A trithiol bifunctional chelate for 72,77As: A matched pair theranostic complex with high in vivo stability

Article abstract of DOI:10.1016/j.nucmedbio.2018.03.001

Introduction: Trithiol chelates are suitable for labeling radioarsenic (⁷²As: 2.49 MeV β⁺, 26 h; ⁷⁷As: 0.683 MeV β^?, 38.8 h) to form potential theranostic radiopharmaceuticals for PET imaging and therapy. To investigate the in vivo stability of trithiol chelates complexed with no carrier added (nca) radioarsenic, a bifunctional trithiol chelate was developed, and conjugated to bombesin(7–14)NH₂ as a model peptide. Methods: A trithiol-BBN(7–14)NH₂ bioconjugate and its arsenic complex were synthesized and characterized. The trithiol-BBN(7–14)NH₂ conjugate was radiolabeled with ⁷⁷As, its in vitro stability assessed, and biodistribution studies were performed in CF-1 normal mice of free [⁷⁷As]arsenate and ⁷⁷As-trithiol- BBN(7–14)NH₂. Results: The trithiol-BBN(7–14)NH₂ conjugate, its precursors and its As-trithiol-BBN(7–14)NH₂ complex were fully characterized. Radiolabeling studies with nca ⁷⁷As resulted in over 90% radiochemical yield of ⁷⁷As-trithiol-BBN, which was stable for over 48 h. Biodistribution studies were performed with both free [⁷⁷As]arsenate and Sep-Pak purified ⁷⁷As-trithiol-BBN(7–14)NH₂. Compared to the fast renal clearance of free [⁷⁷As]arsenate, ⁷⁷As-trithiol-BBN(7–14)NH₂ demonstrated increased retention with clearance mainly through the hepatobiliary system, consistent with the lipophilicity of the ⁷⁷As-trithiol-BBN(714)NH₂ complex. Conclusion: The combined in vitro stability of ⁷⁷As-trithiol-BBN(7–14)NH₂ and the biodistribution results demonstrate its high in vivo stability, making the trithiol a promising platform for developing radioarsenic-based theranostic radiopharmaceuticals.

Full text of DOI:10.1016/j.nucmedbio.2018.03.001

Accepted Manuscript
A Trithiol Bifunctional Chelate for 72,77As: a Matched Pair
Theranostic Complex with High in vivo Stability
Yutian Feng, Anthony J. DeGraffenreid, Michael D. Phipps,
Tammy L. Rold, Nkemakonam C. Okoye, Fabio A. Gallazzi,
Charles L. Barnes, Cathy S. Cutler, Alan R. Ketring, Timothy J.
Hoffman, Silvia S. Jurisson
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DOI:
Reference:
S0969-8051(17)30434-1
doi:10.1016/j.nucmedbio.2018.03.001
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13 February 2018
4 March 2018
Please cite this article as: Yutian Feng, Anthony J. DeGraffenreid, Michael D. Phipps,
Tammy L. Rold, Nkemakonam C. Okoye, Fabio A. Gallazzi, Charles L. Barnes, Cathy S.
Cutler, Alan R. Ketring, Timothy J. Hoffman, Silvia S. Jurisson , A Trithiol Bifunctional
Chelate for 72,77As: a Matched Pair Theranostic Complex with High in vivo Stability.
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ACCEPTED MANUSCRIPT
A Trithiol Bifunctional Chelate for 72,77As: a Matched Pair Theranostic
Complex with High in vivo Stability
Yutian Feng1, Anthony J. DeGraffenreid1,5, Michael D. Phipps1, Tammy L. Rold4, Nkemakonam C. Okoye1, Fabio A.
Gallazzi3, Charles L. Barnes1, Cathy S. Cutler2,5, Alan R. Ketring2, Timothy J. Hoffman4, and Silvia S. Jurisson1,2*
1
2
Department of Chemistry, Research Reactor Center (MURR), 3Molecular Interaction Core, University of Missouri, Columbia, MO
4
5
65211, U.S.A. Research Service, Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO 65201, U.S.A. Medical Isotope
Research and Production Program (MIRP), Collider-Accelerator Department, Brookhaven National Laboratory, PO Box 5000,
Building 801, Upton, NY 11973.
Abstract
Introduction: Trithiol chelates are suitable for labeling radioarsenic (72As: 2.49 MeV β+, 26 h;
77As: 0.683 MeV β−, 38.8 h) to form potential theranostic radiopharmaceuticals for PET imaging
and therapy. To investigate the in vivo stability of trithiol chelates complexed with no carrier
added (nca) radioarsenic, a bifunctional trithiol chelate was developed, and conjugated to
bombesin(7-14)NH2 as a model peptide.
Methods: A trithiol-BBN(7-14)NH2 bioconjugate and its arsenic complex were synthesized and
characterized. The trithiol-BBN(7-14)NH2 conjugate was radiolabeled with 77As, its in vitro
stability assessed, and biodistribution studies were performed in CF-1 normal mice of free
[77As]arsenate and 77As-trithiol- BBN(7-14)NH2.
Results: The trithiol-BBN(7-14)NH2 conjugate, its precursors and its As-trithiol-BBN(7-14)NH2
complex were fully characterized. Radiolabeling studies with nca 77As resulted in over 90%
radiochemical yield of 77As-trithiol-BBN, which was stable for over 48 h. Biodistribution studies
were performed with both free [77As]arsenate and Sep-Pak® purified 77As-trithiol-BBN(7-14)NH2.
Compared to the fast renal clearance of free [77As]arsenate, 77As-trithiol-BBN(7-14)NH2
demonstrated increased retention with clearance mainly through the hepatobiliary system,
consistent with the lipophilicity of the 77As-trithiol-BBN(714)NH2 complex.
Conclusion: The combined in vitro stability of 77As-trithiol-BBN(7-14)NH2 and the biodistribution
results demonstrate its high in vivo stability, making the trithiol a promising platform for
developing radioarsenic-based theranostic radiopharmaceuticals.
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Running Title: A Trithiol Bifunctional Chelate for 72,77As
Key Words: trithiol bioconjugate; arsenic trithiol; no carrier added 77As; radiolabeling;
biodistributions
Correspondence to: Silvia S. Jurisson, Department of Chemistry, University of Missouri,
Columbia, MO 65211, USA; jurissons@missouri.edu; 573-882-2107; 573-882-2754 (fax)
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Introduction
Combined non-invasive radioimaging and radiotherapy (i.e., theranostic) agents are believed to
have many advantages because the diagnostic information provided by the imaging potentially
guides the strategy for radiotherapy [1]. Ideally the radioimaging and radiotherapy counterparts
are two radioisotopes of the same nuclide and thus behave identically in vitro and in vivo,
making them a true “matched pair” [2]. Two arsenic radioisotopes (72,77As) are a potential true
matched pair for positron emission tomography (PET) and radiotherapy, respectively [3].
Arsenic-72 (3.33 MeV maximum β+, 26 h half-life) and 77As (0.683 MeV maximum β-, 38.8 h
half-life) have sufficiently long half-lives compared to most commonly used positron emitters
(18F, 11C, 64Cu, 68Ga) making them useful in radiolabeling antibodies or peptides for
radioimmunoimaging and therapy [4].
Arsenic-72 can be produced directly from proton irradiation of an enriched 72Ge target via the
p,n reaction [5]. Additionally, 72Se decays by electron capture with an 8 day half-life to 72As,
which makes development of a 72Se/72As generator desirable [6-10]. Selenium-72 can be
produced by a number of accelerator based routes on 70Ge, natBr or 75As targets [7-11]. Several
potential separation methods have been reported for such a generator [6-11]. Arsenic-77 is
reactor produced by neutron irradiation of an enriched 76Ge target to form 77Ge, which beta
decays to 77As (Equation 1). The 77As is then separated from the irradiated target as
[77As]arsenate using silica gel and the enriched 76Ge is recovered and recycled [12].
