Novel Molecular Platform Integrated Iron Chelation Therapy for 1H

Feb 7, 2013 - ABSTRACT: Targeting the increased Fe3+ content in tumors, we propose a novel molecular platform integrated cancer iron chelation therapy...
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Novel Molecular Platform Integrated Iron Chelation Therapy for 1 H‑MRI Detection of β‑Galactosidase Activity Xiaojin Li,†,§ Zhongwei Zhang,‡,§ Zijun Yu,‡,∥ Jennifer Magnusson,‡ and Jian-Xin Yu*,‡ †

Xinjiang Institute of Medicinal Development, Chinese Academy of Medical Sciences, 9 Xinming Road, Urumqi, Xinjiang 830002, China ‡ Department of Radiology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75390-9058, United States S Supporting Information *

ABSTRACT: Targeting the increased Fe3+ content in tumors, we propose a novel molecular platform integrated cancer iron chelation therapy for 1H-magnetic resonance imaging (MRI) detection of β-galactosidase (β-gal) activity. Following this idea, we have designed, synthesized, and characterized a series of β-D-galactosides conjugated with various chelators and demonstrated the feasibility of this concept for assessing β-gal activity in solution by 1H-MRI T1 and T2 relaxation mapping. KEYWORDS: responsive Fe-based 1H-MRI agent, T1 and T2 relaxation times, β-galactosidase detection, synthesis



imaging (MRI) and 19F-MRS/MRI approaches.11−27 In this paper, we propose a novel molecular platform incorporated cancer iron chelation therapy for 1H-MRI detection of β-gal activity.

INTRODUCTION Cancer cells exhibit increased uptake and utilization of Fe3+ because it plays a crucial role in malignancy related cellular processes such as proliferation, DNA synthesis, and ribonucleotide reductase functioning, making endogenously abundant Fe3+ in tumors an obvious molecular target for chemotherapeutic agents.1−3 However, the success of chemotherapy is always limited by several drawbacks including systemic toxicity, insufficient drug concentrations in tumors, lack of selectivity for neoplastic cells, and the appearance of drug resistance.4 The promising strategy to overcome these problems is the use of enzyme-activated prodrugs through antibody/gene/virus-directed enzyme prodrug therapy approaches.5,6 The lacZ gene encoding β-galactosidase (β-gal) has been recognized as the most useful marker gene because of its stability, high turnover rate, ease of conjugation, and the absence of endogenous β-gal activity. Its introduction has become a standard means of assaying clonal insertion, transcriptional activation, protein expression and interaction in molecular/cellular biology, small animal investigations, and clinical trials.7−10 So the in vivo assessment of its transfection in terms of spatial extent, gene expression, and progression has attracted much attention ranging from fluorescence, chemiluminescence, positron emission tomography, or single-photon emission computed tomography, to magnetic resonance © 2013 American Chemical Society



RESULTS AND DISCUSSION Probe Design. Cancer cells have a high requirement for Fe3+ as they rapidly replicate. Many studies have shown that depleting cancer cellular Fe3+ results in the disruption of cancer cell proliferation and the inhibition of tumor growth.1,2 Pyridoxal isonicotinoylhydrazone (PIH), salicylaldehyde benzoylhydrazone (SBH), salicylaldehyde isonicotinoylhydrazone (SIH), and salicylaldehyde nicotinoylhydrazone (SNH) (Figure 1) are applied in clinical trials for the treatment of various metastatic and solid cancers.2 From the structural similarities of the Fe-complexes of these clinically applied chelators and the known Fe-based 1H-MRI contrast agents,28,29 we hypothesized that these clinically applied Fe-chelators could act as Fe-based 1 H-MRI contrast agents. If so, when using chelators as aglycones, their β-D-galactosides will work as prodrugs. Upon delivery and cleavage at lacZ transfected tumor through the Received: Revised: Accepted: Published: 1360

November 2, 2012 January 29, 2013 February 7, 2013 February 7, 2013 dx.doi.org/10.1021/mp300627t | Mol. Pharmaceutics 2013, 10, 1360−1367

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Figure 1. Structures of PIH, SBH, SIH, and SNH.

Figure 2. Proposed mechanism of integrated iron chelation therapy for 1H-MRI detection of β-gal activity.

Figure 3. The 1H-MRI of the Fe-complexes PIH/Fe, SBH/Fe, SIH/Fe, and SNH/Fe. MRI acquisition parameters: 1H-MRI, 200 MHz, matrix size: 128 × 128, FOV: 40 mm × 40 mm, slice thickness: 2 mm, receiver bandwidth: 20 kHz, T1-map: saturation recovery spin−echo sequence, TR = 200, 400, 600, 800, 1000, 2000, 3000, and 6000 ms, respectively, TE = 15 ms; T2-map: multiecho SE sequence, TE = 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 ms, respectively, TR = 2000 ms. (A) Control, FAC (12.0 mM); (B) ligand SNH (6.0 mM), FAC (12.0 mM); (C) ligand PIH (6.0 mM), FAC (12.0 mM); (D) ligand SIH (6.0 mM), FAC (12.0 mM); (E) ligand SBH (3.0 mM), FAC (12.0 mM) in 2:1 (v/v) DMSO/PBS (0.1 M, pH = 5.5) at 20−22 °C.

