Norepinephrine-Transporter-Targeted and DNA-Co-Targeted

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

Norepinephrine-Transporter-Targeted and DNA-Co-Targeted Theranostic Guanidines Zbigniew P. Kortylewicz,† Donald W. Coulter,‡ Guang Han,†,§ and Janina Baranowska-Kortylewicz*,† †

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Department of Radiation Oncology, J. Bruce Henriksen Cancer Research Laboratories, University of Nebraska Medical Center, Omaha, Nebraska 68132-6850, United States ‡ Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska 68132-2168, United States § Department of Radiation Oncology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China S Supporting Information *

ABSTRACT: High risk neuroblastoma often recurs, even with aggressive treatments. Clinical evidence suggests that proliferative activities are predictive of poor outcomes. This report describes syntheses, characterization, and biological properties of theranostic guanidines that target norepinephrine transporter and undergo intracellular processing, and subsequently their catabolites are efficiently incorporated into DNA of proliferating neuroblastoma cells. Radioactive guanidines are synthesized from 5-radioiodo-2′-deoxyuridine, a molecular radiotherapy platform with clinically proven minimal toxicities and DNA-targeting properties. The transport of radioactive guanidines into neuroblastoma cells is active as indicated by the competitive suppression of cellular uptake by meta-iodobenzylguanidine. The rate of intracellular processing and DNA uptake is influenced by the agent’s catabolic stability and cell population doubling times. The radiotoxicity is directly proportional to DNA uptake and duration of exposure. Biodistribution of 5-[125I]iodo-3′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine in a mouse neuroblastoma model shows significant tumor retention of radioactivity. Neuroblastoma xenografts regress in response to the clinically achievable doses of this agent.

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meta-[131I]iodobenzylguanidine (131I-MIBG), a functional analog of norepinephrine. Originally developed as the adrenomedullary imaging agent,9 MIBG is taken up by the adrenergic tissues via NET. This radioactive drug has been introduced to the management of NB over 30 years ago.10 In the intervening years, many clinical studies of 131I-MIBG were conducted. Published reports indicate that the treatment is comparatively safe and provides good disease control. However, tumor responses are transient. Even at the maximum tolerated dose of >600 MBq/kg,11 remissions are of short duration.12−14 Failure of 131I-MIBG to produce durable responses can be attributed to the dose limiting radiotoxicities

INTRODUCTION Survival of patients diagnosed with neuroblastoma (NB) depends on the biological characteristics of their tumor.1,2 Current treatment strategies take into account many of these characteristics, resulting in substantial outcome improvements in cases of the low- and intermediate-risk disease.3,4 However, more than half of patients have metastatic disease at diagnosis. Others develop refractory disease or relapse after the initial treatment. The 5-year survival in this group is only ∼30% despite multimodal therapies.5 Still lower survivals of 20% of 17b is converted into 125IUdR (Figure 1B) under the same conditions. None of the tested compounds are stable in mouse serum, which contains four esterases including carboxylesterase. Human and porcine sera do not contain any carboxylesterase activity.39 Half-lives of IDG, the agent selected for the in vivo testing, were estimated at 0.059 h ± 0.021 h, 2.28 h ± 0.08 h, and 3.59 h ± 0.12 h (average ± std err) in mouse, human, and pig serum, respectively. The evaluation of new compounds in porcine serum served a dual purpose. First, we were able to compare the stability in two D

DOI: 10.1021/acs.jmedchem.9b00437 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Determination of the stability of radioactive guanidines in human serum. HPLC analyses of regioisomers IDG and 17b. (A) HPLC analysis of IDG incubated in human serum. (B) HPLC analysis of 17b incubated in human serum. (C) Comparison of the monoexponential degradation curves for tested compounds in human serum. Dotted line indicates 50%. (D) HPLC analyses of IDG incubated for 24 h in cell culture media without cells. (E) HPLC analyses of IDG incubated for 24 h in cell culture media with neuroblastoma cells SK-N-SH. (F) HPLC analyses of 17b incubated for 24 h in cell culture media with neuroblastoma cells SK-N-SH. Peaks highlighted in red indicate the intact compound. Blue peaks correspond to the catabolic product 125IUdR. HPLC conditions: column Columbus C18 100 Å (5 μm; 4.6 mm × 250 mm); flow 0.8 mL/min; linear gradient from 0% to 95% CH3CN over 50 min followed by isocratic 95% CH3CN for 20 min; both solvents with 0.07% TFA.

Table 2. pKa, pI, and log P of Radioactive Guanidines compd

pKa

pI

log P

9 IDG 17a 17b 21 25 MIBG

11.5 12.1 11.9 12.2 12.1 11.9 11.8

9.99 10.29 10.18 10.31 10.30 10.16 nd

−1.30 −0.20 −1.48 −0.20 4.58 0.26 1.63

the passive diffusion across the cell membrane, whereas the active transport and binding to NET appear to be sterically hindered by the deoxyribose residue. While this outcome is disappointing (9 was produced with the expectation that it might mimic the in vivo biological behavior of MIBG52,53 and additionally allow the incorporation and retention of 125I into DNA of proliferating cells54), the lack of uptake of 9 by NB cells confirms that our radioactive guanidines require active, NET-facilitated transport across the plasma membrane. The remaining target compounds IDG, 17a, 17b, 21, and 25 are taken up by NB in a time- and concentration-dependent manner (Figure 2A and Figure 2B). Uptake increases linearly within the tested range of extracellular concentrations, up to 200 kBq/mL (2.45 × 10−9 M), resembling MIBG uptake, which is linear up to ∼1 × 10−7 M and begins to saturate at concentrations >5 × 10−7 M.55 Uptake of new radioactive guanidines is also dependent on the cell population doubling time (TD). When cells are incubated with IDG for 1 h, washed with nonradioactive medium and PBS, and processed immediately, cellular uptake is nearly twice as high in BE cells (TD = 19 h) as it is in SK cells (TD = 40 h), indicating

uptake by passive diffusion.50,51 Cellular uptake of radioactive guanidines was evaluated in two human NB cells lines SK-NSH (SK) and BE(2)-C (BE) and in one murine NB cell line N1E-115 (N1E). Compound 9 is not taken up by NB cells. After 1 h with 9 at the extracellular concentration of 38 kBq/ mL, amounts of 125I barely detectable in a γ-counter were recovered with SK cells (1.0 × 10−5 ± 1.4 × 10−5 mBq/cell). A continuous exposure of SK cells to 9 for 24 h produced only a marginal increase in the cellular 125I levels (5.4 × 10−4 ± 1.8 × 10−5 mBq/cell). This lack of cellular uptake can be attributed to the positive charge of the guanidine residue, which prevents E



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Figure 2. Cellular uptake and retention of radioactive guanidines in neuroblastoma cell lines in vitro. (A) Time dependent uptake of 17b in SK-NSH cells. (B) Concentration dependent uptake of IDG in SK-N-SH cells. (C) Comparison of cellular uptake of IDG by BE(2)-C (orange) and SKN-SH (blue) cells. Cells were treated with IDG for 1 h and harvested and immediately processed (1 h) or were washed with nonradioactive medium and PBS, and returned to the incubator for an additional 23 h (24 h). (D) Subcellular distribution of IDG in SK-N-SH cells. (E) Subcellular distribution of IDG in BE(2)-C cells. Cytoplasm = black; DNA in SK-N-SH = blue; DNA in BE(2)-C cells = orange. (F) Comparison of subcellular distribution of 125I-MIBG (blue) and 17b (orange) in N1E-115 cells. (G) Cellular uptake of 21 in SK-N-SH cells after 1 and 24 h as described above. (H) Surviving fractions of SK-N-SH cells treated with 21 for 1 h, washed, and incubated for additional 23 h. (J) Subcellular distribution of 21 in SK-N-SH cells; cytoplasm = black; DNA = blue. (K) Comparison of cellular uptake of several radioactive guanidines in SK-NSH cells normalized to the extracellular concentration of 37 kBq/mL. All data are shown as average (n ≥ 3) and standard deviation unless otherwise indicated.

greater levels of 125I incorporation into the DNA of rapidly proliferating BE cells. Allowing cells to grow for additional 23 h after the removal of the radioactive media equalizes per cell uptake because the number of BE cells more than doubles in 24 h and now each daughter cell has approximately one-half of the initial radioactivity (Figure 2C). These experiments also confirmed the intracellular processing of these agent with production of 125IUdR, which can diffuse from cells reducing its intracellular pool. Subcellular distribution studies corroborate this notion. After 1 h with IDG, SK cells retain ∼80% of the total recovered 125I in their DNA compared to 93% in DNA of BE cells (Figure 2D, Figure 2E). At 24 h, practically all 125 I is associated with DNA in both cells lines. The intracellular processing of radioactive guanidines and DNA co-targeting is also evident in studies of compound 21. The subcellular distribution of 21, which is slowly catabolized to monophosphate of 125IUdR, reveals the intracellular trapping of this negatively charged catabolite and consequently a higher cellular and cytoplasmic retention of radioactivity at 1 and 24 h, even after repeated washes (Figure 2G, Figure 2J). In the case of 21, which is more stable compared to IDG, the fraction of 125I associated with DNA is less, providing further corroboration of the intracellular trapping and processing of radioactive guanidines. Cell survival is proportional to the levels of 125I incorporation (Figures 2H and 4). Similar results were observed in N1E cells, an NB cell line of murine origin with TD = 36 h similar to SK. The comparison of the

subcellular distribution of 125I-MIBG and 17b in N1E cells is shown in Figure 2F. The majority of 125I-MIBG after 24 h incubation was recovered in the cytoplasmic fraction. Only ∼19% of 125I was recovered in the nuclear fraction and 0% in DNA. In contrast, in cells exposed to 17b, practically all 125I was recovered in the DNA pellet (>99%). The overall comparison of all radioactive guanidines in terms of the total cellular uptake is shown in Figure 2K. Compound 25, structurally closest to MIBG, is retained by SK cells at levels marginally lower than 125I-MIBG. Because of their short halflives in human serum, 25 (t1/2 = 45 min), 17a (t1/2 = 23 min), and 17b (t1/2 = 53 min) were not evaluated further. On the basis of the high cellular uptake, >4× greater compared to 125IMIBG, and good stability in human serum (t1/2 = 137 min), IDG appeared to be the candidate drug most likely to succeed in the in vivo studies. Competition with MIBG. SK and BE cells express high levels of endogenous NET and represent a well-established robust model for the NET-specific MIBG uptake.53,56−59 Two variations of the binding assay were employed to determine the NET-directed uptake of new radioactive guanidines: the cell suspension and the cell monolayer method. Both assays confirmed that the active uptake of radioactive guanidines is competitively inhibited by nonradioactive MIBG indicating the same specific transport mechanism. MIBG inhibits uptake of IDG, 17b, and 21, three compounds evaluated in this assay. Exponentially growing monolayers of BE cells exposed to 21 F



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Figure 3. Dependence of the cellular uptake of new agents on the availability of norepinephrine transporters in neuroblastoma cell lines. (A) Competition of 21 with nonradioactive MIBG in BE(2)-C cells grown as a monolayer. Cells were exposed to 37 kBq/mL 21 without (blue) and with (orange) 100 μM nonradioactive MIBG. (B) Temperature-dependent uptake of IDG (300 kBq/mL) in BE(2)-C cells and competition with nonradioactive MIBG analyzed in a filtration assay. (C) Cell cycle analysis of quiescent SK-N-SH cells. (D) Lack of competition of IDG with nonradioactive MIBG in quiescent SK-N-SH cells. (E) Western blot of norepinephrine transporter expression in cell lysates from SK-N-SH cell in the exponential (exp) growth or in G0. (F) Western blot of β-actin to verify uniform protein loads. (G) Cellular uptake of IDG in SK-N-SH cells at various levels of confluency. All data are shown as average and standard deviation (n ≥ 3).

after 1 h incubation with 100 μM nonradioactive MIBG had uptake of 21 reduced by ∼28% compared to cells treated with 21 alone. The extended 3 h incubation with nonradioactive MIBG reduced the uptake by 40% (Figure 3A). Cellular uptake of radioactive guanidines is also energy dependent confirming their active transport into the NB cell. When cells are cooled on ice to 4 °C and the MIBG inhibition assay is conducted at this temperature, significantly reduced uptake of IDG is observed compared to experiments run at 37 °C (Figures 3B). At low temperatures, precipitation of MIBG was noted at concentrations of >80 μM. Similar to BE cells, uptake in SK cells depends on the temperature. SK cells incubated with 37 kBq/mL IDG at 37 °C take up 1.85 ± 0.01 mBq/cell. In a parallel uptake study conducted at 4 °C, SK cells retained only 0.05 ± 0.02 mBq/cell. The estimated IC50 values are listed in Table 3. We consider the IC50 estimates from

of 0.1 mM and in some instances up to 1 mM are employed.63,64 MIBG can disrupt normal cellular functions at such high concentrations. Moreover, MIBG is far more lipophilic than our new compounds (Table 2) and can cross the plasma membrane unaided by NET. At low concentrations comparable to the physiological levels of norepinephrine, ∼1 × 10−9 to 1 × 10−7 M, MIBG uptake is predominantly via the NET-facilitated active transport process, whereas at nonphysiological levels typically used in the competitive binding assays, MIBG uptake occurs through the passive diffusion.65,66 Concentrations of our radioactive guanidines do not exceed 1 × 10−8 M, i.e., are well within the active transport limits. We have also undertaken studies intended to uncouple MIBG-competed uptake from the DNA targeting. We hypothesized that quiescent SK cells produced by the cell density-dependent inhibition of proliferation will not be able to incorporate 125IUdR, the catabolic product of our compounds, into their DNA but will retain their ability to transport guanidines via the NET function. BE and N1E cells cannot be used in this experiment because their growth is not inhibited at high cell densities. Density-inhibited SK cells were maintained in culture with daily additions of fresh media for 3 days after reaching 100% confluency. Cell cycle analyses indicated nearly 90% of cells in G0 phase with no discernible S phase cells (Figure 3C). However, we unexpectedly found that uptake of IDG by quiescent SK cells is insensitive to MIBG inhibition. Only ∼15% reduction of uptake was detected at the highest tested MIBG concentration (Figure 3D). The Western blot of proteins derived from lysates of exponentially growing SK cells indicates the presence of a single protein band, MW ∼ 68 kDa, detectable with anti-NET antibodies. In the same Western blot, protein lysates from quiescent SK cells show two protein bands, the minor 68 kDa band and a more prominent 47 kDa band (Figure 3E). It appears that allowing SK cells to arrest in G0 not only affects proliferation but also renders a significant fraction of NET inactive. In follow-up experiments, we found that cellular uptake of radioactive guanidines by SK cells is inversely proportional to the cell density (Figure 3G). Consequently, it was not possible to clearly separate MIBGinhibited uptake of new compounds from the DNA cotargeting. An exhaustive literature search did not uncover any information regarding the expression and functional activity of NET in quiescent NB cells. This aspect of NET expression and

Table 3. Half Maximal Inhibitory Concentrations (IC50) of Nonradioactive MIBG in Competition with IDG in BE(2)-C and SK-N-SH Human Neuroblastoma Cell Lines average IC50 (std err), μM conditions monolayer (1 h) cell suspension 37 °C 4 °C

BE(2)-C

SK-N-SH

8.2 (2.1)

76.2 (9.3)

