b function as appendant reference
emphasis on their particular NMR behavior. In order to demon- compounds to analyze the prospective 18F-containing com-
strate the Cu-catalyzed azide–alkyne click reaction (Huisgen pounds. The reaction pathway for all piperazines is illustrated in
1,3-dipolar cycloaddition) and the traceless Staudinger ligation, Scheme 1.
Scheme 1: Preparation of the nitro derivatives 4a and 5a and the fluorine-containing compounds 4b and 5b. Reagents and conditions: a) 1a, Et3N,
CHCl3, 0 °C, 2 h, 0 °C then 2 h, rt; b) 1b, acetonitrile, 1 M HCl, rt, 4 h; c) 4-tosylbutyne, Et3N, THF, 60 °C, 3 d; d) 3-azidopropyl tosylate, Et3N, THF,
60 °C, 3 d.
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Dynamic NMR studies
Two effects are responsible for this behavior. The first arises
During the full characterization of our compounds, we ob- from the presence of two different conformers (rotamers); this
served a quite unusual behavior in the 1H and 13C NMR spec- is caused by the limited interconversion by rotation about the
tra. Four broad singlets (ratio: 1:1:1:1) were observed in the C–N amide bond resulting from the partial double bond char-
1H NMR spectra of all nitro compounds 3a, 4a, and 5a (see acter of N,N-dialkylated amides as shown in Figure 3. This ob-
Supporting Information File 1) and three broad singlets (ratio servation is typical and can be found for symmetrically substi-
1:1:2) were determined for all fluorine compounds 3b, 4b, and tuted amides. The best known and widest investigated example
5b measured in CDCl3 at 25 °C. Normally, under these condi- is DMF. Two distinct signals are observed in the 1H NMR spec-
tions only two signals are expected for the NCH2-protons of un- trum of DMF as a result of two structurally different methyl
symmetrically substituted piperazines [15-17].
groups (R1 ≠ R2) attached to the amide nitrogen [18,19]. In
general, this behavior of symmetric N,N-dialkylamide spin
To investigate this phenomenon, nitro compound 3a was chosen systems is describable as first-order process on the NMR time
and 1H NMR spectra were measured in five different solvents to scale [20].
confirm this behavior. The results are illustrated in Figure 1. As
an example, the spectrum of 3a shows four broad signals The same NMR behavior is observed for 4-nitrobenzoyl amides
(δ = 2.81, 2.96, 3.33, 3.97 ppm in CDCl3 at 25 °C) for the 3a, 4a and 5a. The expected single signal of the CH2 groups at-
piperazine NCH2 groups and evaluation of a H,H-COSY tached to the amide nitrogen is duplicated as seen for 3a in
measured in CDCl3 showed an independent coupling of two Figure 1. The coupling is pointed out by the analysis of a
NCH2 groups (Figure 2). Next, the HSQC spectra showed the H,H-COSY of 3a (Figure 2, small spectrum).
independent coupling of the protons to the appropriate carbon
signals (further detailed NMR spectra can be found in Support- The second effect is related to the reduced flipping of the piper-
ing Information File 1). Additionally, four broad signals for azine ring at the amine nitrogen (Figure 3, bottom). In our case,
the carbons of the NCH2 groups (e.g., 3a: δ = 43.7, 46.0, interconversion of the amine is also reduced at room tempera-
46.3, 49.0 ppm) are found when analyzing the 13C NMR spec- ture. Normally, such formation of conformers is found for
tra.
piperazines [21,22] and morpholines [21,23] only at lower tem-
Figure 1: 1H NMR spectra of compound 3a measured in five different solvents: (A) CDCl3, (B) DMSO-d6, (C) acetone-d6, (D) methanol-d4 and
(E) acetonitrile-d3 (orange: region of interest).
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and 5a as well as for fluorine compound 3b. When monitoring
compound 3a over a minimum range of 45 K, the four signals
of the NCH2 groups gradually disappear and coalesce to the two
expected signals at increased temperatures (>67 °C). At the coa-
lescence temperature Tc, the exchange rate is given by the equa-
tion kexc = π·Δν/21/2 [25]. As a result, the activation energy
(ΔG#exp) to the amide bond rotation can be calculated using the
Eyring equation [14,16].
In general, the difference in chemical shifts Δδ (as well as Δν)
strongly depends on the nature of the solvent [26]. Thus, the
1H NMR spectra seen in Figure 1 were recorded in CDCl3,
DMSO-d6, acetone-d6, methanol-d4 and acetonitrile-d3 at
400 MHz and the results are summarized in Table 1. The NCH2
groups of 3a show the highest difference Δν1 and Δν2 when dis-
solved in CDCl3 and the lowest when dissolved in acetone-d6.
Figure 2: HSQC and H,H-COSY (small spectrum) of compound 3a
measured in CDCl3 at 25 °C. The independent coupling of the NCH2
groups is marked.
