University of Groningen
Metallodrugs for therapy and imaging: investigation of their mechanism of action
Spreckelmeyer, Sarah
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Spreckelmeyer, S. (2018). Metallodrugs for therapy and imaging: investigation of their mechanism of action. University of Groningen.
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98
Chapter A2
H
4neunpa: A Bifunctional Acyclic Chelator with many
Faces
Sarah Spreckelmeyer,a,b Caterina Ramogida,c Hsiou-Ting Kuo,d Ben Woods,
e Valery Radchenko,c Maria Guadalupe Jaraquemada Peláez,a Francois Bénard,d Angela Casinie and Chris Orviga
a Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada
b Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute
of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands
c Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, V6T
2A3, Canada
d BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3, Canada
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1 Abstract
Bifunctional chelators are useful tools in nuclear medicine and they also find great application in the relevant field of personalized medicine. We evaluated the nonadentate chelator H4neunpa as a bifunctional chelator for therapeutic as well as diagnostic application. Several conjugates were synthesized. First, H4neunpa was bifunctionalized with a small biomolecule Glu-ureido-Lys, as biovector that targets the prostate-specific membrane antigen (PSMA) for prostate cancer and can be further radiolabelled with 111In for single photon emission computed tomography (SPECT) imaging. H4neunpa was also bifunctionalized with a supramolecular metallacage precursor for cisplatin encapsulation. This probe was further investigated for La3+ complexation in order to use it in the future for fluorescence imaging and in vitro/in vivo tracking of the supramolecular metallacage. Besides modifying the biovector (Glu-ureido-Lys or metallacage), we also achieved promising radiolabeling results with different radiometals for therapy (225Ac, 213Bi and natSb). 225Ac and 213Bi are interesting alpha emitters for targeted alpha therapy (TAT). 119Sb is a promising Auger electron emitter for targeted radiotherapy.
100
2 Introduction
Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases in 2012, 8.8 million deaths in 2015, and an expected rise by about 70% over the next two decades.1 Of note, in the last decades, the treatment and diagnosis of cancer improved tremendously, but we are also still facing serious obstacles. As individuals are unique, so is each cancer type and differences can be observed on the macroscopic (e. g. tissue invasions, growth rate) as well as microscopic (e. g. protein expression) level. To cope with this, a personalized treatment that is targeted to the needs of a patient based on his/her own genetic, biomarker, phenotypic, or psychosocial characteristics, is absolutely necessary. Among numerous methods for personalized medicine, the use of a bifunctional chelator (BFC, Figure 1) in nuclear medicine is a promising strategy and various types are clinically used, like 131I-tositumomab and 90Y-ibritumomab that are bearing anti-CD20 antibodies for the treatment of B cell lymphoma and leukemia.2 BFCs consist of a biovector, like small molecules or antibodies, a linker, and a chelator that stably binds the radiometal of choice. The antibody can have either a targeting or therapeutic value and the radiometal of interest can be either for imaging or diagnosis.
Figure 1. Illustration of a bifunctional chelator (BFC).
Before the treatment with these BFCs, the expression level of the CD20 antigen needs to be assessed. If the expression level is sufficient, a therapy with anti-CD20 antibodies is promising. Non-responders are directly sorted out. By this, side-effects are limited and costs are kept as low as possible.
101 Overall, the aim of this chapter is to show that the bifunctional chelator H4neunpa can easily be modified for multiple purposes in the field of medicinal inorganic chemistry. This chapter is divided into three sub-chapters, each of them consisting of a short introduction and results and discussion part. The experimental part and the summary are combined. Specifically, the first sub-chapter deals with the prostate-specific membrane antigen (PSMA), which is an attractive target for targeted radiotherapy. A bifunctional chelator, H4neunpa, was linked to a PSMA-specific peptide and radiolabeled with 111In. The second sub-chapter describes the linkage of H4neunpa to a metallacage for therapeutic purposes and in the third sub-chapter, radiolabeling of H4neunpa with different radiometals for imaging or therapeutic purposes is investigated.
2.1 Subchapter 1
An attractive target for prostate cancer radiopharmaceuticals is the prostate-specific membrane antigen (PSMA). PSMA is a well-characterized biomarker for imaging prostate cancer. It is a type II membrane-bound, glutamate-preferring carboxypeptidase and is expressed on prostate tissue, with strong overexpression in prostate cancer. The first antibody targeting PSMA that was published was mAb 7E11 which binds to the receptor intracellularly.3 In the late 1990s, ProstaScint (capromab pendetide, EUSA Pharma, Figure 2) was approved by the FDA for imaging prostate cancer. ProstaScint consists of the chelator GYK-DTPA-HCl that chelates the gamma emitter 111In (thus suitable for SPECT imaging) and is linked to the murine monoclonal antibody 7E11 (capromab).4 Beside antibodies (eg. 7E11) that target PSMA, also aptamers and PSMA inhibitors of low molecular weight have gained interest as targeting devices for prostate cancer. PSMA possesses an enzymatic site in its extracellular domain that cleaves endogenous substrates such as N-acetylaspartylglutamic acid (NAAG) and poly-gamma-glutamyl folic acid. The enzymatic site contains two zinc ions, and is composed of two pockets: the glutamate sensing pocket and the non-pharmacophore pocket that contains an arginine rich region.
