Introduction of Peripheral Carboxylates to Decrease the Charge on

Article pubs.acs.org/jmc

Introduction of Peripheral Carboxylates to Decrease the Charge on Tm3+ DOTAM-Alkyl Complexes: Implications for Detection Sensitivity and in Vivo Toxicity of PARACEST MRI Contrast Agents Mojmír Suchý,†,‡ Mark Milne,† Adam A. H. Elmehriki,† Nevin McVicar,‡ Alex X. Li,‡ Robert Bartha,‡ and Robert H. E. Hudson*,†,§ †

Department of Chemistry, The University of Western Ontario, Chemistry Building, London, Ontario N6A 5B7, Canada Department of Medical Biophysics, The University of Western Ontario, London, Ontario N6A 5K8, Canada § Centre for Advanced Materials and Biomaterials Research, The University of Western Ontario, London, Ontario N6A 5B7, Canada ‡

S Supporting Information *

ABSTRACT: A series of structurally modified Tm 3+ DOTAM-alkyl complexes as potential PARACEST MRI contrast agents has been synthesized with the aim to decrease the overall positive charge associated with these molecules and increase their biocompatibility. Two types of structural modification have been performed, an introduction of terminal carboxylate arms to the alkyl side chains and a conjugation of one of the alkyl side chains with aspartic acid. Detailed evaluation of the magnetic resonance imaging chemical exchange contrast associated with the structurally modified contrast agents has been performed. In contrast to the acutely toxic Tm3+ DOTAMalkyl complexes, the structurally modified compounds were found to be tolerated well during in vivo MRI studies in mice; however, only the aspartic acid modified chelates produced an amide proton-based PARACEST signal.

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INTRODUCTION Currently, there are several groups of magnetic resonance imaging (MRI) contrast agents (CAs) available,1 including Gd3+ chelates,2 Mn2+ chelates,3 superparamagnetic iron oxideparticles (SPIO),4 and hyperpolarized nuclei such as Xe.5 Paramagnetic chemical exchange saturation transfer (PARACEST) MRI CAs6 are complexes of multidentate ligands with lanthanides (Dy3+, Eu3+, Nd3+, Tb3+, Tm3+, Yb3+) or transition metals (Co2+, Fe2+, Ni2+)]7 that offer several advantages compared to the aforementioned groups of MRI CAs. The contrast associated with PARACEST MRI CAs is generated by selective radiofrequency saturation pulses applied to water bound to the metal center or other exchangeable protons within the close proximity of the paramagnetic cation. These exchangeable protons are in dynamic chemical exchange with bulk water.6 PARACEST MRI CAs possess an excellent environmental sensitivity and are capable of sensing important physiological parameters (e.g., temperature,8 pH9,10), the concentration of physiologically important cations and anions (e.g., Zn2+, phosphate),11 the concentration of primary metabolites (e.g., glucose),12 and various enzymatic activities (e.g., caspase 3).13 Moreover the MRI contrast generated when using PARACEST MRI CAs can be turned on and off at will,6 offering a significant advantage in comparison to the widely used Gd3+-based CAs, where the contrast is always on. Despite the many advantages listed above, the in vivo use of PARACEST MRI CAs is hampered by severe limitations. Upon administration to biological system, the PARACEST MRI CAs suffer a significant sensitivity decrease attributable in a large © 2015 American Chemical Society

part to concomitant magnetization transfer (MT) from endogenous macromolecules.14 The MT effect is a competing mechanism that saturates the bulk water spins and consequently lowers the contrast efficiency of PARACEST MRI CAs within the MT frequency range (ca. −100 to 100 ppm). Several research groups including ours have investigated the use of PARACEST MRI CAs for in vivo studies with limited success. To enhance in vivo PARACEST contrast detection, various pulse sequence modifications have been proposed including a method called on resonance paramagnetic chemical exchange effects (OPARACHEE),15 along with the use of different pulse sequences such as fast low angle shot (FLASH),16 fast imaging with steady state precession (FISP),17 and frequency labeled exchange transfer (FLEX).18 Another feasible alternative leading to in vivo contrast enhancement is to increase the payload of PARACEST MRI CAs by utilizing nanoparticle-19 or dendrimer-based CAs20 or discrete “small molecule-based” targeted CAs (e.g., glucose targeted CA 1,21 Figure 1, detected in mouse liver). Considering the effective range of endogenous MT effects (ca. −100 to 100 ppm), the design of PARACEST MRI CAs with exchangeable proton frequencies shifted near or outside of this range might offer a solution to the in vivo sensitivity problems caused by endogenous MT. Toward this end, Aime’s group has recently investigated Yb3+-HPDO3A (2, Figure 1), which has two exchangeable protons at ca. 66 and 92 ppm.22 This CA, a Received: April 23, 2015 Published: July 27, 2015 6516

DOI: 10.1021/acs.jmedchem.5b00621 J. Med. Chem. 2015, 58, 6516−6532

Journal of Medicinal Chemistry

Article

Figure 1. Structures of MRI CAs 1−6.

temperature and pH mapping as shown upon injection of 3 directly into a mouse leg muscle.10 Unfortunately, the in vivo sensitivity associated with CA 3 (amide-based CEST effect at −47 ppm) is severely attenuated by the endogenous MT effect. We have been investigating alternative molecular designs with the aim to increase the chemical shift of the exchangeable amide-proton. Our efforts led to the preparation of two CAs Tm3+ DOTAM-n-hexyl (4a, Figure 1) and Tm3+ DOTAM-t-butyl (4b, Figure 1)26 with exchangeable proton chemical shifts (4a, −87 ppm, 6% and −94 ppm, 17%; 4b, −68 ppm, 10% and −102 ppm, 21%) located near the border of the endogenous MT frequency range in bovine serum albumin (BSA) phantom studies.26 The presence of two exchangeable proton frequencies was attributed to the SAP/TSAP isomerism (SAP, square antiprism; TSAP,

chemical analogue (Yb versus Gd) of the clinically approved CA (Gadoteridol or ProHance) was recently used to map the extracellular pH in murine melanoma. The favorable outcome of these studies support the potential clinical translation of Yb3+-HPDO3A (2) for human use.22 However, promising this approach is, it would be instructive to examine lanthanides that can produce larger hyperfine shifts to avoid overlap with the MT frequency range. As an alternative approach to the aforementioned, we have recently developed a dipeptide decorated PARACEST MRI CA Tm3+ DOTAM-Gly-Lys-OH23 (3, Figure 1) and investigated its potential in vivo application. We have detected the CA (upon intravenous administration of 150 μL of 50 mM solution) in mouse kidneys24 and xenograft mouse brain tumors.25 Moreover, 3 can also be utilized for simultaneous in vivo 6517



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Scheme 1. Preparation of Electrophiles 8a−8c, 9a−9c, 12a−12c, and 13a−13c

water exchange kinetics. Conjugation of Asp-OH with CA 4c was also performed. The overall charge of these Asp-modified CAs was positive (+1) presuming complete ionization; however, we have previously shown that Tm3+ DOTAM-GlyLys-OH (3, Figure 1), bearing an overall positive charge at pH 6−7, is compatible with in vivo studies and can be administered safely in relatively high (50 mM) concentrations.10,24,25 The structures of Asp-modified CAs 6a−6c are depicted in Figure 1. In addition to 6a−6c, we have also prepared the conjugate 6d because the unmodified “parent” Tm3+ DOTAM-propargyl (4d) exhibits a relatively strong CEST effect (16% at −54 ppm),29 comparable in magnitude to the CEST effect associated with 3. Complex 4d also exhibits nonexchangeable proton signals associated with the cyclen moiety whose chemical shift respond sensitively to temperature (1.05 ppm/ °C), offering the potential for 1H magnetic resonance spectroscopy (MRS) temperature mapping.30 Chemical synthesis of CAs 5a−5c and 6a−6d is described along with detailed investigation of the chemical exchange mediated contrast associated with these compounds. As described below, Asp-modified CAs 6a−6d are compatible with in vivo studies, moreover, the CEST sensitivities of 6a−6d are similar to those of the unmodified “parent” CAs 4a−4d, suggesting, that conjugation with Asp-OH might be used in the future as an efficient tool to decrease the overall charge of the CAs while maintaining desirable CEST contrast.

twisted square antiprism) as previously described for other Tm3+ DOTAM-derived complexes.27 Although the detailed BSA phantom studies performed with CA 4b indicated that this agent may be a good candidate for measuring in vivo temperature;26a the results obtained after in vivo administration were quite disappointing. Contrast agent 4b, which possesses a +3 overall charge, was found to be acutely toxic to mice, causing death within seconds of intravenous administration or direct injection into leg muscle. This observation is consistent with the results reported by other researchers, wherein a small series of overall positively charged CAs was found to be acutely toxic.28 We decided to modify the original chemical structure of agents 4a and 4b by introduction of carboxylate functionality on the termini of each side chain. This modification would produce CAs with overall negative (−1) charge presuming a completely ionized state. Similarly, we have also modified the Tm3+ DOTAM-benzyl (4c, Figure 1) that exhibits a strong amide-based CEST effect26b (27% at −51 ppm). The structures of modified CAs 5a−5c are depicted in Figure 1. As shown later, this structural modification had a detrimental effect on chemical exchange magnetic properties (no CEST effect observed for 5a−5c), presumably due to significant changes in water exchange kinetics. Other structural modifications have been considered and the conjugation of one of the four side chains with L-aspartic acid (Asp-OH) presented itself as a feasible alternative. We speculated that this remote modification may, to a large extent, preserve the original coordination geometry (both SAP and TSAP isomers present) of 4a and 4b as well as cause less of a change to the favorable 6518



