University of Groningen
Metallodrugs for therapy and imaging: investigation of their mechanism of action
Spreckelmeyer, Sarah
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Chapter A3
Tetrahydroxamic Acid Bearing Ligands:
EDTA and DTPA Analogues
Sarah Spreckelmeyer,a,b Yang Caoa 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
1 Abstract
Hydroxamic acids are unique cation chelators that find application in the treatment of many diseases. Two tetra hydroxamic acid bearing chelators H4EDT(M)HA and H4EDT(B)HA
were successfully synthesized and characterized as metal chelators. They were tested for their anticancer activity and 89Zr
radiolabeling properties for positron emission tomography (PET) imaging. Although, Fe3+, Zn2+ and Cu2+ cold metal complexation
reactions were achieved as proven by IR, NMR spectroscopies, HR ESI-MS spectrometry and UV-VIS experiments, 89Zr
radiolabeling did not lead to any radiolabeling under the tested conditions. With DFT calculations and UV-VIS stability experiments, metal-EDT(M)HA complexes were found unstable due to the inflexibility of the four hydroxamic acid arms and the formation of complexes in a 2:2 ratio of chelator:metal. A second set of hydroxamic acid bearing chelators was designed and evaluated with density functional theory (DFT) and found to have theoretically a better stability of 89Zr radiolabeling. The synthesis
2 Introduction
Hydroxamic acids have a long history as biological and chemical agents. In 1869, W. Lossen discovered the first hydroxamic acid, oxalohydroxamic acid.1 Beside their use as
insecticides, antimicrobials, plant growth regulators, antioxidants, corrosion inhibitors or redox switches for electronic devices, hydroxamic acids have been identified as a key functional group for the development of pharmacological agents. Several drugs against hypertension, cancer growth, inflammation, infectious agents, asthma and arthritis that contain hydroxamic acid functional groups are used in the clinic. This application versatility is due to their common mechanism of action: their ability to chelate metal ions as well as to form a hydrogen bond at the active site of enzymes. They are selective and potent inhibitors of enzymes such as zinc metalloproteases (e.g. matrix metalloproteinases MMPs, angiotensin converting enzymes ACE, leukotriene A4 hydrolase LTA4), nickel metalloproteases (e.g.
urease) and iron metalloproteases (e.g. lipoxygenase 5-LO, peptide deformilase PDF).2 Furthermore, it is suggested that
hydroxamic acids are nictric oxide (NO) donors, the reason for their use against hypertension. Consequently, it is beyond dispute that hydroxamic acids are attractive tools in drug-design for a variety of diseases. In addition to their use as therapeutics, hydroxamic acids have been recently found to be attractive compounds for imaging purposes due to their chelation properties of radiometals.3
The chemical structure of hydroxamic acids is RC(O)N(R1)OH, where R1 can be either hydrogen or an alkyl
moiety. They belong to a class of organic acids, which are weaker acids (acetohydroxamic acid pKa = 8.70) than structurally related
carboxylic acids (acetic acid pKa = 4.75). Hydroxamic acids can
either act as a monodentate ligand through the deprotonated hydroxyl (OH) moiety, or as bidentate monoanionic di-oxygen ligands, strongly prone to complex di- and trivalent metal cations.
Hydrogen bonds can be formed through their OH group, amine (NH) group and carbonyl (C=O) oxygen. Their iron and copper complexes are highly coloured and are used for spectrophotometric and gravimetric analysis.4 Hydroxamic acids
exhibit keto-iminol tautomerism (Figure 1). If the nitrogen is not substituted, hydroxamic acids exist preferably in the keto-Z or keto-E form over the iminol-Z or iminol-E. N-substituted hydroxamic acids on the other hand, can only exist in keto-Z or keto-E form, since they lack the NH group necessary for hydrogen transfer. In solution, the keto-E form seems to be more stable compared to the keto-Z form by 0.8 kcal/mol.4 In order to chelate
metal ions, the acid should adopt the required Z-conformation; for that, interconversion between the Z and E conformation must take place by rotation about the C-N bond. The rotational barrier for N-substituted and N-non-substituted hydroxamic acids is between 16.6 kcal/mol and 20.2 kcal/mol.
Figure 1. Keto-iminol tautomerism of hydroxamic acids.
A special class of hydroxamic acid bearing compounds are siderophores (Greek: iron carrier), metal chelators that are low molecular weight agents, expressed by microorganisms in order to scavenge insoluble Fe3+ cations.3 Iron is essential for
organisms to maintain DNA synthesis and respiration. The hydroxamic acids bearing agent desferrioxamine B (DFO, Desferal, Figure 2) is FDA approved for the treatment of iron poisoning or
R N H OH O R NH OH O R N OH OH R N OH OH keto-Z keto-E iminol-Z iminol-E
thalassemia.5 Due to their strong chelation to metal ions like Fe3+,
Cu2+ or Ni2+, siderophores have been recently considered for
molecular imaging applications, reviewed by Petrik et al.3
Commonly used radiometals for imaging purposes are
68Ga (t
1/2 = 68 min), 11C (t1/2 = 20 min) or 18F (t1/2 = 109 min) for PET
imaging. The half-lives of these radiometals are quite short. Consequently, a short time for the tracer preparation is thus the limiting step and on-site preparation is often necessary to avoid loss of radioactivity during transportation. Thus, extensive research has been focused on radiometals with longer half-lives for PET imaging. 89Zr is an attractive radionuclide for this purpose,
with a half-life of 3.3 days, which suits the biological half-life of biomolecules like antibodies. The development of a stable chelator for 89Zr is a challenging and a highly pursued goal of
many research groups. To date, 89Zr-DFO-labelled antibodies are
used off-label in the clinic to detect various types of cancer depending on the antibody (e.g. 89Zr-DFO-retuximab for B-cell
non-Hodgkin lymphoma), although it bears major drawbacks like high bone uptake of 89Zr due to an unstable metal-ion chelate.6 It is
hypothesized, that the preferred coordination number of Zr4+ is 8,
thus DFO does not fulfill this requirement with its hexadentate chelating groups (Figure 2).7 Recently, several research groups
have developed cyclic and acyclic chelators bearing hydroxamic acid motifs for 89Zr chelation, summarized in Figure 2.8,9,10,11 They
were tested for 89Zr radiolabeling in vitro and in vivo. Noteworthy,
the acyclic chelator DOTA was successfully labeled with 89Zr and
a crystal structure of the “cold” Zr4+ complex was obtained,
showing an octadentate binding to the metal centre by four nitrogens and four oxygens.12 Most recently in July 2017, C.