28.7 (4.4) 23.9 (1.5)

35.2 (16.6) 22.0 (6.6)

experiments conducted in a monolayer a better representation of the competitive inhibitory effect of MIBG. The higher IC50 in SK cells is a manifestation of the fact that the SK cell line consists of three distinct subpopulations, N-, S- and I-types of which N-type cells express ∼7× higher NET than the I-type.60 In contrast, BE cell line is a homogeneous I-subtype. The cell suspension assay has several shortcomings in the evaluation of compounds that co-target DNA. SK and BE cells in suspension fail to maintain their normal functions after ∼1−2 h and are especially vulnerable at 4 °C. SK cells seem to be a little more resilient. Further complications arise from the cytotoxicity of nonradioactive MIBG when used at high concentrations. MIBG is cytotoxic in a large panel of diverse cancer cell lines without preference against tumor cells of neural origin.61,62 In a typical competitive inhibition assay, high MIBG concentrations G



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Figure 4. Radiotoxicity of guanidines in SK-N-SH and BE(2)-C cells. (A) Surviving fractions of SK-N-SH cells 24 h after treatment with various concentration of IDG. (B) Concentration-dependent clonogenic survival of SK-N-SH cells treated with IDG. (C) Colonies formed by SK-N-SH cells 14 days after plating: (a) untreated controls cells; (b) cells treated with 18 kBq/mL IDG; (c) cells treated with 35 kBq/mL IDG. (D) Number of 125I accumulated in DNA of BE(2)-C cells exposed to the increasing concentrations of IDG for 1 h, extensively washed and harvested with trypsin. (E) Evaluation of clonogenic survival of BE(2)-C as a function of the intracellular content of 125I. (F) Colonies formed by BE(2)-C untreated control cells. (G) Colonies formed by BE(2)-C with the intracellular 125I accumulation of 0.3 mBq/cell after 1 h treatment with IDG. (H) ImageJ analyses of colonies in (F). (J) ImageJ analyses of colonies in (G). All data are shown as an average (n ≥ 3) and standard deviation unless otherwise indicated.

function in NB cells will require comprehensive studies beyond the scope of this report. Radiotoxicity. Radiotherapeutic potential of radioactive guanidines was first evaluated in vitro. The survival of NB cells was assessed at 24 h after treatment using the trypan blue exclusion method followed by the clonogenic assay to measure the reproductive integrity of treated cells. Both end points were dependent on the compound concentration and duration of exposure (Figure 4). Twenty-four hours after the exposure to IDG, only ∼60% of SK cells survive at the extracellular concentration of 180 kBq/mL (Figure 4A). The clonogenic assay of cells harvested in this experiment, replated at 500 live cells/flask and grown in nonradioactive medium for 14 days, indicates that after the exposure to the extracellular concentration of 35 kBq/mL IDG, the majority of SK cells, >90%, lose their reproductive integrity (Figure 4B). Cells treated with concentrations IDG > 100 kBq/mL did not produce any colonies even after 21 days in culture. Typical colony forming assay results are shown in Figure 4C for the SK cells, untreated (a) and after treatment with IDG at the concentration of 18 kBq/mL (b) and 35 kBq/mL (c). BE cells respond to treatment with radioactive guanidines much like SK cells. Given the ∼2.3 h half-life of IDG in human serum, it was

important to determine if the radioactivity retained by BE cells after short treatment of 1 h is sufficient to produce measurable radiotoxicity. To answer this question and relate the cell survival to the duration of exposure, BE cells were treated for 1 h with several concentrations of IDG, washed, harvested and replated for the clonogenic assay. Cellular uptake was measured and the number of 125I atoms per cell in DNA estimated (Figure 4D). A brief 1-h exposure to IDG resulted in the incorporation of >6,000 125I/cell. Extracellular concentrations required to achieve this level of 125I in DNA are expected to be easily attainable in a clinical setting. At the intracellular retention levels of 0.3 mBq/cell and 0.8 mBq/cell, only a fraction of BE cells, 0.6 and 0.4, respectively, survive, retain their reproductive integrity, and form colonies (Figure 4E), which are considerably smaller compared to the untreated cells (Figure 4F, Figure 4G). Image analyses also revealed the significant decrease in the crystal violet intensity in colonies. This parameter is proportional to the number of cells in each colony (Figure 4H, Figure 4J). The estimated extracellular concentration of IDG required to kill 63% of cells (D37) is approximately 25× greater for the 1 h treatment as compared to the 24 h treatment (Table 4). H



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Table 4. Estimated Intracellular and Extracellular 125I Concentrations, Average (std error), Needed To Kill 63% (D37) of BE(2)-C cells treated for 1 h and SK-N-SH cells treated for 24 h with IDG BE(2)-C extracellular (kBq/mL) intracellular (mBq/cell)

escapes into the extracellular spaces, reducing the cellular pool of 125IUdR available for DNA uptake. The cell survival is inversely proportional to the radioactivity located in the cell nucleus. This comparison of 125I-MIBG and IDG confirms a remarkable dependence of 125I radiotoxicity on the subcellular location and highly localized energy deposition around the decay site. This property of 125I also indicates that compared to 131 I-MIBG, a significant reduction of absorbed radiation doses by normal tissues is achievable, a property of great consequence in pediatric patients. In Vivo Evaluation. On the basis of its favorable stability in human serum, in vitro uptake, and radiotoxicity in human NB cells, IDG was selected for biodistribution, whole body clearance, and therapy studies in immunodeficient female and male mice bearing subcutaneous (SQ) NB xenografts of BE cells. Intratumor injections (ITs) were used as the preferred route of administration to avoid rapid degradation of IDG in blood by carboxylesterases. One group of mice was given IT injections of 125I-MIBG to compare its tissue distribution with IDG. Figure 6A and Table 1S (Supporting Information p S118) summarize tissue and tumor uptake of these two radioactive agents at 24 h after administration. The radioactive content of tumors extirpated from mice treated with IDG is on average 3.93% ± 0.23 %ID/g, twice the content of tumors injected with 125I-MIBG, which retained 1.85% ± 0.35 %ID/g (P = 0.02). The gender of mice did not influence tumor retention of IDG (Figure 5B). The corresponding tumor-to-blood ratios were estimated at 161 ± 30 for IDG and 19 ± 7 for 125I-MIBG (Table 1S, Supporting Information p 118). As expected, 125I-MIBG is also taken up by adrenal glands70 at 4.64% ± 1.40 %ID/g 24 h after injection. Many lower mammals including mice have high levels of carboxylesterases in blood, while little or no expression of this enzyme is observed in higher primates and humans.39 Consequently in mice studies, all traces of IDG that leak out from the tumor site are rapidly degraded by carboxylases in the blood, preventing any uptake in tissues other than the tumor. Human NB cells have high levels of carboxylesterase activities.71−73 We attribute greater tumor retention of IDG as compared to MIBG to the intracellular processing of IDG and co-targeting of DNA in proliferating NB cells. To

SK-N-SH

1h

24 h

398 (48) 0.91 (0.29)

15.6 (2.3) 1.20 (0.27)

However, predictably, the estimated intracellular D37 doses are very similar for BE and SK cells. The radiotoxicity of 125I, which produces a cascade of monoenergetic electrons known as conversion and Auger electrons, is dependent on the intranuclear localization; i.e., 125I is radiotoxic when within DNA or in the immediate vicinity of DNA.26,67−69 The cell survival is proportional to levels of 125I incorporation into DNA. After the intracellular processing of radioactive guanidines, both cell lines incorporate 125IUdR into their DNA (Figure 2), and accordingly, the intracellular doses D37 are similar. The requirement for the intranuclear localization of 125I was further confirmed in studies comparing the radiotoxicity of our radioactive guanidine to 125I-MIBG (Figure 5). Cells exposed to 125I-MIBG store this drug predominantly in the cytoplasmic compartments, 87% and 74% of total cell-associated radioactivity after 1 and 24 h, respectively, whereas IDG accumulates predominantly in DNA, ∼70%−80% and >97% after 1 and 24 h, respectively (Figures 2D, 5B, 5C). Number of colonies formed after 1 h and 24 h treatments is significantly smaller in SK cells treated with IDG as compared to 125I-MIBG (Figure 5D, Figure 5E). Cells exposed to 125I-MIBG for 1 h produced 229 ± 13.4 colonies compared to SK cells exposed to IDG, which produced 163 ± 8.3 colonies. The 24 h exposure resulted in 250 ± 18.7 colonies in 125I-MIBG group and 97 ± 4.3 colonies in cells treated with IDG. More colonies were observed in cells in the 1 h group treatment with IDG because when the radioactive medium is removed and cells are washed with PBS, harvested with trypsin, and replated for the clonogenic assay, a substantial amount of 125IUdR already produced and present in the cytoplasm diffuses from cells and

Figure 5. Comparison of cellular uptake and subcellular distribution effects on the reproductive integrity in SK-N-SH cell treated with 125I-MIBG (extracellular concentration 39.3 ± 0.3 kBq/mL) and IDG (extracellular concentration 40 ± 0.5 kBq/mL). (A) Cellular uptake of 125I-MIBG and IDG in SK-N-SH cells. (B) Cytoplasmic distribution of 125I-MIBG and IDG after 1 h and 24 h treatments. (C) Distribution of 125I-MIBG and IDG in the cell nucleus after 1 h and 24 h treatments. 125I activity in the nuclei of cells treated with IDG was recovered in DNA. All cells were exposed to radioactive compounds for 1 h. Cells in the 1 h group were washed with nonradioactive medium, PBS, harvested and replated for the clonogenic assay. Cells in 24 h group were washed with nonradioactive medium, PBS, given fresh nonradioactive medium, and returned to the incubator for 23 h. (D) Colony forming assay results after treatment with125I-MIBG and IDG. Blue bars represent averages for 125I-MIBG; orange bars represent averages for IDG; standard deviations of n = 4. (E) Colonies formed by SK-N-SH treated with 125I-MIBG and IDG. I



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Figure 6. In vivo evaluation of IDG in mice. (A) Biodistribution of IDG (orange) and 125I-MIBG (blue) in athymic Ncr nu/nu female and male mice bearing subcutaneous BE(2)-C xenografts at 24 h after the intratumor administration. (B) Comparison of IDG uptake by BE(2)-C xenografts in female and male mice. (C) Effects of carboxylesterase inhibition with bis-para-nitrophenylphosphate (BNPP) on the retention of IDG in BE(2)C xenografts. (D) Volume of BE(2)-C xenografts in vehicle-injected control mice (blue bar) and in mice treated with 8.4 ± 0.3 MBq IDG (orange bar); average ± std err. (∗) Control mice were killed 7 days after the vehicle injection. (E) Whole body clearance of IDG in mice with BE(2)-C xenografts after injection of therapeutic doses of IDG. (F) Radioactivity retained in tumor, blood, and the whole body at necropsy 14 days after the administration. (G) Hemoglobin levels in blood of control (NT) mice and mice treated with IDG. (∗∗) Normal range of Hb values in adult mice.

weight in the NT group was 3.69 ± 1.48 g. Treated mice were terminated 14 days after the injection of IDG when the average tumor volume was 2.55 ± 1.08 cm3. This tumor volume corresponded to the average tumor weight of 2.71 ± 1.77 g at necropsy. The difference between treatment and control groups is statistically significant (P = 0.01). It is evident that a single dose of 8.4 ± 0.3 MBq/mouse (227 ± 9 μCi/mouse) produced a significant tumor growth delay. The estimated tumor doubling times (TD) from day 0 to day 7 were 2.4 days in the NT group and 14.9 days in mice treated with IDG. The overall TD, calculated from the time 0 to day 14, for tumors treated with IDG was 5.3 ± 0.4 days. The whole body clearance after the injection of therapeutic doses of IDG is biphasic (Figure 6D). The initial rapid decline of radioactivity has the biological half-life of 1.35 ± 0.07 h and is followed by a slow elimination with the biological half-life of 76.2 ± 12.6 h. Over 50% of whole-body radioactivity is eliminated by 2 h postinjection. The remaining activity is primarily tumor-associated. The slow phase of the whole-body elimination parallels the clearance of 125I from the tumor site. On day 14, the tumor radioactive content evaluated in terms of %ID/g was >70× greater compared to blood (Figure 6E). The estimated tumor absorbed radiation dose is 1200 ± 147 mGy/ MBq (Table 5). We also estimated whole body absorbed radiation doses for 1-year-old and 5-year-old pediatric patients. The reported 131I-MIBG whole-body radiation is estimated at 0.23 mGy/MBq,77 approximately 16× higher than doses estimated for new radioactive guanidines. Hb levels in mice

substantiate this notion, we conducted the in vivo tumor uptake studies in BE xenograft-bearing mice injected with bispara-nitrophenylphosphate (BNPP). BNPP is an irreversible carboxylase inhibitor.74 It has been used in vitro and in vivo to inhibit carboxylase activity and alter the pharmacokinetic profiles of drugs hydrolyzed by this enzyme.75,76 Groups of mice were injected with IDG alone or alongside BNPP at two doses: 9 μmol and 18 μmol. In this experiment, 3.31 ± 0.85 % ID/g was found in tumors extirpated from mice receiving only IDG (Figure 6C). Tumors from mice preinjected with 9 and 18 μmol of BNPP retained far less of the radioactive guanidine, 1.63% ± 0.13 %ID/g and 1.66% ± 0.16 %ID/g, respectively. These differences are statistically significant as compared to the compound alone uptake (P = 0.0014). BNPP-inhibited tumor uptake of IDG is nearly identical to tumor uptake of 125I-MIBG (1.85 ± 0.35 %ID/g), confirming the intracellular processing with subsequent incorporation and retention of 125I-1 in DNA. Therapeutic potential of radioactive guanidines was tested in athymic Ncr nu/nu female and male mice bearing SQ xenografts of BE cells randomized via a lottery into two groups: treatment with IDG and control (NT). On the day of treatment, initial tumor volumes were 0.39 ± 0.15 cm3 and 0.42 ± 0.14 cm3 in NT and IDG groups, respectively (P = 0.85). One week after the administration of IDG, tumor volume in mice treated with IDG was 0.54 ± 0.12 cm3 (Figure 6D). All control mice had to be terminated at this time because their tumor volume at 3.26 ± 1.20 cm3 was reaching the 10% body weight limit. At necropsy on day 7, the average tumor J



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IDG sufficient to kill >90% of NB cells is ∼37 kBq/mL. This corresponds to 800 mCi) 125I-A33 antibody,95 which has a far longer half-life in circulation. The whole body doses that might be expected in pediatric patients treated with IDG are also reassuring since the estimates are ∼16× lower compared to 131I-MIBG. Additionally, we should note that radionuclides such as 125I, which emit only low energy photons, are easily shielded, greatly simplifying the radiation protection of medical and nursing staff. In conclusion, radioactive guanidines can be efficiently synthesized from 5-radioiodo-2′-deoxyuridine, a molecular radiotherapy platform with clinically proven minimal toxicities and DNA targeting properties. In vitro and in vivo properties of new compounds indicate their significant potential as novel theranostics for the management of NB.

Table 5. Estimated Tumor and Whole Body Absorbed Radiation Doses tumor mean residence time (MRT) absorbed radiation dose, 1 g tumor whole body dose estimate, 1-year old whole body dose estimate, 5-years old

11.6 (1.4) h 1200 ± 147 mGy/MBq 0.014 mSv/MBq 0.017 mSv/MBq

treated with IDG are within the normal range values (Figure 6F) and support the postulated low normal tissue toxicity of 125 I-labeled compounds.