102
Small molecules have been designed to inhibit PSMA, containing a zinc-binding moiety, a glutamate moiety that can reside in the glutamate sensing pocket and a lipophilic moiety that can reside in the non-pharmacophore pocket.5 Beside phosphonate-, phosphate-, and phosphoramidates and thiols, ureas play an important role in small molecule design of PSMA inhibitors.5 Recently, lysine-glutamate-urea based small molecules gained a lot of interest in PET imaging (Figure 1), but no SPECT tracer is available up to now. Herein, we present the synthesis of a Glu-ureido-Lys based small molecule linked to p-SCN-Bn-H4neunpa, which is called H4neunpa-PSMA-L, as it is aimed to bind to PSMA. Additionally, we performed preliminary radiolabeling experiments with 111In as well as determined stability in human serum and determination of the 111In-neunpa-PSMA-L chelate for SPECT imaging of prostate cancer. The aim of this study was to have a proof-of-principle that also small molecules can easily be linked to H4neunpa without loosing its 111In-chelating properties.
Figure 2. Compounds discussed in this work. 2.1.1 Results and Discussion
2.1.1.1 Synthesis
p-SCN-Bn-H4neunpa was synthesized using a protocol already published in chapter A1.6 The Glu-ureido-Lys moiety (Scheme S 1) was linked via a reaction of the isothiocyanate of p-SCN-Bn-H4neunpa with the amine functional group of Glu-ureido-Lys to yield H4neunpa-PSMA-L (Figure 3). Characterization of the product was achieved via HR ESI-MS (see Experimental). N H NH OH HO O O O NH2 N N N NCS N N OH OH O O OH O O OH p-Bn-SCN-H4neunpa Glu-ureido-Lys N N N N H HO O O O OH O HO O OH ProstaScint O OH NH O HN O NH HO capromab O OH
103 Figure 3. Synthesis scheme of H4neunpa-PSMA-L.
2.1.1.2 Radiolabeling with 111In
Radiolabeling experiments with 111InCl3 (200 µCi) were performed in a concentration range of 10-8 M to 10-4 M H4neunpa-PSMA-L and a maximum of 82 % radiochemical yield (RCY) was achieved at 10-5 M (Figure 4). Unfortunately, increasing the concentration of H4neunpa-PSMA-L did not lead to an increase in RCY. The reason for missing about 18 % RCY for quantitative radiolabeling might be that the three carboxylic acids of Glu-ureido-Lys interfere with 111In chelation of the H4neunpa moiety.
Figure 4. RCY of H4neunpa-PSMA with 111In.
N N N NCS N N OH OH O O OH O O OH NH NH OH HO O O O HN N N N HN N N OH OH O O OH O O OH S OH O N H NH OH HO O O O NH2 OH O DMF, DIEA H4neunpa-p-Bn-NCS H4neunpa-PSMA-L 10-9 10-8 10-7 10-6 10-5 10-4 10-3 0 20 40 60 80 100 ligand [mol/L] % RCY
104
2.1.1.3 Stability of H4neunpa-PSMA-L in human serum
The stability of 111In-neunpa-PSMA-L in human serum was assessed at time points 1, 48 and 120h (Table 1). After 1h incubation, the RCY decreased to 67 % (% of t=0 was taken as 100% reference). The RCY decreased further to 60 % after 120h incubation in human serum albumin. 111In-neunpa-PSMA-L is clearly not stable under these conditions. We assume, that the carboxylic acids of Glu-ureido-Lys might interact with the chelation of 111In, since carboxylic acids are in general good chelators for 111In and transchelation with albumin may take place. The resulting 111 In-neunpa-PSMA-L complex might be less stable than 111In-neunpa (see chapter A1).7 In addition, the distance between the carboxylic acids of the lysine-glutamate-urea and the neunpa-cavity is very small, favoring the removal of the 111In from the chelator by the carboxylic groups.
Table 1. Stability of 111In-neunpa-PSMA-L (10-5M) in human serum,
expressed as % of the %RCY at t=0.
Time [h] % RCY 0 100 1 66.8 ± 2.3 48 65.2 ± 0.1 120 58.9 ± 0.7 2.2 Subchapter 2
Instead of linking a small molecule like Glu-ureido-Lys to p-SCN-Bn-H4neunpa, a metallacage can be added for imaging purposes (111In, SPECT imaging) or therapeutic purposes. Self-assembled metallacages that incorporate a chemotherapeutic drug like cisplatin may represent an attractive drug-delivery system. Exo-functionalized Pd2L4 cages, formed by self-assembly of the four ligands in the presence of Pd ions, are therefore highly promising metallacages (Figure 5), that are proven to encapsulate
105 cisplatin.8 Not much is known about these fairly new compounds. In order to get more information about the mechanism of action and cellular uptake mechanisms of the metallacages, they can be linked to a fluorescent moiety such as lanthanide complexes. Using this approach, their uptake can be visualized by fluorescence microscopy in vitro, provided that the uptake characteristics of the metallacages will not be changed upon bifunctional chelator (BFC) linkage. The use of a (BFC) like
p-SCN-Bn-H4neunpa is very promising for this purpose. p-SCN-Bn-H4neunpa has shown to be a good chelator for various metal ions, like La3+, In3+ and Bi3+.7
Lanthanide cations are known for their unique photonic and magnetic properties associated with their f0-f14 electron configuration. They have wide practical applications in many fields including catalysis, additives in glass, photonic applications, as well as luminescent stains for biomedical analysis, medical diagnosis and cellular optical imaging.9 La3+ ([Kr]4d105s25p6, 1.03 Å) and Eu3+ (([Xe]4f6, 0.95 Å) are metal ions that have fluorescent properties. Here, we report on the synthesis of a self-assembled metallacage functionalized with La3+-H4neunpa to achieve fluorescent properties for cellular imaging.