1
76Ge + n  77Ge  77As + β- + ῡ
(1)
Arsenic toxicity has been well discussed over the last few decades [13]. The high binding affinity
between arsenic and thiols explains the in vivo arsenic accumulation in tissues with high
cysteine content, and hence is the leading cause of arsenic toxicity [14]. Arsenic is typically
observed in two oxidation states, namely +3 (arsenite) and +5 (arsenate), with trivalent arsenite
believed to be the toxic form and reactive toward thiols [13,14].
The thiophilic nature of arsenic was recently used to develop potential no carrier added (nca)
radioarsenic labeled radiopharmaceuticals. Monoclonal antibodies modified with N-succinimidyl
S-acetylthioacetate (SATA) to increase the number of available thiol groups were directly
radiolabeled with 72,74As with high radiolabeling yields achieved (>99%) [15], excellent in vitro
stability in serum [15,16], and rat biodistribution studies of radiolabeled bavituximab showed a
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tumor:liver ratio of 22 at 72 h in tumor bearing rats with minimal release of radioarsenic from
radiolabeled antibodies [16]. Thiol-containing N-(2-hydroxypropyl)-methacrylamide (HMPA)
based polymers radiolabeled with nca 72/74As in high radiochemical yield showed good in vitro
stability [17]. More recently, radioarsenic (72As) labeling strategies using dithiol ligands and thiol-
modified mesoporous silica nanoparticles were reported with reasonable yields [5]. Compared
to the fast renal clearance of free 72As, the blood circulation lifetime of 72As labeled
nanoparticles was enhanced providing a potential PET imaging radiopharmaceutical.
Aryldithiol [3] and trithiol [18] ligand frameworks and their nca radioarsenic complexes were
recently reported. The trithiol ligand framework is being pursued to develop a bifunctional
chelate because its radiochemistry is more straightforward. Since the nca radioarsenic is
isolated as [72/77As]arsenate with the arsenic in its highest oxidation state, the first step in
radiolabeling is reduction of As(V) to As(III). The +3 oxidation state is required for radiolabeling.
A monothiol, namely mercaptoacetate, was previously found to reduce [77As]arsenate to 77As
(III) efficiently, and enabled trithiol radiolabeling to occur readily without interference [18]. The
high in vitro stability observed for [77As]arsenic-trithiol [18] led us to develop a bifunctional
chelate based on the trithiol ligand framework. The initial trithiol bifunctional chelate selected
was based on available starting materials and ease of synthesis (sulfurs are very reactive). Due
to the high lipophilicity of the simple As-trithiol [18], the bifunctional trithiol was conjugated to a
model peptide, namely bombesin(7-14)NH2 (BBN(7-14)NH2), via solid phase peptide synthesis.
Its nca 77As labeled analogue was evaluated in vitro in sterile saline and in vivo in mice for
stability. Bombesin(7-14)NH2 is not the ideal peptide for targeting gastrin-releasing peptide
receptors (GRPr), which are highly expressed in human prostate cancer cells, but it is a well-
characterized system at our institution [19-23]. This study reports the synthesis and
characterization of a trithiol-BBN(7-14)NH2 bioconjugate, the evaluation and biodistribution of
nca 77As radiolabeled trithiol-BBN(7-14)NH2 in CF-1 normal mice, and comparison to free nca
77As (arsenate) in CF-1 normal mice.
Experimental
Materials. Pentaerythritol, triethyl orthoacetate, p-toluene sulfonic acid monohydrate, propargyl
bromide (80% in toluene), dioctyl phthalate, 3-bromopropionic acid, sodium azide, ethyl 3-
bromopropionate, thioglycolic acid, tris(2-carboxyethyl)phosphine (TCEP), 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), N-
ethyl-N-(propan-2-yl)propan-2-amine (DIEA), triisopropylsilane (TIS), protected amino acids,
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silica gel TLC plates, silica gel 60Å, reversed phase C18 125Å, and aluminum backed C18-W
TLC plates were purchased from Fisher Scientific or Sigma-Aldrich and used as received.
Bacteriostatic saline was purchased from Hospira Inc. Sep-Pak® C18 Plus Light Cartridges
were purchased from Waters. All solvents, and reagent grade acids and bases were purchased
from Fisher Scientific or Sigma-Aldrich and used without further purification. Only 18 MΩ water
was used. CAUTION! Arsenic is highly toxic and should be handled with care.
Arsenic-77 was prepared by irradiation of 2-5 mg of enriched 76GeO2 (98.6%; Trace Sciences
International) in a thermal neutron flux of 2.4 x 1014 n/cm2-s at the University of Missouri
Research Reactor (MURR). [77As]arsenate was isolated in aqueous solution as previously
reported [12]. CAUTION! 77As and 77Ge are radioactive and must be handled in laboratories
outfitted and approved for work with radioactive materials. Arsenic-77: 38.8 h, 0.683 MeV
maximum β- , 239 keV γ (1.65%); 77Ge: 11.3 h, 2.7 MeV maximum β-, 211, 215.6, and 264.5 keV
γ emissions.
Physical Measurements. 1H and 13C NMR spectra were obtained in CDCl3 on a Bruker ARX-
500 MHz spectrometer using TMS as an internal standard. Electrospray Ionization Mass
Spectra (ESI-MS) were obtained on a Thermo Finnigan TSQ7000 triple-quadrupole instrument
with an API2 source. Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross,
GA). An ORTEC HPGe detector outfitted with Genie multichannel analysis software was used
to assay 77Ge and 77As liquid samples. Reversed phase HPLC (RP-HPLC) was performed using
a Shimadzu Prominence HPLC system equipped with a pump, controller, and Prominence UV-
Vis detector (model SPD20-AV) set to 220 and 280 nm, and coupled to a Beckman 170 NaI(Tl)
radioisotope detector. The gradient system used for RP-HPLC run on a Phenomenex Jupiter C
18 (5 µm, 150 mm x 4.6 mm) column was as follows: a linear gradient from 20/80 acetonitrile
(ACN)/H2O w/ 0.1 % trifluoroacetic acid (TFA) to 50/50 ACN/H2O w/ 0.1 % TFA in 10 min, then
from 50/50 ACN/H2O to 90/10 ACN/H2O in 3 min, followed by returning to 20/80 ACN/H2O over
5 min, all at a flow rate of 1 mL/min. Peptide analysis and purification were performed on a
Beckmann Coulter System Gold HPLC equipped with a 168 diode array detector, a 507e
autoinjector and the 32 KARAT software package (Beckmann Coulter, Fullerton, CA) using a C-
18 XBridge BEH, 250 x 4.6 mm, 5 m, 130 Å from Waters, Milford, MA. All LC-MS analyses and
MS assisted preparative purifications were performed with an LCQ Fleet from Thermo Fisher,
Waltham, MA.