Table 1: NMR parameters Δν and kexc of nitro compound 3a
measured in dependence of the solvent.
solvent
Δν1 [Hz] Δν2 [Hz] kexc,1 [Hz] kexc,2 [Hz]
CDCl3
59.5
55.2
49.5
53.3
30.4
176.2
162.2
157.6
155.8
133.2
132.2
122.6
110.0
118.4
67.5
391.4
360.3
350.1
346.1
295.9
acetonitrile-d3
DMSO-d6
methanol-d4
acetone-d6
Due to the low boiling point of most of these solvents, DMSO-
d6 was chosen for the measurement of the coalescence tempera-
ture via temperature-dependent 1H NMR (Figure 4). For 3a, the
Tc,1 was determined to be 50 °C (322 K) and Tc,2 was 67 °C
(340 K). Using this data, the activation energies ΔG#exc were
calculated to be 66.7 and 67.1 kJ/mol, respectively (Table 2).
Figure 3: Illustration of the general partial double bond character of an
amide bond and the limited isomerization of the amine nitrogen atom
which results in possible conformers of piperazine compounds.
For fluorine compound 3b, only the CH2 group attached to the
amide nitrogen is split in two signals at room temperature and
peratures (below −10 °C). Additionally, only the protons of its Tc was found to be 33 °C (306 K) resulting in a ΔG#exc of
4-nitrobenzoylamides 3a, 4a and 5a exhibit this behavior at 61.1 kJ/mol. This result nicely demonstrates the influence of the
room temperature. Consequently, this phenomenon is strongly substituent in the ortho-position (F vs NO2) of the benzoate
influenced by the substituent in the para-position of the residue.
benzoate (F vs NO2).
Next, the influence of alkyl groups on Tc and ΔG#exp were in-
Both effects resulted in two different coalescence points de- vestigated using both nitrobenzoylpiperazines 4a and 5a. Both
pendent upon their different energy barriers. In order to further coalescence temperatures are found to be lower than those of
investigate the conformational behavior of the piperazines and the non-alkylated derivative 3a. This effect could arise due to
to determine these energies, temperature-dependent NMR ex- the increased steric demand of the alkyl groups compared to the
periments [24] were performed for all nitro derivatives 3a, 4a, sole hydrogen connected to the amine nitrogen.
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Figure 5: Molecular structure of compound 3a (ORTEP plot with 50%
probability level).
Figure 4: Temperature-dependent 1H NMR spectra of 3a measured in
DMSO-d6 (aliphatic region of the piperazine protons from 2.00 to
4.00 ppm is shown).
When comparing our results with the literature, Tc and ΔG#exp
found for the amide site of the piperazines are in good agree-
ment with the previously published (Tc = 330–340 K,
ΔG#exp = 61–68 kJ/mol) [21,22,27]. In contrast, our values for
Tc,1 and ΔG#exp,1 for the amine residues are much higher as
found in the literature. For instance, N-alkylated morpholines
showed a Tc of 248 K with a ΔG# of 11.1 kJ/mol [23] whereas
piperidine shows a higher ΔG# of 42.3 and N-methylpiperidine
a ΔG# = 49.8 kJ/mol [21,28].
Figure 6: Molecular structure of compound 4b (ORTEP plot with 50%
probability level).
tions. The amine-type N2 atom in 4b has three bonds to the
neighboring carbon atoms of about the same length (N2–C9:
1.466(1) Å, N2–C11: 1.466(1) Å, N2–C12: 1.464(1) Å), but the
amide-type N1 atom has a significantly shorter distance to the
C1 atom (1.350(1) Å), which carries the carbonyl O atom. The
other two contacts of N1 to the C atoms of the piperazine ring
are in the same distance range (N1–C8: 1.461(1) Å and
N1–C10: 1.461(1) Å) as those of the N2 atom.
X-ray structure analyses of 3a and 4b
Single crystals of 3a and 4b were obtained and their molecular
structures determined using single crystal X-ray structure analy-
sis. Crystals of 3a have monoclinic symmetry of the space
group C2/c. Crystals of 4b have monoclinic symmetry of the
space group P21/n. The C14–C15 distance of 1.188(1) Å and
the C13–C14–C15 angle of 178.2(1)° clearly indicate this group
to be an alkyne residue, thus enabling the use of the Huisgen-
click reaction for binding to the target biomacromolecule. The
molecular structures of these compounds and their atom Furthermore, the environment of the N2 atom can best de-
numbering schemes are shown in Figure 5 and Figure 6. scribed as pyramidal with an average C–N2–C angle of 109.8°,
whereas the environment of the N1 atom is almost planar, with
The limited ability of the residues to rotate along the C1–N1 an average C–N1–C angle of 119.9°. All these results indicate a
bond, as observed by NMR spectroscopy (see above), is sup- partial double-bond character for the C1–N1 bond with a
ported by the results of both single-crystal structure determina- limited rotational ability. As discussed above, this results in two
Table 2: Evaluation of the Gibb’s energy ΔG#exc of compounds 3a, 3b, 4a and 5a from NMR data measured in DMSO-d6.