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DISCUSSION AND CONCLUSIONS NB is a heterogeneous disease. Some tumors regress or differentiate spontaneously. Others exhibit extremely malignant behavior with life-threatening progression, very low cure rates, and very few therapeutic options.2,78−80 NBs in children 1 year of age or older are frequently unresectable or metastatic and are fatal in ∼60% of cases despite intensive multimodal therapies. 131I-MIBG has been introduced to the management of NB nearly 30 years ago10 and became an essential component of treatment strategies for relapsed and refractory disease with overall response rates of ∼30%.11,12,18,22,81 However, the therapeutic applications of 131I-MIBG are limited. Tumor responses are transient. Myelosuppression and other adverse effects related to the radiation exposure of normal tissues prevent administration of higher, more effective doses. New therapies must attain a balance between improved survival rates and the morbidity of side effects. Our radioactive guanidines are radiolabeled with Auger electron emitters and are expected to have a more favorable toxicity profile that will permit the administration of curative doses. Low energy electrons from 125I decaying in DNA produce nonrepairable strand breaks and lesions82,83 analogous to those produced by high LET radiations. Each decay of DNA-incorporated 125I produces double DNA strand breaks with the probability of nearly one and several single DNA strand breaks.84−87 Approximately 90% of these DNA breaks are located within 10 nm of the 125I decay site; i.e., nearly all of the electron energy associated with the 125I decay is deposited within a sphere smaller than the cell nucleus,25,26 ensuring minimal irradiation of the surrounding healthy tissues and organs. Synthetic methods and the design of new radioactive guanidines can be altered to produce derivatives with distinct stabilities under physiologic conditions and varied cellular uptakes. The chemical structure accommodates therapeutic as well as diagnostic radionuclides. For these reasons, the clinical potential of radioactive guanidines is high. New compounds mimic MIBG in their mechanism of NB targeting; however, unlike MIBG they subsequently undergo intracellular processing and their catabolites are efficiently incorporated into DNA of NB cells with high proliferation activities, i.e., cells in the high risk and aggressive disease. Moreover, the structure can comprise groups that allow the lock in mechanism,88 such as in compound 21, to trap the catabolites within the cancer cells, ensuring their sustained availability throughout the cell cycle. This feature is important in eradicating slowly proliferating tumor cells89,90 that can be expected to occur in a heterogeneous cell population of NB. Biological properties of radioactive guanidines support their potentially significant role as theranostics for diagnostic and effective nontoxic therapeutic strategies in pediatric patients diagnosed with high risk NB. For example, a concentration of

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EXPERIMENTAL SECTION

Reagents and Instrumentation. Chemicals and reagents purchased from commercial suppliers were used without further purification, unless explicitly indicated. 5-Iodo-2′-deoxyuridine 1 (IUdR) was purchased from Sigma-Aldrich (St. Louis, MO). Reagents 4 and 5 were prepared using routine procedures, and these are described briefly in the Supporting Information (pp S5, S20, S39). Solutions of sodium [125I]iodide in 1 × 10−5 NaOH (pH 8−11), with specific activities of ∼78 000 GBq/mmol (carrier-free) were obtained from PerkinElmer (Billerica, MA). Radioactivity was measured with Minaxi γ-counter (Packard, Waltham, MA) and a dose calibrator (CapIntec Inc., Ramsey, NJ). Analytical TLC was carried out on plastic plates coated with a 0.2 mm layer of silica (normal phase Merck 60 F254), and spots were visualized with either short wave UV K



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or iodine vapors. Flash column chromatography was performed using Merck silica gel 60 (40−60 μM) as a stationary phase. Compounds were resolved, and their purity was evaluated by the HPLC analyses conducted on Gilson (Middleton, WI) and ISCO (Lincoln, NE) systems, with 5 μm, 250 mm × 4.6 mm analytical columns, either Columbus C8 or C18 (Phenomenex, Torrance, CA) and ACE C18 (Advanced Chromatography Technologies, www.ace-hplc.com). All compounds were determined to be ≥95% pure by this method. Columns protected by guard filters were eluted at a rate of 0.8 mL/ min with various gradients of acetonitrile (CH3CN; 10−95%) in water, with or without trifluoroacetic acid (TFA; 0.07%, w/v). Two variable wavelength UV detectors: UVIS-205 (Linear, Irvine, CA) and UV116 (Gilson) were used jointly with the sodium iodide crystal Flow-count radioactivity detector (Bioscan, Washington, DC) connected to the outlet of UV detector. Both signals were monitored and analyzed simultaneously. All target nonradioactive compounds were found to be ≥98% pure by the rigorous HPLC analysis, with the integration of a peak area (detected at 220 and/or 280 nm). Radioiodinated products were identified and evaluated through the independently prepared nonradioactive reference compounds by comparing UV signals of the nonradioactive standards with signals from radio-TLC (Rf) and radio-HPLC (tR) of the radioactive products. Before testing the biological activities and stability, each final target compound was once again purified by HPLC using linear gradient of ethanol (EtOH; 0−70%) in phosphate buffer (10 mM, pH 6.1) to eliminate TFA and CH3CN in tested samples. Solutions containing the product were evaporated with a stream of sterile nitrogen and reconstituted in a preferred solvent at the required concentration and then filtered through a sterile 0.2 μm filter (Millipore) into a sterile evacuated vial. 1H, 13C, and 31P NMR spectra were recorded in (CD3)2SO or CDCl3 at ambient temperature on Bruker Avance III HD 600 MHz spectrometer. All NMR analyses were performed by the University of Nebraska Medical Center Fred & Pamela Buffett Cancer Center Structural Biology Facility Shared Resource (Omaha, NE). Chemical shifts are given as δ (ppm) relative to TMS as internal standard with J in hertz. Deuterium exchange and decoupling experiments were performed to further assist signals assignments of protons. High resolution electrospray ionization (ESIHR) mass spectra (in positive mode) were acquired on a Waters Synapt G2 HDMS mass spectrometer at the Washington University Resource for Biomedical and Bio-Organic Mass Spectrometry (St. Louis, MO). General Procedure A: Preparation of 5-Iodo-5′-N-[N′,N″bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine (6) and 3′-O -and 5′-O-[(N,N′-bis(tert-butyloxycarbonyl)-N″alkylcarboxy)guanidino] Esters of 5-Iodo-2′-deoxyuridine: 10, 14a,b, 18, 22. The Boc group of each starting 5-iodo-3′-O- or −5′-O-(N-tert-butyloxycarbonyl)aminoalkylcarboxy)-2′-deoxyuridine was cleaved with ∼15% TFA in dichloromethane (DCM) or MeCN. When reaction was completed (monitoring by TLC), the mixture was evaporated under a vacuum at room temperature, the resulting oily residue treated with 15 mL of EtOAc/hexane (1:1) and sonicated briefly a few times. The solvent was carefully decanted from a formed solid, a crude product was washed with Et2O, and a solvent was drawn off again. This washing procedure was repeated, and then the remaining TFA salt was kept under a vacuum to dry. A resulting TFA salt of 5-iodo-3′-O- or 5-iodo-5′-O-aminoalkylcarboxy-2′-deoxyuridine (1.0 molar equiv) was suspended in DCM, placed on an ice bath, and to a stirred mixture TEA (1.05 molar equiv) was added, immediately followed by N,N′-bis(tert-butoxycarbonyl)-N″trifluoromethanesulfonylguanidine (1.1 molar equiv). The mixture was stirred for 5 min, and to a resulting clear solution a second portion of TEA (1.0 molar equiv) added. The stirring continued for 2−6 h (TLC monitoring) at room temperature. Upon the completion, an excess of amines and triflic amide were removed by aqueous workup with 5% citric acid and saturated brine. Organic phase was dried over MgSO4, filtered, and evaporated. Crude products were purified using a silica gel column with gradients of MeOH in DCMor EtOAc in hexanes. In preparations conducted with 3′-O-levulinyl-protected deoxyuridines, to remove the O-Lev group,

the resulting solid was directly treated with a solution of hydrazine hydrate (0.5 M, ∼5 equiv) in a pyridine/AcOH mixture (4:1, v/v, 5 mL), cooled on an ice−water bath. The stirring continued for ∼10 min at room temperature, and water (40 mL) and EtOAc (50 mL) were added. The organic layer was separated, washed with 10% aqueous solution of NaHCO3 (20 mL), water (20 mL), dried over MgSO4, and evaporated. General Procedure B: Preparation of Trimethyltin Precursors 7, 11, 15a,b, 19, 23, and 27. Stirred solutions of 5-iodo-3′-Oor 5-iodo-5′-O-[(N,N′-bis(tert-butyloxycarbonyl)-N″-alkylcarboxy)guanidino]-2′-deoxyuridine 10, 14a,b, 18, 22, 5-iodo-5′-N-[N′,N″bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine 6, or N,N′bis(tert-butyloxycarbonyl)-3-iodobenzylguanidine 26 (1.0 equiv), in ethyl acetate or dioxane (depending on solubility), containing hexamethylditin (1.25−1.70 equiv), triethylamine (2−4 equiv), and dichloro-bis(triphenylphosphine)palladium II catalyst (0.10 equiv), were gently refluxed under nitrogen until the starting iodide could be detected (1−3 h). The reaction progress was checked often by TLC. After cooling to ambient temperature, the mixture was filtered through a thin pad of silica (washed with EtOAc/hexanes, 2:1) to separate the remaining catalyst and the solvent was evaporated. Two major products were always present: the corresponding trimethylstannane (with high TLC mobility), isolated in 30−62% yield, and the protodestannylated form of starting iodide (slower on TLC). Crude products were separated and purified by repeating a silica gel flash column chromatography, using mixtures of EtOAc/hexanes (2:1−2, v/v) and/or with various gradients of MeOH in DCM (0.3−0.7:10). In order to reach ≥98% purity of stannylated products, the successive preparative HPLC purifications were usually required. Purified, anhydrous samples of stannanes (100 μg/vial) were stored with the exclusion of light under nitrogen at −20 °C. General Procedure C: Preparation of 125I-Radioiodinated 3′and 5′-Guanidino-N-alkylcarboxy Esters of 5-[125I]Iodo-2′deoxyuridine 13 (IDG), 17a,b, 21, 25, 5-[125I]Iodo-5′-Nguanidino-2′,5′-dideoxyuridine 9, and 3-[125I]Iodobenzylguanidine 29. Into a glass tube containing the selected tin precursor, 7, 11, 15a,b, 19, 23, or 27 (∼100 μg, 0.1−0.2 μmol) in MeCN (50 μL), a solution of Na125I/NaOH (10−50 μL, 22.2−148 MBq) was added, followed by a solution of 30% H2O2 in water (5 μL) and 2 min later, a solution of TFA (50 μL, 0.1% in MeCN). The resulting mixture, briefly vortexed and/or sonicated, was left for 15 min at room temperature and then quenched with a solution of Na2S2O3 (90−100 μg) in water (60 μL). At this point, one of the two pathways was implemented. Pathway 1: The reaction mixture was taken up into a syringe and the reaction tube washed twice with a solution of MeCN/H2O (50 μL, 1:1−3, depending on the stannane solubility). The reaction mixture and washes were combined, injected onto the HPLC system, and separated on the C8 or C18 reverse phase column with a linear gradient of MeCN in water. Eluted fractions with one of the radioiodinated products, 8, 12, 16a,b, 20, 24, 28, were pooled, evaporated with a stream of dried nitrogen, or else dissolved in dry MeCN (∼18.5 MBq/mL) for further analysis or storage. To remove the guanidine Boc-protective groups, neat TFA (100 μL) was added to a dried residue of 125I-iodinated product, and the resulting mixture was vortexed and then heated in a sealed vial at 55−65 °C for 20−35 min. After cooling, the mixture diluted with CH3CN (200 μL) was evaporated repetitively with a stream of nitrogen, each time leaving ∼20 μL of the liquid in the reaction vial, to prevent the adhesion of products to walls of a vial. This process was repeated at least three times to ensure that an excess of TFA was eliminated. The residue was then dissolved in a solution of 50% MeCN in water, injected onto the HPLC system, and separated on the C18 reverse phase column with a gradient of MeCN in water, with both solvents containing 0.07% TFA (v/v), as the eluant. Pathway 2. Alternatively, if the initial separation of Boc-protected radioiodinated product was not essential, a crude radioiodination mixture was evaporated with a stream of nitrogen and/or kept under a high vacuum, then it was directly treated with neat TFA at elevated temperature and separated using HPLC, as described previously. To L



