Improved synthesis of the bifunctional chelator p-SCN-Bn-HOPO

Article abstract of DOI:10.1039/c9ob01068h

The bifunctional ligand p-SCN-Bn-HOPO, which has four 1,2-hydroxypyridinone groups on a spermine backbone with an isothiocyanate linker, has been shown to be an efficient and stable chelator for Zr(iv) and, more importantly, the radioisotope ⁸⁹Zr for use in radiolabeling antibodies for positron emission tomography (PET) imaging. Previous studies of ⁸⁹Zr-HOPO-trastuzumab in mice showed low background, good tumor to organ contrast, and very low bone uptake which show p-SCN-Bn-HOPO to be an important next-generation bifunctional chelator for radioimmunoPET imaging with ⁸⁹Zr. However, the reported synthesis of p-SCN-Bn-HOPO involves nine steps and multiple HPLC purifications with an overall yield of about 1.4%. Herein we report an improved and efficient synthesis of p-SCN-Bn-HOPO in four steps with 14.3% overall yield which will improve its availability for further biological studies and wider application in PET imaging. The new synthetic route also allows variation in linker length and chemistries which may be helpful in modifying in vivo clearance behaviors of future agents.

Full text of DOI:10.1039/c9ob01068h

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Bhupathiraju, A. Younes, M. Cao, J. Ali, H. T. Cicek , K. M. Tully, S. Ponnala, J. W. Babich, M. A. Deri, J. S.
Lewis, L. Francesconi and C. M. Drain, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01068H.
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DOI: 10.1039/C9OB01068H
ARTICLE
Improved Synthesis of the Bifunctional Chelator p-SCN-Bn-HOPO
N. V. S. Dinesh K. Bhupathiraju,a* Ali Younes,a Minhua Cao,a Jafar Ali,a Huseyin T. Cicek,a Kathryn
M. Tully,b,c Shashikanth Ponnala,d John W. Babich,d Melissa A. Deri,e,f Jason S. Lewis,b,c Lynn C.
Francesconi,a,f Charles Michael Draina,f
Received 00th January 20xx,
Accepted 00th January 20xx
The bifunctional ligand p-SCN-Bn-HOPO, which has four 1,2-hydroxypyridinone groups on a spermine backbone with an
isothiocyanate linker, has been shown to be an efficient and stable chelator for Zr(IV) and, more importantly, the
radioisotope 89Zr for use in radiolabeling antibodies for positron emission tomography (PET) imaging. Previous studies of
89Zr-HOPO-trastuzumab in mice showed low background, good tumor to organ contrast, and very low bone uptake which
show p-SCN-Bn-HOPO to be an important next-generation bifunctional chelator for radioimmunoPET imaging with 89Zr.
However, the reported synthesis of p-SCN-Bn-HOPO involves nine steps and multiple HPLC purifications with an overall yield
of about 1.4%. Herein we report an improved and efficient synthesis of p-SCN-Bn-HOPO in four steps with 14.3% overall
yield which will improve its availability for further biological studies and wider application in PET imaging. The new synthetic
route also allows variation in linker length and chemistries which may be helpful in modifying in vivo clearance behaviors of
future agents.
DOI: 10.1039/x0xx00000x
Introduction
Positron Emission Tomography (PET) is a molecular imaging
modality that provides physiological data non-invasively and
quantitatively. PET imaging relies on the decay of positron
emitting radionuclides that are chemically bound to ligands or
targeting vectors designed to probe biochemical phenomena in
vivo. Traditionally, short-lived PET isotopes, such as 18F, 15O, 11C,
and 13N, have been used for labelling peptides and small
molecules that accumulate in target tissue and clear the body
rapidly. For PET imaging with antibodies, called immunoPET,
longer lived radionuclides are required to match the residence
time of the antibodies in vivo. The radionuclide 89Zr has become
a key to the success of immunoPET and the field is rapidly
growing.1, 2
Figure 1. Chemical structures of the 89Zr complexes with bifunctional p-SCN-Bn-
HOPO (left) and p-SCN-Bn-DFO (right) ligands.
accomplished easily and inexpensively using medical cyclotrons
via proton bombardment on natural yttrium targets (100%
abundance 89Y) by the 89Y(p,n)89Zr reaction.
Zr(IV) is an oxophilic, octacoordinate ion, so octadentate
ligands comprised of oxygen donor atoms would be
advantageous. Presently, the only bifunctional chelator used in
the clinic for linking 89Zr to an antibody is desferrioxamine
(DFO), most commonly in the form of commercially available
p-SCN-Bn-DFO3 (Figure 1). DFO consists of three hydroxamate
moieties and is a hexadentate chelator. The two open
coordination sites result in demetallation of 89Zr from the
chelate in vivo that is manifested in high bone uptake in
preclinical studies in mice.
