X-ray Structures of Succinimidyl Halobenzoates

The crystal and molecular structures of five succinimidyl halobenzoates are reported. Corresponding derivatives with the respective halo-radionuclide (18F, 76Br, 123I/124I/125I/131I) were prepared and used for the radiolabeling of biologically active (macro-)molecules (peptides, proteins, antibodies) under mild labeling conditions. All compounds were crystalized from petroleum ether/ethyl acetate mixtures.


Introduction
The radiolabeling of large biologically active molecules such as peptides, proteins or antibodies is an ongoing issue in radiopharmacy [1][2][3][4][5].Harsh reaction conditions (high temperature, organic solvents, oxidizing conditions) for the introduction of the radionuclide and the selective radiolabeling, together with the sensitivity of the applied biomolecules are often the main problems.Thus, novel radiolabeling building blocks were elaborated for a selective and mild insertion of the radionuclide under physiological friendly conditions.Succinimidyl esters which belong to activated esters [6] play a major role for this purpose.mSIB, oSIB, and pSIB with radioiodine were first developed [7][8][9][10] and are the basis of all other (radio)halogenated succinimidyl esters described in this paper.They are important for labeling purposes with radioiodine ( 123 I/ 124 I/ 125 I/ 131 I) [11][12][13][14][15][16][17][18][19][20].[ 18 F]SFB is the most applied building block in fluorine-18 chemistry.Additionally, a carbon-11-containing SFB derivative was synthesized in the past [21].In the meantime, several commercial suppliers deliver the non-radioactive SFB compound.
In this paper, we synthesized the non-radioactive esters SFB 3a, SClB 3b, SBrB 3c, o-SIB 3d, and p-SIB 3e which are commonly in use as non-radioactive standards to analyze radiolabeling via TLC and HPLC analyses and determined the molecular structure of these compounds via single crystal XRD.

Synthesis and Chemistry
In general, the radiolabeling of biomacromolecules (peptides, proteins, and antibodies) follows a two-step procedure.Normally, these compounds were not directly radiolabeled.For this purpose, the radionuclide-containing building block was prepared first.In the case of radiofluorine 18 F, electron-demanding ethyl benzoates were applied [24,25] and the fluorine was introduced via a nucleophilic substitution S N Ar of the trimethylammonium group in most of the cases [2].Newer developments are based on the use of iodonium salts [26,27] or nickel complexes [28] as precursors.Afterwards, the ethyl group was cleaved followed by the introduction of the succinimidyl group.
In the case of radiobromine and radioiodine, both radionuclides were classically inserted by an electrophilic substitution S E Ar (radiohalodestannylation) using stannyl precursors.
The second step involves the actual labeling of the (sensitive) biomacromolecule.Mostly, free amine groups were used for this labeling reaction under mild conditions (room temperature, aqueous solvents, non-oxidizing conditions, short reaction times).The overview is outlined in Scheme 1.
Crystals 2017, 7, 90 2 of 8 developments are based on the use of iodonium salts [26,27] or nickel complexes [28] as precursors.Afterwards, the ethyl group was cleaved followed by the introduction of the succinimidyl group.
In the case of radiobromine and radioiodine, both radionuclides were classically inserted by an electrophilic substitution SEAr (radiohalodestannylation) using stannyl precursors.
The second step involves the actual labeling of the (sensitive) biomacromolecule.Mostly, free amine groups were used for this labeling reaction under mild conditions (room temperature, aqueous solvents, non-oxidizing conditions, short reaction times).The overview is outlined in Scheme 1.
Application of halobenzoyl chlorides or the use of the Steglich esterification are the most convenient synthesis methods with the highest yields and the shortest reaction times.In our case, the synthesis of all succinimidyl esters was accomplished using N-hydroxy succinimide (2) which was treated with the respective halobenzoyl chlorides (1a-e) in anhydrous THF and triethylamine as base [16].All succinimidyl esters 3a-e were obtained in high yields of 77% to 92%.The purification of 3a-e was accomplished via a short column chromatography.The reaction path is outlined in Scheme 2.