Syntheses
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(1-Methyl-2,6,7-trioxabicyclo[2.2.2]octan-4-yl)methanol [C7H12O4], 2. Synthesis of compound 2
was accomplished using a modified literature procedure [24]. Pentaerythritol (60 g, 440 mmol),
was added to a stirring solution of dioctyl phthalate (100 mL) containing p-toluene sulfonic acid
monohydrate (~15 mg, 78 μmol). The reaction outfitted with a distillation apparatus, heated to
120 °C, and triethyl orthoacetate (71.685 g, 441.87 mmol) was added. After stirring for 22 h, 64
mL of ethanol had distilled, 83% of the theoretical amount. Triethylamine (TEA) (2 mL, 14 mmol)
was added, and the reaction mixture was allowed to stir for 10 minutes. The reaction clarified
upon heating to 160 °C, at which time a vacuum was applied to remove the TEA and any
remaining ethanol. The final product, a white solid, was isolated by vacuum distillation at 185 °C
and recrystallized from benzene. Yield: 83%, 58.64 g. 1H NMR (CDCl3; 500 MHz) δ ppm: 1.453
(s, 3H, CH3), 1.58 (t, 1H, OH), 3.454 (d, 2H, CCH2OH), and 4.015 (s, 6 H, OCH2C). 13C NMR
(CDCl3; 125.8 MHz) δ ppm: 23.53 (CH3), 35.71 (CH2CCH2), 61.50 (CCH2OH), 69.41 (OCH2C),
and 108.67 (OCCH3). ESI-MS (m/z): 161.06 (161.07 calc’d for C7H12O4 [M+H]+). Elemental
analysis calc’d (found) for C7H12O4: C, 52.49 (51.58); H, 7.55 (7.67).
1-Methyl-4-((prop-2-yn-1-yloxy)methyl)-2,6,7-trioxabicyclo[2.2.2]octane [C10H14O4], 3. Synthesis
of compound 3 was accomplished using a modified literature procedure [24]. Compound 2 (5.43
g, 33.9 mmol) was added to a stirring solution of anhydrous DMSO (30 mL) and powdered KOH
(7.60 g, 136 mmol). After stirring for 10 minutes, the reaction mixture was cooled in an ice bath
(0 °C). Propargyl bromide (4.04 g, 3.02 mL, 33.9 mmol) was added drop-wise (Caution!
Reaction becomes very hot!), and the reaction mixture rapidly became dark brown. The reaction
was stirred at room temperature for 95 minutes and poured into ice-cold water (200 mL). The
off-white solid was collected by vacuum filtration, washed with water, and dried in vacuo to
obtain the analytically pure product. X-ray quality crystals were obtained by slow evaporation
from chloroform. Yield: 5.44 g, 81%. 1H NMR (CDCl3; 500 MHz) δ ppm: 1.456 (s, 3H, CH3),
2.442 (t, 1H, CCH), 3.286 (s, 2H, CCH2O), 4.006 (s, 6 H, (CH2)3C), and 4.094 (d, 2H,
OCH2CCH). 13C NMR (CDCl3; 125.8 MHz) δ ppm: 23.56 (CH3), 34.85 (CH2CCH2), 58.88
(OCH2CCH), 68.12 (CCH2O), 69.55 (OCH2C), 75.34 (CH), 78.97 (CH2CCH), 108.72 (OCCH3).
ESI-MS (m/z): 199.10 (199.09 calc’d for C10H14O4 [M+H]+). Elemental analysis calc’d (found) for
C7H12O4: C, 60.59 (59.06); H, 7.12 (7.23).
2-(Hydroxymethyl)-2-((prop-2-yn-1-yloxy)methyl)propane-1,3-diol [C8H14O4], 4. Synthesis of
compound 4 was prepared using a modified literature procedure [24]. Deprotection was
accomplished by the addition of crude 3 (2.225 g, 11.22 mmol) to 6 M HCl (12 mL) in methanol
(40 mL) at room temperature. After stirring overnight (16 h), potassium carbonate (5.6 g, 50
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mmol) was added slowly. No starting material was observed after 24 hours, based on silica TLC
using ethyl acetate as the eluent (4, Rf ≈ 0.25; 3, Rf ≈ 0.8). The solvent was removed by vacuum
distillation to give the crude product, a thick dark yellow oil (~3 g). This crude mixture was
reconstituted in ethyl acetate, and filtered to remove any solids. The filtrate was taken to
dryness, dissolved in ethyl acetate and loaded on a silica gel column. Ethyl acetate was used to
elute the purified product. Removal of the solvent under vacuum gave the pure product, a thick
light yellow oil. Yield: 84%, 1.6 g. 1H NMR (CDCl3; 500 MHz) δ ppm: 2.148 (bs, 3H, OH), 2.465
(t, 1H, CH), 3.578 (s, 2H, CCH2O), 3.722 (s, 6 H, OCH2C), and 4.154 (d, 2H, OCH2CCH). 13C
NMR (CDCl3; 125.8 MHz) δ ppm: 45.14 (C), 58.98 (CCH2O), 64.70 (HOCH2C), 71.60
(OCH2CCH), 79.40 (CH2CCH), and 75.15 (CH). ESI-MS (m/z): 174.99 (175.09 calc’d for
C8H14O4 [M+H]+)
2-((Prop-2-yn-1-yloxy)methyl)-2-((tosyloxy)methyl)propane-1,3-diyl bis(4-
methylbenzenesulfonate) [C29H32O10S3], 5. Tosylation of compound 4 was accomplished by the
slow addition of p-toluene sulfonyl chloride (78.76 g, 413.1 mmol) to a stirring solution of
pyridine (90.3 g, 92 mL, 1.135 mol), and 4 (14.38 g, 82.56 mmol) at -5 °C. The reaction was
allowed to slowly come to room temperature, and stirred for approximately 2 days. The reaction
progress was followed by silica gel TLC with dichloromethane as the mobile phase and
visualized using KMnO4 (5, Rf ≈ 0.55). The reaction mixture was poured into 2 M HCl (400 mL)
at 5 °C to give a white solid isolated by filtration. Solids were washed with 2 M HCl (2 x 100 mL),
and cold water (2 x 100 mL). The solid was dissolved in ethyl acetate (200 mL) and washed with
saturated sodium bicarbonate (2 x 50 mL), 2 M HCl (2 x 100 mL), and brine (1 x 50 mL). The
organic layer was dried over magnesium sulfate, filtered, and taken to dryness yielding a clear
light yellow oil of the crude product. The crude product was recrystallized from a mixture of
hexane/dichloromethane (DCM) (70/30), and then washed with hot hexanes (50 °C) until the
hexane wash revealed no UV active material. Residual hexane was removed in vacuo to give
the product as a white solid. Yield: 92%, 48.21 g. 1H NMR (CDCl3; 500 MHz) δ ppm: 2.408 (t,
1H, CH), 2.468 (s, 9H, CH3), 3.358 (s, 2H, CCH2O), 3.890 (m, 8H, OCH2CCH and OCH2C),
7.357 (d, 6H, ArH), and 7.716 (d, 6H, ArH). 13C NMR (CDCl3; 125.8 MHz) δ ppm: 21.87 (CH3),
43.76 (CH2CCH2), 66.11 (OCH2CCH), 66.89 (OCH2C), 75.51 (CH), 78.66 (CH2CCH), 128.16
(ArC), 130.22 (ArC), 132.01 (ArC), and 145.50 (ArC). ESI-MS (m/z): 659.10 (659.11 calc’d for
C29H32O10S3 [M+Na]+). Elemental analysis calc’d (found) for C29H32O10S3: C, 54.70 (54.48); H,
5.07 (5.12); S, 15.10 (15.01).