Figure 5. Synthetic approach to achieve metallacage-NH2 by
self-assembly. N N H2N metallacage-ligand-NH2 N N H2N Pd Pd N N NH2 NH2 N N H2N N N metallacage-NH2 [Pd(NCCH3)4][BF4]2 0.5 eq. rt, 1h, DMSO
106
2.2.1 Results and Discussion
2.2.1.1 Metallacage exo-functionalization
The synthesis of the BFC p-SCN-Bn-H4neunpa has already been published and described in chapter A1.7 The coupling of the metallacage-NH2 to this BFC can be achieved in two ways: i) The first possibility starts with the metallacage precursor metallacage-ligand-NH2 (Figure 5), which bears an amine functional group. This precursor can be coupled to the NCS functional group of p-SCN-Bn-H4neunpa via a thiourea linkage. Thereafter, the metal ion complexation can be performed and in a next step, the metallacage formation will be achieved as shown in Figure 6.8 ii) The second possibility starts with 4 eq. of p-SCN-Bn-H4neunpa that will be coupled directly to the already self-assembled metallacage-NH2 (Figure 5). After that, the metal ion complexation will be performed. In both cases, after successful metal complexation and cage formation, cisplatin can be incorporated.
Here, we describe the results and discussion of the first approach: The metallacage-ligand-NH2 was coupled via the reaction between its amine and the isothiocyanate group from p-SCN-Bn-H4neunpa to form compound 1 (Figure 6). The product was purified by HPLC. In Figure 7, the 1H NMR spectrum of the H4neunpa-cageligand (compound 1) and of p-SCN-Bn-H4neunpa can be seen. All proton peaks can be properly assigned (Figure 8) with the help of 2D-HSQC and 2D-COSY NMR spectra (Figure S2 – Figure S5). The next step was the metal ion complexation. Since Eu3+ complexation was not successful (no results in ESI-MS, no fluorescence), we performed a La3+ complexation. HR ESI-MS and 1H NMR confirmed the successful complexation reaction (Figure 9). In addition, fluorescence was observed (Figure 10); this will be discussed below in more detail.
107
Figure 6. Synthesis scheme of La-neunpa-metallacage (compound 1,
compound 2 and compound 3).
N N N NCS N N OH OH O O OH O O OH N N NH2 N N N HN N N OH OH O O OH O O OH N N HN S DMSO La(NO3)3 N N N HN N N OH OH O O OH O O OH N N HN S La 1 2 3 N N N HN N N OH OH O O HO O O HO S N N N H Pd Pd N N HN H N N N HN N N N N N NH N N OH OH O O HO O O HO S N N N HN N N HO HO O O OH O O OH S N N N H N N N HO HO O O OH O O OH S La La La La 1a [Pd(NCCH3)4[BF4]2 0.5 eq. rt, 1h, DMSO
108
Figure 7. 1H NMR spectra of (A) p-Bn-SCN-H4neunpa and (B) compound 1
(H4neunpa-cageligand ) (400MHz, MeOD, 25°C). 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) 3. 31 C D 3O D 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) 3. 3 C D 3O D A: p-Bn-SCN-H4neunpa B: H4neunpa-cageligand
109
Figure 8. 1H NMR peak assignments of compound 1.
2.2.1.2 La3+ complexation reaction
The La3+ complexation of compound 1 was performed following a standard protocol.7 HR ESI-MS and the 1H NMR spectrum of the product compared to its precursor show clear changes in the aromatic region as well as in the alkyl region (Figure 9). Peak assignments for the La complex are difficult due to the low concentration of the sample.
N N N HN N N OH OH O O OH O O OH N N HN S 1 1 2 2 3 3 4 4 5 6 A A B B C C D D E E F G G H H I I J J K K 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 f1 (ppm) 3. 31 C D 3O D 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 f1 (ppm) A D B+K J E C + FI G H 1 3 5 2 4 6
110
Figure 9. 1H NMR spectra of compound 1 (top, 400 MHz, MeOD, 25°C) and
compound 2 (bottom, 400 MHz, DMSO-d6, 25°C).
Figure 10. A NMR tube showing the fluorescence of compound 2.
2.2.1.3 Fluorescence spectroscopy
The solutions of compound 2, metallacage-ligand-NH2 and La-neunpa-NO2 were prepared at a concentration of 10-6 M in DMSO and the
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 f1 (ppm) 2. 32 2. 50 D M S O -d 6 2. 66 2. 72 2. 88 3. 88 4. 54 6. 64 7. 21 7. 48 7. 54 7. 77 8. 02 8. 60 8. 78 9. 94 9. 98 3. 04 3. 25 3. 31 C D 3O D 3. 48 3. 58 3. 67 4. 03 7. 24 7. 41 7. 51 7. 59 7. 75 7. 92 8. 02 8. 56 8. 74
111 fluorescence was measured at λex = 305 nm. In Figure 11, the emission spectra of the compound 2, metallacage-ligand-NH2 and La-neunpa-NO2 is shown. The compound 2 shows high intensity fluorescence at 430 nm (line 2). La-neunpa-NO2 itself shows a weak emission at 360 nm and 430 nm (line 1 and line 2). The addition of La3+ ions results in successive red shifts of the emission peaks to 380 nm (line 1a) and 430 nm (line 2) accompanied by the enhancement of emission intensity. The enhancement of emission intensity was previously described by F. Wang et al., who studied the effect of a set of lanthanides on the fluorescence intensity of carminic acid, and showed that La3+ results in the best enhancement of fluorescence when compared to Dy3+, Tb3+, Gd3+, Eu3+, Y3+ and Sm3+.10
Figure 11. Emission spectra of compound 2, metallacage-ligand-NH2 and
La-neunpa-NO2.