X-ray Structure Determination
Single crystals of 3a to 3e were obtained by the slow evaporation method.The crystal and experimental as well as the structure refinement parameters for the single crystal X-ray structure determinations are summarized in Table 1.The crystals of all five compounds consist of neutral succinimidyl halobenzoate molecules.The chloro-, bromo-and iodo-derivatives 3b, 3c and 3d, which have the halogen atom attached on the para-position of the benzoate ring, are isotypic.Figures 1-3 show the molecular structures of the five compounds.Scheme 1.General labeling procedure using radiolabeling building blocks based on radiohalogenated ( 18 F, 76 Br, 123/124/125/131 I) succinimidyl benzoates.
Various ways to prepare the halogenated succinimidyl esters are known from the literature.Several are based on the Steglich esterification of N-hydroxy succinimide with the halobenzoic acid and DCC, EDC or TSTU as coupling reagent [11,[29][30][31][32][33].Others used halobenzyl alcohols under radical conditions [34,35] or halobenzoic acid and N,N -succinimidyl carbonate [36].Transition metal catalyzed reactions are also applied such as palladium catalyzed coupling reactions with CO [37] or with formyl derivatives [38] as well as Ru-catalyzed reactions using the respective benzaldehyde [39].
Application of halobenzoyl chlorides or the use of the Steglich esterification are the most convenient synthesis methods with the highest yields and the shortest reaction times.In our case, the synthesis of all succinimidyl esters was accomplished using N-hydroxy succinimide (2) which was treated with the respective halobenzoyl chlorides (1a-e) in anhydrous THF and triethylamine as base [16].All succinimidyl esters 3a-e were obtained in high yields of 77% to 92%.The purification of 3a-e was accomplished via a short column chromatography.The reaction path is outlined in Scheme 2.
Crystals 2017, 7, 90 2 of 8 developments are based on the use of iodonium salts [26,27] or nickel complexes [28] as precursors.Afterwards, the ethyl group was cleaved followed by the introduction of the succinimidyl group.
In the case of radiobromine and radioiodine, both radionuclides were classically inserted by an electrophilic substitution SEAr (radiohalodestannylation) using stannyl precursors.
The second step involves the actual labeling of the (sensitive) biomacromolecule.Mostly, free amine groups were used for this labeling reaction under mild conditions (room temperature, aqueous solvents, non-oxidizing conditions, short reaction times).The overview is outlined in Scheme 1.
Application of halobenzoyl chlorides or the use of the Steglich esterification are the most convenient synthesis methods with the highest yields and the shortest reaction times.In our case, the synthesis of all succinimidyl esters was accomplished using N-hydroxy succinimide (2) which was treated with the respective halobenzoyl chlorides (1a-e) in anhydrous THF and triethylamine as base [16].All succinimidyl esters 3a-e were obtained in high yields of 77% to 92%.The purification of 3a-e was accomplished via a short column chromatography.The reaction path is outlined in Scheme 2.

X-ray Structure Determination
Single crystals of 3a to 3e were obtained by the slow evaporation method.The crystal and experimental as well as the structure refinement parameters for the single crystal X-ray structure determinations are summarized in Table 1.The crystals of all five compounds consist of neutral succinimidyl halobenzoate molecules.The chloro-, bromo-and iodo-derivatives 3b, 3c and 3d, which have the halogen atom attached on the para-position of the benzoate ring, are isotypic.Figures 1-3 show the molecular structures of the five compounds.