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RESULTS AND DISCUSSION

Scheme 2. Preparation of CAs 5a−5c

Chemistry. The approach to CAs 5a−5c is based on the methodology previously developed in our laboratory based on the alkylation of cyclen or its derivatives by amino acid Nhaloacetyl derivatives, Scheme 1.23,26b 6-Aminohexanoic acid (7a, Scheme 1), 3-amino-3-methylbutyric acid (7b, Scheme 1), and 4-aminomethyl benzoic acid (7c, Scheme 1) were converted in quantitative yields to the corresponding esters by treatment with SOCl2 in MeOH. Acylation of the esters derived from acids 7a and 7b with chloroacetyl chloride under previously established conditions was found to be troublesome (i.e. resulting in complex mixtures of products), therefore peptide coupling between chloroacetic acid and corresponding esters31 was used and provided the Nchloroacetyl derivatives 8a in 82% yield (based on 7a, Scheme 1) and 8b (57%, based on 7b). Acylation of 4-aminomethyl benzoic acid (7c) with chloroacetyl chloride produced the desired N-chloroacetyl derivative 8c (Scheme 1), albeit in poor yield (28%). Conversion 8a−8c to the N-iodoacetyl-derived electrophiles 9a−9c via the Finkelstein reaction (NaI/acetone)23 proceeded smoothly and in good yield (58−86%, Scheme 1). 1H and 13C NMR spectra associated with N-haloacetyl derivatives 8a−8c and 9a−9c can be found in the Supporting Information. With electrophiles 9a−9c in hand, the peralkylation of cyclen23 (10, Scheme 2) was carried out, followed by treatment of the corresponding ligands with TmCl3·H2O first under slightly basic conditions (NaOH, pH ∼ 9) to achieve the hydrolysis of the esters and then under slightly acidic conditions (HCl, pH ∼ 6) to achieve the quantitative metalation.9 CAs 5a−5c were obtained in reasonable yields (37−53%, based on 10, Scheme 2), they were purified as described in the Experimental Section, and were characterized by high resolution mass spectrometry (HR-MS) as shown in Experimental and in the Supporting Information. A slightly different methodology has been utilized to prepare the Asp-modified CAs 6a−6c. Amino acids 7a−7c were acylated with chloroacetyl chloride (Scheme 1) as described in the literature.32 The N-chloroacetyl amino acids 11a and 11c were obtained as colorless solids, while 11b, known to be an oil,33 was obtained by solvent extraction (see Experimental Section for details) along with a small amount of chloroacetic acid (formed upon hydrolysis of chloroacetyl chloride), which was ultimately removed by flash column chromatography (FCC). Amino acids 11a−11c were subjected to peptide coupling with L-Asp-(OMe)2·HCl using N-hydroxysuccinimide/dicyclohexylcarbodiimide23 (NHS/DCC, 12a and 12b, Scheme 1) or propanephosphonic acid anhydride34 (T3P, 12c, Scheme 1). N-Chloroacetyl derivatives 12a−12c were prepared in moderate yields (43−66%), and characterization data for 12a−12c can be found in the Experimental Section and Supporting Information. Finkelstein reaction23 of 12a and 12c gave the desired N-iodoacetyl derivatives 13a and 13c (Scheme 1). The same reaction was carried out with 12b, and the desired N-iodoacetyl derivative 13b was detected by HR-MS analysis of the crude reaction mixture but decomposed during FCC purification (Scheme 1). Fortunately, the N-chloroacetyl derivative 12b was found to be sufficiently reactive for the subsequent step. After having prepared the electrophiles, we first investigated the possibility of selective monoalkylation of cyclen35 10 with electrophiles 12b, 13a, and 13c; however, the

results of these studies were rather disappointing. The problem was solved by use of tri-Boc-cyclen36 (14, Scheme 3). Alkylation of 14 with electrophiles 12b, 13a, and 13c proceeded smoothly affording the intermediates 15a−15c in high yields (70−94%, Scheme 3). The Boc groups were removed by trifluoroacetic acid (TFA)/triethylsilane (TES) promoted acidolysis (Scheme 3), followed by alkylation with Niodoacetyl-n-hexylamine26b (to prepare the ligand 16a), Niodoacetyl-t-butylamine26b (to prepare the ligand 16b), or Niodoacetyl-benzylamine26b (to prepare the ligand 16c, Scheme 3). Treatment of ligands 16a−16c with TmCl3·H2O (Scheme 3) was performed as described above, leading to CAs 6a−6c in moderate yields (16−48%) and quantities sufficient for the detailed testing of magnetic properties associated with 6a−6c. The intermediates 15a−15c and 16a−16c were characterized by 1H NMR spectroscopy and HR-MS, and CAs 6a−6c were characterized by HR-MS (see Experimental Section and Supporting Information for further details). A different synthetic methodology has been utilized to prepare the propargyl-containing CA 6d. Literature protocols37 have been applied to convert but-2-yne-1,4-diol (17) to 1,4dichlorobut-2-yne (18, Scheme 4), followed by nucleophilic 6519



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Scheme 3. Preparation of CAs 6a−6c

but-2-yne-1,4-diamine39 (19, Scheme 4) in 18% overall yield (based on 17). Acylation of 19 with chloroacetyl chloride provided an electrophile 20 in 64% yield (Scheme 4, see Experimental Section and Supporting Information for characterization). Our initial intention was to assemble the CA 6d in fashion similar to CAs 6a−6c. To proceed, we first removed the Boc group from 20 (proceeded without difficulties, data not shown), followed by treatment with racemic 2-bromosuccinic acid methyl ester. Unfortunately, this later step proved difficult, and we were unable to obtain the desired alkylation product in reasonable yield. Our results indicated that an alternative synthetic strategy leading to CA 6d was required. Delépine reaction of 1,4-dichlorobut-2-yne (18, Scheme 4) with hexamethylenetetraamine, followed by hydrolysis of the quarternary ammonium salt40 and protection of the amine (Boc),41 afforded N-Boc-4-chlorobut-2-ynylamine (21, Scheme 4) in 45% overall yield (based on 18). LiOH-mediated alkylation42 of Asp(OMe)2·HCl with chloroderivative 21 afforded the conjugate 22 in 46% yield (Scheme 4). t-Butyl cyclen-N-monoacetate43 (23) was alkylated with N-chloroacetyl-propargylamine,9 affording the ligand 2444 in moderate yield (48%, Scheme 5). The acid labile protecting groups (Boc in 22 and t-butyl ester in 24) were removed by TFA-mediated acidolysis to give intermediates 25 and 26, which were coupled together by means of 1-benzotriazolyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole

Scheme 4. Preparation of Intermediates 20 and 22

replacement of the chlorine atoms with NaN3, PPh3-mediated reduction38 and selective Boc protection to afford mono-N-Boc 6520



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possessing the same ligand framework as 4b and 5b have been investigated, albeit for direct water exchange at the metal center.46 The CEST effect due to the metal-bound water of the Eu3+ DOTAM-t-butyl complex was found to be rather weak due to the faster-than-optimal exchange rate between bound and bulk pools. When carboxylate functionalities were introduced (Eu3+ analogue of 5b), the exchange rate decreased to a more favorable regime and the CEST effect became more prominent.47 In our case, although we are considering the exchange at amide protons, the change of exchange rate precipitated by the peripheral modification and attendant changes in solvation appeared to be detrimental, consequently, the CAs 5a−5c did not exhibit any observable CEST effect. To our delight, more favorable results have been obtained for aspartic acid-modified CAs 6a−6d. CEST spectra associated with 6a−6d have been acquired in water (37 °C, pH 7.0) as described in the General Experimental Procedures. The results (Figure 2, Table 1) have been compared to those obtained for unmodified congeners 4a−4d.26,29 Some general trends were observed when comparing the CEST effects associated with 4a−4d to those associated with 6a−6d. In general, conjugation of the Asp-OH moiety causes a slight decrease in the chemical shift of the exchangeable proton relative to bulk water that is set to 0 ppm. Although this decrease is quite small for almost all CEST effects (1−4 ppm, Table 1), it is more prominent in the case of n-hexyl decorated CAs 4a and 6a, wherein the chemical shift of the exchangeable proton closer to bulk water is shifted by 30 ppm upon conjugation with Asp-OH. The chemical shift of the exchangeable proton further from bulk water, on the other hand, follows the general trend outlined above (3 ppm shift). The magnitude of the CEST effect associated with Asp-OH modified agents is also significantly reduced to ca. one-third (Table 1), when compared to the unmodified congener CAs with two notable exceptions. The CEST effects (with the chemical shift closer to bulk water) of 4a and 6a have the same intensity (6%, Table 1). Only a small decrease of the CEST effect associated with CA 6d is observed upon comparison with the “parent” CA 4d29 (22% for 6d versus 25% for 4d, Table 1). Overall, the results indicate that Asp-OH conjugates 6a and 6b produced appreciable CEST contrast using exchangeable protons with chemical shifts near the limit of the endogenous MT effect. The conjugation with Asp-OH maintained the sensitivity of propargyl-decorated CA 6d, while it was decreased for benzyl-decorated CA 6c. As described below, CAs 6a−6d were found to be compatible with in vivo studies, suggesting that the conjugation with Asp-OH might be used as general strategy for the preparation of CAs with diminished overall charged while maintaining a degree of their desirable magnetic properties. Rates of Chemical Exchange. The exchange rate constant of the amide protons (kex) with bulk water were measured by the Ω-plot method48 and are reported as residence lifetimes (τ = 1/kex) in Table 2. The longer the residence lifetime of the amide proton represents slower exchange with the bulk water. The rate of exchange is an important factor for being able to observe a CEST signal and must satisfy the slow-tointermediate exchange regime under the conditions of measurement as expressed by τ*Δω > 1, where Δω represents the frequency difference between exchanging pools of protons.49 Chelates 4a−4d showed clear CEST signals but were acutely toxic to mice. Modification of the ligand structure to include peripheral carboxylate groups, presumably reduced

Scheme 5. Preparation of CA 6d

(HBTU/HOBt)-mediated peptide coupling45 (Scheme 5). Ligand 27 was purified by high pressure liquid chromatography (HPLC) and was obtained in 14% yield. Treatment of 27 with TmCl3·H2O was carried out as described above, providing the CA 6d in 47% yield (Scheme 5, see Experimental Section and Supporting Information for the spectral characterization). CEST Spectra. After having the CAs 5a−5c and 6a−6d in hand, we acquired the corresponding CEST spectra with the intention to compare them to the CEST spectra from the unmodified “parent” CAs 4a−4d.26,29 We first attempted to acquire the CEST spectra associated with tetracarboxylatemodified CAs 5a−5c. Despite of varying the temperature (20− 37 °C), concentration (10−100 mM) and pH (phosphate buffered saline pH 5.0−7.5), we were unable to observe any CEST effect associated with CAs 5a−5c. A possible explanation of this rather discouraging result is based on alteration of amide proton exchange rate, which is presumably strongly influenced by the change in second sphere solvation due to the presence of terminal COOH functionalities. This notion is supported by recent work from Sherry’s group, wherein Eu3+ complexes 6521



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Figure 2. CEST spectra of CAs 4a−4d (left) and 6a−6d (right). The spectra were acquired at 37 °C, saturation power 15 μT, concentration 10 mM, pH 7.0, at 600 MHz.

the overall charge of the complexes from +3 to −1 (considering a fully ionized species) and eliminated the acute toxicity but also undesirably eliminated the CEST signals. Clearly, the peripheral modification had changed the amide proton exchange rate to outside the range that is useful for generating a CEST signal. Because no CEST signal was observed, we could not apply the Ω-plot method to determine the exchange rates.