2.3 Subchapter 3
Instead of labelling H4neunpa with a metal to obtain a fluorescent molecule, the BFC can also be loaded with a radioactive metal for
400 500 600 0 200 400 600 800 1000 wavelength [nm] Emission Compound 2 metallacage-ligand-NH2 La-neunpa-NO2 1 1a 2
112
therapeutic or diagnostic purposes. Considering the periodic table, a large number of radiometals can be identified for either therapeutic or diagnostic purposes. Here, we discuss 225Ac and 213Bi as alpha emitters for targeted alpha therapy (TAT) and 119Sb as Auger-electron emitter for targeted radiotherapy (TRT). Alpha emitters and Auger electrons have a characteristic linear energy transfer (LET). Alpha emitters such as 225Ac (t1/2 = 10 d), 212Pb (t1/2 = 10.6 h), 213Bi (t1/2 = 45.6 min) are gaining popularity for labeling biomolecules in targeted α-therapy (TAT). An essential characteristic of the radiometal for targeted radiotherapy is a short travelling distance in tissue and a radiation with high-energy transfer. 225Ac and 213Bi are attractive radiometals for therapeutic applications due to their high-energy alpha decays (see simplified decay scheme in Figure 12).
119Sb is another interesting isotope that emits Auger-electrons and could be used in targeted radiotherapy of small tumours, micrometastases and single cancer cells. 117Sb can be used in SPECT for visualization of the tumor.11
113
2.3.1 Results and Discussion
2.3.1.1 Radiolabeling with 225Ac
The ISAC (isotope separator and accelerator) facility is a unique and powerful resource that offers the possibility of a superior production method for alpha-emitting isotopes such as 225Ac, 213Bi, and 212Pb. 225Ac and 225Ra were produced in the spallation of an uranium carbide (UCx) target with 480 MeV protons, following a separation of 225Ra/225Ac from other isotopes via a high resolution mass separator. Purification of 225Ac was achieved using solid phase extraction on branched-DGA resin.12 Radiolabeling of p-Bn-NO2-H4neunpa with 225Ac was compared to that of the macrocyclic DOTA. Incubation of 10-5 or 10-4 M of p-Bn-NO2-H4neunpa at ambient temperature for 30 min with 225Ac yielded an 88.8 % RCY and 98.00 % RCY respectively, as determined via iTLC. For DOTA, the same conditions were used and resulted in 0.85 % RCY and 0.96 % RCY, respectively. p-Bn-NO2-H4neunpa has clearly superior radiolabeling kinetics compared to the gold standard DOTA under these conditions. The stability of both 225Ac complexes was determined after 120 min incubation in NH4OAc (0.15 M, pH 5) buffer solution. The RCY of both p-Bn-NO2-H4neunpa and DOTA remained stable over the tested time period (Figure 13).
114
Figure 13. %RCY of the tested chelators 225Ac-H
4neunpa-p-Bn-NO2 and 225Ac-DOTA.
2.3.1.2 225Ac/213Bi iTLC chromatograms
In Figure 14, three sets (1, 10 and 100 µg of chelator) of iTLC chromatograms are shown, representing the results of different times of incubation (0, 15, 30, 60 and 120 min) of neunpa with 225Ac3+. The following features are valid for all iTLC chromatograms shown here. The origin (0-30 mm) represents free radiometal (225Ac and daughters). This is due to the use of 10 mM NaOH/ 10% NaCl as the mobile phase, which causes the free metal ions to form hydroxide species that precipitate at the baseline. Between 30 mm and 120 mm, metal complexes can be detected. The black graph represents the chromatogram analyzed directly after finishing the development of the plate of different concentrations of neunpa-NO2 at different times of incubation with 225Ac3+. The blue chromatogram represents the radioactivity patterns on the plates 3 h later.
0 50 100 0 50 100 Time [min] % RCY Actinium-225 neunpa: 10-4 M neunpa: 10-5 M DOTA 10-4 M DOTA: 10-5 M
115 Figure 14. iTLC chromatograms of neunpa labeled with 225Ac ( (black: 0, 15, 30, 60 and 120 min for 1, 10 or 100 µg chelator; blue: same iTLC plate read 3h later). 0 m in 15 m in 30 m in 60 m in 120 m in 1 µg 10 µg 100 µg 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 200 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 200 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 200 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 200 Distance eluted [mm] Counts 0 50 100 150 0 50 100 150 200 Distance eluted [mm] Counts
116
In general, three scenarios of peak development can be anticipated between the black and blue graphs:13
A) If a peak on the plate is only the daughter of 225Ac, 213Bi, then it will decay within the 3h difference between measurements based on the 46 min half-life of 213Bi, and counts will decrease accordingly.
B) If a peak on the plate is a mixture of 225Ac and 213Bi in equilibrium, the total number of counts will remain more or less constant over a period of time, since as 213Bi decays, more is constantly being produced by the 225Ac decay, hence 225Ac and 213Bi are in equilibrium.
C) If a peak contains primarily 225Ac, then as the 225Ac decays, it forms detectable 213Bi, and we will get an increase in counts over time as 225Ac decays to 213Bi. The counts will increase (so the plate reader will detect the gamma rays from both the 225Ac and the 213Bi resulting in an increase in counts). The counts will increase until the 225Ac reached equilibrium with 213Bi after that point the counts will stay constant as in 'B'.
First, we compare the iTLC chromatograms for the samples taken at 0 min of incubation of neunpa with 225Ac at different concentrations of neunpa-NO2 (1, 10 and 100 µg) with each other. The chromatogram of the lowest concentration of neunpa-NO2 (1 µg) shows two black peaks, one at the origin and one at 100 mm distance eluted. The origin peak stays the same in counts during 3h and the 100mm peak decreases completely within 3h. A third blue peak appeared between 30-60 mm distance eluted. The peak at 0-30 mm can be assigned as free 225Ac and 213Bi being in equilibrium (scenario B), the 100 mm peak can be assigned as Bi-neunpa-NO2 complex (scenario A) and the 30-60mm peak as Ac-neunpa-Bi-neunpa-NO2 peak (scenario C).