combined iron chelation therapy for 1H-MRI detection of β-gal activity, using PIH as a model chelator. This concept of probe design is based on the tumor biology and synergized the prodrug and iron chelation therapy strategies. The mechanism proceeds in the β-gal responsive “turn-on” way to selectively deplete tumor Fe3+ leading to cancer cell cycle arrest and apoptosis as reported,1−3 and

lacZ gene-directed enzyme prodrug therapy strategy, the blocked chelators will be lacZ (or β-gal)-specifically released and activated to capture the tumor-abundant Fe3+. The in situ formed paramagnetic Fe-complexes will (1) generate a 1H-MRI contrast effect and (2) localize and accumulate 1H-MRI signals at the site of β-gal activity. Figure 2 illustrates the design 1361

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Figure 4. Synthetic route and the structures of 1−13. Reaction conditions: (a) CH2Cl2−H2O, pH 8−9, 50 °C, TBAB, N2, 5−6 h, 71%(→4) or 85%(→5), respectively; (b) EtOH, AcOH (20 μL), isoniazide, benzoic hydrazide, or nicotinic hydrazide (1.1 equiv), 80 °C, N2, 3−4 h, 82%(→6), 95%(→7), 90%(→8), and 93%(→9), respectively; (c) 0.5 M NH3-MeOH, 0 °C→r.t., 24 h, 91−94% yields.

contrast agent, we then started the conjugation of β-Dgalactopyranosyl group with the chelators PIH, SBH, SIH, and SNH at the phenolic hydroxyl group. Figure 4 depicts the synthetic strategy and the structures of 1−13. The apparent lower pKa of phenol hydroxyl in pyridoxal 2 (pKa(3‑OH) = 8.23 ± 0.01, whereas pKa(5‑CH2OH) = 13.46 ± 0.01) and salicylaldehyde 3 (pKa(2‑OH) = 8.18 ± 0.01, pKa was calculated using the Advanced Chemistry Development Software, V12) suggested that phase-transfer-catalysis at pH = 8−9 could provide regio- and stereoselective synthesis of mono β-D-galactopyranosides, as we exploited previously for β-gal 19FMRS/MRI reporters.30−32 To the well-stirred solution of 2 or 3 in CH2Cl2−H2O biphasic system (pH 8−9) catalyzed by tetrabutylammonium bromide (TBAB) at 50 °C, an equimolar amount of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide 1 was added dropwise over ∼1 h under N2 atmosphere, and 3O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-pyridoxal 4 (71%) and 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-benzaldehyde 5 (85%) were obtained, respectively. The position of β-D-galactopyranosylation in 4 at 3-phenolic hydroxyl group was confirmed by its 1H NMR spectrum, in which α5-CH2 exhibits doublet with JH‑5,HO‑5 = 6.0 Hz at 5.01 ppm due to the coupling from free α5-OH. Treatment of 4 or 5, respectively, with 1.1 equivalent of isoniazid, benzoic hydrazide, or nicotinic hydrazide in acidic EtOH at 80 °C accomplished the corresponding β-D-galactopyranosylated aroylhydrazones: 3-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-PIH 6, 2O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SBH 7, 2-O(2′,3′,4′,6′-tetra-O-acetyl-β-D-galacto-pyranosyl)-SIH 8 and 2O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SNH 9 in high yields (82−95%), which were deacetylated with NH3/ MeOH from 0 °C to room temperature giving their free β-Dgalactopyranosides: 3-O-(β-D-galactopyranosyl)-PIH 10 (91%), 2-O-(β-D-galacto-pyranosyl)-SBH 11 (94%), 2-O-(β-D-galactopyranosyl)-SIH 12 (93%) and 2-O-(β-D-galacto-pyranosyl)SNH 13 (92%). The anomeric β-D-configuration of 10−13 in the 4C1 chair conformation was confirmed by the 1H NMR chemical shifts at δH‑1′ = ∼4.95 ppm of the anomeric protons, and the coupling constants of J1′,2′ = 8 Hz and J2′,3′ = 10 Hz. The anomeric carbon resonances appeared at δC‑1′ ∼101.40 ppm are in accordance with the β-D-configuration.27