89Zr is ideally suited for pairing with monoclonal antibodies
because its physical half-life of 78.4 h matches the residence
time, generally several days, of intact mAbs in the body. The low
positron energy, max:897 keV, avg: 396.9 keV, provides high
resolution PET images. The production of 89Zr can be
Several chelators were reported with the goal of increasing
89Zr complex stability in vivo. Some chelators contain
hydroxamate moieties; for example, the linear and macrocyclic
tetrahydroxamate chelators reported by Guerrad et al.4, 5, Vugts
and Mindt6, 7 and branched tetrahydroxymates by Rousseau8.
Trihydroxamate chelators,9, 10 and a trihydroxamate possessing
an additional cyclic hydroxamate (DFOcyclo*)11 giving it 8-
oxygen donor atoms have been reported.
a. Department of Chemistry, Hunter College of the City University of New York. New
York, New York 10065 USA
b. Division of Radiopharmaceutical Sciences, Department of Radiology, Weill Cornell
Medicine, New York, NY 10065, USA
c. Department of Radiology, the Program in Pharmacology, and the Radiochemistry
and Molecular Imaging Probes Core, Memorial Sloan Kettering Cancer Center,
New York, New York 10065, USA
d. Radiopharmaceutical Sciences and Department of Radiology, Weill Cornell
Medicine, New York, NY, USA.
e. Lehman College of the City University of New York, Bronx, New York 10468
f. Chemistry Program, Graduate Center of the City University of New York, New
York, New York 10016 USA.
We reported the potential applications of the 3,4,3-LI(1,2-
HOPO) or HOPO ligand,12 which has four 1,2-hydroxypyridinone
groups for metal binding and comes from the actinide
*email: nb216@hunter.cuny.edu
Electronic Supplementary Information (ESI) available: reagent synthesis and
spectroscopic data. See DOI: 10.1039/x0xx00000x
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sequestration literature.13 The 89Zr-HOPO complex showed Unreacted spermine was recovered from the methanol wash
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equal or greater stability compared to 89Zr-DFO in both in vitro and can be recycled. Compound S2b was synthesized by BOC
DOI: 10.1039/C9OB01068H
and in vivo assays.12
protection of 2-(4-aminophenyl)ethan-1-ol followed by
The development of a bifunctional derivative of the HOPO tosylation of the hydroxide group obtaining compound S2b in
ligand was the next logical step and led to the design and quantitative yield (Scheme S1, supporting information).
synthesis of p-SCN-Bn-HOPO.14 The p-SCN-Bn-HOPO conjugated
to trastuzumab achieved similar specific activity, ~2 mCi/mg,
and remained stable (~90%) in human serum (7 d). The greatest
distinction between the two compounds was the amount of
bone uptake seen in the imaging and biodistribution studies.
The activity measured in the bone for 89Zr-HOPO-trastuzumab
was more than 7 times lower than for 89Zr-DFO-trastuzumab.
While the absolute uptake 89Zr-DFO-trastuzumab by BT474
breast cancer tumors was just over twice that of 89Zr-HOPO-
trastuzumab, the tumor to bone ratio was more than 3-fold
greater for the HOPO complex. This improved contrast between
tumor and bone is advantageous for the detection of bone
metastasis and for the general clarity of the images as well as
reducing the radiation dose to sensitive non-target tissues.
Furthermore, the lower bone uptake is the ultimate proof that
the p-SCN-Bn-HOPO ligand forms a more stable complex with
89Zr4+ than p-SCN-Bn-DFO and drastically reduces the bone
accumulation of free 89Zr4+ in vivo.
We reported the 9-step synthesis of p-SCN-Bn-HOPO that
required multiple HPLC purifications with a 1.4% overall yield.14
The long reaction sequence and low yield limits the applications
of this important ligand and is impractical for the synthesis of p-
SCN-Bn-HOPO in large scale. Therefore, a more efficient
synthesis is needed to develop cost effective applications.
Herein we report an improved synthesis consisting of four steps
with a 10-fold improvement in overall yield (14.3%). We also
report modified conditions that improve the yields in our
previously reported synthesis.
Results and Discussion
In order to achieve p-SCN-Bn-HOPO (compound 3 Scheme
1)14 in high yield, we adopted two strategies. In the first
approach, we designed a new 4-step synthetic route requiring
only HPLC purification of the final product (Scheme 1). In the
second approach, we reinvestigated the reaction conditions
and solvents to improve the yields in the original 9-step
synthesis (Scheme 2).