Buchwalder et al. published a paper on an octadentate 3,4-HOPO chelator called THPN, that quantitatively complexates 89Zr4+ and
Figure 2. Hydroxamic acids bearing ligands discussed in this
work and macrocyclic DOTA. The hydroxamic acid functional groups are given in red.
Due to the great success of ethylenediaminetetraacetic acid (EDTA) in stable metal complexation reactions14, we report
here the synthesis, characterization as well as biological evaluation of the two EDTA analogues with four hydroxamic acid moieties (ethylenediaminetetra(methyl)hydroxamic acid H4EDT(M)HA and ethylenediaminetetra(benzyl)hydroxamic acid,
H4EDT(B)HA) (Figure 3, left side).
N OH O HN NOH O NH O O N HO NCS O HN O N HO O N OH O HN N HO O NH O O N HO NH2 O DFO N N HN N O O N OH O O N HO O O N OH N O O N N O N OH O N HO O N HO O N OH NCS -Rousseau et al.9
Boros et al.8 Vugts et al.10
N N N N O O O O O O O O OH OH HO HO Deri et al.7 N N N N DOTA O OH O HO O OH O HO N N O HN O NH O HN O NH N N N N HO O O O O OH OH HO
Figure 3. Novel tetrahydroxamic acids bearing ligands discussed
in this work.
Cold metal complexation reactions with Fe3+, Cu2+ and Zn2+
were studied via HR ESI-MS, IR spectroscopy and UV-VIS spectroscopy. In addition, to study their potential use as anti-cancer agents, the antiproliferative effects of H4EDT(M)HA
compared to desferrioxamine (DFO) and suberanilohydroxamic acid (SAHA, Vorinostat) were tested in HT-29, 7 and MCF-10A cell lines. Moreover, their use as 89Zr chelators was assessed
in radiolabeling experiments. Additionally, density functional theory (DFT) studies were performed to explain the stability found
N N N O N HO O N OH OH O N HO O N N N N N O HO O OH O HO O OH O OH O OH O HO O OH O OH EDTA DTPA H₄EDT(M)HA N N N O N HO O N OH OH O N HO O H₄EDT(B)HA n = 2 DTT(ME)HA = 3 DTT(MP)HA N N N O N HO O N OH OH O N HO O n n n n N H n = 2 DTT(BE)HA = 3 DTT(BP)HA N N N O N HO O N OH OH O N HO O n n n n N H 1-. generation 2ⁿ: generation
for the complexes and to contribute to the design of a second
generation hydroxamic acids bearing
diethylenetriaminepentaacetic acid (DTPA) analogues DTT(ME)HA, DTT(MP)HA, DTT(BE)HA and DTT(BP)HA (Figure 3, right side) as potential 89Zr chelators for PET imaging.
3 Results and Discussion
3.1 Synthesis and characterization
Based on the structure of EDTA, the hydroxamic acid analogue ethylenediaminetetra(methyl)hydroxamic acid, H4EDT(M)HA, was synthesized (Scheme 1). Starting from
N-methylhydroxylamine hydrochloride (Scheme 1a), the secondary amine was protected with a tert-butyloxycarbonyl (BOC) group15 1.
In a next step, the hydroxyl group was deprotonated with sodium hydride under an inert atmosphere, following a benzylation reaction with benzylbromide to yield 2.16 After deprotection of the
BOC group with TFA/DCM 1:1 to yield 3, bromoacetylbromide was added17 to synthesize the “arm“ of H
4EDT(M)HA 4. In a next
reaction (Scheme 1b), ethylenediamine was added to the “arm“ 4 to yield the protected intermediate 5. The product was synthesized by palladium-catalyzed debenzylation to produce H4EDT(M)HA 6 in an overall yield of 1.5 %. The yield limiting step
is most likely the addition of the “arms” to the backbone, since three different side-products were observed, which are either the mono, di or tri functionalized backbone. The product was fully characterized using 1H NMR (Figure 4), 13C NMR, 2D-HSQC
Scheme 1. Synthesis route of H4EDT(M)HA.
H4EDT(B)HA was synthesized starting with
O-benzylhydroxylamine hydrochloride (Scheme 2) and benzaldehyde to form its imine intermediate (not shown). For the reduction of the imine, sodium cyanoborohydride was used at pH 4 to yield 7.18 In a next reaction, bromoacetyl bromide was added
to synthesize the “arm” of H4EDT(B)HA 8. After linking the arm to
ethylenediamine 9 (Scheme 2b), the O-benzyl groups were removed by palladium catalyzed hydrogenation to produce the final ligand H4EDT(B)HA 10 in an overall yield of 0.35 %. 1H NMR
(Figure 4) and 13C NMR confirmed the successful synthesis.
HNOH O NOH O NO O O HNO TEA, DCM (BOC)2O NaH, DMF Br TFA/DCM (1:1) 1 2 3 NO Br O Br X O n n
n = 1 arm for EDT(M)HA, 4
= 2 DTT(ME)HA, 4a = 3 DTT(MP)HA, 4b K2CO3, THF a) b) H2N NH2 K2CO3, CH3CN N N N O N O O N O O O N O O Pd(OH)2/C, H2 MeOH N N N O N HO O N OH OH O N HO O 5 H4EDT(M)HA, 6 4
Scheme 2. Synthesis route of H4EDT(B)HA.
Figure 4. 1H NMR spectra of H
4EDT(M)HA (top, 400 MHz, D2O,
25°C) and H4EDT(B)HA (bottom, 400 MHz, MeOD, 25°C).