X-ray Structure Determination
Single crystals of 3a to 3e were obtained by the slow evaporation method.The crystal and experimental as well as the structure refinement parameters for the single crystal X-ray structure determinations are summarized in Table 1.The crystals of all five compounds consist of neutral succinimidyl halobenzoate molecules.The chloro-, bromo-and iodo-derivatives 3b, 3c and 3d, which rhave the halogen atom attached on the para-position of the benzoate ring, are isotypic.Figures 1-3 show the molecular structures of the five compounds.The interatomic distances for all five compounds are found within the expected ranges.Selected atom distances and mean plane angles are listed in Table 2.A different packing of the molecules is observed only in crystals of the fluoro compound 3a and the ortho-iodobenzoate 3e, resulting in different space groups.The two carbon-oxygen bonds of C5 in all five structures differ significantly in length.Generally, the much shorter C5-O4 lengths compared to C5-O3 indicate a strong double bond character and a single bond character for C5-O3.The mean planes through the halo benzoate moieties are tilted towards the mean planes through the succinimidyl moieties by angles ranging from 70.7 • (3c) to 80.5 • (3d), such that the two ring systems are arranged almost perpendicular to each other.Because of the lack of acidic protons, no classical hydrogen bonds are observed in the structures (see below).Furthermore, the surrounding of the nitrogen atoms of the succinimidyl residue in compounds 3a-e can be described as follows.These atoms show nearly planar bonding geometry, with a maximum deviation of 0.08 Å out of the C1-C4-O3 plane.The presence of an adjacent single bound oxygen atom can act to pyramidalize the N bonding geometries, but in these cases it is minimal due to the strong conjugation between the N atom and two carbonyl groups.This nearly planar behavior can be explained by the partial double bond character of the N1-C1-O1 and the N1-C4-O2 amide function.The interatomic distances for all five compounds are found within the expected ranges.Selected atom distances and mean plane angles are listed in Table 2.A different packing of the molecules is observed only in crystals of the fluoro compound 3a and the ortho-iodobenzoate 3e, resulting in different space groups.The two carbon-oxygen bonds of C5 in all five structures differ significantly in length.Generally, the much shorter C5-O4 lengths compared to C5-O3 indicate a strong double bond character and a single bond character for C5-O3.The mean planes through the halo benzoate moieties are tilted towards the mean planes through the succinimidyl moieties by angles ranging from 70.7° (3c) to 80.5° (3d), such that the two ring systems are arranged almost perpendicular to each other.Because of the lack of acidic protons, no classical hydrogen bonds are observed in the structures (see below).Furthermore, the surrounding of the nitrogen atoms of the succinimidyl residue in compounds 3a-e can be described as follows.These atoms show nearly planar bonding geometry, with a maximum deviation of 0.08 Å out of the C1-C4-O3 plane.The presence of an adjacent single bound oxygen atom can act to pyramidalize the N bonding geometries, but in these cases it is minimal due to the strong conjugation between the N atom and two carbonyl groups.This nearly planar behavior can be explained by the partial double bond character of the N1-C1-O1 and the N1-C4-O2 amide function.

Conclusions
In this paper, we have synthesized four succinimidyl halobenzoate derivatives which are used in radiopharmacy as prostetic groups with the respective halo radionuclides.The structures of all derivatives were elucidated.

General
NMR spectra were recorded on an Agilent DD2 (400 or 600 MHz) with ProbeOne probe.Chemical shifts of the 1 H, 13 C and 19 F spectra were reported in parts per million (ppm) using TMS for 1 H and 13 C spectra and CFCl 3 for 19 F spectra as internal standard.Chromatographic separations and TLC detections were carried out with Merck Silica Gel 60 (63-200 µm) and Merck Silica Gel 60 F 254 sheets, respectively.TLCs were developed by visualization under UV light (λ = 254 nm).Anhydrous THF was purchased from Acros (Geel, Belgium) or SigmaAldrich (Schnelldorf, Germany).N-Hydroxysuccinimide (2), all benzoyl chlorides 1a-e and Et 3 N were used as received without further purification.Crystallographic data were collected with a Bruker-Nonius Apex-X8 CCD-diffractometer (Bruker, Madison, WI, USA) with Mo-K α radiation (λ = 0.71073 Å) at 123 K.The structures were solved by direct methods using SHELXS-97 and refined against F 2 on all data by full matrix least-squares refinements using the program suites from G. M. Sheldrick [40][41][42].Data corrections including multi-scan absorption corrections were applied to the data sets using the Bruker AXS software [43].All non-hydrogen atoms were refined anisotropically; all hydrogen atoms bonded to C atoms were placed on geometrically calculated positions and refined using riding models.CCDC 1524925 (3a), CCDC 1504220 (3b), CCDC 1505323 (3c), CCDC 1505325 (3d), and CCDC 1505324 (3e) contain the supplementary crystallographic data of the compounds.These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/conts/retrieving.html.