At 10 µg neunpa-NO2, one origin peak, one possible peak at 50 mm and one distinct peak at 100 mm can be observed. After a 3 h time period, the origin peak decreased, giving assumption that only free 213Bi was at the origin in its hydroxide form. The peak at 50 mm increased in counts and another peak at 80 mm showed up, giving assumption that two
Ac-
117 neunpa-NO2 complexes are formed. The 100 mm peak decreased, accounting for a Bi-neunpa-NO2 complex.
At 100 µg neunpa-NO2, three peaks can be seen, similar to the 10 µg chromatogram, one at the origin, one at 50 mm and one at 100 mm. After a 3h time period, the origin peak decreased, the 50 mm peak decreased slightly, making it difficult to assign as pure Ac or Bi complex. Another distinct 80 mm peak showed up, being an Ac-neunpa-NO2 complex and the 100 mm peak decreased completely, accounting for a Bi-neunpa-NO2 complex. Overall, comparing the iTLC chromatograms of different concentrations of neunpa-NO2 at 0 min incubation with each other shows that a Bi-neunpa-NO2 complex is already formed at low concentrations, indicating a high affinity to 213Bi. At higher concentrations, two Ac-neunpa-NO2 complexes can be seen as well as the Bi-neunpa-NO2 complex.
Secondly, each row gets analyzed in more detail, showing a fixed neunpa-NO2 concentration after different times of incubation. At 1 µg neunpa-NO2, the 100 mm peak, likely a Bi-neunpa-complex, decreased over the time of incubation. This might be due to the short 213Bi half-life. The peak between 20-60 mm gets into equilibrium after 30 min incubation, since no difference between the black and blue graph can be observed. At 10 µg neunpa-NO2, the counts of each peak decrease over time, due to the half-life of the metals and a new equilibrium between 225Ac and 213Bi. At 100 µg neunpa-NO2, the second Ac-neunpa-NO2 complex at 80 mm is very distinct and is persistent over 120 min, giving assumption to a pure Ac-neunpa-NO2 complex. The 50 mm peak does not change drastically and the black and blue graphs are overlapping, giving assumption to a mixture of Ac-225 and Bi-213 neunpa-NO2 complexes.
118
2.3.1.3 Sb-complexation
Figure 15. Antimony complexes discussed in this work.
In addition to complexation with 225Ac (Figure 14), cold complexation with antimony (Sb) was performed using solid antimony(V)oxide. Desferioxamine (DFO) and suberanilohydroxamic acid (SAHA) consist of hydroxamic acids moieties which are hard donors, meeting theoretically the requirements for complexation of the hard acid Sb. DFO, SAHA and H4neunpa successfully chelated antimony (Figure 15), as proven by HR ESI-MS. DFO and H4neunpa formed a 1:1 complex and SAHA a 2:1 complex. Noteworthy, the HR-ESI-MS showed a single positive charge on each Sb-complex. This suggests that antimony was reduced to Sb(III) upon chelation. The 1H NMR spectrum of Sb-neunpa-p-Bn-NO2 is shown in Figure 16. N O O HN N O O NH O O N O O H2N Sb HO N H O O H N O Sb H N O O N H O N N N NO2 N N OH OH O O O O O O Sb
119
Figure 16. 1H NMR spectra of p-Bn-NO
2-neunpa (top) and Sb-neunpa-p-Bn-NO2 (bottom) (400 MHz, MeOD, 25°C).
3 Summary
H4neunpa is a very versatile chelator. We showed that H4neunpa could easily be bifunctionalized with Glu-ureido-Lys (Scheme S1) to target the PSMA receptor, which is highly expressed in prostate cancer. Unfortunately, the three carboxylic acids of Glu-ureido-Lys seem to interfere with the radiolabeling properties of H4neunpa, resulting in a decreased RCY compared previously radiolabeled H4neunpa-p-Bn-NO2 and its antibody derivative H4neunpa--trastuzumab.7 We hypothesize, that the
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) 1. 48 2. 46 3. 61 3. 43 3. 95 4. 27 2. 00 2. 14 5. 86 3. 25 3. 31 C D 3O D 3. 65 3. 75 3. 90 4. 04 4. 07 4. 57 7. 54 7. 76 8. 13 1. 6 2. 6 4. 5 4. 2 3. 6 3. 7 2. 1 2. 0 6. 5 3. 31 C D 3O D 3. 65 3. 82 3. 95 4. 12 4. 66 7. 57 7. 59 7. 83 7. 84 8. 15 8. 19
120
distance between Glu-ureido-Lys and H4neunpa is too short and an extension of the distance between the functionalization part and the chelator will improve radiolabeling efficiencies. Different lengths of linker are currently being studied at the BC Cancer Agency in Vancouver with the aim of increasing radiolabeling efficiencies with 111In3+.
In addition, bifunctionalization can be performed with a therapeutic metallacage, which is able to incorporate cisplatin for the treatment of specific cancer types. We successfully synthesized compound 2, the precursor of the metallacage, as proven via various 1D and 2D NMR spectroscopies as well as HR ESI-MS. The formation of the metallacage is still ongoing in a collaboration project between UBC and the University of Cardiff. After the successful H4neunpa-metallacage synthesis, extensive studies are needed to confirm the cell uptake of the new compound compared to the metallacage alone as well as the evaluation of the cytotoxic profile.
Furthermore, H4neunpa-p-Bn-NO2 shows stable 225Ac labeling efficiencies >95 % over 120 minutes incubation at room temperature at a chelator concentration of 10-4 M. DOTA showed only 1 % radiolabeling yield under the same conditions. Conversely, H4neunpa-p-Bn-NO2 forms a 213Bi complex at 10-6 M and additionally, at higher concentrations, two 225Ac complexes. These results are extremely promising, since H4neunpa is able to bind to 225Ac as well as its daughter nuclide 213Bi, which makes it a potent chelator. In order to proof complex stability, the stability should be assessed in serum. These experiments are currently underway at TRIUMF.