3.2 Metal complexation reactions
To determine, whether the synthesized ligands H4EDT(M)HA and H4EDT(B)HA are chelators for metal ions, Fe3+,
H2NO HNO O MeOH, HCl, NaCNBH3 NO Br O 7 Br X O K2CO3, THF n n
n = 1 arm for EDT(B)HA, 8 = 2 DTT(BE)HA, 8a = 3 DTT(BP)HA, 8b a) N N N O N O O N O O O N O O 9 H4EDT(B)HA, 10 N N N O N HO O N OH OH O N HO O b) 8 H2N NH2 K2CO3, CH3CN Pd(OH)2/C, H2 MeOH 3. 26 3. 63 4. 36 4. 79 D 2O 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 f1 (ppm) 3. 31 C D 3O D 3. 37 4. 26 4. 76 7. 33 methylen N-CH2-CH2-N N-CH3 O-CH2 methylen N-CH2-CH2-N benzyl H2O
Cu2+ and Zn2+ were chosen as model ions for metal complexation
reactions because of their roles in biological processes. As mentioned above, many enzymes bear these metal ions in their active site, which can be chelated by hydroxamic acids. Fe3+ is a
hard acid, paramagnetic and forms coloured complexes. Cu2+ is a
hard acid and paramagnetic as well. Zn2+ is diamagnetic. As
proven by HR ESI-MS (see Materials and Methods section), H4EDT(M)HA and H4EDT(B)HA form metal-ligand complexes in a
1:1 ratio with each of the three metals. 1H NMR spectra of
Fe(III)-EDT(M)HA and Cu(II)-Fe(III)-EDT(M)HA show the expected changes in chemical shifts (Figure S 2), due to metal complexation. However, the 1H NMR spectra do not allow proper peak assignments due to
the paramagnetism of these two metals. Therefore, IR as well as UV VIS spectroscopy is applied for further characterizations. A 1H
NMR spectrum of Zn-EDT(M)HA was not recorded, the Cu-EDT(M)HA and Fe-(Cu-EDT(M)HA spectra were considered sufficient as a proof of principle.
3.3 Infrared (IR) spectroscopy
IR is a useful tool to determine vibrational modes; symmetric and antisymmetric stretching as well as bending modes are the typical vibrational modes that are observed. The carbonyl bond of hydroxamic acids usually show a characteristic peak around 1650 cm-1 in the IR spectra, and they can easily be
distinguished from other carbonyl bonds, such as carboxylic acids that show absorption above 1700 cm-1. A successful metal
complexation would be indicated by a shift of the characteristic peak of the hydroxamic acids, due to changes in the vibrational modes, to less frequent vibrations.19 For proof of principle, the
infrared spectra of the H4EDT(M)HA, Fe-EDT(M)HA and
Cu-EDT(M)HA are shown in Figure 5 and the characteristic shifts for the functional groups of the hydroxamic acids are summarized in Table 1. The ligand itself shows a typical carbonyl stretch at 1660 cm-1. After complexation, the carbonyl stretches of the
and 1634 cm-1, respectively. This behaviour has been reported
before for hydroxamic acid complexes with Ni2+, Co2+ and Zn2+.20
This finding indicates a coordination of the ligand with the metal ion through the oxygen of the carbonyl functional group. The symmetric N-O stretch shifts to lower frequencies as well, indicating that the hydroxamate complexation is bidentate. These two results give an indication that the metals are chelated via bidentate bis-oxygen chelation. Moreover, an intense stretch at about 1200 cm-1 was observed for the ligand as well as for the
metal complexes, which can be assigned as C-N stretch. This stretch does not shift upon chelation, suggesting that the C-N bond is not involved in the metal complexation. Due to N-substitution of the ligand, the iminol form is not an option, making the involvement of the C-N bond in chelation unlikely (for full spectra, see Supporting Information, Figure S 3).
Figure 5. Partial IR spectra of H4EDT(M)HA, Fe(III)-EDT(M)HA and
Cu(II)-EDT(M)HA at 25°C (solid state).
Table 1. Summarized IR data of the relevant functional groups. Functional group H4EDTMHA [cm-1] Fe(III)-EDTMHA [cm-1] Cu(II)-EDTMHA[cm-1] N-O 1138 1055 1068 C=O 1660 1628 1634
3.4 In vitro cell experiments
Based on the successful metal complexation, H4EDT(M)HA was tested for its possible toxic effects towards two
cancer cell lines (MCF-7: breast cancer and HT-29: colon cancer) compared to a non-tumorigenic cell line (MCF-10A: epithelial
600 800 1000 1200 1400 1600 1800 80 90 100 wavenumber [cm-1] Tr a n s m it ta n c e [ % ] H4EDT(M)HA Fe-EDT(M)HA Cu-EDT(M)HA
healthy breast cells). We hypothesize that H4EDT(M)HA shows
toxicities similar to desferoxamine (DFO, Desferal®) and suberanilohydroxamic acid (SAHA, Vorinostat®) which have been used as controls. DFO has already shown anti-proliferative and cytotoxic effects on several tumour cell lines, due to its chelation ability of iron and thereby removing the iron from an iron-dependent enzyme necessary for cell cycle progression.21 SAHA
is an anticancer agent that was approved in 2006 by the FDA. SAHA binds Zn2+ and thereby stays in the active site of histone
deacetylases.22
The results of three independent cell experiments with H4EDT(M)HA, DFO and SAHA are shown in Table 2. For DFO the
expected low IC50 values of 9.5 ± 2.4 µM, 15.0 ± 2.7 µM and 6.4 ±
0.8 µM were found for HT-29, MCF-7 and MCF-10A cells, respectively.23,24 SAHA shows the highest toxicity, with IC
50
values of 1.0 ± 0.3 µM, 0.8 ± 0.5 µM and 1.5 ± 0.5 µM for these cell lines. H4EDT(M)HA shows very low toxicity towards HT-29,
MCF-7 and MCF-10A cells with IC50 values above 100 µM. A
reliable IC50 determination was not possible above 100 µM of the
compound, since these high concentrations required a DMSO concentration above 1.0 % in the final cell incubation medium, which affected the cell viability.
Table 2. Cytotoxicity of DFO, SAHA and H4EDT(M)HA on HT-29,
MCF-7 and MCF-10A cells, expressed as IC50 [µM] (n = 3 ± SD).
Cell line/compound HT-29 MCF-7 MCF-10A
DFO 9.5 ± 2.4 15.0 ± 2.7 6.4 ± 0.8
SAHA 1.0 ± 0.3 0.8 ± 0.1 1.5 ± 0.5
H4EDT(M)HA >100 >100 >100
The low toxicity of H4EDT(M)HA could have different
reasons among which a low stability of the metal-EDT(M)HA complexes or a low uptake into the cells can be considered. Hydroxamic acids are very hydrophilic functional groups and
H4EDT(M)HA incorporates four hydroxamic acid moieties. Thus,
membrane passage by passive diffusion of H4EDT(M)HA seems
unlikely due to its hydrophilicity. Alternatively the complex might cross the cell membrane via channels or carriers. Further experiments are needed to get more information about the uptake mechanisms of H4EDT(M)HA. As mentioned above, instability of
the metal-EDT(M)HA complexes might be the reason for the low toxicity. Therefore, the stability of the complexes was determined by UV-VIS spectroscopy.