General Synthesis Procedure
N-Hydroxysuccinimide (2, 150 mg, 1.33 mmol) was dissolved in anhydrous THF (10 mL), Et 3 N (197 mg, 1.95 mmol) was added and the mixture was cooled to 0 • C. Next, the respective halobenzoyl chloride 1a-e (1.56 mmol) was added dropwise, the solution was stirred at 0 • C for 60 min and at rt for 2 h.Afterwards, the reaction was quenched with water (15 mL) and extracted with ethyl acetate (3 × 15 mL).The combined organic layers were separated and dried over Na 2 SO 4 .The solvent was removed and the crude product was purified via flash chromatography (petroleum ether/ethyl acetate 2:1) to yield compounds 3a-e (77%-92%) as colorless solids.

Figure 1 .
Figure 1.A view of the molecular structures of 3a (left) and 3b (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 2 .
Figure 2. A view of the molecular structures of 3c (left) and 3d (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 3 .
Figure 3.The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 1 .
Figure 1.A view of the molecular structures of 3a (left) and 3b (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 1 .
Figure 1.A view of the molecular structures of 3a (left) and 3b (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 2 .
Figure 2. A view of the molecular structures of 3c (left) and 3d (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 3 .
Figure 3.The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 2 .
Figure 2. A view of the molecular structures of 3c (left) and 3d (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 1 .
Figure 1.A view of the molecular structures of 3a (left) and 3b (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 2 .
Figure 2. A view of the molecular structures of 3c (left) and 3d (right), showing the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 3 .
Figure 3.The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.Figure 3. The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 3 .
Figure 3.The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.Figure 3. The molecular structure of 3e with the atom labeling scheme.Displacement ellipsoids are drawn at the 50% probability level.

Figure 4
demonstrates exemplarily the packing of the molecules of 3b in a view along the b axis of the unit cell.The dotted lines included in the figure show the shortest center distances of the phenyl rings (brown dotted lines) and the shortest intermolecular O....H distances (green dotted lines).Weak π-π interactions with distances between the planes of the aromatic phenyl rings of 4.181 Å and 4.586 Å as well as weak "non-classical" hydrogen bonds with the shortest acceptor-donor distance of 3.264(1) Å (in 3b) are responsible for the final arrangement of molecules.

Figure 4
demonstrates exemplarily the packing of the molecules of 3b in a view along the b axis of the unit cell.The dotted lines included in the figure show the shortest center distances of the phenyl rings (brown dotted lines) and the shortest intermolecular O....H distances (green dotted lines).Weak π-π interactions with distances between the planes of the aromatic phenyl rings of 4.181 Å and 4.586 Å as well as weak "non-classical" hydrogen bonds with the shortest acceptordonor distance of 3.264(1) Å (in 3b) are responsible for the final arrangement of molecules.

Figure 4 .
Figure 4.The packing of the molecules of 3b in an expanded view of the unit cell along the b axis.The shortest contacts between the phenyl rings are shown as brown dotted lines and the shortest intermolecular O....H distances as green dotted lines.

Figure 4 .
Figure 4.The packing of the molecules of 3b in an expanded view of the unit cell along the b axis.The shortest contacts between the phenyl rings are shown as brown dotted lines and the shortest intermolecular O....H distances as green dotted lines.

Table 1 .
Crystal data and structure refinement for compounds 3a-e.

Table 1 .
Crystal data and structure refinement for compounds 3a-e.

Table 1 .
Crystal data and structure refinement for compounds 3a-e.