To conclude, H4neunpa can be labelled with diverse radiometals for therapy (225Ac, 213Bi) and imaging (111In) and it might thus find application as diagnostic or therapeutic bifunctional chelator. Current studies are ongoing to understand the mechanism of action of 111In-neunpa as well as human serum stability experiments for 225Ac experiments.
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4 Experimental Materials and Methods
All solvents and reagents were from commercial sources (Sigma Aldrich, TCI) and were used as received unless otherwise noted. 1H and 13C NMR spectra were recorded at room temperature on a Bruker AV400 instrument; the NMR spectra are expressed on the δ (ppm) scale and are referenced to the residual solvent signal of the deuterated solvent. All spectra were recorded with sweep widths of 0-14 ppm or -20-220 ppm for 1H and 13C NMR respectively. Assignments of the peaks in the NMR spectra are approximate. Mass spectrometry was performed on a Waters ZQ spectrometer equipped with an electrospray source at the Department of Chemistry, University of British Columbia. The HPLC system used for purification of ligands and precursors consisted of a Waters 600 controller equipped with a Waters 2487 dual λ absorbance detector connected to a Phenomenex synergi hydro-RP 80Å 250mm x 4.60 mm semipreparative column. Fluorescence spectra were recorded on Varian Cary Eclipse fluorescence spectrophotometer.
Synthesis of compounds
Synthesis of H4neunpa-PSMA-L
10 mg (0.015 mmol) of H4neunpa-p-Bn-NCS7 and 10 mg (0.031 mmol, 2 eq.) of Glu-ureido-Lys (Scheme S1) were dissolved in DMF (2 mL). 26.1 µL (0.150 mmol, 10 eq.) of N,N-diisopropylethylamine (DIEA) was then added and the solution was allowed to stir at room temperature for 2 days. Water (1 ml) was then added to the mixture and the mixture was lyophilized. The crude product was purified by RP-HPLC using a semi-preparative column eluted with 18 % acetonitrile with 0.1 % TFA at a flow rate of 4.5 mL/min. The retention time was 13.6 min, and the yield of the product was 13.7 % (1.9 mg). HR ESI-MS: calcd. [M+H]+ for H4neunpa-PSMA-L C43H55N9O15S 970.3617; found [M+H]+ 970.4766
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Labelling of H4neunpa-PSMA-L with non-radioactive Indium (cold
standard)
H4neunpa-PSMA-L (1.5 mg, 1.5 μmol) was suspended in 200 µL water and 200 µL 0.1 M HCl as well as In(NO3)3 (2.3 mg, 7.5 μmol) were then added. The pH of the reaction mixture was adjusted to 4-5 by using 1 M NaOH to achieve precipitation of the product. After 1 hour of stirring the reaction mixture, the reaction mixture was centrifuged to obtain the product as a white precipitate. After dissolving the crude product in H2O/acetonitrile (2 mL, 1:1) with 0.1 % TFA, the product was purified by RP-HPLC using a semi-preparative column eluted with 18 % acetonitrile with 0.1 % TFA at a flow rate of 4.5 mL/min. The retention time was 11.8 min, and the yield of the product was 43.7 % (0.7 mg). HR ESI-MS: calcd. [M+H]+ for In-H
4 -neunpa-PSMA C43H52InN9O15S 1081.2342; found [M+H]+ 1081.2025
Radiolabeling of neunpa-PSMA-L with 111In
The ligand p-NO2-Bn-H4neunpa-PSMA-L was made up as a stock solution (1 mg/mL, ~10-3 M) in deionized water. From this stock solution, serial dilutions were prepared to final ligand concentrations of 10-4 M – 10-8 M. A 100 µL aliquot of each ligand stock (10-4 to 10-8 M) or water (blank control) was added to screw-cap mass spectrometry vials and diluted with sodium acetate buffer (pH 4, 10 mM, 880 µL). An aliquot of diluted 111In stock (20 µL, ~200 µCi) was added to each vial and allowed to radiolabel at ambient temperature for 10 min, then it was analyzed by RP-HPLC to confirm radiolabeling and calculate radiochemical yields. To study the stability of the radiolabelled compound in human serum, undiluted 111InCl3 stock (~20 µL, 5 mCi) was added to the reaction vial containing 10-4 M ligand in sodium acetate buffer. Areas under the peaks observed in the HPLC radio-trace were integrated to determine radiolabeling yields. Elution conditions used for RP-HPLC analysis were gradient: A: 0.1% trifluoroacetic acid (TFA) in water, B: acetonitrile; 0 to 100% B linear gradient 20 min, 1 mL/min. [111In(p-NO2-Bn-neunpa-PSMA)]- (tR = 11.2 min) and “111In3+” (tR = 3.0 min).
123
Synthesis of H4neunpa-cageligand, compound 1
H4neunpa-p-Bn-NCS (0.03 g, 0.35 mmol, 1.1 eq.) and metallocage-arm-NH2 (0.010 g, 0.32 mmol) were dissolved in 2 mL dry DMSO. The reaction mixture was stirred overnight at room temperature and solvents were removed in vacuo. The dry crude powder was washed with ethylacetate and acetone, before purification via semi-prep reverse-phase HPLC (10mL/ min, gradient A: 0.1% TFA in deionized water, B: acetonitrile, A: 95% to B: 100% for 25 min.; tR= 14.4 min) to yield the product as an off-white solid (0.01 g, 40.1 %).