3.5 Stability determinations of Fe-EDT(M)HA and Fe-EDT(B)HA by UV-VIS spectroscopy
UV-VIS spectroscopy is a technique based on electronic transitions in atoms, molecules or ions. Fe(III), in contrast to Zn(II), is a unique cation that changes its colour upon chelation. Thus, the successful iron complexation can easily be seen by a color change of the Fe(III)-solution from yellow to red after adding H4EDT(M)HA or H4EDT(B)HA. The intensely coloured complexes
can be studied by UV-VIS spectroscopy. Fe(III) belongs to the d-block elements, with a partly 3d filled orbital shell. Upon complexation, the d orbitals split into two different energy levels, two orbitals with a higher energy level and three orbitals with a lower energy level. Upon absorption of light, an electron is promoted from the lower energy level to the higher energy level. The greater the difference in energy between the levels, the more energy is needed for this promotion. In Figure 6, the changes in UV-VIS spectra of Fe(III)-EDT(M)HA or Fe(III)-EDT(B)HA at 37 °C in water over a time period of 5 days are shown. An absorption maximum at 490 nm for Fe(III)-EDT(M)HA and at 500 nm for Fe(III)-EDT(B)HA correlates well with the red coloured solutions. Fe(III)-EDT(M)HA shows a sharper absorption peak compared to Fe(III)-EDT(B)HA. The UV VIS spectrum of Fe(III)-EDT(B)HA might be broader due to the co-absorbance of the benzyl groups of the chelator. A decrease of the absorbance over 5 days indicates an unstable Fe-complex for both ligands. Notably, the absorbance of
Fe(III)-EDT(M)HA decreases faster than that of Fe(III)-EDT(B)HA. Concerning the in vitro IC50 values, the low stability of the
Fe(III)-complexes shown here, might at least partly explain the low toxicity in vitro. The cell experiments were conducted after three days, when still 50 % of the Fe-complexes were intact (Figure 6). However we have to consider, that for the cell experiments, other factors may also play a role, like trans-chelation of the Fe-complexes eg. via transferrin. Moreover, the UV VIS experiments were performed in water, with only the tested compound and Fe3+ions present, whereas the cell experiments were performed in
Figure 6. UV-VIS spectra of Fe-EDT(M)HA (left) and Fe-EDT(B)HA
(right) at 37°C over 5 days in H2O.
3.6 89Zr-radiolabeling
Chelation of 89Zr was investigated to explore whether our
ligands could be applied for PET imaging. As mentioned above, hydroxamic acids are hard electron donors, and as such, a good fit for the hard acid cation Zr4+. Zirconium prefers a coordination
number of 8, thus the tetrahydroxamic acid H4EDT(M)HA was
assumed to be a good candidate for 89Zr radiolabeling. However,
we could not observe any labelling of H4EDT(M)HA with 89Zr.
Different radiolabeling media (phosphate buffered saline pH 7.4 (PBS) or 0.9 % NaCl solution pH 7.4 (saline)) were applied. The
400 500 600 700 800 0.0 0.5 1.0 1.5 wavelength [nm] Absorbance 0d 1d 2d 3d 4d 5d 400 600 800 0.0 0.5 1.0 1.5 wavelength [nm] Absorbance 0 d 1 d 2 d 3d 4d 5d
radiochemical yield (RCY) was determined via instant thin layer chromatography (iTLC). Different mobile phases (EDTA pH 7, citrate pH 5.5, DTPA pH 6) were applied for iTLC. Additionally, radio-HPLC was performed as well to get further information. However, 0 % radiolabeling of H4EDT(M)HA with 89Zr was
observed whereas labeling of the gold-standard DFO always gave expected good RCYs. An example of an iTLC chromatogram can be found in the Supporting Information Figure S 4.
3.7 Density Functional Theory (DFT)
In order to find an explanation for the low stability found in UV-VIS experiments for the Fe-EDT(M)HA complexes, and the lack of complexation with 89Zr, various DFT optimized structures were
calculated.
Our aim was to determine, if Fe3+ or Zr4+ complexation can
theoretically take place, taking into consideration the bond lengths and angles of the coordinative bonds between the metal and chelator. The results show that the ligand itself shows all four hydroxamic acids arms pointing away from each other, making it impossible to chelate a single metal ion in a 1:1 ratio (Figure 7, left), due to the inflexibility of the arms. Thus, no DFT structure could be calculated in a 1:1 metal-chelator ratio. The second DFT structure in Figure 7 shows a dimer of H4EDT(M)HA
with two Fe3+ ions, indicating that a complex can theoretically be
formed where the iron is coordinated with only six donor arms of the hydroxamic acids in a 2:2 ratio. The HR ESI-MS does only show the iron-complex in a 1:1 ratio. These findings explain the low stability of metal-EDT(M)HA complexes and can also explain the unsuccessful 89Zr radiolabeling.
In a DFT study of Boros et al.9, hydroxamic acid bearing
cyclic DOTA analogues showed higher stability with metal ions, if the hydroxamic acid arms are longer than acetohydroxamic acid and if the cavity is bigger than ethylenediamine. In that case, incorporation of large metal ions like Zr4+ seems feasible. Thus,
(Figure 8) with longer hydroxamic acid donor arms was evaluated by DFT. By increasing the size of the backbone from ethylenediamine to diethylenetriamine and by increasing the hydroxamic acid arm length from a methylene group to ethylene or propylene, a more ideal geometry of the Zr-complexes were calculated (see bond lengths in the Supporting Information Figure S 5 and Figure S 6).
Additionally, the middle nitrogen atom can be functionalized without interfering with the chelation properties (Supporting Information, Figure S 7), which makes the new generation of DTPA analogues ideal bifunctional chelators for
Figure 7. DFT optimized structures of H4EDT(M)HA (left) and
[Fe2(EDT(M)HA)2] (right) (grey: carbon, blue: nitrogen, red: oxygen,
orange: iron (B3LYP/6-31G* + LANL2DZ and PCM solvation (water) on Gaussian 09).