meanwhile to generate 1H-MRI contrast revealing lacZ gene expression, development, location, and magnitude, which offers a potential for simultaneous cancer imaging and therapy. Confirmation of Fe-Chelator as an 1H-MRI Agent. Following the methods described previously, the clinically applied tridentate chelators PIH, SBH, SIH, and SNH, and their corresponding 2:1 Fe-complexes [Fe(PIH-H)2]+ (PIH/ Fe), [Fe(SBH-H)2]+ (SBH/Fe), [Fe(SIH-H)2]+ (SIH/Fe), and [Fe(SNH-H)2]+ (SNH/Fe) were synthesized.1−3 T1/T2weighted 1H-MRI evaluations were investigated by using a saturation recovery spin−echo sequence and multiecho SE sequence at varying repetition times (TRs) and echo times (TEs); the images were acquired with 5-well plate containing different samples in parallel. Figure 3 displayed the T1 and T2 maps and relaxation times of the complexes PIH/Fe, SBH/Fe, SIH/Fe, and SNH/Fe, in which their relaxivities are r1(PIH/Fe) = 22.22, r2(PIH/Fe) = 11.49 [r1/r2(PIH/Fe) = 1.93], r1(SBH/Fe) = 3.21, r2(SBH/Fe) = 7.75 [r1/r2(SBH/Fe)= 0.41], r1(SIH/Fe) = 1.38, r2(SIH/Fe) = 5.56 [r1/r2(SIH/Fe) = 0.25], r1(SNH/Fe) = 4.22, r2(SNH/Fe) = 7.25 [r1/r2(SNH/Fe)= 0.58] mM−1s−1, respectively. The comparison with ferric ammonium citrate (FAC) [r1(FAC) = 0.35, r2(FAC) = 0.70 mM−1s−1] showed that each complex PIH/Fe, SBH/Fe, SIH/Fe, or SNH/Fe generated substantial T1 and T2 1H-MRI contrast. But, their contrast is different very much upon the structure of ligands, because these tridentate chelators PIH, SBH, SIH and SNH form Fe-complexes with the complete coordination of Fe3+,1−3 which eliminates the possibility of inner-sphere coordination of water, leaving outer-sphere and second-sphere coordination as the only mechanisms for relaxation.28,29 These features result in that the characteristics of ligands are the determinants for the effect of relaxation. The T1, T2 maps also evidently showed the significant differences in both T1 and T2 values of the complexes PIH/Fe and SNH/Fe, which suggested the potential for integrating T1 and T2 measurements as combined effects for the addition of certainty to imaging evaluation, especially where tissue heterogeneity may otherwise be misinterpreted. β-D-Galactopyranosides Synthesis. Escherichia coli (lacZ) β-gal catalyzes the hydrolysis of β-D-galactopyranosides by cleavage of the C−O bond between D-galactose and the aglycones with overall retention of β-anomeric configuration. After demonstration of the Fe-chelation complex as a 1H-MRI 1362

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Figure 5. Structures of mono β-D-galactopyranosides 14−30.

1.87 s−1 were observed. In 11/FAC solution without β-gal the relaxation rate was determined to be T1(11/FAC) = 211 ± 14 ms [r1(11/FAC) = 4.76 ± 0.31 s−1] and T2(11/FAC) = 94 ± 4 ms [r2(11/FAC) = 10.66 ± 0.45 s−1], while in 11/FAC solution with β-gal T1(11/Fe) = 204 ± 13 ms [r1(11/Fe) = 4.92 ± 0.31 s−1] and T2(11/Fe) = 80 ± 3 ms [r2(11/Fe) = 12.53 ± 0.47 s−1] (compared with the concentration of 11 in 2.5 mM), which depended on the increase of Fe-complex 11/Fe formed in situ. The T1 and T2 values with various concentrations are represented as bars at the right sides adjacent to T1 and T2 maps (Figure 7). Similarly, the relaxation times in 13/FAC and 30/FAC solution in the absence of β-gal were T1(13/FAC) = 210 ± 9 ms [r1(13/FAC) = 4.77 ± 0.21 s−1] and T1(30/FAC) = 221 ± 8 ms [r1(30/FAC) = 4.53 ± 0.16 s−1], T2(13/FAC) = 89 ± 2 ms [r2(13/FAC) = 11.24 ± 0.25 s−1] and T2(30/FAC) = 94 ± 1 ms [r2(30/FAC) = 10.64 ± 0.11 s−1], whereas in the presence of β-gal T1(13/Fe) = 154 ± 10 ms [r1(13/Fe) = 6.52 ± 0.42 s−1] and T1(30/Fe)= 197 ± 13 ms [r1(30/FAC) = 5.10 ± 0.34 s−1], T2(13/Fe) = 56 ± 2 ms [r2(13/Fe) = 17.88 ± 0.64 s−1] and T2(30/Fe) = 76 ± 2 ms [r2(30/Fe) = 13.17 ± 0.35 s−1] (with 13 in 3.0 mM Δr1 = 1.75 and Δr2 = 6.64 s−1, and 30 in 2.3 mM Δr1 = 0.57 and Δr2 = 2.53 s−1), respectively (Figure 8, Figure S3 in the Supporting Information).

Given that the specificity and activity of β-gal substrates are expressed upon the various structures of aglycones, we designed and synthesized additional seventeen analogs 14−30 (Figure 5, Figure S1 and S2 in the Supporting Information) of 10−13 with diverse substituents at different positions, according to the methods as described in Figure 4. β-Gal Hydrolysis. The solutions of free β-D-galactopyranosides 10−30 with FAC in 2:1 (v/v) DMSO/PBS (0.1M, pH = 5.5) all are clear and light yellow, but the solutions of their corresponding aglycones with FAC at the same conditions each turn into black upon the formation of thermodynamically stable 2:1 Fe3+-complexes,1−3 which all exhibited the maximum absorption around 480 nm. The colorimetric measurements at λmax = 480 nm following the reaction of 10−30 with β-gal (G5160) in the presence of FAC in DMSO/PBS at 37 °C in different time points indicated that 12 β-D-galactopyranosides 11−13, 16−18, 24, and 26−30 are reactive to β-gal (G5160) with varying hydrolytic rates (Figure 6).