Δν1
Δν2
kexc,1
kexc,2
Tc,1 [K]
Tc,2 [K]
ΔG#exc,1 [kJ/mol] ΔG#exc,2 [kJ/mol]
3a
3b
4a
5a
49.5
n.d.
157.6
106.6
154,0
154.9
110.0
n.d.
350.1
236.8
342.1
344.1
323
n.d.
320
315
340
306
338
338
66.7
n.d.
67.1
61.1
66.7
66.7
48.5
48.6
107.7
108.0
66.1
65.0
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conformers, as shown for solutions of 4b by NMR spectrosco- and was modified with an azide moiety at the C-terminus to
py. In the solid state, the molecules of the asymmetric unit are yield SNEWILPRLPQH-Azp 6 (the synthesis is reported else-
located on a side without any symmetry (besides identity). where) [29]. The structure of 6 and the click reaction is shown
Through the symmetry element besides the molecule (i.e., in Scheme 2. For labeling purposes, building block 4b (10-fold
inversion center) in the solid state, two conformers exist in a excess) was added to peptide 6 which was dissolved in
ratio of 1:1. Figure 7 shows a superimposed figure of the two PBS buffer (pH 7.4), followed by addition of freshly prepared
conformers, where C1, the phenyl ring and F1 of both solutions of Na ascorbate and CuSO4. The mixture was stirred
conformers are fitted on top of each other. The different struc- for 16 h at 40 °C and the desired product 7 was purified by
tural arrangement of both conformers is clearly visible.
semi-preparative HPLC.
The second proof of concept study was performed under
Staudinger conditions using a small inhibitory molecule for the
EphB4 receptor. For this purpose, the azide-containing building
block 5b was reacted with 8 [30] to give 9. The resulting refer-
ence compound 9 was obtained after 3 h reaction time in a high
yield of 76%. The reaction is shown in Scheme 3.
Preparation of bioorthogonal 18F-containing
building blocks
The development of new 18F-based radiotracers remains an
ongoing goal in the field of radiopharmacy and provides tools
for specific cancer diagnostics using positron emission tomog-
raphy (PET) [31-33]. When radiotracers are based on tumor-
specific peptides, proteins or antibodies [34,35], mild and fast
methods for radiolabeling are mandatory because of the short
half-live of [18F]fluoride (t1/2: 110 min). In most cases, indirect
radiolabeling is used for these more sensitive biomacromole-
cules due to the harsh conditions (i.e., organic solvents, high
temperatures and basic conditions) for the direct incorporation
of [18F]fluoride, which can alter the biological/pharmacological
behavior or, at least, destroy sensitive biomolecules [36-38].
For this purpose, bioorthogonal building blocks were de-
veloped using Huisgen-click or the Staudinger ligation.
Figure 7: Superimposition fit of the two conformers, which exist in the
ratio of 1:1 in the solid state structure of compound 4b (label A marks
the atoms of the second conformer).
Using these methods, a synthesis procedure for both bioorthog-
onal 18F-building blocks [18F]4b and [18F]5b was developed
Similar structural features are observed for 3a. The N1–C1 from their precursors 4a and 5a, respectively (Scheme 4). For
atom distance is again much shorter, 1.346(2) Å, than the other this purpose, the nitro group of 4b and 5b was replaced by
N–C bonds within the piperazine moiety, which range from [18F]fluoride in a nucleophilic aromatic exchange reaction. To
1.462(2) to 1.466(1) Å. Furthermore, the average bond angle of evaluate the radiolabeling process, the precursor 4a (approx.
119.8° of N1 indicates double-bond character and thereby 3–4 mg) was first dissolved in anhydrous acetonitrile and added
limited rotational ability of the residues along the N1–C1 bond. to dry [18F]fluoride (typically 0.5–1 GBq), but the radiochem-
As found for 4b, crystals of 3a contain two isomers in a ical yield (RCY) did not exceed 5% after 60 min at 100 °C.
1:1 ratio as imposed by crystal symmetry.
Thus, anhydrous DMSO was chosen. After addition of
[18F]fluoride the resulting mixture was stirred for 30 min at
150 °C and the 18F-building blocks were obtained in 12% RCY.
An elongation of the reaction time to 60 min afforded [18F]4b
Sample ligation using Huisgen-click and
traceless Staudinger
As a proof of labeling concept, fluorine compound 4b was in an elevated RCY of approx. 20% (80–155 MBq, decay
clicked to peptide 6 using the Cu-catalyzed Huisgen-click reac- corrected) after purification. Similar results were obtained for
tion. This SNEW peptide (SNEW: Ser-Asn-Glu-Trp) was the labeling of azide-containing [18F]5b under the same reac-
chosen due to its biologically and pharmacologically activity tion conditions. The radio-TLC of the reaction mixture of
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Scheme 2: Peptide labeling using the Huisgen-click reaction and building block 4b. Reagents and conditions: a) 4b, CuSO4, Na ascorbate,
PBS buffer (pH 7.4), 40 °C, 16 h.