Figure 8. DFT optimized structure of Zr-DETT(ME)HA (left) and
Zr-DETT(MP)HA (right) (grey: carbon, blue: nitrogen, red: oxygen, light blue: zirconium (B3LYP/6-31G* + LANL2DZ and PCM solvation (water) on Gaussian 09).
Based on the DFT calculations, as is discussed above, we aimed to synthesize four second-generation DTPA analogues. The proposed synthesis routes for these four DTPA analogues diethylenetriaminetetra(methylethyl)hydroxamic acid (H4DTT(ME)HA),
diethylenetriaminetetra(methylpropyl)hydroxamic acid (H4DTT(MP)HA), diethylentriaminetetra(phenylethyl)hydroxamic
diethylenetriaminetetra(phenylpropyl)hydroxamic acid (H4DTT(BP)HA) are shown in Scheme 3.
Scheme 3. Proposed synthesis route for f H4DTT(ME)HA,
H4DTT(MP)HA, H4DTT(BE)HA and H4DTT(BP)HA.
Regarding H4DTT(ME)HA and H4DTT(MP)HA, their arms
were synthesized starting with intermediate 3 (see Scheme 1a) and adding either 4-bromobutyryl chloride or 3-bromopropionyl chloride, respectively to the reaction mixture. The successful synthesis of the two hydroxamic acid arms 4a and 4b was characterized by 1H NMR spectroscopy (Figure 9), 13C NMR
spectroscopy (Figure S 8) and HR ESI-MS.
N N N O N O O N O O O N O O n = 2 precursor of DTT(BE)HA = 3 DTT(BP)HA n n n n n = 2 DTT(BE)HA = 3 DTT(BP)HA b) NH N N N O N HO O N OH OH O N HO O n n n n N H Pd(OH)2/C, H2 MeOH H2N N H NH2 N N N O N O O N O O O N O O n = 2 precursor of DTT(ME)HA = 3 DTT(MP)HA n n n n N H n = 2 DTT(ME)HA = 3 DTT(MP)HA N N N O N HO O N OH OH O N HO O n n n n N H a) Pd(OH)2/C, H2 MeOH H2N N H NH2 4a 4b 8a 8b
Figure 9. 1H NMR spectra of arms (4a, top) and (4b, bottom), 400
MHz, CDCl3, 25°C.
The next reaction step, the attachment of the “arms” 4a or
4b to the backbone diethylenetriamine via a SN2 reaction, did not
result in the anticipated products but always led to the E2
side-products (Figure 10). Unfortunately, the conditions for a SN2
reaction are quite similar to the conditions of an E2 reaction. SN2
reactions require a strong nucleophile and E2 reactions require a
strong base. Good nucleophiles are often strong bases. To obtain the required product, a good nucleophile, that is a weak base (eg. K2CO3, NaHCO3), would favour an SN2 reaction over an E2 reaction.
The use of polar, aprotic solvents (eg acetone, DMF, acetonitrile and DMSO) might also increase the nucleophilicity and thus increase the rate of SN2. The conditions tried thus far are
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 f1 (ppm) 7. 26 C D C l3 7. 26 C D C l3 benzyl O-CH2 N-CH3 Br-CH2-CH2 Br-CH2-CH2 benzyl O-CH2 Br-CH2-CH2-CH2 Br-CH2-CH2-CH2 Br-CH2-CH2-CH2 N-CH3
summarized in the Supporting Information (Table S1). Unfortunately, none of the tried conditions resulted in the anticipated products. Further work to synthesize the appropriate compounds is ongoing.
Figure 10. E2 reaction schemes that yielded the unwanted
side-products.
4 Conclusions
Hydroxamic acids are unique metal ion chelators and can be used for either therapy or diagnosis. Our first aim was the evaluation of new chelators for PET imaging, using 89Zr as
radiometal. The synthesis of 89Zr chelating ligands for PET
imaging is theoretically a promising goal but appeared challenging in practice. Hydroxamic acid bearing chelators were suggested to be the perfect functional groups for this purpose, as they are hard donors and perfectly fit the hard acid character of
89Zr. Our second aim deals with the evaluation of hydroxamic acid
bearing ligands as anticancer agents due to the ability of hydroxamic acids to chelate essential metals like Cu2+, Fe3+ and
Zn2+.
In this work, two tetrahydroxamic acid bearing EDTA analogues were synthesized, which differ in their N-substitution. They were successfully characterized using 1H NMR, 13C NMR
N O O N O O Br NO O Br N O O E2 E2 4a 4b
and 2D HSQC NMR spectroscopies. Metal complexations were confirmed via IR spectroscopy, UV-VIS spectroscopy and HR ESI-MS spectroscopy. The stability evaluation of Fe-EDT(M)HA and Fe-EDT(B)HA via UV-VIS shows limited metal complex stability of both ligands, which may explain the relatively low toxicity towards HT-29, MCF-7 and MCF-10A cell lines. DFT calculations further strengthened these findings by showing inflexible hydroxamic acid arms, which makes a metal chelation unlikely to occur. Thus, the synthesized chelators are not suitable as anticancer agents. The same instability hypothesis may explain the 0 % radiochemical yield of 89Zr labeling.
A second set of tetrahydroxamic acid bearing DTPA analogues with diethylentriamine as the backbone and longer hydroxamic acid arms was designed and evaluated by DFT for their potential ability to chelate Zr4+. The DTPA analogues show
greater flexibility of the arms, making a metal complexation more favorable. The synthesis of the arms was successful. However, the linkage of the arms to the backbone was restrained by an E2
elimination reaction instead of SN2 substitution reaction thus far.
Further experiments are currently underway to improve the synthesis of these promising complexes.
5 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. Infrared spectra were recorded using a Frontier FT-IR spectrometer purchased from PerkinElmer. UV absorbance measurements were recorded on an Agilent Technologies Cary 5000 UV-VIS spectrometer.
Synthesis of compounds
N-Methyl-N-Boc- hydroxylamine15 ,1
N-Methylhydroxylamine hydrochloride (0.53 g, 6.4 mmol) was added to dichloromethane (50 mL) and the mixture was cooled to 0°C for 15 min. After that, di-tert-butyl dicarbonate (1.53 g, 7.0 mmol, 1.1 eq) was added in portions to the mixture. Triethylendiamine was added until the solution went clear (1.5 mL) and the reaction mixture was stirred overnight. Dichloromethane was removed by blowing air through the solution without heating, since the product is volatile and has a low boiling point. The resulting colorless oil 1 was dried for one day and used without further purification (70 %, 0.65 g).