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3-(3-Thiocyanatomethyl)propoxy)prop-1-yne [C11H11N3OS3], 6. Synthesis of compound 6 was
accomplished using a modified literature procedure [24]. To a 100 mL round bottom flask
equipped with a condenser, 5 (10.02 g, 15.75 mmol), KSCN (19.86 g, 204.8 mmol) and
anhydrous dimethylformamide (DMF) (70 mL) were heated to 110 °C for 18 hours, 120 °C for 2
hours, and then an additional 8 hours at 110 °C while vigorously stirring. The reaction was
monitored by silica gel TLC, using DCM as the mobile phase, until no starting material remained
(6, Rf ≈ 0.4). The dark brown reaction mixture was poured over water containing crushed ice
(800 mL) and placed in the freezer overnight (-13 °C) to precipitate the crude product, a dark
brown solid. All of the solids were collected, combined, dissolved in ethyl acetate, and purified
through a plug of silica gel. Pure product, a light yellow precipitate, was obtained by
recrystallization from a mixture of ethyl ether and DCM using a dry ice/acetone bath. X-ray
quality crystals were obtained by slow evaporation from ethyl ether. Yield: 2.79 g, 60%. 1H NMR
(CDCl3; 500 MHz) δ ppm: 2.53 (t, 1H, CH), 3.33 (s, 6 H, CH2SCN), 3.70 (s, 2H, CCH2O), and
4.24 (d, 2H, OCH2CCH). 13C NMR (CDCl3; 125.8 MHz) δ ppm: 37.04 (SCH2C), 46.17
(CH2CCH2), 58.69 (CCH2O), 68.97 (OCH2CCH), 76.42 (CH), 78.09 (CH2CCH), and 111.53
(SCN). ESI-MS (m/z): 298.73 (299.01 calc’d for C11H11N3OS3 [M+H]+). Elemental analysis calc’d
(found) for C11H11N3OS3: C, 44.42 (44.72); H, 3.73 (3.67); N, 14.13 (13.86); S, 32.34 (32.51).
Ethyl 3-azidopropionate [C5H9N3O2], 9. The product was prepared using a modified literature
procedure [25]. To a stirring solution of DMSO (200 mL), and ethyl 3-bromopropionate (20.55 g,
113.5 mmol) at 0 °C, NaN3 (9.71 g, 149.3 mmol) was added slowly. The reaction was allowed to
slowly come to room temperature and stirred for 3 days. The reaction was poured into water
(600 mL), and extracted with ethyl acetate (3 x 100 mL). The combined ethyl acetate fractions
were washed with saturated sodium bicarbonate (2 x 50 mL), water (3 x 50 mL), brine (1 x 50
mL), dried over sodium sulfate, filtered, and taken to dryness to give the pure product, a light
yellow liquid. Yield: 71%, 11.5 g. 1H NMR (CDCl3; 500 MHz) δ ppm: 1.468 (t, 3H, CH3), 2.54 (t,
2H, CH2C), 3.54 (t, 2H, N3CH2), and 4.16 (q, 2H, COOCH2). 13C NMR (CDCl3: 125.8 MHz) δ
ppm: 14.28 (CH3), 34.08 (CH2COO), 46.87 (N3CH2), 61.02 (COOCH2), and 170.91 (C).
Ethyl 3-(4-((3-thiocyanato-2,2-bis(thiocyanatomethyl)propoxy)methyl)-1H-1,2,3-triazol-1-
yl)propanoate [C16H20N6O3S3], 7. Synthesis of compound 7 was accomplished using a modified
literature procedure [26,27]. Compounds 6 (1.19 g, 4 mmol) and 9 (1.76 g, 12 mmol) were
added to a stirring solution of copper (II) sulfate pentahydrate (21.9 mg, 0.08 mmol), sodium
ascorbate (270 mg, 1.2 mmol), copper metal (1 g; cut from Cu sheet (Fisher Scientific)),
tetrahydrofuran (THF) (4 mL), ACN (4 mL), H2O (4 mL), and tert-butanol (t-BuOH) (4 mL) at 55
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°C. The reaction was monitored by silica gel TLC using DCM as the mobile phase (9, Rf ≈ 1; 6,
Rf ≈ 0.4; 7, Rf ≈ 0). Once compound 6 was no longer observed, the solvents were removed by
vacuum distillation. The residue was dissolved in DCM and loaded on a plug of silica gel, and
then washed with DCM to remove impurities. Ethyl acetate was then added to elute the crude
product. Further purification was accomplished by silica gel column chromatography (3 x 18 cm,
40 g) using 40/60 hexanes/ethyl acetate to 20/80 hexanes/ethyl acetate to 100% ethyl acetate
to obtain the product in the fractions containing ≥80% ethyl acetate; removal of solvent under
vacuum gave the product as a clear light yellow oil. Yield: 1.73 g, 98%. 1H NMR (CDCl3; 500
MHz) δ ppm: 1.24 (t, 3H, CH3), 2.96 (t, 2H, CH2COO), 3.28 (s, 6H, NCSCH2), 3.65 (s, 2H,
CCH2), 4.15 (q, 2H, COOCH2), 4.64 (t, 2H, NCH2), 4.67 (s, 2H, OCH2C=C), and 7.71 (s, 1H,
CH). 13C NMR (CDCl3; 125.8 MHz) δ ppm: 14.22 (CH3), 34.64 (CH2COO), 36.92(NCSCH2),
45.74 (NCH2), 46.08 (C), 61.36 (COOCH2), 64.18 (CCH2O), 69.33 (OCH2), 111.62 (SCN),
124.64 (C=C), 143.11 (C=C), and 170.53 (C=O). ESI-MS (m/z): 441.06 (440.08 calc’d for
C16H20N6O3S3 [M+H]+).
3-(4-((3-Thiocyanato-2,2-bis(thiocyanatomethyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propanoic
acid [C14H16N6O3S3], 8. Concentrated sulfuric acid (0.5 mL) was added to a stirring solution of
compound 7 (0.40 g, 0.908 mmol) in ACN (5 mL) and water (25 mL) at 70 °C. The reaction was
equipped with a reflux condenser, and monitored by silica gel TLC (20/80 hexane/ethyl acetate :
9, Rf ≈ 1; 7, Rf ≈ 0.5; 8, Rf ≈ 0.25) until no starting material (compound 7) was observed. The
ACN was removed via vacuum distillation, and the remaining material was extracted in DCM (3
x 50 mL). The organic layers were combined, and taken to dryness by vacuum distillation. The
residual oil was dissolved in DCM (10 mL) and applied to a plug of silica gel. DCM (200 mL)
was added to remove any unwanted materials, and then ethyl acetate (200 mL) to elute the
crude product. The ethyl acetate was removed and the product was further purified by reversed
phase C18 chromatography (3 x 15 cm) using 40/60 ACN/H2O as the mobile phase (8, Rf ≈ 0.6
by reversed phase TLC with 40/60 ACN/H2O). The solvent was removed in vacuo to afford a
light yellow oil. X-ray quality crystals (pale yellow) were obtained by slow evaporation from a
40/60 mixture of ACN and H2O. Yield: 50%, 0.187 g. 1H NMR (CDCl3; 500 MHz) δ ppm: 3.03 (t,
2H, CH2COO), 3.29 (s, 6H, NCSCH2), 3.64 (s, 2H, CCH2), 4.65 (t, 2H, NCH2), 4.67 (s, 2H,
OCH2C=C), and 7.76 (s, 1H, CH). 13C NMR (CDCl3; 125.8 MHz) δ ppm: 34.34 (CH2COO), 36.95
(NCSCH2), 45.79 (NCH2), 46.05 (C), 63.94 (CCH2O), 69.38 (OCH2), 111.77 (SCN), 124.95
(C=C), 143.07 (C=C), and 173.92 (C=O). ESI-MS (m/z): 412.02 (412.04 calc’d for C14H16N6O3S3
[M+H]+). Elemental analysis calc’d (found) for C14H16N6O3S3: C, 37.07 (37.15); H, 3.89 (3.94).