However, when only one arm of the ligand was modified with an asparate residue to produce chelates 6a−6d and reduce the overall charge to +1 (for the fully ionized species), CEST signals were observable and exchange rates were measured. The exchanges rates are reported in Table 2 for each signal observed with the exception of the weaker signals in 4a and 6b, which could not be accurately measured. These measurements were 6522



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effect on the exchange of amide protons ascribed the highshifting isomer. Compound 4c (benzyl-type amide substituent) shows a larger effect when compared to 6c, that is, a significant increase in the exchange reflected by a decreased lifetime. However, 4d (propargyl amide substituent), that like 4c shows only a CEST effect associated with a SAP conformer, shows a slight decrease of the exchange rate. Sherry and co-workers have evaluated the effect of polar amide substituents on the water exchange of some similar Eu3+ chelates and determined that more polar substituents slow the exchange rate, presumably by structuring second-sphere water of solvation.46 The present case involves amide−proton exchange rather than direct water exchange at the metal center; however, factors that affect the rate of exchange of the bulk solvent with metal-bound water might also reasonably be expected to affect water access and exchange rates at the amide group in a parallel fashion, yet this is not observed. Besides a change in the exchange rate that could diminish the observed CEST signal intensity, compounds 6a−6d are unsymmetrical and it is a possibility that the overall CEST signal is divided over several peaks, some that may be too weak to observe. Relaxation Evaluation. Another recent study by Sherry and co-workers has shown that PARACEST CAs containing inner sphere water can produce sizable relaxation effects (mainly T2), resulting in image darkening during in vivo PARACEST imaging.50 To overcome this problem, we have recently studied a small series of Tm3+ based anilide PARACEST MRI CAs,51 which do not contain inner sphere water or the water exchange rates lie outside the optimum exchange regime. An in vitro phantom study clearly demonstrated, that T2 relaxation effects are negligible for Tm3+ based anilide PARACEST MRI CAs, while significant image darkening is observed for Tm3+ DOTAM-Gly-Lys-OH (3, Figure 1), known to possess inner sphere water.51 Bearing this knowledge in mind, we decided to evaluate the relaxivities associated with Tm3+ DOTAM-alkyl-based CAs 4a− 4d, 5a−5c, and 6a−6d. The results of our studies are listed in Table 3. Tm3+ DOTAM complexes are known to be poor T1

Table 1. Magnitudes and Chemical Shifts (ppm) of CEST Effects Associated with CAs 4a−4d and 6a−6d at 600 MHza CA no.

CEST (%)

δ (ppm)

4a

6 17 10 21 27 25 6 5 3 7 10 22

−87 −94 −68 −102 −51 −54 −57 −91 −66 −100 −50 −49

4b 4c 4d 6a 6b 6c 6d a

[CA] = 10 mM, 37 °C, pH 7.0, saturation power 15 μT.

Table 2. Chemical Shift (δ), Lifetime (τ), and CEST Signal Strength (at 18μT) for Amide Protons Assigned to Low Shifting (LS) or High Shifting (HS) Complexes at pH 7.0, [CA] = 20 mMa

Table 3. Longitudinal (r1) and Transverse (r2) Relaxivities in (s−1 mM−1) Associated with CAs 4a−4d, 5a−5c, and 6a−6d

a

*At 18 μT.

made as detailed in the Experimental Section; however, it is key to note that the agent concentration and saturation powers for these measurements are different than used for Table 1, and hence the magnitude of the CEST signal varies. The CEST effect signals observed fall into two arbitrary chemical shift ranges labeled as “low shift” (δ < 90 ppm), which is characteristic of known Tm3+ DOTAM derivatives in the square antiprismatic (SAP) conformation, and “high shift” (δ > 90 ppm), which, by analogy,26 may be produced from Tm3+ DOTAM derivatives in the twisted square antiprismatic (TSAP) conformation. Unfortunately, a simple trend cannot be elucidated from the data in Table 2. For instance, introduction of a charged carboxylate group into a hydrophobic alkyl amide (e.g., 4a, hexyl; or 4b, t-butyl) had only a small

compd

r1

r2

4a 4b 4c 4d 5a 5b 5c 6a 6b 6c 6d

0.04 0.01 0.05 0.05 0.06 0.02 0.01 0.05 0.03 0.02 0.06

0.74 0.15 2.28 2.43 1.26 0.16 0.42 0.26 0.28 0.53 3.46

relaxation agents (low longitudinal relaxivity r1) for water; our findings are fully consistent with this observation (r1 ≤ 0.06 s−1 mM−1, Table 3). Transverse relaxivities (r2) of water for chelates 4a−4d, 5a−5c, and 6a−6d range between 0.15 and 3.46 s−1 mM−1 (Table 3), indicating that inner sphere water might be present in some of these CAs (high r2, short T2) while absent or exchanging at rates unfavorable to produce T2 effects in other members of the series (low r2, long T2). The 6523



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propargyl-decorated CAs 4d and 6d possess high r2 values, suggesting the presence of inner sphere water, it appears to be absent (or the water exchange rates lie outside the optimum exchange regime) in t-butyl decorated CAs 4b, 5b, and 6b. The difference in water exchange rates at the metal center can be attributed to different coordination geometries; the Tm3+ center is likely significantly more accessible in the presence of rather small propargyl groups as opposed to bulky lipophilic tbutyl groups. In the case of hexyl- (4a, 5a and 6a) and benzyldecorated (4c, 5c, and 6c) complexes, the r2 values appear to be more dependent on the overall structure of the complex as indicated by results in Table 3. This effect is more prominent for benzyl-decorated CAs, possessing high r2 value (2.28 s−1 mM−1) for unmodified Tm3+ DOTAM-benzyl (4c, Table 3), while introduction of COOH functionalities or conjugation with Asp-OH results in marked decrease of r2 values (5c, 0.42 s−1 mM−1; 6c, 0.53 s−1 mM−1; Table 3). Overall, our results indicate that T2 relaxation effects during in vivo studies should be negligible for CAs 6a−6c, while significant T2 related signal loss can be expected for propargyl-decorated conjugate 6d. In Vivo Toxicity. As noted previously, Tm3+ DOTAMalkyl-based CAs 4a−4d were found to be acutely toxic, causing the death of laboratory mice within seconds of administration by either tail vein or intramuscular (leg) injection of a 10 mM solution. The toxicity was attributed to the overall 3+ charge associated with these complexes; similar problems with positively charged MRI CAs have been observed previously.28 Tetracarboxylate modified CAs 5a−5c (presumed overall charge −1) have been found to be compatible with potential in vivo studies, as the mice injected with 50 mM (phosphate buffered saline) solutions of 5a−5c remained alive and appeared healthy for an extended period of time (up to 1 month). Although no imaging studies were performed with these CAs because they did not produced measurable CEST contrast, the ligand framework present in 5a−5c might however serve for the preparation of Dy3+ based T2 exchange CAs or 1H MRS thermometry probes.52 The conjugation of Asp-OH to 4a−4d to produce 6a−6d eliminated the acute toxic effects associated with the former compounds. CAs 6a−6d bear an overall charge of +1 (completely ionized species) and appear to be fully compatible with in vivo studies, suggesting that charge distribution in 6a− 6d plays a significant role. Tail vein or leg muscle injection of 50 mM solution (phosphate buffered saline) of 6a−6d was tolerated well, and mice remained alive and appeared healthy for a period of several weeks after administration. Herein we suggest that conjugation with Asp-OH or related dianionic moieties (e.g., Glu-OH) might be used as a general strategy for the preparation of in vivo compatible CAs bearing decreased overall positive charge. In Vivo Imaging. Our in vivo PARACEST imaging studies started with CA 6a bearing hexyl side chains. Among the two CAs (6a and 6b) possessing CEST effects around −100 ppm, the synthesis of 6a proceeded with higher overall yield and produced sufficient amounts of 6a to carry out more detailed investigation. However, no in vivo CEST effect (1 h duration of the imaging session) was observed upon tail vein injection of 50 mM solution of 6a in phosphate buffered saline. Neither changes in administration route (leg muscle injection) nor increased concentration (100 mM of 6a in phosphate buffered saline, higher concentrations were not attempted due to solubility issues) improved the outcome of this study. The presence of 6a in kidneys was confirmed by T2 weighted

imaging, wherein darkening of kidneys (Figure 3) was observed 20 min post injection (tail vein) despite the fact that 6a is a

Figure 3. T2 weighted image of mouse kidneys before the injection of 6a (top left) and 20 min post injection of 6a (top right). Wash-in curves of 6a (bottom).

rather poor T2 exchange CA (Table 3). This result indicates that the CEST effects associated with 6a (6% at −57 ppm and 5% at −91 ppm, Table 1) are too weak to be directly detected in vivo. Considering the similar intensity of CEST effects (3% at −66 ppm and 7% at −100 ppm, Table 1), it is not surprising that similar negative results have been observed upon administration (tail vein) of 50 mM solution of t-butylmodified CA 6b in phosphate buffered saline. Higher concentrations of 6b have not been administered due to the limited amount of material available at the time. The in vivo CEST effect associated with the benzyldecorated CA 6c, which displays only a modest 10% CEST at −50 ppm in vitro, was observed when a 50 mM solution (phosphate buffered saline) of 6c was injected into leg muscle, Figure 4. Despite the modest intensity of the signal, this result is a clear demonstration of the observation of a PARACEST effect in vivo. This result also highlights the challenge of PARACEST imaging in the presence of endogenous MT. A similar study with CA 6d bearing propargyl side chains did not result in the in vivo detection of CEST contrast, likely due to the image darkening caused by T2 exchange (Table 3).50

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CONCLUSIONS In the present work, we have synthesized a series of carboxylate- or aspartic acid (Asp-OH)-modified PARACEST MRI CAs based on DOTAM-alkyl-based structures present in 4a−4d. The synthesis was performed either by applying the 6524



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analogous manner failed, likely due to the T2 related image darkening.50 Herein we propose that the conjugation with Asp-OH (or related moiety) might be used as a general strategy for producing in vivo compatible CAs bearing decreased overall positive charge while retaining the ability to produce CEST contrast associated with their simpler, highly positively charged counterparts.

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EXPERIMENTAL SECTION

General Experimental Procedures. Reagents were commercially available and all solvents were HPLC grade except for water (18.2 MΩ cm−1, Millipore), CH2Cl2, dioxane, DMF, Et2O, and THF (dried by passing through columns of activated Al2O3). Solvents were removed under reduced pressure in a rotary evaporator, aqueous solutions were lyophilized, and organic extracts were dried over Na2SO4. Flash column chromatography (FCC) was carried out using silica gel (SiO2), mesh size 230−400 Å. Thin-layer chromatography (TLC) was carried out on Al backed silica gel plate with compounds visualized by I2 vapors, anisaldehyde stain, 5% ninhydrin stain, phosphomolybdic acid stain, and UV light. Melting points were obtained on Fisher−Johns apparatus and are uncorrected. Ultra performance liquid chromatography (UPLC, Waters Acquity) was performed using a BEH C18 column (particle size 1.7 μm; 1.0 id ×100 mm) and HR-ESI-MS detector (Waters/Micromass LCT Premier XE). Mobile phase: method A, 100% H2O−100% MeCN (both solvents containing 0.1% HCOOH) over 5 min, linear gradient, flow rate 0.1 mL/min. HPLC purification (method B) was performed using a Delta-Pak C18 300 Å column (particle size 15 μm; 8 mm × 100 mm radialcompression cartridge). Mobile phase for method B was 0 min, 90% H2O−10% MeCN (both solvents containing 0.1% TFA) to 8 min, 41% H2O−59% MeCN, 3 mL/min. Size exclusion chromatography (SEC) was carried out on Bio-Gel P2, 45−90 μm mesh resin (8 g, per 0.1 mmol of compound). Ten fractions (10 mL each) were collected and identified with I2 vapors. Absence of free Tm3+ was verified by xylenol orange test.53 NMR spectra were recorded on a 400 MHz spectrometer (Varian MERCURY) for 1H NMR spectra δ values were recorded as follows: CDCl3 (7.27 ppm), DMSO-d6 (2.49 ppm), D2O (4.75 ppm); for 13C (125 MHz) δ CDCl3 (77.0 ppm), DMSO-d6 (39.5 ppm). Mass spectra (MS) were obtained using electron impact (EI, Finnigan MAT 8200) or electrospray ionization (ESI, Micromass LCT). The purity of compounds (≥95%) was verified by 1H NMR spectroscopy (small molecules) or UPLC (method A, ligands and complexes). Nuclear Magnetic Resonance Experiments. Chemical exchange saturation transfer (CEST) spectra were acquired on a 600 MHz vertical bore NMR spectrometer (Varian INOVA). The CEST effect was measured by recording the bulk water signal intensity as a function of presaturation frequency. The basic pulse sequence consisted of a 2 s presaturation pulse (B1 = 650 Hz) followed by hard 20° pulse and a 1 s data acquisition. All CEST spectra were recorded in steps of 1 ppm from −110 to +110 ppm at concentration of 10 mM in Millipore, pH = 7.0 and at 37 °C. Each sample was pH adjusted using either 10 mM HCl or 10 mM NaOH; the volumes of acid or base were negligible relative to the total sample volume. T1 relaxation time constant measurements were made for four different concentrations (1, 2, 4, 8 mM, averaged n = 3) of CA (in pH adjusted water) using an inversion recovery sequence comprised of 12 inversion times in the range of 10 ms to 10 s with a 20 s repetition time to ensure full recovery, pH 7.0 and 37 °C. r1 reported are taken from the slope of the linear fit. T2 relaxation time constant measurements were made for four different concentrations (1, 2, 4, 8 mM, averaged n = 3) of CA (in pH adjusted water) using a CPMG pulse sequence comprised of 12 train echo times in the range of 10 ms to 10 s with a 20 s repetition time to ensure full recovery, pH 7.0 and 37 °C. r2 reported are taken from the slope of the linear fit. All proton exchange rates were measured by using a small animal 9.4 T Agilent MRI scanner (Agilent, Santa Clara, CA) at 37 °C. Plots of CEST effect as a function of B1 amplitude were produced by