1H NMR (400 MHz, CDCl
3, 25°C): 3.09-3.06 (m, 3H), 1.38 (s, 9H). 13C NMR (101 MHz, CDCl
3, 25°C): 157.3, 81.0, 38.3, and 28.3.
HR-ESI-MS: calcd. for [C6H13NO3+H]+: 148.0974; found 148.0973
[M+H]+
t-Butyl- N-benzyloxy-N-methylcarbamate16 , 2
Compound 1 (0.65 g, 4.4 mmol) was placed in a two neck round bottom flask that was then evacuated; it was then flushed with argon, dry DMF (10 mL) added and the mixture placed in an ice-bath. Sodium hydride (0.13 g, 5.3 mmol, 1.2 eq.) was slowly added, turning the solution white and the development of foam (H2) was observed. After 30 minutes, benzylbromide (0.63 mL,
5.3 mmol, 1.2 eq.) was slowly added to the solution, turning the solution clear again. The reaction mixture was stirred for 4 h at room temperature. Hexane was added to the solution and adding solvent dropwise with increasing polarity until no gas formation was observed any longer, destroying unreacted sodium hydride. The solvents were evaporated and the product was dried in vacuo and purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 2 as a yellowish oil (48 %, 0.51 g). 1H NMR (400 MHz, CDCl 3, 25°C): 7.46-7.36 (m, 5H), 4.87 (s, 2H), 3.09 (s, 3H), 1.54 (s, 9H). 13C NMR (101 MHz, CDCl 3, 25°C): 156.9, 135.6, 129.4, 128.7, 128.4, 81.1, 76.4, 36.8, and 28.3.
HR-ESI-MS: calcd. for [C13H19NO3+H]+: 238.1443; found 238.1443
[M+H]+
O-Benzyl-N-methylhydroxylamine , 3
Compound 2 (0.51 mg, 2.2 mmol) was dissolved in TFA/DCM (1:1, 2 mL) and the reaction mixture stirred at room temperature for 4 h. The solvent was removed under reduced pressure and purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 3 as a colorless oil (97 %, 0.29 g).
1H NMR (400 MHz, CDCl
3, 25°C): 11.70 (s, 2H) 7.39 (s, 5H), 5.09 (s,
2H), 2.95 (s, 3H). 13C NMR (101 MHz, CDCl
3, 25°C): 135.6, 129.4,
128.7, 128.4, 76.4, and 36.8. HR-ESI-MS: calcd. for [C8H11NO+H]+:
138.0919; found 138.0912 [M+H]+
O-Benzyl-2-bromo-N-methylacetohydroxamic acid17, 4
Compound 3 (0.29 g, 2.1 mmol) and potassium carbonate (0.50 g, 3.6 mmol, 1.75 eq.) were dissolved in dry tetrahydrofuran (3 mL) and after 15 min stirring at 0°C, bromoacetyl bromide (0.22 mL, 2.5 mmol, 1.2 eq.) was added slowly to the solution, forming a white cloudy mixture. After 7 h stirring at room temperature, the
suspension was filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 4 as a colorless oil (26 %, 0.14 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.42 (s, 5H), 4.95 (s, 2H), 3.93 (s,
2H), 3.27 (s, 3H). 13C NMR (101 MHz, CDCl
3, 25°C): 164.2, 135.6,
129.4, 128.7, 128.4, 76.4, 42.3 and 36.8. HR-ESI-MS: calcd. for [C10H12BrNO2+H]+: 259.0130; found 259.0132 [M+H]+
O-Benzyl-3-bromo-N-methylpropiohydroxamic acid17, 4a
Compound 3 (0.5 g, 3.7 mmol) and potassium carbonate (0.89 g, 6.4 mmol, 1.75 eq.) were dissolved in dry tetrahydrofuran (10 mL) and after 15 min stirring at 0°C, 4-bromobutyryl chloride (0.44 mL, 4.4 mmol, 1.2 eq.) was added slowly to the solution, forming a white cloudy mixture. After 7 h stirring at room temperature, the suspension was filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 4 as a colorless oil (32 %, 0.32 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.39 (s, 5H), 4.95 (s, 2H), 4.85 (s,
2H), 3.56 (t, 2H), 3.22 (s, 3H), 2.95 (t, 2H). 13C NMR (101 MHz,
CDCl3, 25°C): 171.9, 134.2, 129.4, 129.2, 128.8, 76.4, 35.6, 33.5 and
26.6. ESI-MS: calcd. for [C11H14BrNO2+H]+: 272.01; found 272.1
[M+H]+
O-Benzyl-4-bromo-N-methylbutylhydroxamic acid17, 4b
Compound 3 (0.15 g, 1.1 mmol) and potassium carbonate (0.26 g, 1.9 mmol, 1.75 eq.) were dissolved in dry tetrahydrofuran (10 mL) and after 15 min stirring at 0°C, 3-bromopropionyl chloride (0.15 mL, 1.3 mmol, 1.2 eq.) was added slowly to the solution, forming a white cloudy solution. After 7 h stirring at room temperature, the suspension was filtered and the filtrate evaporated under reduced pressure. The residue was purified by
silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 4 as a colorless oil (20 %, 0.06 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.39 (s, 5H), 4.83 (s, 2H), 3.44 (dt,
2H), 3.19 (s, 3H), 2.55 (t, 2H), 2.11 (t, 2H). 13C NMR (101 MHz,
CDCl3, 25°C): 173.8, 134.4, 129.4, 129.1, 128.8, 128.6, 128.5, 127.5,
127.0, 76.3, 65.1, 33.8, 30.4, 28.3 and 27.3. ESI-MS: calcd. for [C11H14BrNO2+H]+: 272.01; found 272.1 [M+H]+
N-Benzyl-O-benzylhydroxylamine, 5