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Trithiocyanate-BBN(7-14)NH2 synthesis. Bombesin(7-14)NH2 was synthesized by solid phase
peptide synthesis (model AAPPTEC 396 Omega, Louisville, KY) using Fmoc chemistry on
Sieber Resin. The protecting groups used for the amino acid side chains were: Trityl (Gln, His);
tert-butyloxycarbonyl (Boc) (Trp). The Fmoc protecting groups were removed at each
subsequent cycle by treatment with 20% piperidine for 10 min. The peptide chain was
assembled by sequential acylation (20 min for coupling) with "in situ" activated Fmoc-amino
acids. Re-coupling was automatically performed at every cycle. The "in situ" activation of Fmoc-
amino acids (3 eq. compared to the resin amount) was carried out using uronium salts (HBTU,
2.7 eq., HOBt 3 eq.) and DIEA (6 eq.). Compound 8 (700 mg, 2.4 mmol) was conjugated to the
N terminus of the Bombesin(7-14)NH2 peptide on the resin using the same standard procedure
used for the other amino acids [28]. The peptidyl-resin was cleaved and deprotected in a single
reaction (2 hours) with the following mixture: TFA, phenol, water and TIS (85:5:5:5). Thiol
scavengers were avoided as they react with thiocyanate groups. Precipitation and multiple
washings with diethyl ether gave the final crude product. The product was HPLC purified as the
trithiocyanate-BBN(7-14)NH2 precursor, characterized by LC-MS (1333 (1333 calc’d for
C57H79O11N19S4 [M+H+])), lyophilized and stored for further use.
Trithiol-BBN(7-14)NH2. The trithiocyanate-BBN precursor (1 mg, 0.75 μmol, 0.75 mM) was
dissolved in 20% ACN in water (1 mL), to which tris(2-carboxyethyl)phosphine (TCEP, 2 mg, 7.5
μM) was added. The solution was placed in a 55 °C water bath for 2 h to yield the trithiol-
BBN(7-14)NH2 (residual TCEP remained), which was characterized by LC-MS (1258 (1258
calc’d for C54H82O11N16S4 [M+H+]), 90% yield) and used without further purification as the trithiol-
BBN stock solution.
As-trithiol-BBN(7-14)NH2. As-trithiol-BBN(7-14)NH2 was synthesized from natural As2O3 and
characterized by LC-ESI-MS. The trithiol-BBN(7-14)NH2 stock solution (500 μL, 0.5 mg, 0.4
μmol) was added to a vial. An arsenic trioxide stock solution was prepared by dissolving As2O3
(10.6 mg, 0.054 mmol) in water (13.4 mL, As2O3 concentration 0.004 M). NaOH solution (50 μL,
10 M) was added to assist the dissolution of As2O3. The arsenic trioxide stock solution (100 μL,
0.4 μmol) was added to the trithiol-BBN(7-14)NH2 stock solution (0.4 μmol) and the reaction vial
was placed in a 55 °C water bath for 40 min. The As-trithiol-BBN(7-14)NH2 reaction solution
was characterized by LC-ESI-MS (1330 (1330 calc’d for C54H79O11N16S4As for [M+H+]), over
95% yield). HPLC: tr = 12.3 min for trithiol-BBN(7-4)NH2 and tr = 13.3 min for As-trithiol-BBN(7-
14)NH2.
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X-ray Crystal Structures. Intensity data for compound 3 was obtained at -173 °C on a Bruker
Prospector equipped with a Cu IµS tube and an Apex II CCD detector. Data for 6 and 8 were
collected at -173 ºC on a Bruker Smart system with an Apex II CCD detector and Mo K
radiation from a sealed tube and a graphite monochromator. Intensities were corrected for
Lorentz and polarization effects. Equivalent reflections were merged, and absorption corrections
were made using the multi-scan method. The structures were solved by direct methods with full-
matrix least-squares refinement, using the SHELX package [29]. All non-hydrogen atoms were
refined with anisotropic thermal parameters. The hydrogen atoms were placed at calculated
positions and included in the refinement using a riding model, with fixed isotropic U, except for
the OH and water H atoms of 8, which were located and refined. Data were corrected for decay
and absorption using the program SADABS [30]. Final difference maps contained no features of
chemical significance.
Radiotracer synthesis of no carrier added 77As-trithiol-BBN. No carrier added (nca) 77As
-
([77As]H2AsO4 , 370 MBq/mL (10 mCi/mL)) in aqueous solution was obtained from the University
of Missouri Research Reactor (MURR) as a stock solution. An aliquot of the 77As stock solution
(200 μL, 74 MBq (2 mCi)) was added to a 2 mL sterile centrifuge tube along with a 30%
ethanol/water solution (581 μL). Ammonium mercaptoacetate (18.2 μL, 5.5 M, 100 μmol) was
then added, and the reaction was placed in a 55 °C water bath for 45 min. The trithiol-BBN(7-
14)NH2 stock solution (100 μL, 0.75 mM, 0.1 mg) was added to the reaction mixture and
heating continued in the 55 °C water bath for 45 min. The reaction was cooled to room
temperature and diluted with 10 mL of water. A Sep-Pak® C18 Plus Light cartridge was
preconditioned with ethanol and rinsed with water, and then the diluted reaction mixture was
loaded on the cartridge, and washed with 10 mL of water. No carrier added 77As-trithiol-BBN(7-
14)NH2 was eluted from the cartridge with 1 mL of ethanol to which ascorbic acid (30 μg) was
added. The solution was brought to dryness under a gentle N2 stream at 25 °C, and then
reconstituted with 5 mL of sterile saline solution. The final yield was determined to be 44 MBq
(1.2 mCi; 60%) in 5 mL of bacteriostatic saline solution. The specific activity of the Sep-Pak
purified product was determined to be 20.91 MBq/nmol based on HPLC analysis.
Stability in Saline (sterile saline). An aliquot of the Sep-Pak® purified radiolabeled 77As-
trithiol-BBN in sterile saline solution at a concentration of 11.1 MBq/mL (300 μCi/mL) was set
aside at room temperature. Ascorbic acid (6 μg/mL) was added to prevent radiolysis from the
decay of 77As. The 77As-trithiol-BBN solution was monitored over time by HPLC. It remained
intact at least to 48 h with no sign of free 77As (arsenate) observed by HPLC (Figure S3).
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Biodistribution studies of no carrier added 77As-arsenate and 77As-trithiol-BBN. Male CF-1
mice at 5-6 weeks of age (Charles River Laboratories, Wilmington, MA) were used for
pharmacokinetic studies. Mice were fed ad libitum rodent chow (Lab Diet 5008; Lab Diet, Inc.,
St. Louis, MO) and ad libitum acidified water in a humidity-controlled environment while housed
four mice per cage in a ventilated rack system on a 12-hour light/12-hour dark light cycle in an
AAALAC accredited facility. All studies were approved by the HS Truman Memorial Veterans’
Hospital Subcommittee for Animal Studies (SAS).
-
Biodistribution studies of both uncomplexed 77As ([77As]H2AsO4 ) and [77As]As-trithiol-BBN(7-
-
14)NH2 were performed in CF-1 mice. An 77As ([77As]H2AsO4 ) solution was prepared by diluting
-
60 μL of nca 77As ([77As]H2AsO4 , 370 MBq (10 mCi/mL)) stock solution with 2 mL of sterile
saline solution. Animals were administered 100 μL via the lateral tail vein with 0.74 MBq (~20
-
μCi) administered of the uncomplexed 77As ([77As]H2AsO4 ) and 0.37 MBq (10 μCi) administered
of the complexed [77As]As-trithiol-BBN(7-14)NH2. Mice were sacrificed at 15 min, 1 h, 4 h and
24 h post-injection for uncomplexed 77As and 1 h and 4 h p.i. for 77As-trithiol-BBN(7-14)NH2.