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same column and the elution rate; eluent, solvent A water, solvent B CH3CN; 45% B for 30 min, then a linear gradient of B to 95% over 30 min, continued for 30 min. 1H NMR (CDCl3, 600 MHz) δ: 10.62 (bs, 1H, NH-uridine), 9.05 (bs, 1H, NH), 8.31 (t, 1H, NH, J = 5.72 Hz), 7.49 (s, 1H, H6-uridine, 3JSn,H = 19.5 Hz), 5.98 (dd, 1H, H1′, 3J1′,2′= 6.2 Hz, 3J1′,2″ = 4.6 Hz), 4.21−4.15 (m, 1H, H4′), 3.78−3.73 (m, 2H, H3′, OH), 3.63−3.59 (m, 1H, H5″), 2.34−2.28 (m, 1H, H2′), 2.19− 2.04 (m, 1H, H2″), 1.44 (s, 9H, 3 × CH3-t-Bu), 1.41 (s, 9H, 3 × CH3-t-Bu), 0.32 (s, 9H, 3 × CH3, 2JSn,H = 29.1 Hz) ppm. 13C NMR (CDCl3, 100 MHz) δ: 165.6 (C4), 160.46 (C-guanidine), 158.4 (C1Boc-N′), 156.2 (C1-Boc-N″), 150.9 (C2), 143.4 (C6), 112.7 (C5), 86.8 (C1′), 85.2 (C4′,), 79.7 (C2-Boc-N′), 75.3 (C2-Boc-N″), 71.6 (C3′), 67.0 (C5′), 38.9 (C2′), 28.1 (C3-Boc-N′), 28.1 (C3-Boc-N″), −7.15 (3 × CH3-Sn) ppm. HDMS (m/z): [M + H]+ calcd for C23H39N5O8112Sn, 626.1919; found 626.1909 using the 112Sn isotope signal. 5-[ 1 2 5 I]Iodo-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine (8). A total of four radioiodinations was carried out according to general procedure C, with ∼100 μg of stannane 7 and Na125I/NaOH, within the 18.5−77.7 MBq range, to give overall 140.6 MBq of 8. An average yield of the purified product was 84%. The HPLC purification of a crude reaction mixture proceeded on ACE C8 100 Å (5 μm, 4.6 mm × 250 mm), eluted at 0.8 mL/min initially with 45% MeCN in water for 30 min, then with a linear gradient of MeCN from 45% to 95% MeCN over 30 min, and 95% MeCN was held for 15 min longer. The product 8 eluted within 19.5−22.0 min after the injection and was collected in three fractions (>98% pure). An excess of unreacted stannane 7, eluting between 46.5 and 49.0 min, was completely separated from the product. Two HPLC co-injections of purified 8 and nonradioactive analog 6 (Bioscan NaI(T) detector and UV signal at 220/280 nm) confirmed the same mobility of both compounds: (1) tR= 20.1 min, ACE C8 100 Å (5 μm, 4.6 mm × 250 mm), eluted at 0.8 mL/min at first with 45% MeCN in water for 30 min, then a linear gradient of MeCN from 45% to 95% MeCN over 30 min and kept at 95% MeCN for 15 min. (2) tR = 37.4 min, ACE C18 100 Å (5 μm, 4.6 mm × 250 mm), eluted at 0.8 mL/min of the eluant: a linear gradient of MeCN in water from 5% to 95% over 60 min, then kept at 95% MeCN for 30 min. 5-[125I]Iodo-5′-N-guanidino-2′,5′-dideoxyuridine (9). Applying general procedure C, the radioiodination reaction mixture of 7 or dried residue of purified 8 was treated with TFA (neat) and kept at 75 °C for 15 min to completely cleave Boc-protection of guanidine. The deprotection progress of 8 using a solution of 40% TFA in MeCN was also monitored by HPLC at room temperature and was completed within 70 min. An average yield of the purified 9 was 91%. Product was separated on ACE C18 100 Å (5 μm, 4.6 mm × 250 mm), starting with 5% MeCN in water for 10 min and eluted at 0.8 mL/ min, followed by a linear gradient of MeCN from 5% to 95% over 30 min and 95% MeCN was kept constant for 15 min longer. Both solvents contained 0.07% TFA (v/v). The product (tR = 10.4 min, ≥98% purity, Bioscan/UV 280 nm) eluting within 9.5−12.0 min after injection was collected in three fractions, which were combined, evaporated, and to the tube containing the product residue 80 μL of ethanol was added, followed by potassium phosphate buffer (100 μL, 10 mM PB, pH 6.1). The resulting solution of 9 was injected again on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm), a reverse phase column eluted at 0.8 mL/min with the two consecutive gradients of EtOH in potassium phosphate buffer (10 mM PB, pH 6.1): 0−5% in 10 min and 10−60% over the next 30 min. The product was collected within 21.5−23.0 min after the injection. HPLC analysis: tR= 22.5 min (≥98% purity, Bioscan/UV 280 nm). 5 - I o d o - 3 ′ - O- [ ε - ( N, N ′- b i s( t e r t - ( b u t y l o xy c a rb on yl ) guanidino))hexanoyl]-2′-deoxyuridine (10). General procedure A was carried out with 5-iodo-3′-O-(6-N-Boc-aminohexanoyl)-2′deoxyuridine (1.12 g, 1.97 mmol). The Boc protecting group was cleaved, and the separated dried TFA salt after addition of TEA (279 μL, 2.0 mml) reacted immediately with 0.82 g (2.10 mmol) of N,N′bis(tert-butoxycarbonyl)-N″-trifluoromethanesulfonylguanidine in DCM (23 mL) in the presence of TEA (280 μL, 2.0 mmol). A crude product was purified on a silica gel column using a gradient of

fully eliminate the presence of TFA and CH3CN, the combined HPLC fractions containing a final separated product were evaporated with a stream of nitrogen and to the residue, ethanol (80 μL) was added, followed by potassium phosphate buffer (100 μL, 10 mM PB, pH ∼ 6.1). The resulting mixture was injected for a second time on HPLC reverse phase column and eluted with a linear gradient of EtOH in potassium phosphate buffer. In every HPLC separation or analysis of radiolabeled products, the eluate from a column was monitored with a radioactivity detector connected to an outlet of UV detector (detection at 220 and 280 nm). Solutions containing the product were reconstituted in a preferred solvent to the required concentration and then filtered through a sterile filter (Millipore, 0.22 μm) into a sterile evacuated vial. Identities of the radiolabeled products were confirmed by the evaluation of the UV signals of nonradioactive iodo analogs with both signals of radiolabeled compounds and by comparing Rf obtained from the radio-TLC and tR from the radio-HPLC analysis. All radiolabeled products, if kept in a solution overnight at ambient temperature, were purified one more time shortly before conducting further experiments, although the HPLC analysis rarely indicated less than 95% of the radiochemical purity. 5-Iodo-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)guanidino]2′,5′-dideoxyuridine (6). To a stirred solution of 5-iodo-5′-amino2′,5′-dideoxyuridine (1.91 g, 5.39 mmol) in 30 mL of dioxane/water mixture (12:1, v/v), N,N′-bis(tert-butyloxycarbonyl)-N″trifluoromethanesulfonylguanidine (2.11 g, 5.39 mmol) was added, followed by TEA (750 μL, 5.4 mmol). The stirring continued for 6 h, dioxane was evaporated, water (60 mL) added, and the mixture extracted with DCM (2 × 40 mL). The organic phase was washed with brine, then dried over MgSO4 and evaporated. The residue of a crude product was purified on a silica gel column (DCM/MeOH, 10:0.4), followed by a second silica gel purification (EtOAc/nhexanes, 2:1) to yield 6 (2.57 g, 79%) as colorless foam. HPLC analysis: (1) tR = 19.89 min (98.2% pure at 254 nm) on Columbus C8 column, eluted at the rate of 0.8 mL/min, with 45% MeCN in water for 30 min and then with a linear gradient of MeCN from 45% to 95% over 30 min. (2) tR = 34.56 min (98.2% pure at 280 nm) on ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 10% to 95% over 35 min, then 95% MeCN for 25 min. 1H NMR (CDCl3, 600 MHz) δ:11.40 (s, 1H, NH-uridine), 9.13 (bs, 1H, NH), 8.07 (t, 1H, NH, J = 5.85 Hz), 7.81 (s, 1H, H6-uridine), 6.08 (dd, 1H, H1′, 3J1′,2’= 6.4 Hz, 3J1′,2″ = 4.8 Hz), 4.31−4.25 (m, 2H, H3′, C3′-OH), 3.78−3.73 (m, 2H, H4′, H5′), 3.63−3.59 (m, 1H, H5″), 2.24−2.18 (m, 1H, H2’), 2.13−2.06 (m, 1H, H2″), 1.51 (s, 9H, 3 × CH3-BocN′), 1.47 (s, 9H, 3 × CH3-BocN″) ppm. 13C NMR (CDCl3, 100 MHz) δ: 162.4 (C4), 159.8 (C1-Boc-N′), 157.7 (C1-Boc-N″), 153.0 (C2), 149.66 (C-guanidine), 144.2 (C6), 118.3 (C5), 84.7 (C1′), 84.3 (C4′,), 77.3 (C2-Boc-N′), 77.0 (C2-Boc-N″), 73.5 (C3′), 67.0 (C5′), 38.9 (C2′), 28.2 (C3-Boc-N′), 28.1 (C3-Boc-N″) ppm. HDMS (m/ z): [M + H]+ calcd for C20H30IN5O8, 596.1212; found, 596.1219. 5-Trimethylstannyl-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine (7). General procedure B was carried out twice with (520 mg, 0.873 mmol and 712 mg, 1.196 mmol) 5-iodo-5′-N-[(N′,N″-bis-Boc)guanidinyl]-2′,5′-dideoxyuridine 6 in EtOAc containing hexamethylditin 486 mg (1.48 mmol) and 670 mg (2.05 mmol) respectively, TEA (300 μL, 2.15 mmol), and the palladium catalyst (30 mg, 0.043 mmol). Combined crude products were purified on a silica gel column using a mixture of EtOAc/hexanes (2:1, v/v) to give 7 as a pale foam (680 mg) in 52% yield. Rf = 0.51 (DCM/MeOH, 10:0.3). The final HPLC purification (160 mg, ∼11 mg per injection) was done on a semipreparative Columbus C18, 100 Å (10 mm × 250 mm) column, eluted at 2.2 mL/min with 55% MeCN in water for 20 min, then with a linear gradient of MeCN from 55% to 95% over 15 min and 95% MeCN kept further 30 min. HPLC analysis: (1) tR = 30.02 min (97.3% pure at 280 nm) on ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min with 50% MeCN in water for a 20 min, then with a linear gradient from 50% to 95% of MeCN over 15 min and 95% MeCN continued for next 30 min. (2) tR = 47.06 min (98.4% pure at 280 nm), using the M



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MeOH in DCM (0.3−0.5:10) to give 938 mg of 10 in 67% yield. Rf = 0.42 (DCM/MeOH, 10:0.4). HPLC analysis: tR = 29.9 min (≥96.6% pure at 254/280 nm) on ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column, at the elution rate of 0.8 mL/min with a linear gradient of MeCN in water from 40% to 95% over 45 min, then 95% MeCN kept for 15 min. 1H NMR (DMSO-d6, 600 MHz) δ: 11.61 (s,1H, NHuridine), 8.32 (bs, 1H, NH), 8.16 (t, 1H, NH, J = 5.52 Hz), 8.06 (s, 1H, H6-uridine), 6.28 (dd, 1H, H1′, 3J1′,2′ = 6.40 Hz, 3J1′,2″ = 4.85 Hz), 5.15−4.87 (m, 2H, H3′, OH), 4.19−4.02 (m, 1H′, H4′), 3.77− 3.56 (m, 4H, H5′, H2-Ac), 2.89−2.76 (m, 2H, H6-Hex)′ 2.58−2.46 (m, 1H, H2′), 2.38−2.29 (m, 1H, H2″), 1.72−1,64 (m, 2H, H3Hex), 1.61−1.54 (m, 2H, H5-Hex), 1.52 (s, 9H, H3-BocN′, 1.46 (s, 9H, H3-BocN″), 1.36−1.29 (m, 2H, H4-Hex) ppm. 13C NMR (DMSO-d6, 100 MHz) δ:172.8 (C1-Hex), 162.8 (C4), 158.8 (C1Boc-N′), 156.7 (C1-Boc-N″), 153.5 (C2), 149.52 (C-guanidine), 142.5 (C6), 110.3 (C5), 85.19 (C1′), 84.4 (C4′), 80.8 (C2-Boc-N′), 79.7 (C2-Boc-N″), 74.7 (C3′), 61.4 (C5′), 41.3 (C2-hex), 40.5 (C6Hex), 37.7 (C2′), 31.8 (C5-Hex), 28.4 (C3-Boc-N′), 28.3 (C3-BocN″), 26.5 (C4-Hex), 24.7 (C3-Hex) ppm. HDMS (m/z): [M + H]+ calcd for C26H40IN5O10, 710.1892; found, 710.1906. 5-Trimethylstannyl-3′-O-[ε-(N,N′-bis(tert(butyloxycarbonyl)guanidino))hexanoyl)]-2′-deoxyuridine (11). Stannylation of 5-iodo-3′-O-[ε-(N,N′-bis(tert(butyloxycarbonyl)guanidino))hexanoyl)]-2′-deoxyuridine (10) was conducted two times, with 230 mg (0.324 mmol) and repeated with 316 mg (0.445 mmol) of 10, and hexamethylditin 160 mg (0.488 mmol) and 219 mg (0.668 mmol), respectively, with the palladium catalyst (25 mg, 0.036 mmol) and TEA (200 μL, ∼1.4 mmol). Crude products were purified on a silica gel column using a gradient of MeOH in DCM (0.3−0.5:10) to give 11 (89 mg and 136 mg) in 37% and 41% yield, respectively. Rf = 0.47 (DCM/MeOH, 10:0.4). HPLC analyses: tR = 36.8 min (95.9% pure at 220/280 nm) on ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 40% to 95% over 50 min, then 95% MeCN was kept further 20 min. 1H NMR (DMSO-d6, 600 MHz) δ: 11.66 (s,1H, NH-uridine), 8.72 (bs, 1H, NH), 8.37 (t, 1H, NH, J = 5.48 Hz), 7.76 (s, 1H, H6-uridine, 3JSn,H = 18.42 Hz), 6.32 (dd, 1H, H1′, 3J1′,2′ = 6.45 Hz, 3J1′,2″ = 4.80 Hz), 4.95−4.81 (m, 1H, H4′), 4.39−4.22 (m, 1H′ H3′), 3.77−3.64 (m, 3H, H5′,OH), 2.89−2.77 (m, 2H, H6-Hex), 2.59−2.42 (m, 3H, H2′, H2-Hex), 2.38−2.29 (m, 1H, H2″), 1.70−1,63 (m, 2H, H3-Hex), 1.58−1.50 (m, 2H, H5-Hex), 1.41 (s, 9H, H3-BocN′, 1.37 (s, 9H, H3-BocN″), 1.30−1.27 (m, 2H, H4-Hex), 0.27 (s, 9H, 3 × SnCH3, 2 JSn,H = 29.2 Hz) ppm. 13C NMR (DMSO-d6, 100 MHz) δ:173.2 (C1Hex), 163.37 (C4), 160.1 (C1-Boc-N′), 158.3 (C1-Boc-N″), 153.5 (C2), 149.6 (C-guanidine), 142.3 (C6), 110.2 (C5), 85.9 (C1′), 84.3 (C4′), 80.9 (C2-Boc-N′), 79.9 (C2-Boc-N″), 74.5 (C3′), 61.5 (C5′), 41.2 (C6-hex), 40.5 (C2-Hex), 37.7 (C2′), 31.9 (C5-Hex), 28.4 (C3Boc-N′), 27.4 (C3-Boc-N″), 26.5 (C4-Hex), 24.7 (C3-Hex), −7.9 (3 × CH 3 -Sn) ppm. HDMS (m/z): [M + H] + calcd for C29H49N5O8112Sn, 740.2600; found 740.2589 using the 112Sn isotope signal. 5-[ 125 I]Iodo-3′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidine))hexanoyl)]-2′-deoxyuridine (12). Standard radioiodination (general procedure C) of stannane 11 (∼100 μg) was carried out two times, with 40.7 and 57.4 MBq of Na125I/NaOH, to give overall 82 MBq of 12. An average yield of the isolated product was 85%. A crude reaction mixture was separated and purified by HPLC on Jupiter C18 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min, using a linear gradient of MeCN in water from 40% to 95% over 45 min, then 95% MeCN was held for further 25 min. The product was collected within 29.3−30.8 min after the injection, and an excess of unreacted tin precursor 11 was fully separated, eluting ∼7 min later. HPLC analysis: tR = 29.90 min (≥98% purity, Bioscan/UV 280 nm). 5-[125I]Iodo-3′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine (13, IDG). The deprotection (HPLC monitoring) of 12 to IDG in a solution of 40% TFA in MeCN at room temperature required ∼250 min. A treatment of 12 ( dried residue, 21.8 MBq) with neat TFA (30 μL) at 75 °C accelerated the elimination of the guanidine Boc-