O-Benzylhydroxylamine hydrochloride (1.3 g, 8.12 mmol) was dissolved in methanol (20 mL) and triethylamine was added until complete dissolution. Benzaldehyde (0.91 mL, 8.93 mmol, 1.1 eq.) was added at room temperature and after 2 h the imine intermediate was confirmed via ESI-MS. After that, the white mixture was cooled to 0°C and acidified with conc. HCl. Sodium cyano- borohydride (1.53 g, 24.4 mmol, 3 eq.) was added slowly under cooling and the reaction mixture left stirring overnight. After removing the solvents in vacuo, 0.1 M sodium hydroxide solution (10 mL) was added to the residue and extracted with dichloromethane. The organic phase was dried and purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 5 as a white solid (58 %, 1.02 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.39 (m, 10H), 4.62 (s, 4H) 13C
NMR (101 MHz, CDCl3, 25°C): 141.0, 128.5, 127.5, 127.1, 64.8, and
64.7. HR-ESI-MS: calcd. for [C14H15NO+H]+: 214.1232; found
214.1233 [M+H]+
O-Benzyl-2-bromo-N-benzyl-acetohydroxamic acid, 6
Compound 5 (1.02 g, 4.7 mmol) and potassium carbonate (0.71 g, 5.2 mmol, 1.1 eq.) were dissolved in dry tetrahydrofuran (20 mL) and after 15 min stirring at 0°C, bromoacetyl bromide (0.45 mL, 5.2 mmol, 1.1 eq.) was added slowly to the solution,
forming a white cloudy suspension. After 7 h stirring at room temperature, the suspension was filtered and the filtrate evaporated under reduced pressure. The residue was purified by silica chromatography (CombiFlash Rf automated system; 12 g HP silica; A: hexane, B: ethyl acetate, 100% A to 100% B gradient) to yield product 6 as a white solid (43 %, 0.67 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.42-7.32 (m, 10H), 4.89 (s, 4H),
3.98 (s, 2H). 13C NMR (101 MHz, CDCl
3, 25°C): 168.4, 135.7, 133.9,
129.4, 129.3, 128.9, 128.7, 128.6, 128.0, 77.2, 50.6 and 2.9. HR-ESI-MS: calcd. for [C16H16BrNO2+Na]+: 356.0262; found 256.0257
[M+H]+
N,N,N,N-Tetra-(O-benzyl-N-methylacetohydroxamic acid)-diaminoethane , 7
Compound 4 (0.06 g, 0.23 mmol, 4.4 eq.) was dissolved in acetonitrile (1 mL) and potassium carbonate (0.03 g, 0.23 mmol, 4.4 eq.) and ethylenediamine (3.5 μL, 0.05 mmol) were added to the solution. After 16 h stirring at room temperature, the solution was dried under reduced pressure and the residue purified by semi-prep RP HPLC (10 mL/ min, gradient A: 0.1% TFA in deionized water, B: acetonitrile, A: 95 % to B: 80 % for 10 min., followed by A: 20 % to B: 100 % for additional 20 min, tR= 15.25
min) to yield product 5 as a colorless oil (58.7 %, 0.003 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.37 (s, 20H), 4.94 (s, 8H), 3.92 (s,
8H), 3.26 (s, 12H), 3.22 (m, 2H), 3.16 (m, 2H). 13C NMR (101 MHz,
CDCl3, 25°C): 168.1, 133.7, 129.4, 128.7, 128.4, 76.3, 33.7 and 25.3.
HR-ESI-MS: calcd. for [C42H52N6O8+H]+: 769.3925; found 769.3928
[M+H]+
H4EDT(M)HA
(Ethylenediaminetetra(methylene-N-methylhydroxamic acid), 8
Compound 5 (0.03 g, 0.04 mmol) was dissolved in methanol (2 mL) and Pd(OH)2/C (20 w/w %, 0.007 g) was added to
the solution. Charging the flask with a H2-filled balloon gave the
(74 %, 0.001 g).
1H NMR (400 MHz, CDCl
3, 25°C): 4.36 (s, 8H), 3.63 (s, 4H), 3.26 (s,
12H). 13C NMR (101 MHz, CDCl
3, 25°C): 166.5, 55.5, 51.9, 35.6.
HR-ESI-MS: calcd. for [C14H28N6O8+H]+: 409.2047; found 409.2047
[M+H]+
N,N,N,N-Tetra-(O-benzyl-N-benzylacetohydroxamic acid)-diaminoethane, 9
Compound 6 (0.67 g, 2.00 mmol, 5 eq.) was dissolved in acetonitrile (15 mL) and potassium carbonate (0.28 g, 2.00 mmol, 5 eq.) and ethylenediamine (26.7 μL, 0.40 mmol) were added to the solution. After 16 h stirring at room temperature, the solution was dried under reduced pressure and the residue purified by semi-prep RP HPLC (10 mL/ min, gradient A: 0.1% TFA in deionized water, B: acetonitrile, A: 50 % to B: 100 % for 20 min., tR=
13.8 min) to yield product 9 as a colorless solid (33.5 %, 0.14 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.28 (m, 40H), 4.75 (s, 16H), 4.20
(s, 8H), 3.23 (s, 4H). 13C NMR (101 MHz, CDCl
3, 25°C): 135.5, 133.8,
129.6, 129.2, 128.7, 128.6, 128.0, 77.2, 55.4, 50.6 and 50.1 HR-ESI-MS: calcd. for [C66H68N6O8+H]+: 1073.5177; found 1073.5181
[M+H]+
H4EDT(B)HA, 10
Compound 9 (0.14 g, 0.13 mmol) was dissolved in methanol (5 mL) and Pd(OH)2/C (20 w/w %, 0.067 g) was added to
the solution. After charging the flask with a H2-filled balloon, the
solution was stirred for 16 h at room temperature. The crude oil was purified by semi-prep RP HPLC (10 mL/ min, gradient A: 0.1% TFA in deionized water, B: acetonitrile, A: 95 % to B: 100 % for 25 min., tR= 20.8 min) as a colorless oil (20.9 %, 0.02 g).