Tissues and organs were collected, weighed, and residual radioactivity was quantified using a
NaI(Tl) well detector. Data were analyzed to determine the percent injected dose (%ID) and
percent injected dose per gram (% ID/g) of each tissue/organ. Blood volume was calculated
based on a blood volume of 6.5% of the total body weight.
Results and Discussion
No carrier added 72As and 77As are of interest as a potential matched pair for theranostic
radiopharmaceuticals [3,18]. Previously a trithiol ligand, its arsenic complex, and its nca 77As
complex were reported as the basis of potential theranostic radiopharmaceuticals [18]. Now a
functionalized trithiol ligand conjugated to a model peptide (BBN(7-14)NH2) and its arsenic (75As
and 77As) complex were synthesized with the goal of determining the in vivo stability of the nca
77As-trithiol framework. A linkable trithiol ligand was synthesized and then conjugated to the N
terminus of the BBN(7-14)NH2 peptide via solid phase peptide synthesis to afford the
trithiocyanate-BBN(7-14)NH2 precursor. The trithiol-BBN(7-14)NH2 conjugate was readily
generated by reducing the trithiocyanate-BBN(7-14)NH2 precursor with TCEP, and then
radiolabeled with 77As for in vivo stability evaluation in normal mice.
Trithiol Precursor Syntheses. Synthesis of the first trithiol precursors, namely 3-(3-
thiocyanatomethyl)propoxy)prop-1-yne, 6, and 3-(4-((3-thiocyanato-2,2-
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bis(thiocyanatomethyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propanoic acid, 8, were prepared in
yields of 31% and 50%, respectively, using modified literature procedures (Scheme 1) [18].
Scheme 1. Synthetic scheme for the linkable trithiol precursor, Compound 8.
(1-Methyl-2,6,7-trioxabicyclo[2.2.2]octan-4-yl)methanol, 2, was synthesized by the reaction of
pentaerythritol, 1, with triethyl orthoacetate and a catalytic quantity (0.025% wt.) of p-toluene
sulfonic acid monohydrate in dioctyl phthalate at 120 °C. A key detail omitted in many literature
preparations is the addition of TEA or another trialkyl amine base prior to distillation of the
product. This compound is acid sensitive; rapid decomposition occurs if the amine base is
absent due to the catalytic quantity (0.025% wt.) of p-toluene sulfonic acid present. Reaction of
propargyl bromide with the bicyclic orthoester in dry DMSO containing powdered KOH yielded
the bicyclic orthoester alkyne, 1-methyl-4-((prop-2-yn-1-yloxy)methyl)-2,6,7-
trioxabicyclo[2.2.2]octane, 3. Attempts to purify this by silica gel proved futile because of the
inherent acidity of silica. Recrystallization from benzene or ethyl ether is possible, however
typically the crude product was immediately added to a stirring solution of methanol containing
HCl followed by the addition of base, 24 hours later, to generate 2-(hydroxymethyl)-2-((prop-2-
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yn-1-yloxy)methyl)propane-1,3-diol, 4. Reaction of 4 with excess p-toluene sulfonyl chloride in
pyridine yielded the 2-((prop-2-yn-1-yloxy)methyl)-2-((tosyloxy)methyl)propane-1,3-diyl bis(4-
methylbenzenesulfonate), 5. The first protected trithiol precursor, 3-(3-
thiocyanatomethyl)propoxy)prop-1-yne, 6, was synthesized by reacting the tris-tosylate (5) with
excess potassium thiocyanate in dry DMF.
Further reaction of ethyl 3-azidopropionate, 9, with the alkyne, 6, through a Huisgen 1,3-dipolar
cycloaddition or ‘click’ reaction provided ethyl 3-(4-((3-thiocyanato-2,2-
bis(thiocyanatomethyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propanoate, 7, in good yield.
Subsequent acid catalyzed hydrolysis of the ester was carried out to give 3-(4-((3-thiocyanato-
2,2-bis(thiocyanatomethyl)propoxy)methyl)-1H-1,2,3-triazol-1-yl)propanoic acid, 8.
Saponification of the ester was avoided due to the instability of the thiocyanate in alkali base.
The key intermediates, 2, 5, 6, and 8, were characterized by elemental analysis, while
compounds, 2-8 were characterized by 1H and 13C NMR spectroscopy and ESI-MS. Compound
9 was characterized by 1H and 13C NMR spectroscopy. The molecular ions for compounds 2-8
were observed in the ESI-MS spectra at the calculated m/z values. The 1H and 13C NMR
spectra of the compounds reported, 2-9, were characteristic of the functional groups present
and comparable to available literature [31].
Single Crystal X-ray Structures. Compounds 3, 6, and 8 were characterized by single crystal
X-ray diffraction analysis. Crystal refinement data, bond angles, and distances are summarized
in Tables S1 and S2 (Supplemental Material). Figures 1 show the structures of 8 (structures of
3 and 6 are shown in Figures S1 and S2)
Figures 1
Bond distances and angles for compounds 3, 6 and 8 were consistent with literature values [18].
The CN distances observed for compounds 6 and 8 ranged from 1.142(3) to 1.149(3) are
comparable to another reported trithiocyanate (1.145(2) to 1.147(2)) [11]. The S-CN distances
range from 1.693(2) to 1.7004(16) and are similar to the reported trithiocyanate (S-CN bond
range of 1.6932(2) Å to 1.6977(2) Å) [18]. The CH2-SCN distances for compounds 6 and 8 have
a bond distance range of 1.827(2) Å to 1.8374(14) Å and are in agreement with the distances
reported for the trithiocyanate (1.8289(1) to 1.8332(1) Å. The SCN angles reported here ranged
from 176.4(2)° to 179.12(14)°, which fall within the range of the previously reported
trithiocyanates (175.87(2)° to 179.19(2)°) [18].
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Trithiol Precursor Bombesin analogue synthesis and Trithiol-Bombesin deprotection.
Bombesin (BBN(7-14)NH2) is an amphibian peptide analogue of the mammalian regulatory
gastrin-releasing peptide (GRP) [32], which targets gastrin-releasing peptide receptors (GRPr)
with high binding affinity, specificity and in vivo stability (Figure 2) [20]. GRPr are found highly
expressed in human prostate cancer cells [19,33]. Although BBN(7-14)NH2 is no longer
considered the ideal GRP targeting peptide, it was used as the model peptide for our studies as
we have a lot of pharmacokinetic data for this peptide with other chelators and metals [21-
23,34]. A trithiol-BBN(7-14)NH2 complex was synthesized for developing an 77As radiolabeled
BBN(7-14)NH2 analogue (Scheme 2) for evaluation. The trithiol-BBN(7-14)NH2 complex was
stored as its protected precursor, trithiocyanate-BBN(714)NH2 because thiol groups tend to
oxidize over time. The synthesis of the trithiol-BBN(7-14)NH2 precursor followed standard
peptide synthesis procedures by coupling the protected trithiol ligand (compound 8) to the
Bombesin(7-14)NH2 peptide on resin (Figure 2) [22]. The “click” chemistry to synthesize 8 was
performed prior to peptide synthesis to maximize yield of the Bombesin(7-14)NH2 bioconjugate
on resin. An in situ activation with hydroxybenzotriazole (HOBt) and 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) was carried out and the trithiol ligand
was coupled twice with a three times stoichiometric excess to the resin to ensure maximum
coupling. The final product was cleaved from the resin using a TFA, phenol, water and
triisopropylsilane (TIS) (85:5:5:5) mixture, purified by HPLC and lyophilized for storage. The
product was characterized by LC-MS ([M+H+] 1333, 1333 calculated).