Scheme 3: The traceless Staudinger ligation to yield compound 9. Reagents and conditions: a) acetonitrile/water 10:1 (v/v), 60 °C, 3 h.
Scheme 4: Preparation of the radiolabeling building blocks [18F]4b and [18F]5b. Reagents and conditions: a) K[18F]F, Kryptofix K 2.2.2, DMSO,
150 °C, 60 min.
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[18F]5b and the appropriate radio-TLC and (radio-)HPLC chro- found in the spectra of the fluorine compounds whereas two dif-
matograms are shown in Figure 8 and Figure 9. Compared to ferent coalescence points were found for nitro compounds. A
other alkyne and azide functionalized building blocks described partial double bond in the amide residue and the limited ring
in the literature [39], the radiochemical yields and the reaction conversion of the amine site led to rotational conformers. Acti-
times of [18F]4b and [18F]5b are similar.
vation energies ΔG#exc were calculated from coalescence tem-
peratures Tc which were determined from dynamic 1H NMR
measurements. The formation of conformers resulting from the
partial double bond was additionally determined and verified
using single crystal X-ray structure analysis. The second coales-
cence point is caused by the reduced flipping of the amine part
in the nitro compounds. Furthermore, the coalescence tempera-
ture for the amine residue is higher than expected for amines. In
contrast, the reduced flipping of the amine part was not found
for the fluorine compounds. Thus, the coalescence is dependent
on the substituent of the benzoyl moiety. Furthermore, a proof
of concept was accomplished with a pharmacologically active
peptide using the Huisgen-click reaction and compound 4b. Ad-
ditionally, 5b was introduced in a small molecule using the
traceless Staudinger ligation. The synthesis of the 18F-labeled
building blocks [18F]4b and [18F]5b was accomplished using
the SNAr concept. The appropriate precursors and reference
compounds were prepared in two steps from simple commer-
cially available starting materials in high yields, but at this stage
the building blocks are not appropriate for further labeling
purposes.
Figure 8: Radio-TLC (eluent: ethanol) of [18F]5b (Rf = 0.50; reaction
mixture).
Experimental
General information
All chemicals were purchased from commercial suppliers and
used without further purification unless otherwise specified.
Anhydrous THF was purchased from Acros. NMR spectra of all
compounds were recorded on an Agilent DD2-400 MHz NMR
spectrometer. Chemical shifts of the 1H, 19F, and 13C spectra
were reported in parts per million (ppm) using TMS as internal
standard for 1H/13C and CFCl3 for 19F spectra. Mass spectro-
Figure 9: (Radio)HPLC of 5a (tR = 7.7 min, UV trace, red),
5b (tR = 6.7 min, UV trace: blue) and [18F]5b (tR = 7.1 min, γ-trace,
green). The differences in the retention times of 5b and [18F]5b occurs
due to the setup of UV- and γ-detector of the HPLC device.
Finally, the partition coefficients (log P) were determined to be metric (MS) data were obtained on a Xevo TQ-S mass spec-
1.26 ± 0.01 (n = 3) for compound 4b and 1.45 ± 0.01 (n = 3) for trometer (Waters) by electron spray ionization (ESI). The
5b. Compared to other standard building blocks like [18F]SFB melting points were determined on a Galen III melting point
(log P = 1.8) [40], FBAM (log P = 2.7) [41], 4-fluorobenzalde- apparatus (Cambridge Instruments & Leica) and are uncor-
hyde (log P = 1.9) [42] or the click labeling building block rected. Microanalyses were carried out with an LECO CHNS
p-[18F]F-SA (log P = 1.7) [43], the lipophilicity of our 932 elemental analyzer. Chromatographic separations and TLC
18F-building blocks is reduced. The hydrophilic character of the detections were performed using Merck Silica Gel 60
building blocks enables a radiolabeling in aqueous solutions and (63–200 μm) and Merck Silica Gel 60 F254 sheets, respectively.
the influence of molecular size of the compounds to be labeled TLCs were developed by visualization under UV light
is negligible.
(λ = 254 nm). Diffraction data were collected with a Bruker-
Nonius Apex-II-diffractometer using graphite-monochromated
Mo Kα radiation (λ = 0.71073 Å). The diffraction measure-
Conclusion
Novel piperazine derivatives were successfully synthesized with ments were performed at −150 °C. The unit cell dimensions
high yields using a convenient synthesis procedure. Evaluation were recorded and refined using the angular settings of
of the NMR spectra showed doubled signals of the NCH2 7728 reflections for 4b and 6451 reflections for 3a. The struc-
groups of the piperazine moiety. One coalescence point was tures were solved by direct methods and refined against F2 by
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full-matrix least-squares using the program suites from G. M. 4-fluorobenzoyl chloride (1b, 1.1 g, 6.94 mmol) dissolved in
Sheldrick [44,45]. All non-hydrogen atoms were refined acetonitrile (5 mL) was added dropwise and the resulting mix-
anisotropically; all hydrogen atoms except the one attached to ture was stirred for 4 h at rt. Afterwards, additional 9 mL of
the alkyne group (4b) were placed on geometrically calculated 1 M HCl were added and the aqueous layer was extracted with
positions and refined using a riding model. The alkyne-H atom ethyl acetate (2 × 20 mL). Then, KOH was added to the
was refined isotropically. CCDC 1479835 and CCDC 1445857 aqueous phase until pH 8 was reached. The aqueous layer was
contain the supplementary crystallographic data for compounds again extracted with chloroform (2 × 25 mL), the combined
3a and 4b. These data can be obtained free of charge from organic layers were dried over Na2SO4 and the solvent was re-
The Cambridge Crystallographic Data Centre via http:// moved to yield 931 mg of compound 3b. Spectra are in agree-
www.ccdc.cam.ac.uk/data_request/cif. Analytical (radio-)HPLC ment with those found in the literature [46].