protection and was completed in 20 min. An average yield from four preparations of the purified IDG was 89%. The product (19.37 MBq, first preparation) was separated on ACE C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min with a linear gradient of MeCN from 0% to 95% over 50 min and 95% MeCN kept for an additional period of 20 min, and both solvents contained 0.07% TFA (v/v); tR = 23.67 min (≥98% radiochemical purity). The product eluting within 23.2−24.5 min after the injection was collected in three fractions, which were combined and evaporated. To eliminate the presence of TFA and MeCN in the sample, ethanol (80 μL) was added to a portion of the product residue (9.1 MBq), followed by potassium phosphate buffer (100 μL, 10 mM PB, pH 6.1). The resulting solution was injected again on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm) column and eluted at the rate of 0.8 mL/min with 15% EtOH in potassium phosphate buffer (10 mM PB, pH 6.1) for a period of 10 min, then with a linear gradient of 15− 50% EtOH in PB over a 20 min period. The product (8.2 MBq, 90% yield) was collected within 19.3−22.5 min after the injection (tR = 19.9 min, ≥97% purity Bioscan/UV 280 nm). 5-Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″propionyl)guanidino]-2′-deoxyuridine (14a). As described in general procedure A, the Boc protecting group of 5-iodo-5′-O-(6-NBoc-aminopropionyl)-3′-O-levulinyl-2′-deoxyuridine (1.51 g, 2.42 mmol) was cleaved in MeCN (22 mL) containing 3.5 mL of TFA and the separated TFA salt was suspended in DCM (27 mL) reacted directly with 0.95 g (2.45 mmol) of N,N′-bis(tert-butoxycarbonyl)N″-trifluoromethanesulfonylguanidine in the presence of TEA (720 μL, 5.16 mmol) added in two portions. The residue was purified by chromatography on a silica gel column using a gradient of MeOH in DCM (0.3−0.5:10) to give 1.57g (74%) of the product (Rf = 0.67 in DCM/MeOH 10:0.3). The 3′-O-Lev group was cleaved with a mixture of N2H4·H2O/pyridine/AcOH (0.3/9.5/2.4 mL) and a crude product purified again on a silica gel column (DCM/MeOH, 10:0.35), to give 1.01g (73% yield) of 14a as colorless foam; Rf = 0.60 (DCM/MeOH, 10:0.5). HPLC analysis: (1) tR = 35.6 min (≥98.81% pure at 280 nm) on Columbus C18 100 Å, (5 μm, 4.6 mm × 250 mm) column, eluent solvent A water, solvent B CH3CN; eluted at the rate of 0.8 mL/min with a linear gradient of B from 10% to 90% over 40 min, 90−95% within 40−45 min and 95% B kept over 15 min. On ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column. (2) tR = 34.5 min, (≥98.35% pure at 220 nm); eluent solvent A water, solvent B CH3CN; eluted at the rate of 0.8 mL/min with a linear gradient of B from 40% to 80% over 65 min, then 95% of B kept over 25 min. 1H NMR (CDCl3, 600 MHz) δ: 11.46 (s,1H, NH-uridine), 8.71 (bs, 1H, NH), 8.66 (t, 1H, NH, J = 4.52 Hz), 7.96 (s, 1H, H6uridine), 6.21 (t, 1H, H1′, J = 5.55 Hz), 4.48−4.42 (m, 2H, H3′, OH), 4.37−4.31 (m, 1H, H4′), 3.75−3.53 (m, 4H, H5′, H2-Prop), 2.85−2.74 (m, 2H, H3-Prop), 2.58−2.48 (m, 1H, H2′), 2.24−2.19 (m, 1H, H2″), 1.49 (s, 9H, H3-BocN′), 1.46 (s, 9H, H3-BocN″) ppm. 13C NMR (CDCl3, 100 MHz) δ:171.7 (C1-Prop), 163.3 (C4), 160.0 (C1-Boc-N′), 156.2 (C1-Boc-N″), 153.9 (C2), 149.9 (Cguanidine), 143.4 (C6), 111.7 (C5), 85.7 (C1′), 84.3 (C4′), 77.2 (C2-Boc-N′), 77.1 (C2-Boc-N″), 74.3 (C3′), 67.5 (C5′), 44.2 (C2Prop), 42.4 (C3-Prop), 38.7 (C2′), 28.3 (C3-Boc-N′), 28.0 (C3-BocN″) ppm. HDMS (m/z): [M + H]+ calcd for C23H34IN5O10, 668.1423; found, 668.1398. 5 - I o d o - 5 ′ - O- [ ε - ( N, N ′- b i s( t e r t - ( b u t y l o xy c a rb on yl ) guanidino))hexanoyl]-2′-deoxyuridine (14b). General procedure A was implemented, starting with 5-iodo-5′-O-(6-N-Boc-aminohexanoyl)-3′-levulinyl-2′-deoxyuridine (2.25 g, 3.38 mmol). The Boc protecting group was cleaved, and the separated TFA salt was reacted without delay with 1.36 g (3.47 mmol) of N,N′-bis(tertbutoxycarbonyl)-N″-trifluoromethanesulfonylguanidine in the presence of TEA (970 μL, 6.95 mmol) added in two portions. The product residue dissolved in 60 mL of EtOAc was washed with solution of citric acid and brine, organic phase was dried over MgSO4, evaporated and the residue purified on silica gel column (EtOAc/nhexane, 2:1). The 3′-O-Lev group was cleaved with a mixture of N2H4·H2O (0.26 mL, 5.36 mmol) in pyridine/AcOH (4:1, 5 mL) and a crude product was purified again on a silica gel column (DCM/ N



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Columbus C18, 100 Å, (5 μm, 10 mm × 250 mm) semipreparative column, eluted with a solution of 65% MeCN in water for 25 min, followed by a linear gradient of MeCN from 65% to 95% over 15 min and finally, 95% MeCN for the last 15 min, at the rate of 2.5 mL/ min.HPLC analysis confirmed its high purity: tR = 31.1 min (≥98.2% pure, UV at 220/280 nm); ACE 100 Å, 5 μm, 4.6 mm × 250 mm), eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 40% to 95% over 40 min and 95% for 15 min. 1H NMR (DMSO-d6, 600 MHz) δ: 11.16 (s,1H, NH-uridine), 8.44 (bs, 1H, NH), 8.14 (t, 1H, NH, J = 5.50 Hz), 7.81 (s, 1H, H6-uridine, 3JSn,H = 19.4 Hz), 6.32 (dd, 1H, H1′, 3J1′,2′ = 6.45 Hz, 3J1′,2″ = 4.64 Hz), 4.92− 4.84 (m, 1H, H4′), 4.35−4.12 (m, 2H, H5′), 3.59−3.54 (m, 3H, H3′, OH), 2.87−2.74 (m, 2H, H6-Hex), 2.39−2.32 (m, 3H, H2′, H2Hex), 2.18−2.09 (m, 1H, H2″), 1.74−1,66 (m, 2H, H3-Hex), 1.57− 1.51 (m, 2H, H5-Hex), 1.40 (s, 9H, H3-BocN′, 1.38 (s, 9H, H3BocN″), 1.33−1.26 (m, 2H, H4-Hex), 0.29 (s, 9H, 3 × SnCH3, 2JSn,H = 29.8 Hz) ppm. 13C NMR (DMSO-d6, 100 MHz) δ: 173.4 (C1Hex), 163.5 (C4), 158.9 (C1-Boc-N′), 156.5 (C1-Boc-N″), 152.7 (C2), 149.8 (C-guanidine), 143.2 (C6), 110.4 (C5), 87.3 (C1′), 85.2 (C4′), 83.5 (C2-Boc-N′), 79.7 (C2-Boc-N″), 71.3 (C3′), 62.3 (C5′), 41.9 (C6-hex), 40.5 (C2-Hex), 38.6 (C2′), 30.3 (C5-Hex), 28.6 (C3Boc-N′), 27.3 (C3-Boc-N″), 26.1 (C4-Hex), 24.3 (C3-Hex), −7.3 (3 × CH 3 -Sn) ppm. HDMS (m/z): [M + H] + calcd for C29H49N5O10112Sn, 740.2600; found 740.2601 using the 112Sn isotope signal. 5-[ 125 I]Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″propionyl)guanidino]-2′-deoxyuridine (16a). Radioiodination (general procedure C) of stannane 15a (∼100 μg) was done twice, with 40.1 and 34.4 MBq of Na125I/NaOH, to give overall 67.8 MBq of 16a. An average yield of the isolated product was 91%. The reaction mixture was separated and purified by HPLC using Columbus C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 0% to 95% over 60 min, then 95% MeCN was held further 30 min. The product was collected in three fractions (within 39−41 min after the injection) and an excess of unreacted stannane 15a was sufficiently separated eluting ∼4.5 min later. HPLC analysis: tR = 39.9 min, (≥98% purity). 5-[ 125 I]Iodo-5′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidino))hexanoyl]-2′-deoxyuridine (16b). Radioiodination (general procedure C) of stannane 15b (∼100 μg) was completed with ∼34.1 MBq of Na125I/NaOH giving 28.3 MBq of the isolated product in 83% yield. The reaction mixture was separated and purified by HPLC on Jupiter C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 40% to 95% over 40 min, then 95% MeCN held for additional 20 min. The product was collected in two fractions (within 25.1−26.5 min after the injection), and an excess of unreacted tin precursor was fully separated, eluting ∼4.5 min later. Further HPLC analysis of the product confirmed its high purity: tR = 36.4 min, (≥98% purity, Bioscan/UV 280 nm). 5-[ 125 I]Iodo-5′-O-propionylguanidino-2′-deoxyuridine (17a). The cleavage process of N-Boc guanidine protecting groups of 16a was conducted initially (monitored by HPLC) in a solution of 40% TFA in MeCN at room temperature and required ∼190 min. To complete the N-Boc cleavage faster (general procedure C), to a dried residue of 16a (31.5 MBq) under nitrogen, TFA (40 μL) was added and the solution was kept in a tightly covered vial at 75 °C for 20 min. An average yield of the purified 17a was 83%. The product (26.1 MBq) was separated on Columbus C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min with a linear gradient of MeCN in water from 0% to 95% over 60 min and 95% MeCN held 10 min longer. Both solvents contained 0.07% TFA (v/v); tR = 21.2 min (≥98% purity, Bioscan/UV 280 nm). The product eluting within 20.8−22.0 min after the injection was collected in three fractions, which were combined and evaporated. To the portion of the product dry residue (17.95 MBq) ethanol (80 μL) was added, followed by potassium phosphate buffer (100 μL, 10 mM, pH 6.1). The resulting solution was injected again on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm) column eluted at the rate of 0.8 mL/min with 15% EtOH in potassium phosphate buffer (10 mM PB, pH 6.1)

MeOH, 10:0.6) to give 1.65 g (69% overall yield) of 14b as pale foam, Rf = 0.35 (DCM/MeOH, 10:0.6), and was 93.7% pure by HPLC analysis (at 254/280 nm). Further HPLC purification of this product (106 mg, ∼5 mg per injection) was performed on a Columbus C18, 100 Å, (5 μm, 10 mm × 250 mm) column eluted with a linear gradient of MeCN in water from 50% to 95% over 30 min at the rate of 2.7 mL/min. Subsequent HPLC analysison ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column, confirmed an acceptable purity (≥95.4% at 280 nm) of a final product: tR = 40.6 min, eluent solvent A 10% CH3CN in water, solvent B CH3CN; run with a linear gradient of B from 10% to 95% over 45 min, then 95% B for 25 min, elution rate 0.8 mL/min. 1H NMR (CDCl3, 600 MHz) δ: 11.48 (s,1H, NHuridine), 8.45 (bs, 1H, NH), 8.29 (t, 1H, NH, J = 4.54 Hz), 7.95 (s, 1H, H6-uridine), 6.23 (t, 1H, H1′, J = 5.50 Hz), 4.47−4.39 (m, 2H, H3′, OH), 4.30−4.18 (m, 1H, H4′), 3.42−3.38 (m, 2H, H5′), 2.85− 2.74 (m, 2H, H2-Hex), 2.54−2.47 (m, 3H, H2′, H6-Hex), 2.20−2.15 (m, 1H, H2″), 1.75−1.69 (m, 2H, H3-Hex), 1.63−1.58 (m, 2H, H5Hex), 1.51 (s, 9H, H3-BocN′), 1.49 (s, 9H, H3-BocN″), 1.44−1.36 (m, 2H, H4-Hex) ppm. 13C NMR (CDCl3, 100 MHz) δ: 173.1 (C1Hex), 163.5 (C4), 159.6 (C1-Boc-N′), 156.2 (C1-Boc-N″), 153.3 (C2), 149.6 (C-guanidine), 144.2 (C6), 111.7 (C5), 85.8 (C1′), 84.5 (C4′), 81.0 (C2-Boc-N′), 79.5 (C2-Boc-N″), 70.9 (C3′), 68.4 (C5′), 41.1 (C2-hex), 40.6 (C6-Hex), 38.7 (C2′), 31.2 (C5-Hex), 28.3 (C3Boc-N′), 28.1 (C3-Boc-N″), 26.3 (C4-Hex), 24.5 (C3-Hex) ppm. HDMS (m/z): [M + H]+ calcd for C26H40IN5O10, 710.1892; found, 710.1906. 5-Trimethylstannyl-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)N″-propionyl)guanidino]-2′-deoxyuridine (15a). General procedure B was carried out with 336 mg (0.504 mmol) of 5-iodo-5′-O[(N,N′-bis(tert-(butyloxycarbonyl)-N″-propionyl)guanidino]-2′-deoxyuridine (14a) in EtOAc containing TEA (300 μL, 2.15 mmol) in the presence of the palladium catalyst (35 mg, 0.05 mmol). A crude product was initially purified on a silica gel column (EtOAc/nhexanes, 3:1) to give this stannane as colorless foam in 41% yield, Rf = 0.69 (DCM/MeOH, 10:0.5). The product was only ∼91.3% pure by HPLC analysis (at 254/280 nm). Further HPLC purification (177 mg, ∼8 mg per injection) was performed on a Columbus C18, 100 Å, (5 μm, 10 mm × 250 mm) column eluted with a solution of 65% MeCN in water for 28 min, then with a linear gradient of MeCN from 65% to 95% over 7 min and 95% MeCN for a further 25 min, at the rate of 2.4 mL/min. Subsequent HPLC analysis (ACE 100 Å, 5 μm, 4.6 mm × 250 mm) confirmed high purity of the final product (99.6% at 220/280 nm): tR = 40.85 min, eluent solvent A water, solvent B CH3CN; eluted with a linear gradient of B from 5% to 95% over 45 min and then 95% B for 15 min, at the rate of 0.8 mL/min. 1H NMR (CDCl3, 600 MHz) δ: 11.51 (s,1H, NH-uridine), 8.64 (bs, 1H, NH), 8.49 (t, 1H, NH, J = 5.50 Hz), 7.16 (s, 1H, H6-uridine, 3JSn,H = 18.6 Hz), 6.19 (t, 1H, H1′, J = 6.56 Hz), 4.49−4.38 (m, 2H, H3′, OH), 4.34−4.29 (m, 1H, H4′), 4.07−3.78 (m, 2H, H5′), 3.26−3.14 (m, 2H, H3-Prop), 2.68−2.57 (m, 2H, H2-Prop), 2.46−2.40 (m, 1H, H2′), 2.27−2.18 (m, 1H, H2″), 1.50 (s, 9H, H3-BocN′), 1.49 (s, 9H, H3-BocN″), 0.28 (s, 9H, 3 × SnCH3, 2JSn,H = 27.9 Hz) pm. 13C NMR (CDCl3, 100 MHz) δ: 171.7 (C1-Prop), 166.1 (C4), 163.4 (Cguanidine), 156.1 (C1-Boc-N′), 153.2 (C1-Boc-N″), 150.9 (C2), 143.4 (C6), 113.3 (C5), 86.2 (C1′), 84.0 (C4′), 83.4 (C2-Boc-N′), 79.5 (C2-Boc-N″), 71.6 (C3′), 64.0 (C5′), 39.9 (C2′), 36.0 (C3Prop), 33.9 (C2-Prop), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″), −9.28 (3 × CH3-Sn) ppm. HDMS (m/z): [M + H]+ calcd for C26H43N5O10112Sn, 698.2131; found 698.2115 using the 112Sn isotope signal. 5-Trimethylstannyl-5′-O-[ε-(N,N′-bis(tert(butyloxycarbonyl)guanidino))hexanoyl]-2′-deoxyuridine (15b). Stannylation of the iodide 14b (0.386 g, 0.54 mmol) was conducted with hexamethylditin 267 mg (0.82 mmol) with the palladium catalyst (38 mg, 0.05 mmol) and TEA (150 μL, ∼1.0 mmol) in EtOAc (15 mL). A crude product was purified on a silica gel column using EtOAc/n-hexanes (2:1) to give 134 mg of 15b in 29% yield as yellow foam, Rf = 0.56 (DCM/MeOH, 10:0.4) and was 91.4% pure by HPLC analysis (at 220/280 nm). Further HPLC purification (96 mg, ∼4 mg per injection) was performed on a O