1H NMR (400 MHz, CDCl
3, 25°C): 7.33 (m, 20H), 4.76 (s, 8H), 4.26
(s, 8H), 3.37 (s, 4H). 13C NMR (101 MHz, CDCl
3, 25°C): 168.4, 135, 8,
128.2, 127.4, 54.6, 51.8 HR-ESI-MS: calcd. for [C38H44N6O8+H]+:
General procedure for metal complexations
H4EDT(M)HA or H4EDT(B)HA was dissolved in 1 mL water
at neutral pH, FeCl3 6H2O, ZnSO4 7 H2O or Cu(ClO4)2 6 H2O
were added in a 1:1 ratio. Extraction with ethylacetate gave the following metal complexes, analyzed by HR-ESI-MS: Fe-EDT(M)HA calcd. for [C14H25FeN6O8+H]+: 462.1162; found
462.1162 [M+H]+ ; Cu-EDT(M)HA calcd. for [C
14H26CuN6O8+H]+:
470.1186; found 470.1186 [M+H]+; Zn-EDT(M)HA calcd. for
[C14H26N6O8Zn+H]+: 471.1182; found 471.1182 [M+H]+;
Fe-EDT(B)HA calcd. for [C38H41FeN6O8+H]+: 766.2414; found
766.2442 [M+H]+ ; Cu-EDT(B)HA calcd. for [C
38H42CuN6O8+H]+:
774.2438; found 774.2440 [M+H]+; Zn-EDT(B)HA calcd. for
[C38H42N6O8Zn+H]+: 775.2434; found 775.2439 [M+H]+
DFT calculations
Density functional theory (DFT) calculations were carried out using the Gaussian 09 Rev.D01 suite of Programs.25 The
B3LYP hybrid functional26 with 6-31G* basis set27 (for C, H, O and
N atoms) and the LANL2DZ effective-core pseudopotential28 (for
Zr and Fe) was employed to simulate the ground state structures of both the ligands and their metal complexes. For all simulations, solvation effect of water (ε = 78.3553) was implemented using the polarizable continuum model (PCM) on Gaussian 09.29
Optimized structures were confirmed to be the minimum on the potential energy surface by vibrational frequency calculations.
Cell viability assay
All three human cell lines were obtained from ATCC, American Type Culture Collection, Manassas, USA. The human colon cancer cell line HT29 (HTB-38), the human breast cancer cell line MCF7 (HTB-22) and the human non-tumorigenic breast epithelial cell line (CRL-10317) were cultured in McCoy’s 5A
(Invitrogen 1660082), DMEM (Invitrogen 11965) + 10% FBS or DMEM/F12 (Sigma Aldrich D6421) + Pen/Strep 1x + 2 mM glutamine + hEGF 0.01 µg/mL + hydrocortisone 0.5 µg/mL + human insulin 10 µg/mL, respectively, at 37°C in a humidified atmosphere of 95 % of air and 5 % CO2 respectively. For
evaluation of growth inhibition, cells were seeded in 96-well plates (Corning, Fisher Scientific Co Ltd, Edmonton, Canada) at a concentration of 10000 cells/well (MCF7, MCF10A and HT29) and grown for 24 h in complete medium. Solutions of the compounds were prepared by diluting a freshly prepared stock solution (10-2
M in DMSO, or distilled H2O for DFO) of the corresponding
compound to water. Afterwards, the intermediate dilutions of the compounds were added to the wells (200 µL) to obtain a final concentration ranging from 0 to 200 µM, and the cells were incubated for 72 h. Following 72 h drug exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was added to the cells at a final concentration of 0.5 mg ml-1
incubated for 2 h, then the culture medium was removed and the violet formazan (artificial chromogenic precipitate of the reduction of tetrazolium salts by dehydrogenases and reductases) dissolved in DMSO. The optical density of each well (96-well plates) was quantified three times in tetraplicates at 550 nm using a multi-well plate reader, and the percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. The IC50 value was calculated as the
concentration reducing the proliferation of the cells by 50 % and it is presented as a mean (± SD) of at least three independent experiments.
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Supporting Information
Chapter A3:
Tetrahydroxamic Acid Bearing Ligands:
EDTA and DTPA analogues
Sarah Spreckelmeyer,
a,bYang Cao
aand Chris Orvig
aa
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
Figure S 1. 2D-HSQC of H
4EDT(M)HA (400 MHz, D
2O, 25°C)
Figu
re S 2. 1H NMR spectra of A: H4EDT(M)HA B: Fe-EDT(M)HA C:
Cu-EDT(M)HA (400 MHz, D2O, 25°C). 0 1 2 3 4 5 6 7 8 9 10 11 12 f2 (ppm) 0 20 40 60 80 100 120 140 f1 (p pm ) {4.28,55.81} {3.55,52.65} {3.19,36.23} 4. 79 D 2O 4. 79 D 2O 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 f1 (ppm) A B C
Figure S 3. Full IR spectra of H4EDT(M)HA, Fe-EDT(M)HA and
Cu-EDT(M)HA.
Figure S 4. iTLC chromatograms of free 89Zr4+ (black, all radioactivity at
about 150 mm), DFO (green, all radioactivity at the origin) and EDT(M)HA (pink, same as control); mobile phase: EDTA pH 7.0.
1000 2000 3000 4000 80 85 90 95 100 wavenumber [cm-1] Tr a n s m it ta n c e [ % ] EDT(M)HA Fe-EDT(M)HA Cu-EDT(M)HA 0 100 200 300 0 100 200 300 Distance eluted [mm] Counts Ctrl DFO EDT(M)HA
Figure S 5. DFT optimized structure (B3LYP/6-31G* + LANL2DZ and
PCM solvation (water) on Gaussian 09) and bond lengths of Zr-DTT(ME)HA.
O-Zr2 bond length [A]
O7 2.2269 O9 2.4204 O3 2.2378 O5 2.3933 O8 2.2045 O10 2.1334 O6 2.2019 O4 2.1374
O-Zr2 bond length [A] O51 2.2509 O70 2.2275 O3 2.271 O22 2.2294 O53 2.199 O65 2.1914 O25 2.209 O6 2.1981
Figure S 6. DFT optimized structure (B3LYP/6-31G* + LANL2DZ and
PCM solvation (water) on Gaussian 09) and bond lenghs of Zr-DTT(MP)HA.
Figure S 7. DFT optimized structure of bifunctional Zr-DTT(MP)HA
(B3LYP/6-31G* + LANL2DZ and PCM solvation (water) on Gaussian 09).
Figure S 8. 13C NMR spectra of arms (4a, top) and (4b, bottom), 400
MHz, CDCl3, 25°C. 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 f1 (ppm)
Table S 1. Conditions tried for adding the "arm" to diethylenetriamine. Attempt Arm Salt Solvent
1 4a 9 eq. K2CO3 Acetonitrile
2 4a 6 eq. NaHCO3 Acetone
3 4b 5 eq. K2CO3 Acetonitrile
4 4b Acetone (+/- reflux)
5 4b 6 eq. K2CO3 DMF