was performed on a VWR/Hitachi Elite LaChrome HPLC
system, equipped with a reverse-phase column (Nucleosil 100- 4-(But-3-yn-1-yl)-1-(4-nitrobenzoyl)piperazine (4a): Com-
5C18 Nautilus, Machery Nagel), a UV diode array detector pound 3a (110 mg, 0.47 mmol), but-3-yn-1-yl tosylate (150 mg,
(250 nm), and a scintillation detector (Raytest, Gabi Star) at a 0.67 mmol) and Et3N (100 mg, 0.98 mmol) were dissolved in
flow rate of 1 mL/min. The radioactive compounds were identi- anhydrous THF (10 mL) and the resulting mixture was stirred at
fied with analytical radio-HPLC by comparison of the retention 60 °C for 3 d. After reaction control by TLC, THF was changed
time of the reference compounds. Decay-corrected radiochem- by ethyl acetate (15 mL), water (15 mL) was added and the
ical yields (RCYs) were quantified by integration of radioac- aqueous layer was extracted with ethyl acetate (3 × 15 mL). The
tive peaks on a radio-TLC using a radio-TLC scanner (Fuji, combined organic layers were dried over Na2SO4, the solvent
BAS2000). [18F]Fluoride was produced using the PET was removed and the crude product was purified via automated
cyclotron Cyclone 18/9 (IBA). [18O]H2O was irradiated with column chromatography (Biotage: 10 g KP-Sil, Gradient: petro-
protons (18 MeV, 30 µA) exploiting the 18O(p,n)18F nuclear leum ether → EtOAc) to yield 4a (112 mg, 84%) as yellow
reaction.
solid. Rf = 0.59 (ethanol); mp 76 °C; analytical HPLC:
tR = 6.0 min (eluent: CH3CN/H2O, 15:85 + 0.1% TFA);
CAUTION! Hazard warning for organic azides: risk of explo- 1H NMR (400 MHz, CDCl3) δ 1.97 (t, 4J = 2.5 Hz, 1H, ≡CH),
sion by shock, friction, and fire upon heating. Store these com- 2.36 (dt, 3J = 7.3 Hz, 4J = 2.5 Hz, 2H, CH2C≡), 2.43 (br s, 2H,
pounds in a cool location.
Pip-H), 2.51–2.65 (m, 4H, Pip-H, NCH2), 3.36 (br s, 2H, Pip-
H), 3.79 (br s, 2H, Pip-H), 7.55 (d, 3J = Hz, 2H, H-o), 8.25 (d,
3J = Hz, 2H, H-m); 13C NMR (101 MHz, CDCl3) δ 17.0
Synthetical procedures
1-(4-Nitrobenzoyl)piperazine (3a): Piperazine (2, 1.0 g, (CH2C≡), 42.2, 47.6, 52.4, 53.1 (4 × Pip-C), 56.7 (NCH2), 69.5
11.6 mmol) and Et3N (1.17g, 11.6 mmol) were dissolved in (≡CH), 82.4 (CH2C≡), 124.0 (C-m), 128.2 (C-o), 142.0 (C-i),
chloroform (30 mL) and the mixture was cooled to 0 °C. At this 148.4 (C-p), 167.9 (C=O); MS (ESI+) m/z (%): 288 (100) [M+ +
temperature, a solution of 4-nitrobenzoyl chloride (1a, 1.0 g, H]; Anal. calcd for C15H17N3O3 (287.31): C, 62.71; H, 5.96; N,
5.4 mmol) dissolved in 30 mL of chloroform was added drop- 14.63; found, C, 62.51; H, 5.95; N, 14.65.