1H NMR (400MHz, MeOD): 8.74 (s, 2H), 8.56-8.54 (d, 2H), 8.02-7.97 (m, 4H), 7.92-7.90 (t, 2H), 7.75 (s, 2H), 7.60-7.58 (t, 3H), 7.52-7.50 (m, 2H), 7.41-7.39 (d, 2H), 7.24-7.22 (d, 2H) 13C NMR (100MHz, MeOD): 172.9, 150.6, 147.7, 139.6, 138.7, 130.7, 129.0, 127.4, 126.8, 124.5, 123.9, 123.9, 57.4, 55.1, 54.4, 50.8, 48.5, 47.9 and 29.1. HR-ESI-MS calcd. for [C51H47N9O8S+H]+ 946.3347; found: 947.3359 [M+H]+.
Labelling of neunpa-cageligand with La (cold complexation), compound 2
H4neunpa-cageligand (0.01 g, 0.13 mmol) was dissolved in 1 mL dist. H2O and La(ClO4)3 * 6 H2O (0.01 g, 0.14 mmol) was added and the pH adjusted with 0.1 M NaOH to pH 4. The product precipitated as a white solid, filtered off and washed with water.
HR-ESI-MS calcd. for [C51H44LaN9O8S+H]+ 1082.2175; found:1082.2163 [M+H]+
Fluorescence Spectroscopy
The emission spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer. For each compound, dilutions in DMSO at a concentration of 10 mM were prepared. First UV/vis spectra of the compounds were recorded in DMSO, to determine the wavelength of the absorbance maximum. The measured absorbance wavelength was used as the excitation wavelength for the emission spectra.
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Radiolabeling of H4neunpa-p-Bn-NO2 with 225Ac
10 µL of a ligand stock solution (10 and 1 mg/mL in water) was added to 130 µL of NH4OAc (0.15 M, pH 5), to this solution 10 µL of 225Ac3+ (~750 nCi) was added, mixed gently with a pipette, and left to react at room temperature. 5 µL aliquots were removed at 0, 15, 30, 60 min, and 2h or 4 h and spotted on iTLC-SG plates. Plates were developed in 10% NaCl/10mM NaOH. With this mobile phase, 'free' Ac3+ and Bi3+ remain at the base line (Rf = 0), and complexed metal migrates up the plate (Rf > 0). Plates were counted on a TLC plate reader immediately after development, and 3 hours later.
Sb-complexation (cold)
DFO (5.7 mg) was dissolved in 2 mL dist. H2O, Sb2O5 (s) was added and the pH adjusted to pH 4. A white precipitation was observed. The precipitate was filtered off, washed with water and the Sb-complex was confirmed via HR-ESI-MS calcd. for [C25H46N6O9Sb]+ 695.2364; found: 695.2358.
SAHA (11.1 mg) was dissolved in 2 mL DMSO. After adding Sb2O5 (s), the solution changed its color from colorless to yellow. HR-ESI-MS calcd. for [C28H38N4O6Sb]+ 647.1830; found: 647.1833.
H4neunpa-p-Bn-NO2 (5.6 mg) was dissolved in 2 mL dist. H2O and Sb2O5 (s) was added and the pH adjusted to pH 4. White precipitation was observed. The precipitate was filtered off and washed with water. HR-ESI-MS calcd. for [C30H32N6O10Sb]+ 757.1218; found: 757.1212.
5 References
1. http://www.who.int/mediacentre/factsheets/fs297/en/ (accessed
17th June 2017).
2. Bourgeois, M.; Bailly, C.; Frindel, M.; Guerard, F.; Cherel, M.; Faivre-Chauvet, A.; Kraeber-Bodere, F.; Bodet-Milin, C., Radioimmunoconjugates
125 for treating cancer: recent advances and current opportunities. Expert Opin.
Biol. Ther. 2017, 17 (7), 813-819.
3. Chang, S. S., Overview of Prostate-Specific Membrane Antigen. Rev.
Urol. 2004, 6 (Suppl 10), S13-S18.
4. Bouchelouche, K.; Choyke, P. L.; Capala, J., Prostate Specific Membrane Antigen—A Target for Imaging and Therapy with Radionuclides.
Discov. Med. 2010, 9 (44), 55-61.
5. Mease, R. C.; Foss, C. A.; Pomper, M. G., PET Imaging in Prostate Cancer: Focus on Prostate-Specific Membrane Antigen. Curr. Topics Med.
Chem. 2013, 13 (8), 951-962.
6. Spreckelmeyer, S.; Ramogida, C. F.; Rousseau, J.; Arane, K.; Bratanovic, I.; Colpo, N.; Jermilova, U.; Dias, G.; Dude, I.; Jaraquemada Pelaez, M. G.; Benard, F.; Schaffer, P.; Orvig, C., p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-SPECT Imaging.
Bioconjug. Chem. 2017.
7. Spreckelmeyer, S.; Ramogida, C. F.; Rousseau, J.; Arane, K.; Bratanovic, I.; Colpo, N.; Jermilova, U.; Dias, G.; Dude, I.; Jaraquemada Peláez, M. d. G.; Benard, F.; Schaffer, P.; Orvig, C., p-NO2-Bn-H4neunpa and H4neunpa-Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and 111In Immuno-SPECT Imaging.
Bioconjug. Chem. 2017.
8. Schmidt, A.; Molano, V.; Hollering, M.; Pothig, A.; Casini, A.; Kuhn, F. E., Evaluation of New Palladium Cages as Potential Delivery Systems for the Anticancer Drug Cisplatin. Chemistry 2016, 22 (7), 2253-6.
9. Zhao, Q.; Liu, X.-M.; Li, H.-R.; Zhang, Y.-H.; Bu, X.-H., High-performance fluorescence sensing of lanthanum ions (La3+) by a polydentate pyridyl-based quinoxaline derivative. Dalton Trans. 2016, 45 (26), 10836-10841.
10. Wang, F.; Huang, W.; Li, K.; Li, A.; Gao, W.; Tang, B., Study on the fluorescence enhancement in Lanthanum(III)–carminic acid– cetyltrimethylammonium bromide system and its analytical application.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2011, 79 (5), 1946-1951.