Figure 2
A trithiol-BBN(7-14)NH2 solution was prepared by reducing the trithiocyanate-BBN(7-14)NH2
precursor with tris(2-carboxyethyl)phosphine (TCEP), and was evaluated for quality control prior
to radiolabeling (Scheme 2). The yield of the trithiol-BBN(7-14)NH2 was determined to be
greater than 90% and was thus used in radiolabeling without further purification, with the
impurity identified as the trithiocyanate-BBN(7-14)NH2 precursor.
Scheme 2. Synthesis of trithiol-BBN(7-14)NH2 from the trithiocyanate precursor.
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Trithiol-BBN(7-14)NH2 radiolabeling. The nca 77As radiolabeling of trithiol-BBN(7-14)NH2 was
carried out in 3 h in over 90% radiochemical yield (Scheme 3) [18]. The first step in the
radiolabeling involves reduction of [77As]arsenate from the +5 to the +3 oxidation state to allow
synthesis of the desired complex. Separation of 77As from its irradiated GeO2 target yields
[77As]arsenate. Mercaptoacetate (SR-1; 111 mM) is used to reduce and stabilize the nca 77As in
the +3 oxidation state as [77As]As(SR)3. The trithiol-BBN(7-14)NH2 (0.075 mM) is then added to
displace the mercaptoacetate groups (monothiols) taking advantage of the chelate effect. The
radiolabeling reaction was analyzed by RP-HPLC and purified using a Sep-Pak® C18 cartridge.
Compared to the concentration of nca 77As, the ammonium mercaptoacetate (monothiol) and
trithiol-BBN(7-14)NH2 were in great excess. After radiolabeling, the solution was passed through
a 0.2 μm syringe filter, and approximately 30% of the radioactivity remained on the filter due to
the hydrophobic nature of the 77As-trithiol-BBN(7-14)NH2. The HPLC data is shown below
(Figure 3). In Figure 3, purified 77As-trithiol-BBN(7-14)NH2 was injected and showed over 90%
radiochemical yield. Peaks observed at 3.28 min, 4.18 min, 4.4 min and 6.3 min by UV analysis
were associated with the excess ammonium mercaptoacetate and TCEP. The peak at 12.4 min
was unreacted trithiol-BBN(7-14)NH2, which was in good agreement with the standard trithiol-
BBN(7-14)NH2 solution (1 mg/mL). A Sep-Pak® C18 cartridge was used to purify the 77As-
trithiol-BBN(7-14)NH2. Both radiolabeled 77As-trithiol-BBN(7-14)NH2 and trithiol-BBN(7-14)NH2
were retained on the cartridge because of their high lipophilicities, while the mercaptoacetate
and TCEP eluted in the mobile phase during loading and the water wash. Purified 77As-trithiol-
BBN(7-14)NH2 was eluted from the cartridge with ethanol, and the peaks associated with the
monothiol and TCEP were much less intense (less than 0.1% remaining). Surprisingly the
concentration of unlabeled trithiol-BBN(7-14)NH2 was also reduced because a significant
amount of both labeled and unlabeled trithiol-BBN(7-14)NH2 was retained on the cartridge with
the ethanol elution (~40% of the activity also was retained). To further determine the total
16

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concentration of trithiol-BBN(7-14)NH2 in the purified product solution, the UV profile at 280 nm
of purified 77As-trithiol-BBN(7-14)NH2 solution was compared with a standard trithiol-BBN(7-
14)NH2 solution (1 mg/mL) and the total trithiol-BBN(7-14)NH2 concentration was determined to
be 5.5 μg/mL (below detection limit). Ascorbic acid was added to the Sep-Pak® purified 77As-
trithiol-BBN(7-14)NH2 ethanol solution to prevent radiolysis; significant radiolysis resulting in the
formation of 77As-arsenate was previously observed when the volume was reduced to near
dryness [18]. Following addition of ascorbic acid, the 77As-trithiol-BBN(7-14)NH2 solution was
brought to near dryness under a gentle N2 stream and reconstituted with bacteriostatic saline.
Quality control was performed by RP-HPLC, with no sign of the free 77As formation observed,
and the retention time of the product peak was in good agreement with 77As-trithiol-BBN(7-
14)NH2 standard (Figure 3).
Scheme 3. Radiotracer synthesis of no carrier added 77As-trithiol-BBN(7-14)NH2.
Figure 3
Stability of 77As-trithiol-BBN(7-14)NH2. The radiolabeling was carried out in an excess of
monothiol (ammonium mercaptoacetate) to ensure that the 77As was present in oxidation state
+3 prior to addition of the trithiol-BBN(7-14)NH2. Previously, we observed that the trithiol ligand
was not sufficient for reducing arsenate to As(III) [18]. The 77As-trithiol-BBN(7-14)NH2 remained
in the radiolabeling solution (monothiol concentration 100 mM) for up to 48 h. In vitro stability of
the 77As-trithiol-BBN in the radiolabeling solution (monothiol concentration = 100 mM) was
assessed up to 48 hours by HPLC. The formation of free 77As (arsenate) was not observed.
Similar results were previously reported for the non-functionalized trithiol arsenic [18]. This
result indicates that it is unlikely for thiols found in vivo (i.e., glutathione, cysteine, albumin) to
displace the As from the conjugate.
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Biodistribution studies of no carrier added free 77As and 77As-trithiol-BBN(7-14)NH2. A
summary of the biodistribution of 77As-arsenate and 77As-trithiol-BBN(7-14)NH2 is shown in
Figure 4 and the detailed data are shown in Tables 1 and 2. A fast renal clearance of free As
-
was expected [5]. Arsenate acts as a phosphate (HPO4 ) mimic and can be reduced to As(III) in
vivo [35,36]. Generally arsenite has better in vivo retention due to multiple protein binding sites
compared to arsenate, however a fast renal excretion is typical for both species [36]. 77As
(arsenate) was excreted through the renal system with 86.90 ± 2.53% of the injected dose (ID)
excreted by 4 h p.i. At 4 h, 1.00% ± 0.12% ID remained in the kidneys, which indicates the
majority of 77As (arsenate) was not binding to free thiols from proteins present in blood plasma.
The rapid renal clearance of 77As-arsenate could potentially avoid radiotoxicity because if 77As
was to dissociate from the chelate, it should be excreted in a short period of time (<4 h). The
biodistribution of 77As-trithiol-BBN(7-14)NH2 is vastly different from that of 77As (arsenate) as
77As-trithiol-BBN(7-14)NH2 was primarily cleared through the hepatobiliary system. By 4 h p.i.,
39.83 ± 9.26% ID was located in the large intestine, 2.70 ± 1.71% ID in the small intestine, 6.84
± 2.61% ID in the liver, 1.07 ± 0.68% ID in the kidneys, and 9.46 ± 2.25% ID in the residual
carcass. Renal system excretion accounted for only 38.04 ± 8.76% of the injected dose, which
is lower than typically observed [21-23,34].
Figure 4
Tables 1 and 2
The different clearance pathway of 77As-trithiol-BBN(7-14)NH2 suggests high in vivo stability of
77As-trithiol-BBN(7-14)NH2 complex. At 4 hr, 3.52% ± 1.14% ID/g was retained in the liver,
compared with 0.42 ± 0.07% ID/g for 77As-arsenate. However the effective clearance via liver
and intestines was expected due to the high lipophilicity of 77As-trithiol-BBN(7-14)NH2.