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twice on a silica gel column EtOAc/n-hexanes (1:1) was separated as yellow foam (390 mg, 30% yield), Rf = 0.71 (DCM/MeOH, 10:0.5) and was ≥93.7% pure by HPLC analysis (at 220/280 nm). The HPLC purification (168 mg, ∼11 mg per injection) was performed on a Columbus C18, 100 Å (5 μm, 10 mm × 250 mm) semipreparative column, eluted with a linear gradient of MeCN in water, from 65% to 95% over 60 min at the rate of 2.4 mL/min. HPLC analysis confirmed acceptable purity of the product, a mixture of diastereomers: tR1 = 57.8 and tR2 = 58.1 min (≥98% pure, UV at 220/280 nm); ACE C18 column 100 Å (5 μm, 4.6 mm × 250 mm); eluted with a linear gradient of MeCN in water, from 70% to 95% MeCN over 60 min, then 95% B for 30 min, at the rate of 0.8 mL/min. 1H NMR (DMSOd6, 600 MHz) δ: 11.27, 11.19 (2s,1H, HN-uridine), 8.23 (bs, 1H, NH), 8.12 (t, 1H, NH, J = 4.35 Hz), 7.79, 7.74 (2s, 1H, H6-uridine, 3 JSn,H = 19.47 Hz), 7.35−7.29 (m, 1H, aryl-H4), 5.91−5.84 (m, 1H, H1′), 5.44−5.39 (m, 2H, benzyl), 4.95−4.81 (m, 1H, H4′), 4.29− 4.15 (m, 3H, H5′, H3′), 2.90−2.84 (m, 2H, H6-Hex), 2.49−2.30 (m, 3H, H2′, H2-Hex), 2.26−2.10 (m, 1H, H2″), 1.72−1.63 (m, 2H, H3Hex), 1.61−1.55 (m, 2H, H5-Hex), 1.48 (s, 9H, H3-BocN′), 1.40 (s, 9H, H3-BocN″), 1.39 (s, 9H, 3 × CH3-t-Bu), 1.37−1.28 (s, 9H, 3 × CH3-t-Bu; m, 2H, H4-Hex), 0.29 (s, 9H, 3 × CH3−Sn, 2JSn,H = 29.7 ppm. 13C NMR (DMSO-d6, 100 MHz) δ:173.5 (C1-Hex), 163.6 (C4), 160.3 (C1-Boc-N′), 158.6 (C1-Boc-N″), 155.8 (C2-aryl), 153.42 (C2), 149.8 (C-guanidine), 143.9 (C6-aryl), 143.3 (C6), 137.3 (C5-aryl), 130.5 (C3-aryl), 127.4 (C4-aryl), 111.2 (C5), 110.2 (C1-aryl), 89.5 (C1′), 85.2, (C4′), 82.6 (C2-Boc-N′), 79.8 (C2-BocN″), 73.7 (C3′), 68.4 (C-benzyl), 67.8 (C5′), 41.5 (C2-Hex), 40.7 (C6-Hex), 38.0 (C2′), 34.7, 34.6 (2 × C1 t-Bu), 30.8 (C5-Hex), 29.9, 29.8 (C2 t-Bu), 28.9 (C3-Boc-N′), 28.5 (C3-Boc-N″), 26.6 (C4Hex), 24.6 (C3-Hex), −7.62 (3 × CH3-Sn) ppm. 31P NMR (DMSOd6) δ: −8.73, −8.81 (2s, diastereomers) ppm. HDMS (m/z): [M + H]+ calcd for C44H69N5O13112Sn, 1038.3735; found 1038.3723 using the 112Sn isotope signal. 5-[125I]Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-fluorosaligenyl]3′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidino))hexanoyl)]-2′-deoxyuridine Monophosphate (20). Radioiodination (general procedure C) of stannane 19 (∼100 μg) was conducted twice with 59.2 and 42.6 MBq of Na125I/NaOH and gave 89.6 MBq of 20 with an average yield of 88%. The reaction mixture was separated and purified by HPLC using ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 80%−95% over 60 min, and 95% MeCN held constant for additional 30 min. The product was collected in three fractions (within 35−38 min after the injection). An excess of unreacted tin precursor 19 was separated sufficiently, eluting ∼14.5 min later. The HPLC analysis of the product showed the diastereomeric mixture: tR1 = 34.62 min, tR2 = 35.35 min (≥98% purity, Bioscan/UV 280 nm). 5-[125I]Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-fluorosaligenyl]3′-O-(ε-guanidinohexanoyl]-2′-deoxyuridine Monophosphate (21). The removal of guanidine N-Boc protecting groups of compound 20 was tested initially in a solution of 50% TFA in MeCN at room temperature and required ∼140 min. To complete the N-Boc cleavage faster (general procedure C), to a dried residue of 20 (three preparations conducted with 24.15, 31.5, and 16.65 MBq) under nitrogen, TFA (40 μL) was added and the solution was kept in a tightly covered vial, at 75 °C for 20 min. An excess of TFA was evaporated with a stream of dry nitrogen. The product was separated on ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at 0.8 mL/min with a linear gradient of MeCN in water from 0% to 95% over 60 min and 95% MeCN was kept for an additional 10 min; both solvents contained 0.07% TFA (v/v). The product eluted within 43.2−44.5 min after the injection. An average yield of the purified 21 was 83%; diastereomeric mixture: tR‑1 = 42.1, tR‑2 = 42.4 min (≥98% purity, Bioscan/UV 280 nm). To eliminate an excess of TFA, the solution containing the product was evaporated again and to the dry residue of 21 (64.4 MBq) 80 μL of ethanol was added, followed by potassium phosphate buffer (100 μL, 10 mM, pH 6.1). The resulting solution was injected on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm) column and was eluted with phosphate

for a period of 10 min and then with a linear gradient of EtOH in PB, from 15% to 70% over 30 min. The product (16.1 MBq, 89%) was collected within 16.5−20.5 min after the injection (tR = 16.8 min, ≥97% purity Bioscan/UV 280 nm). 5-[125I]Iodo-5′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine (17b). The standard radioiodination (general procedure C) of stannane 15b (∼100 μg) was carried out three times with 30.7, 47.5, and 50.5 MBq of Na125I/NaOH and gave overall 87.5 MBq of 17b in an average yield of 68%. A crude reaction mixture, quenched with a solution of Na2S2O3, was evaporated with a stream of nitrogen and briefly kept under a high vacuum. The intermediate separation of Bocprotected product 16b was omitted, and to the resulting dried residue TFA (100 μL) was added and the mixture heated in a sealed vial at 70 °C for ∼25 min. After cooling, the mixture was diluted with CH3CN (200 μL) and evaporated with a stream of nitrogen, leaving ∼20 μL of the liquid in the reaction vial. This process was repeated three times. To the tube containing the reaction mixture residue, ethanol (80 μL) was added, followed by potassium phosphate buffer (100 μL, 10 mM PB, pH 6.1). The resulting solution was injected on HPLC reverse phase column eluted with a linear gradient of EtOH in potassium phosphate buffer and was separated and purified by HPLC on Luna CN 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min with a linear gradient of EtOH in potassium phosphate buffer (10 mM PB, pH 6.1) from 15% to 70% over 60 min, then 70% EtOH in PB was kept for further 10 min. The product was collected within 17.5−20.5 min after the injection. The HPLC analysis of the product 17b (tR = 20.1 min) showed its satisfactory ≥98% purity (Bioscan/UV 280 nm). 5-Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-fluorosaligenyl]-3′-O[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidino))hexanoyl)]-2′deoxyuridine Monophosphate (18). General procedure A was carried out with 5-iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-fluorosaligenyl]-3′-O-(6-N-Boc-aminohexanoyl)-2′-deoxyuridine monophosphate (1.20 g, 1.39 mmol), the Boc protecting group was cleaved, and the separated TFA salt reacted directly with 0.60 g (1.53 mmol) of N,N′bis(tert-butoxycarbonyl)-N″-trifluoromethanesulfonylguanidine in DCM (35 mL) in the presence of TEA (390 μL, 2.80 mmol) added in two portions. A crude product purified twice on a silica gel column (DCM/MeOH, 10:0.3−0.5) was obtained as colorless rigid foam: 1.11 g (79% yield), Rf = 0.52 (DCM/MeOH, 10:0.5). The HPLC analysis showed a mixture of diastereomers: tR1 = 42.74 and tR2 = 43.40 min (≥96% pure, UV at 254/280 nm); ACE C18 column 100 Å (5 μm, 4.6 mm × 250 mm); eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 70% to 95% MeCN over 60 min, then 95% B kept for 30 min. 1H NMR (DMSO-d6, 600 MHz) δ: 11.39, 11.31 (2s,1H, NH-uridine), 8.56 (bs, 1H, NH), 8.23 (t, 1H, NH, J = 4.4 Hz), 7.96, 7.94 (2s, 1H, H6-uridine), 7.30−7.24 (m, 1H, aryl-H4), 6.26−6.20 (m, 1H, H1′), 5.54−5.37 (m, 2H, benzyl), 4.45− 4.21 (m, 2H, H3′, H4′), 3.55−3.41 (m, 2H, H5′), 2.90−2.82 (m, 2H, H2-Hex), 2.44−2.34 (m, 3H, H2′, H6-Hex), 2.22−2.16 (m, 1H, H2″), 1.76−1.68 (m, 2H, H3-Hex), 1.64−1.57 (m, 2H, H5-Hex), 1.54 (s, 9H, H3-BocN′), 1.49 (s, 9H, H3-BocN″), 1.46 (s, 9H, 3 × CH3-t-Bu), 1.39−1.36 (s, 9H, 3 × CH3-t-Bu; m, 2H, H4-Hex) ppm. 13 C NMR (DMSO-d6, 100 MHz) δ: 173.2 (C1-Hex), 163.4 (C4), 160.15 (C1-Boc-N′), 158.3 (C1-Boc-N″), 155.3 (C6-aryl), 154.2 (C2), 149.62 (C-guanidine), 148.6 (C2-aryl), 144.2 (C6), 133.4 (C5aryl), 130.5 (C3-aryl), 127.2 (C4-aryl), 111.7 (C5), 110.6 (C1-aryl), 88.7 (C1′), 84.6, (C4′), 81.6 (C2-Boc-N′), 80.1 (C2-Boc-N″), 71.2 (C3′), 69.3 (C-benzyl), 68.4 (C5′), 41.3 (C2-Hex), 40.8 (C6-Hex), 38.9 (C2′), 34.6, 34.6 (2 × C2 t-Bu), 31.2 (C5-Hex), 29.9, 29.9 (CH3-t-Bu), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″), 26.3 (C4-Hex), 24.5 (C3-Hex) ppm. 31P NMR (DMSO-d6) δ: −8.69, −8.89 (2s, diastereomers) ppm. HDMS (m/z): [M + H] + calcd for C41H60IN5O13, 1008.3027; found, 1008.3049. 5-Trimethylstannyl-5′-O-[cyclo-3,5-di(tert-butyl)-6-fluorosaligenyl]-3′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidine))hexanoyl)]-2′-deoxyuridine Monophosphate (19). The iodide 18 (1.26 g, 1.25 mmol) was reacted with hexamethylditin (0.69 g, 2.19 mmol) with the palladium catalyst (90 mg, 0.13 mmol) and TEA (950 μL, 6.82 mmol) in C6H6 (32 mL). A crude product purified P



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(C3-Boc-N″), −7.74 (3 × CH3-Sn) ppm. HDMS (m/z): [M + H]+ calcd for C31H45N5O10112Sn, 760.2287; found 760.2293 using the 112 Sn isotope signal. 5-[ 125 I]Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″methyl-4-benzoyl)guanidino]-2′-deoxyuridine (24). Stannane 23 (∼100 μg) was radioiodinated twice (general procedure C) with 20.3 and 37.6 MBq of Na125I/NaOH and gave ∼47 MBq of the product 24 with an average yield 81%. The reaction mixture was separated on HPLC using ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 50% to 85% over 50 min, then from 85% to 95% in 10 min and 95% MeCN held for further 10 min. The product was collected in three fractions within 19−21 min after the injection. An excess of unreacted stannane 23 was well separated, eluting ∼10 min later. The HPLC analysis of the separated product (tR = 18.8 min) confirmed its adequate radiochemical purity (≥98%, Bioscan/ UV 280 nm). 5-[125I]Iodo-5′-O-methyl-4-benzoylguanidino-2′-deoxyuridine (25). Cleavage of guanidine N-Boc protecting groups of compound 24 carried out in a solution of 40% TFA in MeCN at room temperature was completed within ∼220 min. Faster cleavage rate (general procedure C) was achieved when a dried residue of 24 (conducted twice with 13.2 and 30.7 MBq) was treated with neat TFA (40 μL) under nitrogen and kept in a tightly covered vial at 75 °C. After 30 min, the reaction was completed (checked by HPLC). An excess of TFA was evaporated with a stream of dry nitrogen and the product was separated on HPLC using ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min with a linear gradient of MeCN in water from 0% to 95% over 45 min and with 95% MeCN held for an additional 25 min; both solvents contained 0.07% TFA (v/v). The product eluted within 18−24 min after the injection, and an average yield of separated 25 was 76%. The HPLC analysis: tR = 22.3 min, ≥98% purity, Bioscan/UV 280 nm). Purified 25 was evaporated again with a stream of nitrogen, and to the dry residue (∼33.0 MBq) ethanol (80 μL) was added, followed by potassium phosphate buffer (100 μL, 10 mM, pH 6.1). The resulting solution was injected on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm) column and was eluted at the rate of 0.8 mL/ min with a linear gradient of EtOH in potassium phosphate buffer (10 mM PB, pH 6.1); from 10% to 15% over 10 min, next from 15% to 60% over a period of 30 min and 60% EtOH for further 30 min. The product freed from an excess of TFA (28.4 MBq, 86% yield) was collected within 28.5−30.0 min after the injection and analyzed on HPLC: tR= 28.1 min (≥98% purity, Bioscan/UV 280 nm). N,N′-Bis(tert-butyloxycarbonyl)-3-iodobenzylguanidine (26). To a stirred solution of 3-iodobenzylamine (0.81g, 3.47 mmol) in DCM (40 mL), placed in an ice bath, TEA (490 μL, 3.51 mmol) and N,N′-bis(tert-butoxycarbonyl)-N″-trifluoromethanesulfonylguanidine (1.37g, 3.50 mmol) were added, and the mixture was stirred at room temperature for ∼4 h (TLC monitoring), filtered and the solvent evaporated under reduced pressure. To the remaining residue, 100 mL of EtOAc/n-hexane (4:1) was added, and an excess of amines and triflic amide were removed by aqueous workup with 5% citric acid and saturated brine. The organic phase was dried over MgSO4, filtered, and evaporated. A crude product was separated and purified twice by column chromatography on a silica gel (DCM/ hexanes, 2:0.5−1) to yield 1.26 g (76%) of the guanidine 26; Rf = 0.62 in DCM/MeOH 10:0.5. HPLC analysis: tR = 23.39 (≥97% pure, UV at 280 nm) on ACE C18 column 100 Å (5 μm, 4.6 mm × 250 mm); eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 70% to 95% MeCN over 45 min, then 95% MeCN was kept for 15 min. 1H NMR (CDCl3, 600 MHz) δ: 11.47 (bs, 1H, NH-Boc), 8.58 (bs, 1H, NH-benzyl), 7.69−7.61 (m, 2H, aryl-H4, aryl-H2), 7.29−7.09 (m, aryl-H5, aryl-H6), 4.58 (s, 2H, benzyl), 1.53 (s, 9H, H3-BocN), 1.48 (s, 9H, H3-BocN′) ppm. 13C NMR (DMSO-d6, 100 MHz) δ: 163.5 (C1-BocN), 156.2 (C1BocNH), 153.1 (C-guanidine), 140.4 (C1-aryl), 139.8 (C2-aryl), 136.9 (C4-aryl), 130.4 (C5-aryl), 127.1 (C6-aryl), 94.5 (C3-aryl), 83.4 (C2-BocNH), 79.5 (C2-BocN), 44.1 (C-benzyl), 28.3 (C3-