Figure 4. In vivo CEST signal observed for 6c intramuscular leg injection. Top and bottom regions of interest in the mouse leg (circled) show a weak signal at approximately −50 ppm, indicated by the arrowheads.

“cyclen tetraalkylation” strategy23 (5a−5c) or by use of suitably protected cyclen derivatives35 such as tri-Boc-cyclen (for CAs 6a-6c) or t-butyl cyclen-N-monoacetate (for CA 6d). The primary goal of this work was to modify the chemical structure of acutely toxic CAs 4a−4d while retaining their interesting and potentially useful magnetic properties. Both chemical modifications lead to CAs, which appear to be nontoxic and are well tolerated during in vivo studies. The presence of terminal carboxylate groups on each arm leads to a complete obliteration of the CEST effect associated with CAs 5a−5c, presumably due to the unfavorable water exchange kinetics. On the other hand, the CEST effect (CAs 6a−6d) is partially retained upon conjugation with the Asp-OH moiety. In vivo imaging studies were performed with CAs 6a−6d. Although both 6a and 6b produced highly chemical shifted exchangeable protons near the boundary of the endogneous MT effect,26 their in vivo detection by CEST imaging was not possible, presumably due to low local accumulation of agent. Benzyl-decorated Asp-OH conjugate 6c was detected (CEST imaging) upon injection of the CA into the leg muscle. The detection of propargyl-substituted CA 6d administered in an 6525



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ration of eluates afforded N-chloroacetyl ω-aminocaproic acid methyl ester (8a) and N-chloroacetyl 3-amino-3-methylbutyric acid methyl ester (8b). A small amount of dicyclohexyl urea (DCU) eluted along with desired products 8a and 8b; it was removed by dissolving the separate residues in ice-cold acetone (ca. 5−10 mL) and filtering the insoluble DCU off by using a Pasteur pipet with a cotton plug. A suspension of the carboxylic acid ester hydrochloride derived from 7c in dry DMF (2.5 mL) was cooled to 0 °C, followed by the addition of Na2CO3 (636 mg, 6 mmol). Chloroacetyl chloride (310 μL, 3.9 mmol) was added dropwise (over ca. 1 min), the cooling bath was removed, and the mixture was stirred for 18 h at RT. The mixture was taken up into brine (50 mL) and was extracted with EtOAc (30 mL + 20 mL), and the combined organic extract was consecutively washed with saturated NaHCO3 solution (2 × 50 mL) and brine (2 × 50 mL) and was concentrated. The residue was subjected to FCC on 30 g of SiO2 eluting 8c with hexanes/acetone (2:1), concentration of eluate resulted in crystallization, the mixture was diluted with hexanes, and was set aside for 1 h at −10 °C. Separated crystals were filtered off, washed with hexanes, and dried to leave N-chloroacetyl p-aminomethylbenzoic acid methyl ester (8c). N-Chloroacetyl ω-Aminocaproic Acid Methyl Ester (8a, 545 mg, 82%). Colorless solid. 1H NMR (CDCl3) δ 6.65 (s, D2O exch, 1H), 4.05 (s, 2H), 3.66 (s, 3H), 3.31 (m, 2H), 2.32 (m, 2H), 1.65 (m, 2H), 1.55 (m, 2H), 1.36 (m, 2H). 13C NMR (CDCl3) δ 173.9, 166.0, 51.5, 42.6, 39.6, 33.8, 28.9, 26.2, 24.4. HRMS (EI) m/z; found 221.0815 [M]+ (calcd 221.0819 for C9H16ClNO3). LRMS (EI) m/z (rel abundance): 221 [M+] (5), 186 (100), 172 (18), 144 (45). N-Chloroacetyl 3-Amino-3-methylbutyric Acid Methyl Ester (8b, 355 mg, 57%). Colorless oil. 1H NMR (CDCl3) δ 7.01 (s, D2O exch, 1H), 3.95 (s, 2H), 3.68 (s, 3H), 2.73 (s, 2H), 1.46 (s, 6H). 13C NMR (CDCl3) δ 171.4, 165.4, 52.5, 51.6, 43.9, 42.8, 26.7. HRMS (EI) m/z; found 207.0655 [M]+ (calcd 207.0662 for C8H14ClNO3). LRMS (EI) m/z (rel abundance): 207 [M+] (14), 192 (13), 176 (24), 160 (60), 134 (82), 114 (100), 83 (68). N-Chloroacetyl p-Aminomethylbenzoic Acid Methyl Ester (8c, 206 mg, 28%). Colorless crystals; mp 90−92 °C. 1H NMR (CDCl3) δ 8.01 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.03 (s, D2O exch, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.12 (s, 2H), 3.91 (s, 3H). 13C NMR (CDCl3) δ 166.7, 166.0, 142.4, 130.0, 129.5, 127.5, 52.1, 43.4, 42.5. HRMS (ESI) m/z: found 242.0572 [M + H]+ (calcd 242.0584 for C11H13ClNO3). Acylation of Carboxylic Acids 7a−7c with Chloroacetyl Chloride. Carboxylic acids 7a (1.31 g, 10 mmol), 7b (117 mg, 1 mmol), and 7c (1.51 g, 10 mmol) were dissolved in separate solutions of NaOH (800 mg, 20 mmol, acids 7a and 7c; 80 mg, 2 mmol, acid 7b) in water (7a, 5 mL; 7b, 500 μL; 7c, 10 mL). Resulting solutions were cooled to 0 °C, followed by a dropwise addition (over ca. 5 min) of the solution of chloroacetyl chloride (950 μL, 12 mmol, acids 7a and 7c; 95 μL, 1.2 mmol, acid 7b) in dry Et2O (2.5 mL, acids 7a and 7c; 250 μL, acid 7b). The mixtures were stirred for 10 min at 0 °C and 40 min at RT. The mixtures were cooled to 0 °C, the pH was adjusted to ca. 2−3 (1 M HCl), and Et2O was evaporated. N-Chloroacetyl ωaminocaproic acid (11a) and N-chloroacetyl p-aminomethylbenzoic acid (11c) precipitated upon acidification; mixtures were set aside for 1 h at 0 °C, separated precipitates were filtered off with suction, washed with water, and dried. The mixture containing N-chloroacetyl 3-amino-3-methylbutyric acid (11b) was extracted with Et2O (4 × 5 mL), and the combined organic extract was dried and concentrated. The residue was subjected to FCC on 15 g SiO2, eluting with hexanes/ acetone (5:1). Evaporation of the eluate afforded N-chloroacetyl 3amino-3-methylbutyric acid (11b). N-Chloroacetyl ω-Aminocaproic Acid (11a, 995 mg, 48%).32 Colorless solid. 1H NMR (DMSO-d6) δ 12.02 (s, D2O exch, 1H), 8.20 (s, D2O exch, 1H), 4.02 (s, 2H), 3.06 (m, 2H), 2.19 (m, 2H), 1.48 (m, 2H), 1.40 (m, 2H), 1.25 (m, 2H). 13C NMR (DMSO-d6) δ 174.5, 165.9, 42.8, 38.9, 33.7, 28.7, 26.0, 24.3. HRMS (EI) m/z; found 207.0659 [M]+ (calcd 207.0662 for C8H14ClNO3). LRMS (EI) m/z (rel abundance): 207 [M+] (5), 172 (100), 140 (34), 112 (35), 69 (29).