wise and the resulting mixture was allowed to stir at 0 °C for
2 h and at rt for 2 h. Afterwards, the precipitate was filtered and 4-(But-3-yn-1-yl)-1-(4-fluorobenzoyl)piperazine (4b): Com-
the chloroform solution was washed with saturated hydrogen pound 3b (70 mg, 0.34 mmol), but-3-yn-1-yl tosylate (90 mg,
carbonate solution (30 mL), with water (2 × 30 mL) and dried 0.40 mmol) and Et3N (51 mg, 0.50 mmol) were dissolved in an-
over Na2SO4. Finally, the solvent was removed to yield com- hydrous THF (6 mL) and the resulting mixture was stirred at
pound 3a (700 mg, 55%) as yellowish solid. mp 74 °C; 60 °C for 3 d. After reaction control by TLC, THF was changed
1H NMR (400 MHz, CDCl3) δ 1.72 (s, 1H, NH), 2.81 (br s, 2H, by ethyl acetate (15 mL), water (15 mL) was added and the
Pip-H), 2.96 (br s, 2H, Pip-H), 3.33 (br s, 2H, Pip-H), 3.77 (br aqueous layer was extracted with ethyl acetate (3 × 15 mL). The
s, 2H, Pip-H), 7.56 (d, 3J = 8.7 Hz, 2H, H-o), 8.27 (d, 3J = 8.7 combined organic layers were dried over Na2SO4, the solvent
Hz, 2H, H-m); 13C NMR (101 MHz, CDCl3) δ 43.5, 46.0, 46.6, was removed and the crude product was purified via automated
49.0 (4 × Pip-C), 124.0 (C-m), 128.2 (C-o), 142.2 (C-i), 148.5 column chromatography (Biotage: 10 g KP-Sil, Gradient: petro-
(C-p), 163.1 (C=O); MS (ESI+) m/z (%): 258 (13) [M+ + Na], leum ether → EtOAc) to yield 4b (72 mg, 82%) as colorless
236 (100) [M+ + H]; Anal. calcd for C11H13N3O3 (235.24): C, solid. Rf = 0.60 (ethanol); analytical HPLC: tR = 5.5 min
56.16; H, 5.57; N, 17.86 %; found, C, 56.41; H, 5.35; N, 17.65. (eluent: CH3CN/H2O, 15:85 + 0.1% TFA); 1H NMR
(400 MHz, CDCl3) δ 1.98 (t, 4J = 2.7 Hz, 1H, ≡CH), 2.38 (dt,
1-(4-Fluorobenzoyl)piperazine (3b): Piperazine (2, 2.9 g, 3J = 7.4 Hz, 4J = 2.7 Hz, 2H, CH2C≡), 2.50 (br s, 4H, Pip-H),
33.67 mmol) was dissolved in HCl (1 M, 50 mL). A solution of 2.62 (t, 2H, 3J = 7.4 Hz, NCH2), 3.45 (br s, 2H, Pip-H), 3.75 (br
2486

Beilstein J. Org. Chem. 2016, 12, 2478–2489.
s, 2H, Pip-H), 7.08 (“t”, 3JH,F = 3Jo,m = 8.7 Hz, H-m), 7.40 (dd, (CH2N3), 53.0, 53.5 (2 × Pip-C), 55.2 (CH2N), 115.7 (d, 2JC,F =
2JH,F = 5.4 Hz, 3Jo,m = 8.7 Hz, H-o); 13C NMR (101 MHz, 21.8 Hz, C-m), 129.5 (d, 3JC,F = 8.4 Hz, C-o), 131.9 (d, 4JC,F =
CDCl3) δ 17.0 (CH2C≡), 42.4, 47.8, 52.8 (4 x Pip-C), 56.9 3.3 Hz, C-i), 163.5 (d, 1JC,F = 148.6 Hz, C-p), 163.5 (C=O);
(NCH2), 69.4 (≡CH), 82.5 (CH2C≡), 115.7 (d, 2JC,F = 21.8 Hz, 19F NMR (376 MHz, CDCl3) δ −110.4; MS (ESI+) m/z (%):
C-m), 129.5 (d, 3JC,F = 8.5 Hz, C-o), 131.9 (d, 4JC,F = 3.3 Hz, 292 (100) [M+ + H]; Anal. calcd for C14H18FN5O (291.32): C,
C-i), 163.5 (d, 1JC,F = 249.9 Hz, C-p), 169.5 (C=O); 19F NMR 57.72; H, 6.23; N, 24.04; found, C, 57.90; H, 6.21; N, 24.24.
(376 MHz, CDCl3) δ −110.3; MS (ESI+) m/z (%): 261 (100)
[M+ + H]; Anal. calcd for C15H17FN2O (260.31): C, 69.21; H, SNEW-peptide (7): Azide-functionalized peptide 6 (4.55 mg,
6.58; N, 10.76; found, C, 68.99; H, 6.61; N, 10.80.
2.8 µmol) and 4b (1.00 mg, 3.84 µmol) were dissolved in
PBS buffer (400 µL). Na ascorbate (50 µL, 0.6 M) and CuSO4
4-(3-Azidopropyl)-1-(4-nitrobenzoyl)piperazine (5a): Com- (50 µL, 0.4 M) were added and the reaction mixture was main-
pound 3a (150 mg, 0.64 mmol), 3-azidopropyl tosylate tained at 40 °C for 16 h. Peptide 7 was obtained as colorless
(195 mg, 0.77 mmol) and Et3N (129 mg, 1.28 mmol) were dis- powder (4.01 mg, 76%) after purification using semi-prepara-
solved in anhydrous THF (10 mL) and the resulting mixture tive HPLC and lyophilization; MS (ESI+) m/z: calcd, 1886
was stirred at 60 °C for 3 d at rt. After reaction control by TLC, [M]+; found, 944 [M + 2H]2+.