CONCLUSION

We present here a novel molecular platform combined cancer iron chelation therapy for the detection of β-gal activity. Following the design, we have successfully synthesized and characterized a series of β-D-galactopyranosides with different chelators as aglycones and demonstrated the feasibility of this concept to assess β-gal activity in solution in the presence of Fe3+ ions by 1H-MRI T1 and T2 relaxation mapping. Aroylhydrazones are in clinical use as Fe3+ scavenging agents; thus, this approach also suggests a potential for clinical theranostic application.

Figure 6. Kinetic hydrolysis time courses of β-D-galactopyranosides. Colorimetric measurements at λmax = 480 nm following addition of βgal (G5160, 20 units) to solutions of 11−13, 16−18, 24, and 26−30 each (2.0 mM) with FAC (1.0 mM) in 2:1 (v/v) DMSO/PBS (0.1 M, pH = 5.5) at 37 °C in different time points. 11 (⧫), 12 (■), 13 (▲), 16 (□), 17 (*), 18 (×), 24 (), 26 (○), 27 (●), 28 (◊), 29 (+), 30 (△).



EXPERIMENTAL SECTION General Methods. NMR spectra were recorded on a Varian Unity INOVA 400 spectrometer (400 MHz for 1H, 100 MHz for 13C). 1H and 13C chemical shifts are referenced to TMS as internal standard with CDCl3, or DMSO-d6 as solvents; chemical shifts are given in ppm. All compounds were characterized by NMR at 25 °C. Microanalyses were performed on a Perkin-Elmer 2400CHN microanalyser. Colorimetric measurements were used on a Hitachi U-2900 spectrophotometer. Solutions in organic solvents were dried with anhydrous sodium sulfate and concentrated in vacuo below 45 °C. 2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl bromide 1 was purchased from the Sigma Chemical Company. β-Gal (G5160) was purchased from the Sigma Chemical Company

H-MRI Detection of β-Gal Activity. Having β-gal substrates in hand prompted us to move on to the detection of β-gal activity using 1H-MRI. As an initial proof of principle, we chose three most reactive β-D-galactopyranosides 11, 13, and 30 as the favored for further evaluation. The T1 and T2 maps were measured for a 4-well plate containing various amounts of β-D-galactopyranosides 11, 13, or 30 to a fixed concentration of FAC (10.0 mM), without or with β-gal (G5160). After addition of β-gal (G5160, 20 units) to the mixture solution of 11 and FAC in DMSO/PBS at 37 °C in 4 h, significant differences in relaxivities Δr1 = 0.16 and Δr2 = 1

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Figure 7. 1H-MRI detection of β-gal activity. 1H-MRI acquisition: using the same parameters as in Figure 3. (A) FAC (10.0 mM); (B) reporter 11 (2.5 mM), FAC (10.0 mM); (C) reporter 11 (2.5 mM), FAC (10.0 mM), β-gal (G5160, 20 units); (D) reporter 11 (3.0 mM), FAC (10.0 mM), βgal (G5160, 20 units) in 2:1 (v/v) DMSO/PBS (0.1 M, pH = 5.5) at 37 °C in 4 h.

Figure 8. 1H-MRI detection of β-gal activity. 1H-MRI acquisition: Using the same parameters as in Figure 3. (A) FAC (10.0 mM); (B) reporter 13 (3.0 mM), FAC (10.0 mM); (C) reporter 13 (3.0 mM), FAC (10.0 mM), β-gal (G5160, 20 units); (D) reporter 13 (3.5 mM), FAC (10.0 mM), βgal (G5160, 20 units) in 2:1 (v/v) DMSO/PBS (0.1 M, pH = 5.5) at 37 °C in 4 h.

and enzymatic reactions were performed at 37 °C in 2:1 (v/v) DMSO/PBS (0.1 M, pH = 5.5) solution. Column chromatography was performed on silica gel (200−300 mesh) and silica

gel GF254 used for analytical thin-layer chromatography (TLC) was purchased from the Aldrich Chemical Co. Detection was effected by spraying the plates with 5% ethanolic H2SO4 1364