varying the amplitude (6, 9, 12, 15, and 18 μT) of a 5 s saturation pulse that preceded a standard fast spin−echo pulse sequence (TR = 4000 ms, echo-train length 4, effective echo time 10 ms, averages 5, dummy scans 2, matrix 64 × 64, FOV 30 mm × 30 mm). The results were then fitted by using Matlab, and the exchange rates were calculated based on the linear fit parameters. These were acquired at 20 mM chelate concentration in 20 mM NaH2PO4 buffer (pH 7.0, 37 °C). In Vivo Imaging. T2*-weighted MRI images of the mouse kidneys were acquired on a 9.4 T horizontal bore Agilent MRI scanner equipped with a 30 mm millipede radio frequency coil (Agilent, Santa Clara, CA). Images were acquired using a two-dimensional fast lowangle shot (FLASH) pulse sequence (field of view 32.0 × 32.0 mm2, data matrix 128 × 128, pulse repetition time (TR) 40 ms, echo time (TE) = 8 ms, slice thickness = 1 mm). The contrast agent, 200 μL, 2 mg, was injected over 5 min (40 uL/min) into the tail vein. Imaging began 3 min after injection. In vivo CEST imaging was performed on an Agilent (Santa Clara, CA, USA) 9.4 T MRI scanner equipped with a 3 cm diameter custombuilt radio frequency surface coil. A C57BL/6 mouse (3 months of age, weighing 25 g) was anesthetized (induced using 4% isoflurane in oxygen and maintained using 1.5%−2.5% isoflurane in oxygen). The mouse was secured on a magnetic resonance imaging (MRI)compatible stage with a rectal temperature probe and a respiratory sensor pad for monitoring during MRI. Body temperature was maintained at 37 °C during the imaging procedure by blowing warm air over the animal using a model 1025 small-animal monitoring and gating system (SA Instruments Inc., Stony Brook, NY). Approximately 15 min before acquisition of CEST images, 50 μL of 100 mM 6c dissolved in phosphate buffered saline was injected intramuscularly (at a depth of 2 mm) in the left leg. Injection was performed at a rate of 25.0 μL/min using a 27-gauge needle and a syringe pump (Harvard Apparatus, Holliston, MA). The mouse was recovered following imaging. The animal procedure was performed according to a protocol approved by the Western University Animal Use Subcommittee. CEST images were acquired using a 2 mm thick single slice positioned through the injection site that was identified using T2-weighted scout images. For in vivo CEST imaging, a standard fast spin echo pulse sequence was used (field of view = 19.2 × 19.2 mm2, matrix = 64 × 64, repetition time (TR) = 5000 ms, echo train length (ETL) = 32, effective echo time = 10 ms, 2 pre scans and 1 average) preceded by a continuous wave presaturation pulse (amplitude = 14 μT, duration = 5.0 s). CEST spectra were acquired using saturation frequency offsets −1000 ppm, −65 to −30 ppm with 1 ppm increments along with −1, −0.5 to +0.5 ppm with 0.1 ppm increments, + 35 to +65 ppm with 1 ppm increments and +1000 ppm) in ascending order. In vivo CEST spectra were B0-corrected on a pixel-by-pixel basis by first interpolating the water peak to 1 Hz resolution, then fitting it to a twelfth-order polynomial. Water resonance (0 ppm) was defined at the frequency where the interpolated fitted polynomial was lowest. Esterification of Carboxylic Acids 7a−7c and Subsequent Reactions with Chloroacetic Acid or Chloroacetyl Chloride. Separate solutions of carboxylic acids 7a−7c (7a, 394 mg; 7b, 352 mg; 7c, 453 mg; 3 mmol each) in MeOH (3 mL) were cooled to 0 °C, followed by a dropwise addition (over ca. 1 min) of SOCl2 (265 μL, 3.6 mmol). The cooling baths were removed, and the mixtures were stirred for 24 h at room temperature (RT). Volatiles were evaporated to leave corresponding carboxylic acid esters as hydrochloride salts (quantitative yields). Chloroacetic acid (285 mg, 3 mmol) was dissolved in dry CH2Cl2 (15 mL), NHS (345 mg, 3 mmol) was added, and the mixture was cooled to 0 °C. DCC (743 mg, 3.6 mmol) was added, the cooling bath was removed, and the stirring continued for 24 h at RT. The solvent was evaporated; the solid residue was treated separately with solutions (in dry THF, 15 mL) of carboxylic acid ester hydrochlorides derived from 7a and 7b. Et3N (840 μL, 6 mmol) was added, and the mixtures were stirred for 18 h at RT. Resulting reaction mixtures were diluted with 1 M HCl (30 mL) and were extracted with EtOAc (3 × 20 mL). Combined organic extracts were concentrated; the residues were subjected to FCC on 40 g of SiO2 eluting 8a with hexanes/acetone (2:1) and 8b with hexanes/acetone (3:1). Evapo6526



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N-Chloroacetyl p-Aminomethylbenzoic Acid-Asp-(OMe)2 (12 c, 438 mg, 59%). Colorless solid. 1H NMR (CDCl3) δ 7.76 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.03 (d, D2O exch, J = 8.0 Hz, 1H), 7.10 (s, D2O exch, 1H), 5.03 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H), 4.10 (s, 2H), 3.78 (s, 3H), 3.69 (s, 3H), 3.13 (dd, J = 17.0, 4.5 Hz, 1H), 2.97 (dd, J = 17.0, 4.5 Hz, 1H). 13C NMR (CDCl3) δ 171.6, 171.1, 166.4, 166.0, 141.5, 132.9, 127.7, 127.6, 52.9, 52.1, 48.8, 43.3, 42.5, 35.9. HRMS (ESI) m/z: found 371.1011 [M + H]+ (calcd 371.1010 for C16H20ClN2O6). Finkelstein Reaction of N-Chloroacetyl Derivatives 8a−8c, 12a and 12c. NaI (450 mg, 3 mmol) was added to separate solutions of N-chloroacetyl derivatives (8a, 222 mg; 8b, 207 mg; 8c, 242 mg; 12a, 351 mg; 12c, 371 mg; 1 mmol each) in acetone (8 mL, 8a−8c, 12a; 15 mL, 12c). The mixtures were stirred for 18 h at RT, the acetone was evaporated, and separate residues were partitioned between 10% solution of Na2SO3 (40 mL) and EtOAc (2 × 30 mL). Combined organic extracts were dried and concentrated. The residues were either subjected to FCC on 30 g of SiO2, eluting 9a with hexanes/acetone (5:1), eluting 9b with hexanes/acetone (2:1), eluting 12a with CH2Cl2/MeOH (98:2), or crystallized from acetone/hexanes (9c) or CH2Cl2/hexanes (12c). Evaporation of eluates afforded Niodoacetyl ω-aminocaproic acid methyl ester (9a), N-iodoacetyl 3amino-3-methylbutyric acid methyl ester (9b), and N-iodoacetyl ωaminocaproic acid-Asp-(OMe)2 (13a). The crystallizations were set aside for 3 h at −10 °C, and the crystals were filtered off, washed with hexanes, and dried to leave N-iodoacetyl p-aminomethylbenzoic acid methyl ester (9c) and N-iodoacetyl p-aminomethylbenzoic acid-Asp(OMe)2 (13c). N-Iodoacetyl ω-Aminocaproic Acid Methyl Ester (9a, 241 mg, 77%). Colorless solid. 1H NMR (CDCl3) δ 6.17 (s, D2O exch, 1H), 3.70 (s, 2H), 3.68 (s, 3H), 3.28 (m, 2H), 2.33 (m, 2H), 1.66 (m, 2H), 1.55 (m, 2H), 1.38 (m, 2H). 13C NMR (CDCl3) δ 174.0, 167.0, 51.5, 40.0, 33.7, 28.8, 26.1, 24.3, −0.4. HRMS (EI) m/z; found 313.0174 [M]+ (calcd 313.0175 for C9H16INO3). LRMS (EI) m/z (rel abundance): 313 [M+] (5), 240 (20), 112 (72), 72 (27). N-Iodoacetyl 3-Amino-3-methylbutyric Acid Methyl Ester (9b, 256 mg, 86%). Colorless oil. 1H NMR (CDCl3) δ 6.43 (s, D2O exch, 1H), 3.71 (s, 3H), 3.64 (s, 2H), 2.73 (s, 2H), 1.45 (s, 6H). 13C NMR (CDCl3) δ 171.6, 166.4, 52.7, 51.7, 43.7, 26.6, −0.6. HRMS (EI) m/z; found 299.0022 [M]+ (calcd 299.0018 for C8H14INO3). LRMS (EI) m/z (rel abundance): 299 [M+] (47), 114 (100), 73 (38). N-Iodoacetyl p-Aminomethylbenzoic Acid Methyl Ester (9c, 193 mg, 58%). Colorless crystals; mp 142−144 °C. 1H NMR (CDCl3) δ 7.97 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.78 (s, D2O exch, 1H), 4.48 (d, J = 6.0 Hz, 2H), 3.90 (s, 3H), 3.74 (s, 2H). 13C NMR (CDCl3) δ 167.3, 166.7, 142.8, 130.0, 129.4, 127.4, 52.1, 43.9, −1.0. HRMS (ESI) m/z: found 333.9952 [M + H]+ (calcd 333.9940 for C11H13INO3). N-Iodoacetyl ω-Aminocaproic Acid-Asp-(OMe)2 (13a, 252 mg, 57%). Colorless solid. 1H NMR (CDCl3) δ 6.66 (s, D2O exch, 1H), 6.62 (d, D2O exch, J = 11.5 Hz, 1H), 4.86 (m, 1H), 3.74 (s, 3H), 3.69 (s, 2H), 3.68 (s, 3H), 3.25 (m, 2H), 3.01 (dd, J = 17.0, 4.5 Hz, 1H), 2.84 (dd, J = 17.0, 4.5 Hz, 1H), 2.24 (m, 2H), 1.65 (m, 2H), 1.53 (m, 2H), 1.36 (m, 2H). 13C NMR (CDCl3) δ 172.7, 171.5, 171.2, 167.2, 52.8, 52.0, 48.3, 39.9, 36.0, 35.9, 28.5, 25.9, 24.7, −0.4. HRMS (ESI) m/z: found 443.0664 [M + H]+ (calcd 443.0679 for C14H24IN2O6). N-Iodoacetyl p-Aminomethylbenzoic Acid-Asp-(OMe)2 (13 c, 384 mg, 83%). Colorless crystals; mp 127−129 °C. 1H NMR (CDCl3) δ 7.70 (d, J = 8.0 Hz, 2H), 7.32 (s, D2O exch, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.06 (t, D2O exch, J = 6.0 Hz, 1H), 5.04 (m, 1H), 4.46 (d, J = 6.0 Hz, 2H), 3.79 (s, 3H), 3.75 (s, 2H), 3.70 (s, 3H), 3.13 (dd, J = 17.5, 4.5 Hz, 1H), 2.97 (dd, J = 17.5, 4.5 Hz, 1H). 13C NMR (CDCl3) δ 171.6, 171.1, 167.5, 166.7, 141.9, 132.6, 127.6, 127.5, 52.9, 52.1, 48.9, 43.6, 36.0, −0.9. HRMS (ESI) m/z: found 463.0360 [M + H]+ (calcd 463.0366 for C16H20IN2O6). N-Chloroacetyl-N′-Boc 1,4-Diaminobut-2-yne (20). A solution of but-2-yne-1,4-diol (17, 6.5 g, 75.5 mmol) in pyridine (11 mL) was cooled to 0 °C, followed by a dropwise addition (over 1 h) of SOCl2 (13.2 mL, 181.2 mmol).37 The cooling bath was removed, and the stirring continued for 2 h at RT. The mixture was then cooled to 0 °C