11. Thisgaard, H.; Jensen, M., Sb119—A potent Auger emitter for targeted radionuclide therapy. Med. Phys. 2008, 35 (9), 3839-3846.
12. Robertson, A. K. H.; Ramogida, C. F.; Rodríguez-Rodríguez, C.; Stephan, B.; Peter, K.; Vesna, S.; Paul, S., Multi-isotope SPECT imaging of
126
the 225 Ac decay chain: feasibility studies. Physics Med. Biol. 2017, 62 (11), 4406.
13. Chappell, L. L.; Deal, K. A.; Dadachova, E.; Brechbiel, M. W., Synthesis, Conjugation, and Radiolabeling of a Novel Bifunctional Chelating Agent for 225Ac Radioimmunotherapy Applications. Bioconj.
Chem. 2000, 11 (4), 510-519.
127
Supporting Information
Chapter A2
H
4neunpa: A Bifunctional Acyclic Chelator with
many Faces
Sarah Spreckelmeyer,a,b Caterina Ramogida,c Ting Kuo,d Ben Woods, e
Valery Radchenko,c Maria Guadalupe Jaraquemada Peláez,a Francois Bénard,d Angela Casinie and Chris Orviga
a Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada
b Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands c Life Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia, V6T 2A3, Canada
d BC Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3, Canada
e School of Chemistry, Cardiff University, Park Place, CF103AT Cardiff, United Kingdom
128
Scheme S 1. Synthesis of Glu-ureido-lys (here: HTK-01068).
Synthesis of of HTK-01018: A solution of L-glutamic acid di-tertbutyl
ester hydrochloride (1.5 g, 5.07 mmol) and triethylamine (2.31 mL, 16.63 mmol) in CH2Cl2 (40 mL) was cooled to −78 °C in a dry ice/acetone bath. Triphosgene (525 mg, 1.77 mmol) dissolved in CH2Cl2 (10 mL) was added dropwise to the reaction. After the addition was complete, the reaction was allowed to warm to room temperature and stirred for 30 minutes. H-Lys(cbz)-OtBu hydrochloride (1.5 g, 4.06 mmol) was then added to the reaction mixture, followed by triethylamine (566 μL, 4.06 mmol). After stirred overnight for 17 h, the reaction mixture was diluted with CH2Cl2 (50 mL) and washed with H2O (60 mL × 2). The organic phase was then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography on silica gel eluted with 3:2 hexane/EtOAc to obtain the desired product HTK-01018 as colorless oil (2.32 g, 92.3 %).
Synthesis of of HTK-01027: A solution of HTK-01018 (2.32 g, 4.47
mmol) in MeOH (45 mL) was slowly added Pd/C (117 mg, wet by 5~10 mL MeOH) to the reaction. The reaction mixture was hydrogenated at room temperature under 1 atm. After stirred overnight, the solution was
H2N O O t-Bu O O t-Bu a. Triphosgene Et3N, CH2Cl2, -78oC, 30 min b. H-Lys(Cbz-OtBu), dropwise warm to room Temp, Et3N, 17 hr
N H NH NHCbz O O t-Bu O O O t-Bu O O t-Bu N H NH NH2 O O t-Bu O O O t-Bu O O t-Bu Pd/C MeOH HTK-01018 HTK-01027 HCl TFA 3 % Anisole N H NH NH2 HO O O O OH OH O 4 hr HTK-01068
129
filtered through celite and concentrated under reduced pressure to obtain HTK-01027 as viscous oil (1.81 g).
The crude product of HTK-01027 was used in next step without further purification.
Synthesis of of HTK-01068: A solution of HTK-01027 (203 mg, 0.32
mmol) in TFA (5 mL) followed by 3% anisole was stirred at room temperature. After 4 h, the reaction mixture was concentrated under reduced pressure. The concentrate diluted with water (1 mL) and extracted with hexane (1 mL × 3) to remove anisole. The water phase was then iced and lyophilized to obtain crude HTK-01068 as a yellow oil. The crude product of HTK-01068 was used in next step without further purification.
Figure S1. 13C NMR spectrum of compound 1.
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 f1 (ppm) 53 .2 7 54 .3 9 55 .1 1 57 .4 6 12 3. 87 12 6. 73 12 7. 40 12 9. 00 13 8. 55 13 9. 66 13 9. 87 15 0. 81 17 2. 92
130
Figure S1. 2D-HSQC NMR spectrum of the aromatic region of
compound 1 (400MHz, MeOD, 25°C). 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 f2 (ppm) 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150 152 f1 ( pp m ) {8.75,150.58} {8.57,147.72} {8.05,139.62} {7.94,138.69} {7.59,130.73} {7.25,129.02} {7.76,127.36} {7.61,126.78} {7.42,124.48} {8.04,123.87} {7.53,123.87}
131
Figure S2. 2D-HSQC NMR spectrum of the alkyl region of compound 1
(400MHz, MeOD, 25°C). 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 f2 (ppm) 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 f1 ( pp m ) {4.05,57.44} {3.59,55.06} {3.50,54.42} {3.68,50.82} {3.27,48.52} {3.32,47.93} {3.06,29.08}
132
Figure S3. 2D-COSY NMR spectrum of the aromatic region of
compound 1 (400MHz, MeOD, 25°C). 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 f2 (ppm) 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 f1 ( pp m ) {7.50,8.56} {7.51,8.04} {7.60,7.92} {7.94,7.60} {8.55,7.51} {8.04,7.50}
133
Figure S4. 2D-COSY NMR spectrum of the aromatic region of
compound 1 (400MHz, MeOD, 25°C). 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 f2 (ppm) 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 f1 ( pp m ) {7.50,8.56} {7.51,8.04} {7.60,7.92} {7.94,7.60} {8.55,7.51} {8.04,7.50}