THF was changed by ethyl acetate (15 mL), water (15 mL) was
added and the aqueous layer was extracted with ethyl acetate 5-((3-((4-((5-Chlorobenzo[d][1,3]dioxol-4-yl)amino)pyrim-
(3 × 15 mL). The combined organic layers were dried over idin-2-yl)amino)phenyl)sulfonyl)-N-(3-(4-(4-fluoro-
Na2SO4, the solvent was removed and the crude product was benzoyl)piperazin-1-yl)propyl)pentanamide (9): Compounds
purified via automated column chromatography (Biotage: 10 g 5b (63 mg, 0.22 mmol) and 8 (165 mg, 22 mmol) were dis-
KP-Sil, Gradient: petroleum ether → EtOAc) to yield 5a solved in a mixture of acetonitrile and water (5.5 mL, 10:1 v:v)
(176 mg, 87%) as yellow solid. Rf = 0.49 (ethanol); analytical and the resulting solution was maintained at 60 °C for 3 h.
HPLC: tR = 7.7 min (eluent: CH3CN/H2O, 15:85 + 0.1% TFA); Afterwards, the solvent was removed and the crude product was
mp 64 °C; 1H NMR (400 MHz, CDCl3) δ 1.76 (qi, 3J = 6.8 Hz, purified via column chromatography to give 9 (112 mg, 69%)
1H, CH2), 2.27–2.60 (m, 6H, Pip-H, NCH2), 3.25–3.47 (m, 4H, as a pale yellow syrup. Rf = 0.2 (EtOAc); 1H NMR (400 MHz,
Pip-H, CH2N3), 3.80 (br s, 2H, Pip-H), 7.56 (d, 3J = Hz, 2H, CDCl3) δ 1.41–1.52 (m, 2H, CH2), 1.57–1.67 (m, 2H, CH2),
H-o), 8.27 (d, 3J = Hz, 2H, H-m); 13C NMR (101 MHz, CDCl3) 1.72–1.81 (m, 2H, CH2), 1.93–2.00 (t, 3J = 7.2 Hz, 2H,
δ 26.2 (CH2), 42.3, 47.7, 52.7, 53.5 (4 × Pip-C), 49.5 (CH2N3), CH2C=O), 2.33–2.54 (m, 6H, 3 × CH2N), 2.98 (t, 3J = 7.6 Hz,
55.1 (NCH2), 124.0 (C-m), 128.2 (C-o), 142.1 (C-i), 148.5 2H, CH2S), 3.30–3.89 (m, 6H, 3 × CH2N), 5.93 (s, 2H,
(C-p), 168.0 (C=O); MS (ESI+) m/z (%): 319 (100) [M+ + H]; OCH2O), 6.05 (d, 3J = 5.9 Hz, 1H, Hdiox), 6.69 (d, 3J = 8.7 Hz,
Anal. calcd for C14H18FN6O3 (318.33): C, 52.82; H, 5.70; N, 1H, Hpyr), 6.92 (d, 3J = 8.7 Hz, 1H, Hpyr), 6.97 (s, 1H, NH),
26.40; found, C, 52.79; H, 5.66; N, 26.11.
7.07 (t, 3J = 8.5 Hz, 2H, H-o), 7.38–7.50 (m, 5H, H-m + HAr),
7.97 (d, 3J = 7.9 Hz, 1H, HAr), 8.05–8.10 (m, 2H, Hdiox + NH);
4-(3-Azidopropyl)-1-(4-fluorobenzoyl)piperazine (5b): Com- 13C NMR (101 MHz, CDCl3) δ 22.0, 22.7, 26.1, 32.8 (4 ×
pound 3b (83 mg, 0.40 mmol), 3-azidopropyl tosylate (122 mg, CH2), 42.3, 47.8 (br. s, 2 × CH2Pip), 49.3 (CH2N), 52.8, 53.3
0.48 mmol) and Et3N (61 mg, 0.60 mmol) were dissolved in an- (br. s, 2 × CH2Pip), 55.0, 55.6 (2 × CH2), 97.9 (CHAr), 102.2
hydrous THF (6 mL) and the resulting mixture was stirred at (OCH2O), 106.7, 115.5 (d, 2JC,F = 21.5 Hz, C-m), 117.7, 118.6,
60 °C for 3 d. After reaction control by TLC, THF was changed 120.4, 122.0, 123.3, 123.7, 129.3 (d, 3JC,F = 8.6 Hz, C-o),
by ethyl acetate (15 mL), water (15 mL) was added and the 129.5, 131.9 (d, 4JC,F = 3.3 Hz, C-i), 139.2, 141.2, 143.5, 147.5,
aqueous layer was extracted with ethyl acetate (3 × 15 mL). The 156.9, 159.3, 161.1, 163.3 (d, 1JC,F = 251.0 Hz, C-p), 169.3
combined organic layers were dried over Na2SO4, the solvent (C=O); 19F NMR (376 MHz, CDCl3) δ −110.3; MS (ESI+) m/z
was removed and the crude product was purified via automated (%): 752 (80) [M + H]+; Anal. calcd for C36H39ClFN7O6S
column chromatography (Biotage: 10 g KP-Sil, Gradient: petro- (752.25): C 57.48, H 5.23, N 13.03; found, C 57.66, H 5.21, N
leum ether → EtOAc) to yield 5b (94 mg, 81%) as colorless 13.31.