N-Chloroacetyl 3-Amino-3-methylbutyric Acid (11b, 76 mg, 39%).33 Colorless oil. 1H NMR (DMSO-d6) δ 12.35 (s, D2O exch, 1H), 7.93 (s, D2O exch, 1H), 3.97 (s, 2H), 2.66 (s, 2H), 1.33 (s, 6H). 13 C NMR (DMSO-d6) δ 172.1, 165.2, 51.5, 43.3, 42.9, 26.5. HRMS (EI) m/z; found 193.0507 [M]+ (calcd 193.0506 for C7H12ClNO3). LRMS (EI) m/z (rel abundance): 193 [M+] (30), 178 (21), 160 (65), 134 (91), 94 (100). N-Chloroacetyl p-Aminomethylbenzoic Acid (11c, 1.46 g, 64%). Colorless solid. 1H NMR (DMSO-d6) δ 11.78 (s, D2O exch, 1H), 8.93 (t, D2O exch, J = 6.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H), 4.16 (s, 2H). 13C NMR (DMSO-d6) δ 167.3, 166.3, 144.0, 129.7, 129.5, 127.3, 42.7, 42.3. HRMS (EI) m/z; found 227.0345 [M]+ (calcd 227.0349 for C10H10ClNO3). LRMS (EI) m/z (rel abundance): 227 [M+] (24), 192 (100), 151 (22), 135 (26), 107 (27). Conjugation of Carboxylic Acids 11a−11c with L-Asp(OMe)2·HCl. NHS (136 mg, 1.18 mmol, acid 11a; 62 mg, 0.54 mmol, acid 11b) was added to separate stirred solutions of Nchloroacetyl ω-aminocaproic acid (11a, 246 mg, 1.18 mmol) and Nchloroacetyl 3-amino-3-methylbutyric acid (11b, 105 mg, 0.54 mmol) in dry THF (10 mL, acid 11a; 4 mL, acid 11b). The solutions were cooled to 0 °C, followed by the addition of DCC (317 mg, 1.54 mmol, acid 11a; 145 mg, 0.7 mmol, acid 11b) and subsequent stirring for 20 min at 0 °C. L-Asp-(OMe)2·HCl (246 mg, 1.24 mmol, acid 11a; 107 mg, 0.54 mmol, acid 11b) and Et3N (330 μL, 2.36 mmol, acid 11a; 150 μL, 1.08 mmol, acid 11b) were added, the cooling baths were removed, and the mixtures were stirred for 18 h at RT. The mixtures were diluted with CH2Cl2 (50 mL, acid 11a; 20 mL, acid 11b) and were washed with 1 M HCl (30 mL, acid 11a; 20 mL, acid 11b). The aqueous phases were extracted with an additional amount of CH2Cl2 (30 mL, acid 11a; 20 mL, acid 11b), and the combined organic extracts were dried and concentrated. The residues were subjected to FCC on 30 g of SiO2 eluting 12a and 12b with CH2Cl2/MeOH (95:5). Evaporation of the eluates afforded N-chloroacetyl ωaminocaproic acid-Asp-(OMe)2 (12a) and N-chloroacetyl 3-amino-3methylbutyric acid-Asp-(OMe)2 (12b) containing small amount of DCU, which was removed by dissolving the separate residues in icecold acetone (ca. 5−10 mL) and filtering the insoluble DCU off by using a Pasteur pipet with a cotton plug. A stirred solution of N-chloroacetyl p-aminomethylbenzoic acid (11c, 455 mg, 2 mmol) and L-Asp-(OMe)2·HCl (395 mg, 2 mmol) in dry DMF (2 mL) was cooled to 0 °C, followed by the addition of Nmethylmorpholine (NMM, 990 μL, 9 mmol). A commercial solution of T3P in DMF (1.75 mL, 3 mmol) was the added dropwise over ca. 5 min. The mixture was stirred for 1 h at 0 °C and for 18 h at RT. The mixture was then diluted with saturated NaHCO3 solution (50 mL) and was extracted with EtOAc (3 × 30 mL). Combined organic extract was washed with brine (4 × 50 mL), dried, and concentrated. The residue was subjected to FCC on 40 g of SiO2, eluting 12c with CH2Cl2/MeOH (98:2) later replaced with CH2Cl2/MeOH (95:5). Evaporation of the eluate afforded N-chloroacetyl p-aminomethylbenzoic acid-Asp-(OMe)2 (12c). N-Chloroacetyl ω-Aminocaproic Acid-Asp-(OMe)2 (12a, 276 mg, 66%). Colorless solid. 1H NMR (CDCl3) δ 6.68 (s, D2O exch, 1H), 6.53 (d, D2O exch, J = 8.0 Hz, 1H), 4.85 (m, 1H), 4.03 (s, 2H), 3.74 (s, 3H), 3.68 (s, 3H), 3.29 (m, 2H), 3.02 (dd, J = 17.0, 4.5 Hz, 1H), 2.83 (dd, J = 17.0, 4.5 Hz, 1H), 2.23 (m, 2H), 1.65 (m, 2H), 1.55 (m, 2H), 1.36 (m, 2H). 13C NMR (CDCl3) δ 172.5, 171.5, 171.2, 165.8, 52.7, 52.0, 48.3, 42.6, 39.5, 36.0 (2 × C), 28.9, 26.1, 24.8. HRMS (ESI) m/z: found 351.1321 [M + H]+ (calcd 351.1323 for C14H24ClN2O6). N-Chloroacetyl 3-Amino-3-methylbutyric Acid-Asp-(OMe)2 (12b, 79 mg, 43%). Colorless oil. 1H NMR (CDCl3) δ 7.20 (s, D2O exch, 1H), 6.70 (d, D2O exch, J = 8.0 Hz, 1H), 4.87 (m, 1H), 4.07 (d, J = 15.0, 1H), 3.97 (d, J = 15.0, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.04 (dd, J = 17.0, 4.5 Hz, 1H), 2.82 (dd, J = 17.0, 4.5 Hz, 1H), 2.77 (d, J = 13.5, 1H), 2.55 (d, J = 13.5, 1H), 1.49 (s, 3H), 1.47 (s, 3H). 13C NMR (CDCl3) δ 171.4, 170.9, 170.2, 166.2, 53.2, 52.8, 52.1, 48.2, 46.0, 43.0, 35.9, 27.1 (2 × C). HRMS (ESI) m/z: found 337.1155 [M + H]+ (calcd 337.1166 for C13H22ClN2O6). 6527



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and was slowly quenched by addition of water (30 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (10 mL). Combined organic extract was successively washed with 10% H2SO4 solution, saturated NaHCO3 solution, and water (30 mL each), dried, and concentrated to leave 1,4-dichlorobut-2-yne (18, 5.23 g, 56%) as a pale-brown oil of sufficient purity to be used for the next step. A solution of NaN3 (517 mg, 7.95 mmol) in water (10 mL) was added to a solution of 1,4-dichlorobut-2-yne (18, 468 mg, 3.81 mmol) in THF (10 mL).38 The mixture was stirred for 24 h at RT. Pulverized (pestle and mortar) PPh3 (2.1 g, 7.96 mmol) was added, and the stirring continued for further 24 h at RT.38 The pH of the reaction mixture was adjusted to ca. 10 (2.5 M NaOH solution), and a solution of Boc2O (277 mg, 1.27 mmol) in MeOH (3 mL) was added dropwise over 15 min.39 The pH 10 was maintained during the addition and subsequent stirring for 30 min at RT. THF was evaporated; the residue was diluted with brine (30 mL) and was extracted with CH2Cl2 (3 × 20 mL). Combined organic extract was dried and was concentrated; the residue was subjected to FCC on 40 g of SiO2, eluting with CH2Cl2/MeOH/NH4OH (90:10:1). Evaporation of the eluate afforded N-Boc-1,4-diaminobut-2-yne (19, 42 mg, 18% based on 18 and 0.33 eq of Boc2O used). Na2CO3 (48 mg, 0.46 mmol) was added to a solution of N-Boc-1,4-diaminobut-2-yne (19, 42 mg, 0.23 mmol) in dry DMF (1 mL). Chloroacetyl chloride (24 μL, 0.3 mmol) was added, and the mixture was stirred for 3 h at RT. It was then diluted with brine (30 mL) and was extracted with EtOAc (20 mL + 2 × 10 mL). Combined organic extract was washed with brine (2 × 40 mL), dried, and concentrated. The residue was subjected to FCC on 15 g of SiO2, eluting 20 with hexanes/acetone (2:1). Evaporation of the eluate afforded N-chloroacetyl-N′-Boc 1,4-diaminobut-2-yne (20, 38 mg, 64%). Colorless oil. 1H NMR (DMSO-d6) δ 8.65 (t, D2O exch, J = 4.5 Hz, 1H), 7.25 (t, D2O exch, J = 5.5 Hz, 1H), 4.07 (s, 2H), 3.90 (m, 2H), 3.73 (m, 2H), 1.37 (s, 9H). 13C NMR (DMSO-d6) δ 165.6, 155.2, 79.9, 78.2, 78.0, 42.4, 29.4, 28.5, 28.2. HRMS (ESI) m/z: found 283.0818 [M + Na]+ (calcd 283.0825 for C11H17ClN2O3Na). N-Succinyl-N′-Boc 1,4-Diaminobut-2-yne Dimethyl Ester (22). A solution of 1,4-dichlorobut-2-yne (18, 2.98 g, 24.2 mmol) and hexamethylenetetramine (3.39 g, 24.2 mmol) in CHCl3 (25 mL) was stirred under reflux for 6 h.40 The mixture was cooled to RT, separated precipitate was filtered off with suction, washed with CHCl3, and dried to leave 6.04 g (95%) of the corresponding quaternary ammonium salt. The salt (6.01 g, 22.85 mmol) was suspened in EtOH (46 mL), conc HCl (8 mL) was added, and mixture was stirred under reflux for 30 min and at RT for 18 h.40 Separated precipitate (NH4Cl) was filtered off with suction and washed with EtOH, and the filtrate was concentrated and redissolved in 20 mL of EtOH. The solution was set aside for 2 h at 3 °C; an additional amount of NH4Cl was filtered off with suction and was washed with EtOH. The filtrate was concentrated, redissolved in water, and lyophilized to leave 1-chloro-4aminobut-2-yne hydrochloride (2.93 g, 86% based on 18) as a yellow solid. A 10 M solution of NaOH in water (380 μL, 3.82 mmol) was added to a solution of 1-chloro-4-aminobut-2-yne hydrochloride (535 mg, 3.82 mmol) in water (6 mL). A solution of Boc2O (917 mg, 4.2 mmol) in dioxane (12 mL) was added, and the mixture was stirred for 2 h at RT.41 Dioxane was evaporated; the residue was diluted with water (20 mL) and extracted with EtOAc (2 × 20 mL). Combined organic extract was dried and concentrated. The residue was subjected to FCC on 25 g of SiO2 eluting with hexanes/acetone (5:1). Evaporation of the eluate afforded the product containing a small amount (ca. 10% of Boc2O); this was removed by repeating the FCC purification under identical conditions. 1-Chloro-4-N-Boc-aminobut-2yne (21, 426 mg, 55%). Colorless solid. 1H NMR (CDCl3) δ 4.78 (s, D2O exch, 1H), 4.13 (m, 2H), 3.97 (m, 2H), 1.44 (s, 9H). 13C NMR (CDCl3) δ 155.2, 82.9, 80.1, 77.7, 30.6, 30.4, 28.3. LiOH (53 mg, 2.2 mmol) was added to a solution of 1-chloro-4-NBoc-aminobut-2-yne (21, 204 mg, 1 mmol) and L-Asp-(OMe)2·HCl (198 mg, 1 mmol) in dry DMF (2.5 mL). The mixture was stirred for 18 h at 50 °C, cooled to room temperature, diluted with brine (40 mL), and extracted with EtOAc (20 mL + 2 × 10 mL). Combined organic extract was washed with brine (3 × 40 mL), dried, and