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(followed by heating at 110 °C for 10 min.) or by direct UV illumination of the plate. The purity of the final products was determined by HPLC with ≥95%. 3-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)pyridoxal 4 and 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-benzaldehyde 5. General Procedure. A solution of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide 1 (1.23 g, 3.0 mmol) in CH2Cl2 (20 mL) was added dropwise to a vigorously stirred CH2Cl2−H2O biphasic mixture (pH 8− 9) of pyridoxal 2 or salicylaldehyde 3 (3.0 mmol) and tetrabutylammonium bromide (TBAB) (322 mg, 1.0 mmol) in CH2Cl2−H2O (40 mL, 1:1 v/v) over a period of 1 h at 50 °C under N2 atmosphere, and the stirring continued for additional 4−5 h until TLC showed that the reaction was completed. The products were extracted with CH2Cl2 (4 × 35 mL), washed (H2O), dried (Na2SO4), and evaporated under reduced pressure to give a syrup, which was purified by column chromatography on silica gel to give 3-O-(2′,3′,4′,6′-tetra-Oacetyl-β-D-galactopyranosyl)-pyridoxal 4 or 2-O-(2′,3′,4′,6′tetra-O-acetyl-β-D-galactopyranosyl)-benzaldehyde 5. 3-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-pyridoxal 4 (1.06 g, 71%) as white crystals, Rf = 0.52 (3:3:1 cyclohexane−EtOAc−EtOH). Like pyridoxal, 3-O-(2′,3′,4′,6′tetra-O-acetyl-β-D-galactopyranosyl)-pyridoxal 4 also shows no −CHO signal in its 1H/13C NMR. δH (CDCl3): 8.26−6.26 (1 H, m, Ar−H), 5.18 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 5.52 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 5.09 (1 H, dd, J3′,4′ = 4.0 Hz, H-3′), 5.42 (1 H, d, J4′,5′ = 4.0 Hz, H-4′), 4.12 (1 H, m, H-5′), 4.19 (2 H, m, H-6′), 5.28 (1 H, t, JH‑5,HO‑5 = 6.0 Hz, α5-OH), 5.01 (2 H, d, α5-CH2), 2.44 (3 H, s, 2-CH3), 2.21, 2.11, 2.10, 2.02 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 170.96, 170.50, 170.38, 169.63 (4 × CH3CO), 150.12−134.92 (Py-C), 100.10 (C-1′), 69.03 (C-2′), 71.08 (C-3′), 67.35 (C-4′), 71.41 (C-5′), 62.47 (C-6′), 61.58 (α5-CH2), 21.08, 20.93, 20.89, 20.79 (4 × CH3CO), 13.86 (2-CH3) ppm. Anal. Calcd. for C22H27O12N (%): C, 54.12, H, 5.47, N, 2.82; Found: C, 54.10, H, 5.44, N, 2.80. 2-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-benzaldehyde 5 (1.20 g, 85%) as white crystals, Rf = 0.34 (3:2 cyclohexane−EtOAc). δH (CDCl3): 10.36 (1 H, s, −CHO), 7.85 - 7.17 (4 H, m, Ar−H), 5.19 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 5.59 (1 H, dd, J2′,3′ = 10.4 Hz, H-2′), 5.17 (1 H, dd, J3′,4′ = 3.6 Hz, H-3′), 5.49 (1 H, d, J4′,5′ = 3.2 Hz, H-4′), 4.12 (1 H, m, H5′), 4.23 (1 H, dd, J5′,6a′ = 4.4 Hz, J6a′,6b′ = 8.8 Hz, H-6a′), 4.17 (1 H, dd, J5′,6b′ = 6.0 Hz, H-6b′), 2.20, 2.11, 2.06, 2.03 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 189.33 (−CHO), 170.37, 170.25, 170.17, 169.41 (4 × CH3CO), 158.89−115.83 (Ar−C), 99.47 (C-1′), 68.46 (C-2′), 70.66 (C-3′), 66.85 (C-4′), 71.36 (C-5′), 61.39 (C-6′), 20.69, 20.59, 20.54, 20.50 (4 × CH3CO) ppm. Anal. Calcd. for C21H24O11 (%): C, 55.75, H, 5.35; Found: C, 55.73, H, 5.34. 3-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)PIH 6 and 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SBH, -SIH, and -SNH 7−9. General Procedure. A solution of 3-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-pyridoxal 4 or 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-benzaldehyde 5 (0.50 mmol) in anhydrous EtOH (15 mL) containing acetic acid (20 μL) was vigorously stirred respectively with benzoic hydrazide, nicotinic hydrazide, or isoniazid (0.55 mmol, 1.1 equiv) at 80 °C under N2 atmosphere until TLC showed that the reaction was completed and coevaporated with toluene to dryness in vacuo. Crystallization