syrup. Rf = 0,48 (ethanol); analytical HPLC: tR = 6.7 min
(eluent: CH3CN/H2O, 15:85 + 0.1% TFA); 1H NMR 4-(But-3-yn-1-yl)-1-(4-[18F]fluorobenzoyl)piperazine
(400 MHz, CDCl3) δ 1.76 (qi, 2H, 3J = 6.7 Hz, CH2), 2.30–2.56 ([18F]4b): Similar as described in [47] an anion-exchange
(m, 6H, NCH2+Pip-H), 3.35 (t, 2H, 3J = 6.7 Hz, CH2N3), cartridge (Waters, Sep-Pak Light Accell Plus QMA) was acti-
3.38–3.88 (br d, 4H, Pip-H), 7.08 (“t”, 3JH,F = 3Jo,m = 8.8 Hz, vated by rinsing with 5 mL of a 1 M NaHCO3 solution and
H-m), 7.40 (dd, 2JH,F = 5.4 Hz, 3Jo,m = 8.8 Hz, H-o); 13C NMR 10 mL of deionized H2O. It was charged with [18F]fluoride
(101 MHz, CDCl3) δ 26.3 (CH2), 42.4, 48.0 (2 × Pip-C), 49.5 (0.5–1 GBq) and eluted with 1.5 mL of a solution of Kryptofix
2487

Beilstein J. Org. Chem. 2016, 12, 2478–2489.
3. Emir, N.; Bilge, M.; Tursun, M.; Keşan, G.; Parlak, C.
2.2.2 (10 mg/mL) and K2CO3 (13 mM) in 7 mL CH3CN and
43 mL H2O. The solvents were evaporated azeotropically by
subsequent addition of three portions of 1 mL each of an-
hydrous CH3CN under a stream of nitrogen at 110 °C. Precur-
sor 4a (3–4 mg) was dissolved in 400 µL of anhydrous DMSO,
and the mixture was added to the [18F]fluoride-containing
sealed vial. The resulting solution was heated at 150 °C for
60 min. Afterwards, the mixture was treated with 25 mL de-
ionized H2O and then passed through a C18 cartridge
(LiChrolut RP-18/Merck, 500 mg) to yield 80–155 MBq
(≈20% RCY, dc) of [18F]4b. Samples for analytical radio-TLC
and radio-HPLC were taken. Analytical radio-TLC: Rf = 0.50
(ethanol). Analytical radio-HPLC: tR = 5.7 min (eluent:
CH3CN/H2O, 15:85 + 0.1% TFA).
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4-(3-Azidopropyl)-1-(4-[18F]fluorobenzoyl)piperazine
([18F]5b): Similar as described in [47] an anion-exchange
cartridge (Waters, Sep-Pak Light Accell Plus QMA) was acti-
vated by rinsing with 5 mL of a 1 M NaHCO3 solution and
10 mL of deionized H2O. It was charged with [18F]fluoride
(0.5–1 GBq) and eluted with 1.5 mL of a solution of Kryptofix
2.2.2 (10 mg/mL) and K2CO3 (13 mM) in 7 mL CH3CN and
43 mL H2O. The solvents were evaporated azeotropically by
subsequent addition of three portions of 1 mL each of an-
hydrous CH3CN under a stream of nitrogen at 110 °C. Precur-
sor 5a (3–4 mg) was dissolved in 400 µL of anhydrous DMSO,
and the mixture was added to the [18F]fluoride-containing
sealed vial. The resulting solution was heated at 150 °C for
60 min. Afterwards, the mixture was treated with 25 mL de-
ionized H2O and then passed through a C18 cartridge
(LiChrolut RP-18/Merck, 500 mg) to yield 69–134 MBq
(≈17% RCY, dc) of [18F]5b. Samples for analytical radio-TLC
and radio-HPLC were taken. Analytical radio-TLC: Rf = 0.49
(ethanol). Analytical radio-HPLC: tR = 7.1 min (eluent:
CH3CN/H2O, 15:85 + 0.1% TFA).
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Supporting Information
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Supporting Information File 1
Copies of NMR spectra of investigated piperazines,
radioHPLC chromatograms, and separation methods for
piperazines.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-242-S1.pdf]
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doi:10.1002/cmdc.201200264
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doi:10.3762/bjoc.12.242
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