concentrated. The residue was subjected to FCC on 30 g of SiO2 eluting 22 with hexanes/acetone (2:1). N-Succinyl-N′-Boc 1,4diaminobut-2-yne dimethyl ester (22, 152 mg, 46%): Colorless oil. 1 H NMR (CDCl3) δ 4.76 (s, D2O exch, 1H), 3.92 (m, 2H), 3.78 (m, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.47 (m, 2H), 2.77 (dd, J = 16.0, 5.5 Hz, 1H), 2.72 (dd, J = 16.0, 6.5 Hz, 1H), 2.02 (s, D2O exch, 1H), 1.44 (s, 9H). 13C NMR (CDCl3) δ 173.6, 171.2, 155.2, 80.3, 80.0, 79.9, 56.2, 52.3, 51.9, 37.5, 37.1, 30.6, 28.3. HRMS (ESI) m/z: found 329.1723 [M + H]+ (calcd 329.1713 for C15H25N2O6). Tetraalkylation of Cyclen with Electrophiles 9a−9c and Subsequent Metalation with TmCl3·H2O. Ethyldiisopropylamine (DIPEA, 90 μL, 0.5 mmol) was added to separate solutions of cyclen (22 mg, 0.125 mmol) in MeCN (2 mL). Electrophiles (0.5 mmol each) 9a (157 mg), 9b (150 mg), and 9c (165 mg) were added, and the mixtures were allowed to stir for 18 h at 50 °C (electrophiles 9a and 9b) or 60 °C (electrophile 9c). The mixtures were cooled to RT, diluted with water (30 mL), and extracted with EtOAc (30 + 20 mL). Combined organic extracts were dried, concentrated, and residues triturated in hexanes to provide the ligands of sufficient purity for the subsequent steps in nearly quantitative yields. Methyl DOTAM-ω-Aminocaproate. Pale-yellow solid. 1H NMR (CDCl3) δ 7.47 (m, D2O exch, 4H), 3.61 (m, 20H), 3.23−2.88 (br m, 24 H), 2.30 (m, 8H), 1.61 (m, 8H), 1.53 (m, 8H), 1.33 (m, 8H). HRMS (ESI) m/z: found 913.5982 [M + H]+ (calcd 913.5974 for C44H81N8O12). Methyl DOTAM-3-amino-3-methylbutyrate. Orange oil. 1H NMR (DMSO-d6) δ 6.94 (m, D2O exch, 3H), 3.67 (m, 12H), 3.42−2.72 (br m, 32 H), 1.44 (m, 24H). HRMS (ESI) m/z: found 857.5380 [M + H]+ (calcd 857.5348 for C40H73N8O12). Methyl DOTAM-p-aminomethylbenzoate. Colorless solid. 1H NMR (CDCl3) δ 7.90 (m, 8H), 7.54 (m, D2O exch, 4H), 7.20 (m, 8H), 4.29 (m, 8H), 3.88 (s, 12 H), 2.97 (m, 8 H), 2.53−2.34 (br m, 16H). HRMS (ESI) m/z: found 993.4716 [M + H]+ (calcd 993.4722 for C52H65N8O12). TmCl3·H2O (40 mg, 0.15 mmol) was added to separate suspensions of above-mentioned ligands (0.12 mmol each) in water (2 mL). The pH was adjusted and maintained at ca. 10 (1 M NaOH), while the mixtures were stirred for 18 h at RT. The complete hydrolysis of ester functionalities was confirmed by HR-ESI-MS. In the case of methyl DOTAM-p-aminomethylphenyl tetracarboxylate, the hydrolysis of ester functionalities was found incomplete, therefore the mixture was stirred for additional 6 h at 60 °C. DOTAM-ω-aminocaproic Acid. HRMS (ESI) m/z: found 857.5380 [M + H]+ (calcd 857.5348 for C40H73N8O12). DOTAM-3-amino-3-methylbutyric Acid. HRMS (ESI) m/z: found 801.4745 [M + H]+ (calcd 801.4722 for C36H65N8O12). DOTAM-p-aminomethylbenzoic Acid. HRMS (ESI) m/z: found 937.4078 [M + H]+ (calcd 937.4096 for C48H57N8O12). The pH was then adjusted to ca. 5.5 (1 M HCl), and the stirring of the mixtures continued for further 18 h at RT or at 60 °C (DOTAMp-aminomethylphenyl tetracarboxylate). The mixtures containing the complexes derived from DOTAM-n-hexyl tetracarboxylate and DOTAM-t-butyl teracarboxylate were subjected to SEC as described in General Experimental Procedures. The fractions containing the products were lyophilized to leave the complexes 5a and 5b. The mixture containing the complex derived from DOTAM-p-aminomethylphenyl tetracarboxylate was cooled to 0 °C and was left at 0 °C for 1 h. Resulting solid (complex 5c) was filtered off with suction, washed with water, and dried. Tm3+ DOTAM-ω-aminocaproic Acid (5a, 68 mg, 53%, Based on 10). Yellow waxy solid. HRMS (ESI) m/z: found 1023.4425 [M − 2H]+ (calcd 1023.4455 for C40H70N8O12Tm). Tm3+ DOTAM-3-amino-3-methylbutyric Acid (5b, 45 mg, 37%, Based on 10. Yellow waxy solid. HRMS (ESI) m/z: found 967.3815 [M − 2H]+ (calcd 967.3829 for C36H62N8O12Tm). Tm3+ DOTAM-p-aminomethylbenzoic Acid. (5c, 69 mg, 51%, based on 10). Colorless solid. HRMS (ESI) m/z: found 1103.3232 [M − 2H]+ (calcd 1103.3203 for C48H54N8O12Tm). Trialkylation of Tri-Boc-cyclen (14) with Electrophiles 13a, 12b, and 13c. DIPEA (90 μL, 0.5 mmol) was added to separate 6528



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solutions of tri-Boc-cyclen36 (14, 118 mg, 0.25 mmol) and 0.25 mmol of electrophiles 13a (111 mg), 12b (84 mg), and 13c (116 mg) in DMF (2 mL). The mixtures were stirred for 18 h at 90 °C (electrophiles 13a and 13c) or 24 h at 110 °C (electrophile 12b). The mixtures were cooled to RT, diluted with brine (40 mL), and extracted with EtOAc (20 mL+ 2 × 10 mL). Combined organic extracts were washed with brine (2 × 40 mL), dried, and concentrated. The residues were triturated with hexanes, affording the intermediates 15a−15c of sufficient purity to be used in the next step. Tri-Boc-mono-N-acetyl-ω-aminocaproyl-Asp-(OMe) 2 Cyclen (15a, 186 mg, 94%). Pale-yellow oil. 1H NMR (CDCl3) δ 6.75 (m, D2O exch, 2H), 4.87 (m, 1H), 3.75 (s, 3 H), 3.69 (s, 3H), 3.53−2.55 (br m, 22H), 2.22 (m, 2H), 1.65 (m, 2H), 1.51 (m, 2H), 1.47 (s, 9H), 1.45 (s, 9H), 1.34 (m, 2H). HRMS (ESI) m/z: found 787.4794 [M + H]+ (calcd 787.4817 for C37H67N6O12). Tri-Boc-mono-N-acetyl-3-amino-3-methylbutyryl-Asp-(OMe)2 Cyclen (15b, 120 mg, 70%). Pale-brown oil. 1H NMR (CDCl3) δ 6.76 (m, D2O exch, 2H), 4.87 (m, 1H), 3.74−2.78 (br m, 28H), 1.46 (m, 33H). HRMS (ESI) m/z: found 773.4626 [M + H]+ (calcd 773.4660 for C36H65N6O12). Tri-Boc-mono-N-acetyl-p-aminomethylbenzoyl-Asp-(OMe)2 Cyclen (15c, 187 mg, 93%). Pale-brown oil. 1H NMR (CDCl3) δ 7.76 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.25 (m, D2O exch, 2H), 5.06 (m, 1H), 4.46 (d, J = 6.5 Hz, 2H), 3.79 (s, 3H), 3.70 (s, 3H), 3.46−2.63 (br m, 20H), 1.47 (m, 27H). HRMS (ESI) m/z: found 807.4472 [M + H]+ (calcd 807.4504 for C39H63N6O12). Removal of Boc groups from intermediates 15a−15c and peralkylation with N-ioodoacetyl-n-hexylamine, N-ioodoacetyl-t-butylamine, and N-ioodoacetyl-benzylamine. TES (200 μL) and TFA (500 μL) were added to separate solutions of 0.2 mmol of intermediates 15a (157 mg), 15b (155 mg), and 15c (161 mg) in CH2Cl2 (1.5 mL). The mixtures were stirred for 18 h at RT, the volatiles were evaporated, the excess TFA was removed by successive coevaporation with CH2Cl2 and toluene (30 mL each), and the residues were used for subsequent alkylation without further purification. Complete deprotection was confirmed by HR-ESI-MS. N-Acetyl-ω-aminocaproyl-Asp-(OMe)2 Cyclen. Pale-brown oil. HRMS (ESI) m/z: found 487.3223 [M + H]+ (calcd 487.3244 for C22H43N6O6). N-Acetyl-3-amino-3-methylbutyryl-Asp-(OMe) 2 Cyclen. Palebrown oil. HRMS (ESI) m/z: found 473.3104 [M + H]+ (calcd 473.3088 for C21H41N6O6). N-Acetyl-p-aminomethylbenzoyl-Asp-(OMe)2 Cyclen. Pale-brown oil. HRMS (ESI) m/z: found 507.2910 [M + H]+ (calcd 507.2918 for C24H39N6O6). DIPEA (870 μL, 5 mmol) was added to separate solutions of Bocdeprotected intermediates described above in MeCN (6 mL), followed by the addition of 0.66 mmol of the corresponding electrophiles as follows: N-ioodoacetyl-n-hexylamine26b (178 mg), N-ioodoacetyl-tbutylamine26b (159 mg), and N-ioodoacetyl-benzylamine26b (182 mg). The mixtures were stirred for 18 h at 50 °C, the solvent was evaporated, and the residues were partitioned between water (30 mL) and EtOAc (2 × 30 mL). Combined organic extracts were washed with brine (2 × 30 mL), dried, and concentrated. The residues were triturated with hexanes, affording the desired ligands 16a−16c of sufficient purity for metalation. DOTAM-triacetyl-n-hexyl-monoacetyl-ω-aminocaproyl-Asp(OMe)2 (16a, 180 mg, 99%). Pale-brown solid. 1H NMR (CDCl3) δ 8.35 (br m, D2O exch, 1H), 7.60 (br m, D2O exch, 2H), 7.23−6.62 (br m, D2O exch, 2H), 4.85 (m, 1H), 3.75 (s, 3 H), 3.70 (s, 3H), 3.58− 2.24 (br m, 36H), 1.51 (m, 10H), 1.28 (m, 20H), 0.88 (m, 9H). HRMS (ESI) m/z: found 910.6678 [M + H]+ (calcd 910.6705 for C46H88N9O9). DOTAM-triacetyl-t-butyl-monoacetyl-3-amino-3-methylbutyrylAsp-(OMe)2 (16b, 143 mg, 88%). Pale-brown oil. 1H NMR (CDCl3) δ 6.94 (m, D2O exch, 3H), 6.55 (m, D2O exch, 2H), 4.84 (m, 1H), 3.74 (s, 3 H), 3.69 (s, 3H), 3.56−3.09 (br m, 28H), 1.36 (m, 43H). HRMS (ESI) m/z: found 812.5625 [M + H]+ (calcd 812.5610 for C39H74N9O9).