from EtOH−H2O or chromatography of the crude syrups on silica gel with appropriate eluents accomplished the corresponding 3-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)PIH, 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SBH, 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SIH, and 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-SNH 6−9 in 82−95% yields. 3-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-PIH 6 (264 mg, 82%) as white crystals, Rf = 0.47 (1:1:1 cyclohexane−EtOAc−EtOH). δH (CDCl3): 11.39 (1 H, br, NH, exchangeable with D2O), 8.76 (1 H, s, CHN), 8.74−7.79 (5 H, m, Py-H), 4.83 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 5.53 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 5.14 (1 H, dd, J3′,4′ = 4.0 Hz, H-3′), 5.43 (1 H, d, J4′,5′ = 4.0 Hz, H-4′), 3.91 (1 H, m, H-5′), 4.68 (1 H, dd, J5′,6a′ = 8.2 Hz, J6a′,6b′ = 13.4 Hz, H-6a′), 3.93 (1 H, dd, J5′,6b′ = 6.6 Hz, H-6b′), 5.60 (1 H, br, α5-OH, exchangeable with D2O), 4.73 (2 H, d, JH‑5,HO‑5 = 8.0 Hz, α5-CH2), 2.51 (3 H, s, 2CH3), 2.18, 2.14, 2.02, 1.90 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 171.05, 170.47, 169.99, 169.85 (4 × CH3CO), 162.89 (PhCO), 152.54 - 121.62 (CH=N, Ar−C), 102.43 (C1′), 68.88 (C-2′), 70.61 (C-3′), 67.33 (C-4′), 71.92 (C-5′), 61.58 (C-6′), 61.26 (α5-CH2), 21.03, 20.73, 20.62, 20.45 (4 × CH3CO), 19.70 (2-CH3) ppm. Anal. Calcd. for C28H32N4O12 (%): C, 54.54, H, 5.23, N, 9.09; Found: C, 54.52, H, 5.22, N, 9.07. 2-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-SBH 7 (257 mg, 95%) as white crystals, Rf = 0.67 (2:2:1 cyclohexaneEtOAc-EtOH). δH (CDCl3): 9.84 (1 H, br, NH, exchangeable with D2O), 8.51 (1 H, s, CHN), 8.14−7.01 (9 H, m, Ar−H), 5.07 (1 H, d, J1′,2′ = 7.6 Hz, H-1′), 5.52 (1 H, t, J2′,3′ = 10.0 Hz, H-2′), 5.16 (1 H, dd, J3′,4′ = 3.2 Hz, H-3′), 5.48 (1 H, d, J4′,5′ = 3.2 Hz, H-4′), 4.10 (1 H, m, H-5′), 4.34 (1 H, m, H-6a′), 4.14 (1 H, m, H-6b′), 2.19, 2.07, 2.05, 2.03 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 170.68, 170.31, 170.25, 170.14 (4 × CH3CO), 164.06 (PhCO), 155.79−115.52 (CH=N, Ar−C), 100.53 (C-1′), 69.03 (C-2′), 70.66 (C-3′), 67.02 (C-4′), 71.45 (C-5′), 61.49 (C-6′), 21.05, 20.78, 20.74, 20.71 (4 × CH3CO) ppm. Anal. Calcd. for C28H30N2O11 (%): C, 58.94, H, 5.30, N, 4.91; Found: C, 58.92, H, 5.27, N, 4.89. 2-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-SIH 8 (257 mg, 90%) as white crystals, Rf = 0.56 (2:2:1 cyclohexane−EtOAc−EtOH). δH (CDCl3): 10.43 (1 H, br, NH, exchangeable with D2O), 8.78 (1 H, s, CHN), 8.56−6.70 (8 H, m, Ar−H), 5.07 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 5.54 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 5.16 (1 H, dd, J3′,4′ = 3.2 Hz, H-3′), 5.49 (1 H, d, J4′,5′ = 2.8 Hz, H-4′), 4.12 (1 H, m, H-5′), 4.45 (1 H, dd, J5′,6a′ = 9.2 Hz, J6a′,6b′ = 13.2 Hz, H-6a′), 4.27 (1 H, dd, J5′,6b′ = 6.8 Hz, H-6b′), 2.20, 2.14, 2.10, 2.07 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 171.81, 170.54, 170.30, 170.15 (4 × CH3CO), 162.44 (PyCO), 155.80−115.53 (CH=N, Ar−C), 100.27 (C-1′), 68.99 (C-2′), 70.59 (C-3′), 67.00 (C-4′), 71.52 (C-5′), 61.48 (C-6′), 21.09, 20.79, 20.76, 20.71 (4 × CH3CO) ppm. Anal. Calcd. for C27H29N3O11 (%): C, 56.74, H, 5.11, N, 7.35; Found: C, 56.72, H, 5.10, N, 7.33. 2-O-(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)-SNH 9 (266 mg, 93%) as white crystals, Rf = 0.57 (2:2:1 cyclohexaneEtOAc-EtOH). δH (CDCl3): 10.18 (1 H, s, NH, exchangeable with D2O), 9.15 (1 H, s, CHN), 8.75−6.96 (8 H, m, Ar−H), 5.02 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 5.49 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 5.11 (1 H, dd, J3′,4′ = 4.0 Hz, H-3′), 5.41 (1 H, d, J4′,5′ = 4.0 Hz, H-4′), 4.06 (1 H, m, H-5′), 4.45 (1 H, dd, J5′,6a′ = 1365