Additional indications of stability come from the fact that even in the presence of a 1300-fold
excess of monothiol (mercaptoacetate) compared to trithiol-BBN97-14)NH2 present in the
formulation, no free [77As]arsenate or [77As]As(mercaptoacetate)3 were observed over time by
HPLC here or for the previously reported nca [77As]As-trithiol [18]. The Sep-Pak® purified 77As-
trithiol-BBN(7-14)NH2 was stable for at least 24 h as determined by HPLC analysis.
Although this was a proof of principle study to determine the stability of the trithiol-arsenic
complex under in vivo conditions, some pancreatic uptake was anticipated but none (<2% ID/g)
was observed at 1 h p.i. [23,36,37]. This was not due to excess unlabeled trithiol-BBN(7-14)NH2
as only 0.1 mg were originally used and the HPLC trace (Figure 3) shows that following Sep-
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Pak® purification, the amount remaining was below the detection limit (bottom vs upper UV
trace). It has been reported that the spacer length between the bombesin(7-14)NH2 and the
radiolabeled chelate should be a chain length of 5-8 atoms with less than 5 or more than 8
being detrimental to receptor targeting and thus pancreatic uptake [34,37]. A spacer length of
either 2 or 9 is present in the 77As-trithiol-BBN(7-14)NH2 complex depending on whether the
spacer count begins at the bridgehead carbon or after the sterically rigid triazole ring. The
crystal structure of 8 suggests that the spacer is quite rigid between C5 and C12 due to the
bridgehead and planarity of the C9 through C12 triazole-containing unit and this may impact
receptor binding.
Conclusion
A trithiol ligand and its 77As radiochemistry were previously reported [18]. To investigate the in
vivo utility of the trithiol ligand framework for nca radioarsenic, an 77As trithiol complex, namely
nca 77As-trithiol-BBN(7-14)NH2, was synthesized in >90% radiochemical yield. Following Sep-
Pak® purification, normal mouse biodistribution studies of uncomplexed 77As-arsenate and 77As-
trithiol-BBN(7-14)NH2 demonstrated that the trithiol is a promising ligand for developing 72,77As
matched pair theranostic radiopharmaceuticals. The trithiol ligand forms an in vivo stable
radioarsenic complex and development of a more hydrophilic linker that does not involve click
chemistry (i.e., a triazole unit) to reduce the lipophilicity and rigidity of the 77As complex is
underway.
Acknowledgments
The authors gratefully acknowledge support from the US Department of Energy, Office of
Science, Isotope Research Program under grants DE-SC0003851 and DE-SC0010283, and
trainee support from NIBIB Training Grant 5 T32-EB004822 (AJD). The 77As was produced at
the University of Missouri Research Reactor Center; Dr. Nathan Leigh from the University of
Missouri Mass Spectrometry Facility ran the ESI-MS spectra; and Dr. Wei Wycoff from the
University of Missouri NMR Facility assisted with NMR studies. This work was supported in part
by the H S Truman VA Hospital and support from a US Department of Veterans Affairs
Research Career Scientist award (TJH).
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Tables
-
Table 1. Biodistribution of free 77As ([77As]H2AsO4 ) in CF-1 normal mice at 15 min, 1 h, 4 h and
24 h post injection, n=4. Data are presented as %ID/g ± SD.
Organ/Tissue
Heart
15 min
1.21 ± 1.73
3.43 ± 2.81
4.39 ± 0.73
40.80 ± 5.52
0.00
1 h
4 h
24 h
1.33 ± 2.04
2.87 ± 0.98
3.31 ± 0.42
13.77 ± 1.42
1.07 ± 1.13
0.62 ± 0.31
1.59 ± 0.17
1.31 ± 0.51
1.02 ± 0.70
0.63 ± 0.83
0.35 ± 0.46
1.30 ± 0.82
0.52 ± 0.19
1.25 ± 0.11
54.82 ± 1.69
0.65 ± 1.14
0.60 ± 1.10
0.42 ± 0.07
1.74 ± 0.42
0.00
0.31 ± 0.45
0.67 ± 0.69
0.15 ± 0.21
0.66 ± 0.88
0.57 ± 1.14
0.11 ± 0.21
0.13 ± 0.18
0.03 ± 0.05
0.00
Lung
Liver
Kidneys
Spleen
Stomach
S. Intestine
L. Intestine
Muscle
0.82 ± 1.03
1.67 ± 0.26
1.28 ± 0.28
2.04 ± 2.44
0.95 ± 1.89
0.00
0.16 ± 0.32
0.40 ± 0.16
1.02 ± 0.37
0.30 ± 0.46
0.50 ± 0.99
0.50 ± 0.38
0.25 ± 0.25
0.28 ± 0.42
0.41 ± 0.15
86.90 ± 2.53
Bone
0.00
Brain
0.16 ± 0.21
0.00
Pancreas
Blood
2.29 ± 1.95
1.95 ± 0.74
2.26 ± 0.19
22.79 ± 2.20
0.38 ± 0.32
0.04 ± 0.04
97.13 ± 1.37
Carcass
Excretion*
* Excretion includes bladder, urine, cage paper, and feces (24 h) and is presented as %ID ± SD.
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Table 2. Biodistribution of 77As-trithiol-BBN in CF-1 normal mice at 1 h and 4 h post injection,
n=5. Data are presented as %ID/g ± SD.
Organ/Tissue
Heart
1 h
4 h
0.88 ± 0.36
2.06 ± 0.25
8.03 ± 1.39
4.08 ± 0.72
2.09 ± 0.53
0.44 ± 0.15
13.77 ± 8.24
27.12 ± 13.76
0.60 ± 0.27
0.50 ± 0.60
0.54 ± 0.78
1.51 ± 0.52
0.58 ± 0.21
0.42 ± 0.03
8.07 ± 0.93
0.28 ± 0.39
2.65 ± 0.63
3.53 ± 1.14
1.67 ± 0.85
2.87 ± 1.09
0.80 ± 1.04
1.51 ± 0.93
30.45 ± 10.05
0.20 ± 0.18
0.21 ± 0.46
0.04 ± 0.08
0.66 ± 0.67
0.24 ± 0.25
0.44 ± 0.11
38.04 ± 8.76
Lung
Liver
Kidneys
Spleen
Stomach
S. Intestine
L. Intestine
Muscle
Bone
Brain
Pancreas
Blood
Carcass
Excretion*
* Excretion includes bladder, urine and cage paper and is presented as %ID ± SD. Feces
included in intestinal values.
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Figures
Figure 1. X-Seed representation of (8) (CCDC# 1585889) with 50% probability ellipsoids.
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Figure 2. The structure of trithiol-BBN (7-14)NH2 conjugate.
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Figure 3. RP-HPLC chromatograms of 77As-trithiol-BBN using both gamma and UV detectors
(wavelengths 220 nm and 280 nm) and comparison with trithiocyanate-BBN, trithiol-BBN and
As-trithiol-BBN standards. Upper left diagram: γ peak of 77As-trithiol-BBN (top trace) matched
the UV peak of As-trithiol-BBN standard (middle trace) and was distinguished from the UV peak
of trithiol-BBN standard (bottom trace). Lower diagram: UV signals of monothiols and TCEP
impurities were reduced after purification (second and third traces); no UV signals related to the
γ signals of 77As-trithiol-BBN (first trace) were observed, indicating the low concentration of 77As-
trithiol-BBN in the product solution.
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-
Figure 4. Biodistribution of free 77As ([77As]H2AsO4 ) and 77As-Trithiol-Bombesin in CF-1 normal
mice. (A) Free 77As at 15 min, 1 h, 4 h and 24 h, n=4. (B) 77As-Trithiol-Bombesin at 1 h and 4 h,
n=5.
(A) Free 77As(Arsenate)
49.90
39.90
15 min
1 h
4 h
24 h
29.90
%ID/g
19.90
9.90
-0.10
29