The new strategy to make p-SCN-Bn-HOPO starts with
commercially available spermine, which was reacted with 2-(4-
tertiarybutyl-aminophenyl)ethyl tosylate (S2b, supporting
information) under reflux for 8-10 h resulting in a mixture of
free spermine and the mono substituted 2-(4-teritiarybutyl-
aminophenyl)ethyl group on spermine (Scheme 1). The mono
substituted spermine was easily isolated using preparative silica
gel TLC (GF254) with methanol as the eluent. Though free
spermine is not visible, the mono substituted spermine can be
visualized using a UV lamp. The product was collected by
scraping the preparative silica gel plates followed by washing
the methanol. We observed that there was some quantity of
free spermine obtained along with the substituted product.
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C9OB01068H
Scheme 2. Improvements to the original 9-step synthesis of p-SCN-Bn-HOPO are outlined in red. (a) Ethyl trifluoroacetate, MeOH, −78 °C, 1 h; (b) (BOC)2O, MeOH,
r.t, 18 h; (c) conc. NH4OH, r.t, 15 h (52% over 3 steps); (d) 4-nitrophenethyl 4-methylbenzenesulfonate, K2CO3, CH3CN, reflux, 12 h, 46%; (e) 4 M HCl in Dioxane, r.t,
2 h; (f) 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylic acid chloride, NEt3, DMAP, dichloromethane, 0−25 °C, 12 h, 56% (over 2 steps); (g) Raney Ni, H2, MeOH,
3 h; (h) BCl3 in p-xylene, dichloromethane, -20 ̊C to RT, 15 h, 99%; (i) di-2-pyridyl thiocarbonate, NEt3, CH3CN, H2O, r.t, 1 h, 36%.
Mono-substituted spermine-Bn-NH-BOC, was treated with spermine led to the mono-, di-, and tri- substituted spermine
4.2 equivalents of 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2- derivatives. To avoid the formation of these undesirable side
carbonyl chloride under nitrogen to obtain compound 1 in good products, ethyl trifluoroacetate was added by aid of syringe
yield (42%). However, there was about 20% benzyl protected- pump (2 h at 20 mL/h), followed by addition of five equivalents
3,4,3-(LI-1,2-HOPO)12 that can be separated from the reaction of di-tert-butyldicarbonate with prolonged reaction time (2 d)
mixture as a side product, which suggests that some unreacted to ensure the protection all free amines. Ammonium hydroxide
spermine remained from the previous step. The O-benzyl was then added until a pH of 14 was reached and stirred for two
protecting groups and BOC protecting group on compound 1 days to complete the de-protection of the ethyl trifluoroacetate
were all deprotected in one step using the mild Lewis acid, BCl3 group.
in toluene, to obtain compound 2 in quantitative yield (95%).
Although the reaction of an amine with a primary alkyl
Compound 2 precipitates as the reaction proceeds, and was halide is well-established, the elimination product is often
filtered and washed with acetone several times obviating the formed along with the desired substituted products. By
need for HPLC purification. Finally, bifunctional (p-SCN-Bn- following the previously published procedure for the synthesis
HOPO) was obtained from compound 2 using the published of compound 5, the reaction of compound 4 with 4-
procedure including reaction with di-2-pyridyl thiocarbonate nitrophenylethylbromide, we observed the formation 4-
and purification by preparative HPLC.14 The overall yield nitrostyrene as major byproduct drastically reducing the yield
obtained was 14.3%. It is important to note that about 20% of compound 5. This prompted us to investigate the effect of
3,4,3-(LI-1,2-HOPO) and unreacted spermine are side products. solvents in the particular reaction. We screened several polar
HOPO is a useful ligand and can be isolated for testing with aprotic solvents at different temperatures (Table 2) in
radiometals. The unreacted spermine can be recycled.
preparation of compound 5. Using 2 equiv. of 4-
The second approach improves several key steps in the prior nitrophenylethyl bromide and compound 4 containing 2
9-step synthesis of p-SCN-Bn-HOPO (Scheme 2). Compound 4 equivalents of potassium carbonate in acetonitrile at r.t. for 7
was synthesized in a three-step reaction sequence starting from days, afforded the expected compound 5 in yields of 40% (Entry
the commercially available spermine. We studied the effect of 6 in Table 2) and no elimination product was observed. In
reaction time and reagent stoichiometry to improve the attempts to reduce the reaction time the reaction was carried
reaction yields (Table 1). Optimum yields (46-50%) were out in a microwave reactor at various temperatures (40-153 ̊C)
obtained by using conditions in entry 4, Table 1. In this case, the and times (10-60 min), but the yields significantly decreased
reaction of ethyl trifluoroacetate with amine groups on the (supporting information, Table S1).
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