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Molecular Pharmaceutics

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8.0 Hz, J6a′,6b′ = 12.0 Hz, H-6a′), 4.27 (1 H, dd, J5′,6b′ = 6.0 Hz, H-6b′), 2.18, 2.04, 2.03, 2.01 (12 H, 4 s, 4 × CH3CO) ppm. δC (CDCl3): 171.84, 170.42, 170.32, 170.18 (4 × CH3CO), 162.32 (PyCO), 156.81−114.78 (CHN, Ar−C), 100.55 (C1′), 68.93 (C-2′), 70.54 (C-3′), 66.95 (C-4′), 71.54 (C-5′), 61.48 (C-6′), 21.13, 20.84, 20.76, 20.70 (4 × CH3CO) ppm. Anal. Calcd. for C27H29N3O11 (%): C, 56.74, H, 5.11, N, 7.35; Found: C, 56.71, H, 5.09, N, 7.32. 3-O-(β-D-Galactopyranosyl)-PIH 10 or 2-O-(β-D-galactopyranosyl)-SBH, -SIH, and -SNH 11−13. General Procedure. A solution of 3-O-(2′,3′,4′,6′-tetra-O-acetyl-β-Dgalactopyranosyl)-PIH 6 or 2-O-(2′,3′,4′,6′-tetra-O-acetyl-β-Dgalactopyranosyl)-SBH, -SIH, and -SNH 7−9 (250 mg) in anhydrous MeOH (50 mL) containing 0.5 M NH3 was vigorously stirred from 0 °C to room temperature overnight until TLC showed that the reaction was completed, and evaporated to dryness in vacuo. Chromatography of the crude syrup on silica gel with ethyl acetate−methanol afforded the corresponding 3-O-(β-D-galactopyranosyl)-PIH 10 or 2-O-(βD-galactopyranosyl)-SBH 11, 3-O-(β-D-galactopyranosyl)-SIH 12, and 3-O-(β-D-galactopyranosyl)-SNH 13 in high yields (91−94%). 3-O-(β-D-Galactopyranosyl)-PIH 10 (166 mg, 91%), Rf = 0.36 (1:3 MeOH-EtOAc). δH (DMSO-d6): 11.87 (1 H, s, NH, exchangeable with D2O), 8.89 (1 H, s, CHN), 8.81−7.58 (5 H, m, Py-H), 4.52 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 3.69 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 3.57−3.17 (5 H, m, H-3′, 4′, 5′, 6′), 5.40−4.90 (5 H, br, HO-2′,3′,4′,6′ and α5-OH, exchangeable with D2O), 4.64 (2 H, d, JH‑5,HO‑5 = 4.4 Hz, α5-CH2), 2.58 (3 H, s, 2-CH3) ppm. δC (DMSO-d6): 162.50 (PyCO), 152.32− 121.63 (CHN, Py-C), 105.56 (C-1′), 71.09 (C-2′), 73.09 (C-3′), 67.80 (C-4′), 75.22 (C-5′), 60.34 (C-6′), 59.75 (α5CH2), 19.68 (2-CH3) ppm. Anal. Calcd. for C20H24N4O8 (%): C, 53.57, H, 5.40, N, 12.49; Found: C, 53.54, H, 5.38, N, 12.46. 2-O-(β-D-Galactopyranosyl)-SBH 11 (190 mg, 94%) as white crystals, Rf = 0.46 (1:3 MeOH−EtOAc). δH (DMSO-d6): 11.89 (1 H, s, NH, exchangeable with D2O), 8.84 (1 H, s, CHN), 7.95−7.09 (9 H, m, Ar−H), 4.97 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 3.69 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 3.61 (1 H, dd, J3′,4′ = 4.0 Hz, H-3′), 3.57 (1 H, d, J4′,5′ = 3.6 Hz, H-4′), 3.42 (1 H, m, H5′), 3.74 (1 H, m, H-6a′), 3.48 (1 H, m, H-6b′), 5.09 (1 H, d, JH‑2′,OH‑2′ = 4.0 Hz, HO-2′, exchangeable with D2O), 4.62 (1 H, d, JH‑3′,OH‑3′ = 4.0 Hz, HO-3′, exchangeable with D2O), 4.99 (1 H, d, JH‑4′,OH‑4′ = 4.0 Hz, HO-4′, exchangeable with D2O), 4.73 (1 H, t, JH‑6′,OH‑6′ = 4.0, 6.0 Hz, HO-6′, exchangeable with D2O) ppm. δC (DMSO-d6): 163.64 (PhCO), 156.22−115.85 (CH N, Ar−C), 101.51 (C-1′), 70.60 (C-2′), 73.29 (C-3′), 68.13 (C-4′), 75.71 (C-5′), 60.43 (C-6′) ppm. Anal. Calcd. for C20H22N2O7 (%): C, 59.70, H,5.51, N, 6.96; Found: C, 59.68, H,5.50, N, 6.95. 2-O-(β-D-Galactopyranosyl)-SIH 12 (162 mg, 92%) as white crystals, Rf = 0.35 (1:3 MeOH−EtOAc). δH (DMSO-d6): 8.89 (1 H, s, NH, exchangeable with D2O), 8.79 (1 H, s, CHN), 7.93−7.10 (8 H, m, Ar−H), 4.98 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 3.69 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 3.48 (1 H, dd, J3′,4′ = 4.0 Hz, H-3′), 3.42 (1 H, d, J4′,5′ = 3.8 Hz, H-4′), 3.52 (1 H, m, H5′), 3.62 (1 H, dd, J5′,6a′ = 4.0 Hz, J6a′,6b′ = 8.0 Hz, H-6a′), 3.55 (1 H, dd, J5′,6b′ = 6.2 Hz, H-6b′), 5.20−4.50 (4 H, br, HO2′,3′,4′,6′, exchangeable with D2O) ppm. δC (DMSO-d6): 161.67 (PyCO), 156.39−115.68 (CHN, Ar−C), 101.39 (C1′), 70.60 (C-2′), 73.34 (C-3′), 68.14 (C-4′), 75.75 (C-5′), 60.42 (C-6′) ppm.