buffer (10 mM PB, pH 6.1) and EtOH at the rate of 0.8 mL/min with a linear gradient of EtOH from 50% to 80% over 30 min, then 80% EtOH held for 10 min. The product (57.3 MBq, 89%) collected within 23.3−24.6 min after the injection was analyzed on HPLC: tR= 23.5 min, ≥97% purity Bioscan/UV 280 nm. 5-Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″-methyl-4benzoyl)guanidino]-2′-deoxyuridine (22). Starting with 5-iodo5′-O-[4-(N-Boc-aminomethyl)benzoyl]-3′-levulinyl-2′-deoxyuridine (2.76 g, 4.03 mmol), general procedure A was carried out as follows: the Boc protecting group was cleaved, and the separated TFA salt was reacted right away with 1.23 g (4.89 mmol) of N,N′-bis(tertbutoxycarbonyl)-N″-trifluoromethanesulfonylguanidine in the presence of TEA added in two portions (2 × 680 μL, 9.75 mmol). The crude product dissolved in 60 mL of EtOAc was washed with a solution of citric acid and brine, organic phase was dried over MgSO4, evaporated and the remaining residue purified on a silica gel column (EtOAc/n-hexane, 2:1) to give 2.57 g of white foam. This was treated with a mixture of N2H4·H2O (0.5 M, 0.26 mL) in pyridine/AcOH (4:1, 5 mL) to cleave the 3′-O-Lev group. A crude product purified on a silica gel column (DCM/MeOH, 10:0.4) was obtained as a white amorphous solid: 1.52 g, 67% yield, Rf = 0.54 (DCM/MeOH, 10:0.5). HPLC analysis: tR = 41.39 (≥96% pure, UV at 280 nm) on ACE C18 column 100 Å (5 μm, 4.6 mm × 250 mm); eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water, from 10% to 95% MeCN over 45 min, then 95% B kept for 15 min. 1H NMR (DMSOd6, 600 MHz) δ: 11.46 (s,1H, NH-uridine), 8.63 (bs, 1H, NH), 8.21 (t, 1H, NH, J = 4.5 Hz), 7.97 (s, 1H, H6-uridine), 7.87−7.74 (m, 2H, aryl-H2, aryl-H6), 7.53−7.48 (m, 2H, aryl-H3, aryl-H5), 6.19−6.12 (m, 1H, H1′), 4.34−4.27 (m, 1H, H4′), 4.24−4.19 (m, 1H, H3′, OH), 3.94−3.87 (m, 2H, benzyl), 3.76−3.68 (m, 2H, H5′), 2.34− 2.26 (m, 1H, H2′), 2.20−2.15 (m, 1H, H2″), 1.50 (s, 9H, H3BocN′), 1.46 (s, 9H, H3-BocN″) ppm. 13C NMR (DMSO-d6, 100 MHz) δ: 171.4 (C1-benzoyl), 163.4 (C4), 160.3 (C1-Boc-N′), 158.4 (C1-Boc-N″), 154.4 (C2), 149.7 (C-guanidine), 144.2 (C4-aryl), 139.3 (C2-aryl), 134. (C6), 127.3 (C3-aryl), 126.9 (C5-aryl), 111.6 (C1-aryl), 110.2 (C5), 89.8 (C1′), 86.5, (C4′), 82.3 (C2-Boc-N′), 81.3 (C2-Boc-N″), 71.7 (C3′), 69.3 (C-benzyl), 68.4 (C5′), 38.9 (C2′), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″) ppm. HDMS (m/z): [M + H]+ calcd for C28H36IN5O10, 730.1580; found, 730.1610. 5-Trimethylstannyl-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)N″-methyl-4-benzoyl)guanidino]-2′-deoxyuridine (23). Stannylation of the iodide 22 (0.748 g, 1.025 mmol) was done with hexamethylditin 605 mg (1.846 mmol), the palladium catalyst (72 mg, 0.01 mmol), and TEA (700 μL, 5.0 mmol) in EtOAc (20 mL). A crude product 23 (286 mg, 36% yield) separated on a silica gel column using EtOAc/n-hexanes (2:0.5−1), Rf = 0.62 (DCM/MeOH, 10:0.5), and was ∼92% pure by HPLC analysis (at 220/280 nm) and was further purified on a Columbus C18, 100 Å (5 μm, 10 mm × 250 mm) semipreparative column (166 mg, ∼7 mg per injection), eluted at the rate of 2.4 mL/min with a linear gradient of MeCN from 50% to 67% over 30 min, then from 67% to 95% over 10 min and 95% MeCN kept for 20 min. Following HPLC, analysis confirmed high purity of the product: tR = 24.6 min (97.7% pure at 220/280 nm) on Columbus C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluent solvent A water, solvent B CH3CN; eluted with a linear gradient of B from 50% to 95% over 30 min and then 95% B for 30 min and the elution rate 0.8 mL/min. 1H NMR (DMSO-d6, 600 MHz) δ: 11.14 (s,1H, NH-uridine), 8.24 (bs, 1H, NH), 8.12 (t, 1H, NH, J = 4.46 Hz), 7.94 (s, 1H, H6-uridine, 3JSn,H = 19.42 Hz), 7.84−7.72 (m, 2H, aryl-H2, aryl-H6), 7.48−7.36 (m, 2H, aryl-H3, aryl-H5), 6.10−5.89 (m, 1H, H1′), 4.87−4.62 (m, 1H, H4′), 4.49−4.27 (m, 1H, H3′, OH), 3.97−3.87 (m, 2H, benzyl), 3.68−3.53 (m, 2H, H5′), 2.38− 2.29 (m, 1H, H2′), 2.16−2.08 (m, 1H, H2″), 1.47 (s, 9H, H3BocN′), 1.44 (s, 9H, H3-BocN″), 0.29 (s, 9H, 3 × CH3-Sn, 2JSn,H = 29.5 ppm. 13C NMR (DMSO-d6, 100 MHz) δ:170.2 (C1-benzoyl), 163.4 (C4), 159.8 (C1-Boc-N′), 158.5 (C1-Boc-N″), 153.7 (C2), 149.6 (C-guanidine), 144.6 (C4-aryl), 143.8 (C6), 139.7 (C2-aryl), 127.5 (C5-aryl), 126.7 (C1-aryl), 111.9 (C3-aryl), 110.3 (C5), 90.37 (C1′), 86.2, (C4′), 84.2 (C2-Boc-N′), 81.8 (C2-Boc-N″), 70.7 (C3′), 69.12 (C-benzyl), 68.8 (C5′), 39.5 (C2′), 28.5 (C3-Boc-N′), 28.1 Q



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vacuum. To a resulting dried residue, neat TFA (100 μL) was added, and the mixture remained in a sealed vial at 65 °C for ∼35 min. After cooling, it was diluted with CH3CN (200 μL) and evaporated with a stream of nitrogen leaving ∼20 μL of the liquid. This process was repeated twice. To the final residue ethanol (80 μL) was added followed by potassium phosphate buffer (100 μL, 10 mM PB, pH 6.1) and the solution injected on HPLC equipped with Luna CN 100 Å (5 μm, 4.6 mm × 250 mm) column eluted with a linear gradient of EtOH in potassium phosphate buffer as applied in method A. Using this direct preparation three times, starting with 40.7, 38.9, and 24.1 MBq of Na125I/NaOH, the guanidine 29 was attained with an average yield of 87%. HPLC analysis: tR = 24.7 min, ≥98% radiochemical purity (Bioscan/UV 280 nm. Cells. Two human NB cell lines SK-N-SH (SK) and BE(2)-C (BE) and one murine NB cell line N1E-115 (N1E) were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were maintained according to the vendor’s instructions. The subcultivation ratio of 1:3 was routinely applied. For animal studies, cells were harvested when ∼70% confluent and cryopreserved. When sufficient cell numbers were available, cells were thawed, washed twice with full medium, resuspended in the appropriate growth medium without fetal bovine serum, cell viability was determined using the trypan blue assay and cell suspension distributed into sterile 1 mL syringes equipped with 22 gauge needle. Proteins, Sera, Buffers, and Cell Culture Reagents. ATCCformulated Eagle’s minimum essential medium and DMEM:F-12 medium were from the American Type Culture Collection (ATCC; Manassas, VA). Fetal bovine serum (FBS) was purchased from Rockland Immunochemicals Inc. (Limerick, PA) and ThermoFisher Scientific (Waltham, MA). L-Glutamine, penicillin−streptomycin, and sodium pyruvate were from GE Healthcare Life Sciences (Marlborough, MA) and ThermoFisher Scientific. Pierce NE-PER nuclear and cytoplasmic extraction reagents and Micro BCA protein assay kit were from ThermoFisher Scientific. Mice. Research involving animals was performed in accordance with the UNMC institutional guidelines and protocols as defined by the Institutional Animal Care and Use Committee for U.S. institutions. Athymic NCr nude mice (spontaneous mutant) were purchased from Taconic (Rensselaer, NY). The intratumoral route (IT) was used for the dose administration. All experiments were conducted in mice of both genders 4- to 8 weeks-old. Biodistribution studies were conducted in groups of 14 male and 12 female mice. Twenty mice received the radioactive compounds alone, and six mice also received BNPP (3 male, 3 female). The experiment was conducted using two independent production lots of IDG. The evaluation of the therapeutic potential was conducted in groups of six female and six male mice. Gel Electrophoresis and Western Immunoblotting. All analyses were conducted on 4−20% or Any kD Mini-PROTEAN TGX precast protein gels (10-well, 30 μL) using the Mini-PROTEAN electrophoresis cell (Bio-Rad, Hercules, CA). Protein concentration in cell lysates was measured using Micro BCA protein assay kit (ThermoFisher Scientific). Cell lysate aliquots were added to the 2× sample buffer. Protein samples were denatured at 95 °C for 5 min, cooled, and loaded onto gels at 100 μg total protein per well. Gels were run at the constant voltage (150 or 190 V) for ∼1 h. Proteins were transferred onto a Hybond-P 0.45 μm PVDF membrane (Amersham Biosciences, Piscataway NJ) in a cold room using a constant current of 30 mA for 18 h. Kaleidoscope prestained standards (Bio-Rad Laboratories, Hercules, CA) were used as molecular weight markers and to monitor the efficiency of protein transfer. The membrane was incubated for 1 h in the blocking buffer containing 5% w/v nonfat dry milk. The blocked membrane was incubated in the same buffer containing primary antibodies overnight at 4 °C. Norepinephrine transporter antibody PA5-42080 (1:2,000 dilution, v/v) and Pierce goat anti-rabbit poly-HRP 32260 (1:5000 dilution, v/v) were purchased from ThermoFisher Scientific (Rockford, Il). Rabbit anti-β-actin mAb 13E5 (Cell Signaling Technology, Danvers, MA; 1:2000 dilution, v/v) was used as a marker of the protein load. Antigens were detected using the chemiluminescence

BocNH), 28.1 (C3-BocN) ppm. HDMS (m/z): [M + H]+ calcd for C18H26IN3O4, 476.1041; found, 476.1033. N,N′-Bis(tert-butyloxycarbonyl)-3-trimethylstannylbenzylguanidine (27). A mixture of N,N′-bis(tert-butyloxycarbonyl)-3-iodobenzylguanidine 26 (0.206 g, 0.433 mmol), hexamethylditin 295 mg (0.90 mmol), palladium catalyst (30 mg, 0.043 mmol), and TEA (150 μL, 1.8 mmol) was refluxed in EtOAc (17 mL) under argon atmosphere for as long as the starting iodide was detectable (∼3 h, checked often by TLC). After cooling to room temperature and evaporation of the mixture, a crude product 27 was separated (98 mg, 44% yield) on a silica gel column using EtOAc/n-hexanes (1:4), Rf = 0.65 (DCM/MeOH, 10:0.4), ∼91% pure by HPLC analysis (at 220/280 nm) and was further purified on the Columbus C18, 100 Å (5 μm, 10 mm × 250 mm) semipreparative column: (60 mg, ∼5 mg per injection), eluted at the rate of 2.4 mL/min with a linear gradient of MeCN from 50% to 85% over 45 min, then from 85% to 95% over 15 min and 95% MeCN kept for another 10 min. The HPLC analysis confirmed higher purity of the product: tR = 35.4 min (≥98.5% pure at 220/280 nm) on a Columbus C18 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 70% to 95% over 45 min and then 95% MeCN kept for another 15 min. 1H NMR (CDCl3, 600 MHz) δ: 11.53 (bs, 1H, NH-Boc), 8.62 (bs, 1H, NH-benzyl), 7.40−7.19 (m, 4H, aryl), 5.12 (s, 2H, benzyl), 1.52 (s, 9H, H3-BocN), 1.49 (s, 9H, H3-BocN′), 0.31 (s, 9H, 3 × CH3-Sn) ppm. HDMS (m/z): [M + H]+ calcd for C21H35N3O4112Sn, 506.1749; found 506.1728 using the 112Sn isotope signal. N,N′-Bis(tert-butyloxycarbonyl)-3-[ 1 2 5 I]iodobenzylguanidine (28). Radioiodination (general procedure C) of stannane 27 (∼100 μg) was done twice with 59.2 and 58.1 MBq of Na125I/ NaOH to give overall 104.4 MBq (89% average yield) of the isolated product. The reaction mixture was separated and purified by HPLC on Columbus C8 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in water from 50% to 95% over 45 min, then 95% MeCN was held for further 35 min. The product was collected in three fractions (within 40.5−42.5 min after the injection), and an excess of unreacted tin precursor was fully separated, eluting ∼7.5 min later. Performed HPLC analysis of the product confirmed its high purity: tR = 40.7 min (≥98% purity, Bioscan/UV 280 nm). 3-[125I]Iodobenzylguanidine (29). Method A. A dried residue of 28, treated with neat TFA (60 μL) under nitrogen, was kept in a tightly covered vial at ∼65 °C. After 30 min the reaction (monitored by HPLC) was completed. An excess of TFA was evaporated with a stream of dry nitrogen, and 100 μL of 50% MeCN in water added. The mixture was injected on HPLC fitted with ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column and eluted at 0.8 mL/min with a 10% MeCN in water for 10 min, then by a linear gradient of MeCN in water from 10% to 95% over 40 min and 95% MeCN held for an additional 50 min; both solvents contained 0.07% TFA (v/v). The product was collected within 26−31 min after the injection. An average yield of 29 (from three preparations carried out with 31.8, 44.4, and 50.3 MBq of 28) was 86%. The subsequent HPLC analysis showed tR = 28.7 min and ≥97% purity (Bioscan/UV 280 nm). To eliminate the presence of TFA, the solution of separated product 29 (∼20.7 MBq) was evaporated and ethanol (80 μL) added, followed by potassium phosphate buffer (100 μL, 10 mM, pH 6.1). The resulting solution was again injected on HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm) column and eluted at the rate of 0.8 mL/min with a linear gradient of EtOH in potassium phosphate buffer (10 mM PB, pH 6.1); from 30% to 70% over 60 min and 70% EtOH kept for further 10 min. The product freed from an excess of TFA (18.3 MBq, 88% yield) was collected within 24.0−33.0 min after the injection and was analyzed on HPLC: tR= 24.8 min, ≥98% radiochemical purity (Bioscan/UV 280 nm). Method B. Alternatively, the typical radioiodination (general procedure C) of stannane 27 (∼100 μg) was carried out, but the separation of Boc-protected guanidine 28 was omitted. A crude radioiodination mixture, quenched with a solution of Na2S2O3, was evaporated with a stream of dried nitrogen and kept under a high R