Anal. Calcd. for C19H21N3O7 (%): C, 56.57, H, 5.25, N, 10.42; Found: C, 56.54, H, 5.22, N, 10.39. 2-O-(β-D-Galactopyranosyl)-SNH 13 (166 mg, 94%) as white crystals, Rf = 0.33 (1:3 MeOH−EtOAc). δH (DMSOd6): 12.04 (1 H, br, NH), 8.85 (1 H, s, CHN), 8.79−7.10 (8 H, m, Ar−H), 4.98 (1 H, d, J1′,2′ = 8.0 Hz, H-1′), 3.63 (1 H, dd, J2′,3′ = 10.0 Hz, H-2′), 3.75 (1 H, dd, J3′,4′ = 3.8 Hz, H-3′), 3.47 (1 H, d, J4′,5′ = 4.0 Hz, H-4′), 3.68 (1 H, m, H-5′), 3.57 (1 H, dd, J5′,6a′ = 4.0 Hz, J6a′,6b′ = 12.0 Hz, H-6a′), 3.50 (1 H, dd, J5′,6b′ = 6.6 Hz, H-6b′), 5.12 (1 H, d, JH‑2′,OH‑2′ = 8.0 Hz, HO-2′, exchangeable with D2O), 4.64 (1 H, d, JH‑3′,OH‑3′ = 4.0 Hz, HO3′, exchangeable with D2O), 5.01 (1 H, d, JH‑4′,OH‑4′ = 4.0 Hz, HO-4′, exchangeable with D2O), 4.74 (1 H, t, JH‑6′,OH‑6′ = 4.0, 8.0 Hz, HO-6′, exchangeable with D2O) ppm. δC (DMSO-d6): 161.75 (PyCO), 156.30−115.70 (CHN, Ar−C), 101.35 (C1′), 70.61 (C-2′), 73.36 (C-3′), 68.15 (C-4′), 75.74 (C-5′), 60.46 (C-6′) ppm. Anal. Calcd. for C19H21N3O7 (%): C, 56.57, H, 5.25, N, 10.42; Found: C, 56.55, H, 5.23, N, 10.40. MRI. 1H-MRI studies were performed using a 4.7 T horizontal bore magnet equipped with a Varian INOVA Unity system (Palo Alto, CA, USA). All images were collected with a matrix size of 128 × 128, a 2 mm slice thickness without gap, a 40 mm field of view (FOV), and bandwidth of 20 kHz. A saturation recovery spin−echo sequence was performed for quantitative T1 measurement with varying repetition times (TR = 200, 400, 600, 800, 1000, 2000, 3000, and 6000 ms) and echo times (TE = 15 ms), respectively. The T1 maps were obtained on a voxel-by-voxel basis using nonlinear least-squares fit the equation M = M0(1 − e−TR/T1) from the eight images taken at each TR. The T2 values were evaluated based on the multiecho SE images collected at 16 different TE choices ranging from 10 ms to 160 ms with 10 ms spacing, TR = 2000 ms. The T2 maps were obtained on a voxel-by-voxel basis using nonlinear leastsquares fit the equation M = M0e−TE/T2 from the 16 images taken at each echo time. Images were reconstructed and analyzed by using MatLab (Mathworks, Natick, MA, USA).



ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3, syntheses and molecular characterization of T1−T17, M1−M10, 14−30, and 1H-MRI detection of β-gal activity using reporter 30. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: 214-648-2716. Fax: 214-648-4538. E-mail: jian-xin. [email protected]. Present Address ∥

Summer student from Jasper High School, 6800 Archgate Dr., Plano, Texas 75024, USA. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by the DOD Prostate Cancer New Investigator Award W81XWH-05-1-0593. NMR & MRI experiments were performed at the Advanced Imaging Research 1366

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Molecular Pharmaceutics

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Center. We are grateful to Jennifer McAnally for expert technical assistance.



ABBREVIATIONS MRI,magnetic resonance imaging; NMR,nuclear magnetic resonance; TR,repetition time; TE,echo time; β-gal,β-galactosidase; PIH,pyridoxal isonicotinoylhydrazone; SBH,salicylaldehyde benzoylhydrazone; SIH,salicylaldehyde isonicotinoylhydrazone; SNH,salicylaldehyde nicotinoylhydrazone; DMSO,dimethyl sulfoxide; FAC,ferric ammonium citrate; PBS,phosphate buffered saline; TLC,thin layer chromatography



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dx.doi.org/10.1021/mp300627t | Mol. Pharmaceutics 2013, 10, 1360−1367