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substrate ChemiGlow according to the manufacturer’s instructions (Alpha Innotech Corporation, San Leandro, CA). Stability Studies. Into 1980 μL of 1:1 diluted serum or cells culture medium, 370−740 kBq of the radioactive compound in 20 μL of 25% EtOH/PB (100 mM, pH 6.1) was added. This mixture was briefly vortexed and kept at ambient temperature. At each time point, a volume of 0.5 mL was withdrawn and processed as follows. Each aliquot of the incubated mixture was mixed with 0.5 mL of CH3CN, vortexed, and centrifuged (2000 rpm, 15 min). Aliquots of supernatant (500 μL) were acidified to pH ∼ 6 with 0.05 N TFA (2−8 μL). The excess of CH3CN was evaporated with a stream of nitrogen and 100 μL water added. A mixture was passed through a 0.2 μm filter and each sample (∼100 μL, 62.5−130 kBq) injected onto the HPLC system. Time-Dependent Cellular Uptake. For each compound tested, cells were plated in TPP T25 flasks (Midwest Scientific, Valley Park, MO) at 5 × 105 cells/well in 5 mL of growth medium. Radioactive compounds were diluted in fresh medium to produce the required concentration, and 5 mL of this solution was added to each flask after cells were maintained in culture for 24−48 h, depending on the doubling time of the cell line. Aliquots of the radioactive medium were counted in a γ counter to confirm the concentration. Cells were incubated with radioactive compounds for 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 24 h. Each point in time was tested in triplicate. At the end of incubation, aliquots of media (0.5 mL) were removed from each flask for γ counting. The remaining radioactive medium was aspirated and disposed as a radioactive material. Cells were washed twice with 3 mL of ice-cold PBS. Aliquots of wash PBS (0.5 mL) were also taken for γ counting. Cells were trypsinized with trypsin/EDTA, enumerated using a Cellometer disposable cell counting chambers (Nexcelom Bioscience, Lawrence, MA), and centrifuged at 1200 rpm for 10 min, and cell pellets were washed with PBS. All washes and spent medium were counted in a γ counter alongside the cell pellet to determine the cell-associated radioactivity. Uptake in Exponential and Confluent SK Cells. SK cells were plated into cell culture flasks (n = 14), allowed to attach for 24 h, given fresh media, and grown until ∼60%, ∼80%, >90%, and 100% confluent. Cells in 100% confluent flasks (G0) were given fresh medium and incubated for additional 3 days with daily media replacement. Cell in the remaining flasks were given media containing IDG, incubated for 1 h at 37 °C in the radioactive medium, and harvested by scraping to avoid losses of the membrane-bound radioactivity. Cells were counted, centrifuged at 1500 rpm at 4 °C for 10 min, media were collected, and 1 mL aliquot was counted in a γ counter. Cell pellets were washed with 5 mL of ice-cold PBS, centrifuged at 1500 rpm at 4 °C for 10 min, PBS was collected, and 1 mL was counted in a γ counter. This procedure was repeated twice. Cell pellets were resuspended in 1 mL of PBS, transferred into γ counter tubes, and the cellular radioactivity was measured. Cells from three 100% confluent flasks and two ∼60% confluent flasks were also harvested. These cells were enumerated and aliquoted. One aliquot of 3 × 106 cells was processed for the cell cycle analyses. The remaining cells were lysed for Western immunoblotting. The protein content of lysates was determined using the Micro BCA method. Clonogenic Assay (Reproductive Integrity). BE and SK cells were plated in T75 flasks. After ∼18−48 h in culture, the growth medium was removed from all flasks and replaced with either 15 mL of fresh medium containing various concentrations of radioactive guanidines or 15 mL of fresh nonradioactive medium containing PBS in amounts identical to these added with the radioactive compounds. Triplicate 0.1 mL aliquots of medium were withdrawn from each flask and counted in a γ counter to determine the extracellular concentration of radiolabeled compounds. Cells were processed after 1 or 24 h in the incubator at 37 °C. Medium was removed from all flasks in 1 h and 1 h + 23 h groups. Monolayers were washed once with full nonradioactive medium and PBS. Cells in a 1 h group were harvested, enumerated, and replated in T25 flasks for clonogenic assay at 200 and 500 live cells/flask. Each cell density was tested in quadruplicate. To cells in the 1 h + 23 h group, 15 mL of fresh nonradioactive medium was added and cells were allowed to grow

undisturbed for an additional 23 h, at which time cells were washed with PBS, trypsinized, and counted and their viability was determined. Cells were replated in T25 flasks at plating densities of 200 and 500 live cells/flask. Each cell density was tested in quadruplicate. Radioactive medium from cells in a 24 h group was removed after 24 h of incubations. Cells were washed, harvested, and replated as described above. The clonogenic assay cells were allowed to grow undisturbed for 2−3 weeks. The medium was changed once a week. At 14−21 days later, colonies were washed with 5 mL of ice-cold PBS followed by 5 mL of PBS/methanol (1:1; v/v) and fixed in 5 mL of methanol for 10 min. Methanol was removed, and flasks were left open to dry for a few hours. Crystal violet (5 mL; 0.25% in 1:1 PBS/ methanol) was added to each flask to stain colonies. After approximately 10 min, the dye was removed, and flasks were rinsed thoroughly with tap water followed by distilled water and were left to dry. Colonies were counted by two or three independent readers with the aid of ImageJ software (http://rsb.info.nih.gov/ij/). Subcellular Fractionation. NB cells were plated into six T75 or T150 flasks and allowed to attach overnight. Cells were grown as a monolayer to approximately 60−70% confluency. The nonradioactive growth medium was removed and replaced with 10 mL of fresh medium containing tested radioactive compounds. Cells were exposed for 1 h after which time the radioactive medium was removed and replaced with fresh medium. Aliquots of all radioactive growth media were counted in the γ counter before and after the cell culture. Cells in three flasks were processed immediately. Cells in the remaining three flasks were cultured in fresh nonradioactive medium for 24 h and then processed. Cell monolayers were rinsed with 5 mL of PBS, trypsinized, cells were enumerated, and their viability was determined. By use of NE-PER nuclear and cytoplasmic extraction reagents or Mem-PER Plus membrane protein extraction kit (ThermoFisher Scientific), the cell content was fractionated and counted in a γ counter to determine the compound distribution in various compartments of the cancer cell. Competition with MIBG. Monolayer Method. Cells were plated into T25 flasks and allowed to grow until ∼60−70% confluent. Fresh medium containing variable concentrations of nonradioactive MIBG was added in duplicate to flasks and incubated for up to 3 h. A constant amount of radioactive guanidines was added to each flask and cells were returned into the incubator for 1−3 h at which time media were removed, monolayer washed 1× with full medium, 2× with PBS, and cells were harvested with the nonenzymatic cell dissociation solution (Sigma-Aldrich). N1E cells were harvested by flushing the monolayer with spent media. Cell numbers were determined, and their radioactive content was measured in a γ counter. Filtration Method. Cells in the exponential growth phase were harvested with the nonenzymatic cell dissociation solution, enumerated, centrifuged, washed, and resuspended in PBS. 1 mL aliquots were dispensed into glass tubes and various concentrations of nonradioactive MIBG added, and cells were incubated on a Labquake shaker (Barnstead Thermolyne) at either 37 or 4 °C. To determine nonspecific binding, a parallel set of tubes was prepared without cells. Radioactive guanidines diluted in PBS were added, and the tubes were gently vortexed. Aliquots (0.1 mL) were withdrawn to determine the total radioactivity. Both sets of tubes were incubated on a Labquake shaker for 1 h. Using the vacuum manifold, cells were filtered, washed with PBS, and the radioactive content was measured in a γ counter. The content of tubes without cells was also filtered to measure nonspecific binding of radioactivity to filters. Biodistribution. Groups of athymic NCr nu/nu male and female mice bearing SQ BE xenografts received an intratumor average dose of 0.33 ± 0.05 MBq of IDG in PBS containing 0.1% HSA. Control mice were injected with 125I-MIBG (0.44 ± 0.1 MBq per tumor). For the carboxylase inhibitions studies, mice bearing BE xenografts were given IT injection of BNPP (8.8 and 17.6 μmol) followed by IDG, an average dose 0.83 ± 0.09 MBq. Control mice in the BNPP study received only IDG at the same dose. Weights of the syringe, with the dose and empty after the injection, were determined. Aliquots of the injected dose were counted in a γ-counter before the dose S



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administration and later alongside all extirpated tissues. Mice were euthanized 24 h after the administration. Lungs, heart, liver, spleen, pancreas, stomach, small intestine, cecum, large intestine, kidneys, adrenals, urinary bladder, genitourinary tract (male, en bloc), uterus and ovaries (female), brain, skin, ears, muscle, thyroid, bone, and tumor were dissected, rinsed in PBS, and patted dry, and their wet weight was determined using the analytical balance. Blood, dissected tissues, and tumors were placed in γ-counter tubes and the radioactive content was measured using 15−80 keV channel of the Packard Cobra II model 5003 or Packard Minaxi A5530 γ-counter. Tissue uptake values were calculated in terms of the percent injected dose per gram tissue (%ID/gram) for each mouse. Therapy. Four-week-old NCr nu/nu mice, male (n = 6) and female (n = 5), received SQ implant of 5 × 106 BE cells in BD Matrigel basement membrane matrix (BD Biosciences, San Jose, CA). Three weeks later, mice were randomly assigned via a lottery to either control group or treatment group. Control mice were given IT injection of the vehicle. Mice in the therapy group received a bolus IT injection of 8.41 ± 0.32 MBq IDGper tumor. Whole body radioactivity was measured at 15 min, 2 h, 20 h, 90 h, 142 h, and 166 h after injection. Necropsy was performed as described in the biodistribution protocol. Blood was collected via cardiac puncture, and aliquots were reserved for determination of Hb (HemoCue, Ä ngelholm, Sweden). The whole body radioactivity was used for the construction of the clearance curve and the area under the curve (AUC) and mean residence time (MRT) determinations. AUC was determined using GraphPad Prism 7 for Windows, version 7.04 (GraphPad Software, La Jolla, CA). Absorbed Radiation Dose Estimates. The AUCs for the radioactivity−time curves were integrated from the time of injection of IDG to 166 h after injection, not corrected for radioactive decay, using previously described by us methods.31 AUC from 166 h after injection to infinity was estimated, assuming elimination only by the radioactive decay, by setting tfinal to at least 2 times the half-life. MRT of radioactivity in tumor and whole body was calculated by dividing the total AUC0h→∞ by the average injected dose. The radiationabsorbed doses (mGy/MBq) in 1-year-old (9.8 kg) and 5-year0-old (19 kg) phantoms were estimated on the basis of the whole-body residence times with the aid of the MIRDOSE 3.0 computer program (copyright by Oak Ridge Associated Universities).96 Statistical Analyses. All variables are expressed as average ± standard deviation or ±standard error. The cellular uptake, cell survival and other biological properties of new compounds were analyzed using two-sided Student’s t test. Two-sided P values of 0.8 was used as the lowest acceptable goodness-of-fit criterion. AUC was integrated by the trapezoidal method (Prism). Tumor growth curves were used to determine TD values. Tumor weights at the necropsy were used as the final measure of the tumor response. These data were analyzed using ANOVA with the Tukey−Kramer multiple comparisons. All differences were considered significant when P < 0.05. SigmaPlot/SigmaStat from Systat Software, Inc., Point Richmond, CA, and GraphPad InStat computer software from GraphPad Software, Inc., La Jolla, CA, were used for these analyses.

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HPLC analyses of all compounds from 3 through 29 (PDF) Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Janina Baranowska-Kortylewicz: 0000-0002-2254-0791 Author Contributions

All authors contributed to the data acquisition, analyses, and manuscript and have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Ed Ezell of the Fred & Pamela Buffett Cancer Center Structural Biology Facility Shared Resource (National Cancer Institute Award P30 CA036727) for his help with the NMR analyses. Mass spectrometry analyses were conducted by the NIH/NIGMS Mass Spectrometry Resource, Washington University, in St. Louis School of Medicine, St. Louis, MO. Authors also acknowledge Dr. S. Tian’s technical assistance in the uptake experiments. These studies were funded in part by the National Cancer Institute Grant R21CA187548 (J.B.-K., PI), the National Institutes of Health/National Institute of General Medical Sciences Grant P41GM103422 (Gross, PI), and the State of Nebraska Pediatric Cancer Research Grant LB905 (D.W.C., PI).

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ABBREVIATIONS USED BDDC, bis[[4-(2,2-dimethyl-1,3-dioxolyl)]methyl]carbodiimide; Boc, tert-butyloxycarbonyl; DCC, 1,3-dicyclohexylcarbodiimide; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, dimethylformamide; DMPA, 4(dimethylamino)pyridine; DMSO, dimethyl sulfoxide; DMTrCl, 4,4′-dimethoxytrityl (triphenylmethyl) chloride; EtOAc, ethyl acetate; EtOH, ethanol; HPLC, high performance liquid chromatography; IDG, 5-[125I]iodo-3′-O-(εguanidinohexanoyl)-2′-deoxyuridine; IUdR, 5-iodo-2′-deoxyuridine; Lev, levulinyl (4-oxopentanoyl); MeCN, acetonitrile; MIBG, 3-iodobenzylguanidine; PB, potassium phosphate buffer; TBAF, tetrabutylammonium fluoride; TBDMSCl, tertbutyldimethylsilyl chloride; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; TMS, tetramethylsilane

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ASSOCIATED CONTENT

S Supporting Information *

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