DOTAM-triacetyl-benzyl-monoacetyl-p-aminomethylbenzoylAsp-(OMe)2 (16c, 68 mg, 36%). Pale-brown solid. 1H NMR (CDCl3) δ 7.68−7.01 (br m, D2O exch, 24H), 5.02 (m, 1H), 4.53−4.12 (br m, 8H), 3.75−3.67 (br m, 6 H), 3.44−2.18 (br m, 26H). HRMS (ESI) m/z: found 948.4946 [M + H]+ (calcd 948.4984 for C51H66N9O9). Alkylation of t-butyl cyclen-N-monoacetate (23) with N-chloroacetyl-propargylamine and subsequent conjugation with N-succinylN′-Boc 1,4-diaminobut-2-yne dimethyl ester (22). DIPEA (700 μL, 4 mmol) and N-chloroacetyl-propargylamine9 (530 mg, 4 mmol) were added to a solution of t-butyl cyclen-Nmonoacetate43 (23, 372 mg, 1.3 mmol) in MeCN (10 mL). The mixture was stirred for 18 h at 80 °C, the solvent was evaporated, and the residue was partitioned between water (30 mL) and EtOAc (30 mL + 2 × 20 mL). Combined organic extract was dried and concentrated. The residue was triturated with hexanes, affording the intermediate 24. t-Butyl 2-{4,7,10-Tris[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]-cyclen-1-yl Acetate (24, 359 mg, 48%). Pale-orange oil. Spectral data were in agreement with those previously reported.44 TFA (1 mL) and TES (200 μL) were added to separate solutions of 0.61 mmol of intermediates 22 (350 mg) and 24 (201 mg) in CH2Cl2. The mixtures were stirred for 1 h (intermediate 22) or 18 h (intermediate 24) at RT, the volatiles were evaporated, the excess TFA was removed by successive coevaporation with CH2Cl2 and toluene (30 mL each), and the residues (intermediates 25 and 26) were used for the subsequent step without further purification. A solution of intermediate 25 in dry DMF (1 mL) was cooled to 0 °C, followed by the addition of HOBt (25 mg, 0.18 mmol) and HBTU (232 mg, 0.61 mmol). The mixture was stirred for 5 min at 0 °C, followed by the addition of the solution of intermediate 26 in dry DMF (2 mL). The cooling bath was removed and the stirring continued for 18 h at 50 °C. The mixture was cooled to RT, diluted with brine (50 mL), and extracted with EtOAc (30 mL + 2 × 20 mL). Combined organic extract was washed with brine (3 × 50 mL), dried, and concentrated to leave a pale-brown oily residue (470 mg), which was dissolved in MeOH (3 mL) and subjected to semipreparative HPLC purification as described in the General Experimental Procedures. The fractions containing the product were combined and concentrated to leave ligand 27 as trifluoroacetate salt. DOTAM-triacetyl-propargyl-monoacetyl-1,4-diamonobuty-2ynyl-Asp(OMe)2·4CF3COO− (27, 110 mg, 14%). Colorless solid. HPLC, method B, tR 4.6 min. 1H NMR (D2O) δ 4.23 (m, 1H), 3.76 (m, 4H), 3.67 (m, 6H), 3.50 (s, 3H), 3.39 (s, 3H), 3.31−2.35 (br m, 26H), 2.30 (m, 3H). HRMS (ESI) m/z: found 726.3925 [M + H]+ (calcd 726.3939 for C35H52N9O8). Metalation of Ligands 16a−16c and 27 with TmCl3·H2O. TmCl3·H2O (30 mg, 0.11 mmol) was added to separate solutions of ligands 16a−16c and 27 (0.1 mmol each) in MeOH/water as follows: ligands 16a and 16c (2 mL MeOH/1 mL water), ligand 16b (1 mL MeOH/2 mL water), and ligand 27 (3 mL water). The pH was adjusted and maintained at ca. 10 (1 M NaOH), while the mixtures were stirred for 18 h at RT. The pH was then adjusted to ca. 5.5 (1 M HCl), and the stirring of the mixtures continued for further 18 h at RT, MeOH was evaporated, and remaining aqueous solutions were subjected to SEC as described in General Experimental Procedures. The fractions containing the products were lyophilized to leave the complexes 6a−6d. Tm3+ DOTAM-triacetyl-n-hexyl-monoacetyl-ω-aminocaproylAsp-(OH)2 (6a, 50 mg, 48%). Pale-yellow waxy solid. HRMS (ESI) m/z: found 1048.5483 [M − 2H] + (calcd 1048.5500 for C44H81N9O9Tm). Tm3+ DOTAM-triacetyl-t-butyl-monoacetyl-3-amino-3-methylbutyryl-Asp-(OH)2 (6b, 15 mg, 16%). Pale-brown solid. HRMS (ESI) m/z: found 950.4406 [M − 2H]+ (calcd 950.4404 for C37H67N9O9Tm). Tm3+ DOTAM-triacetyl-benzyl-monoacetyl- p-aminomethylbenzoyl-Asp-(OH)2 (6c, 40 mg, 37%). Pale-yellow solid. HRMS (ESI) m/ z: found 1086.3760 [M − 2H] + (calcd 1086.3778 for C49H59N9O9Tm). 6529



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Tm3+ DOTAM-triacetyl-propargyl-monoacetyl-1,4-diamonobuty2-ynyl-Asp(OH)2 (6d, 41 mg, 47%). Colorless solid. HRMS (ESI) m/ z: found 864.2704 [M − 2H]+ (calcd 864.2733 for C33H45N9O8Tm).

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TSAP, twisted square antiprism; UPLC, ultrahigh pressure liquid chromatography; UV, ultraviolet

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ASSOCIATED CONTENT

S Supporting Information *

(1) (a) Yoo, B.; Pagel, M. D. An overview of responsive MRI contrast agents for molecular imaging. Front. Biosci., Landmark Ed. 2008, 13, 1733−1752. (b) Geraldes, C. F. G. C.; Laurent, S. Classification and basic properties of contrast agents for magnetic resonance imaging. Contrast Media Mol. Imaging 2009, 4, 1−23. (2) (a) Yang, C. T.; Chuang, K. H. Gd(III) chelates for MRI contrast agents: from high relaxivity to “smart”, from blood pool to blood-brain barrier permeable. MedChemComm 2012, 3, 552−565. (b) Tu, C.; Louie, A. Y. Strategies for the development of gadolinium-based ‘q’activatable MRI contrast agents. NMR Biomed. 2013, 26, 781−787. (c) Shen, C.; New, E. J. Promising strategies for Gd-based responsive magnetic resonance imaging contrast agents. Curr. Opin. Chem. Biol. 2013, 17, 158−166. (3) Pan, D.; Schmieder, A. H.; Wickline, S. A.; Lanza, G. M. Manganese-based MRI contrast agents: past, present, and future. Tetrahedron 2011, 67, 8431−8444. (4) Wang, Y. X. J. Superparamagnetic iron oxide based MRI contrast agents: current status of clinical application. Quant. Imaging Med. Surg. 2011, 1, 35−40. (5) Spence, M. M.; Rubin, S. M.; Dimitrov, I. E.; Ruiz, E. J.; Wemmer, D. E.; Pines, A.; Yao, S. Q.; Tian, F.; Schultz, P. G. Functionalized xenon as a biosensor. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10654−10657. (6) (a) Zhang, S.; Merritt, M.; Woessner, D. E.; Lenkinski, R. E.; Sherry, A. D. PARACEST agents: modulating MRI contrast via water proton exchange. Acc. Chem. Res. 2003, 36, 783−790. (b) Viswanathan, S.; Kovacs, Z.; Green, K. N.; Ratnakar, S. J.; Sherry, A. D. Chem. Rev. 2010, 110, 2960−3018. (7) (a) Dorazio, S. J.; Tsitovich, P. B.; Siters, K. E.; Spernyak, J. A.; Morrow, J. R. Iron(II) PARACEST MRI contrast agents. J. Am. Chem. Soc. 2011, 133, 14154−14156. (b) Olatunde, A. O.; Dorazio, S. J.; Spernyak, J. A.; Morrow, J. R. The NiCEST approach: nickel(II) paraCEST MRI contrast agents. J. Am. Chem. Soc. 2012, 134, 18503− 18505. (c) Dorazio, S. J.; Olatunde, A. O.; Spernyak, J. A.; Morrow, J. R. CoCEST: cobalt(II) amide-appended paraCEST MRI contrast agents. Chem. Commun. 2013, 49, 10025−10027. (d) Dorazio, S. J.; Olatunde, A. O.; Tsitovich, P. B.; Morrow, J. Comparison of divalent transition metal ion paraCEST MRI contrast agents. J. Biol. Inorg. Chem. 2014, 19, 191−205. (8) Li, A. X.; Wojciechowski, F.; Suchý, M.; Jones, C. K.; Hudson, R. H. E.; Menon, R. S.; Bartha, R. A sensitive PARACEST contrast agent for temperature MRI: Eu3+-DOTAM-glycine (Gly)-phenylalanine (Phe). Magn. Reson. Med. 2008, 59, 374−381. (9) Suchý, M.; Li, A. X.; Milne, M.; Bartha, R.; Hudson, R. H. E. DOTAM-type ligands possessing arginine pendant groups for use in PARACEST MRI. Contrast Media Mol. Imaging 2012, 7, 441−449. (10) McVicar, N.; Li, A. X.; Suchy, M.; Hudson, R. H. E.; Menon, R. S.; Bartha, R. Simultaneous in vivo pH and temperature mapping using PARACEST-MRI contrast agent. Magn. Reson. Med. 2013, 70, 1016− 1025. (11) (a) Trokowski, R.; Ren, J.; Kálmán, F. K.; Sherry, A. D. Selective sensing of zinc ions with a PARACEST contrast agent. Angew. Chem., Int. Ed. 2005, 44, 6920−6923. (b) Huang, C. H.; Morrow, J. R. A PARACEST agent responsive to inner- and outer-sphere phosphate ester interactions for MRI applications. J. Am. Chem. Soc. 2009, 131, 4206−4207. (12) Zhang, S.; Trokowski, R.; Sherry, A. D. A paramagnetic CEST agent for imaging glucose by MRI. J. Am. Chem. Soc. 2003, 125, 15288−15289. (13) Yoo, B.; Pagel, M. D. A PARACEST MRI contrast agent to detect enzyme activity. J. Am. Chem. Soc. 2006, 128, 14032−14033. (14) Li, A. X.; Hudson, R. H. E.; Barrett, J. W.; Jones, C. K.; Pasternak, S. H.; Bartha, R. Four-pool modeling of proton exchange processes in biological systems in the presence of MRI-paramagnetic

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00621. 1 H NMR spectra of compounds 4a, 4c, 5a−c, 6a−d, 8a− 8c, 9a−9c, 11a−11c, 12a−12c, 13a, 13c, 15a−15c, 16a−16c, 20, 21, 22, 27, methyl DOTAM-ω-aminocaproate, methyl DOTAM-3-amino-3-methylbutyrate, and methyl DOTAM-p-aminomethylbenzoate; 13NMR spectra of compounds 8a−8c, 9a−9c, 11a−11c, 12a− 12c, 13a, 13c, 20, 21, and 22; UPLC chromatograms and HR-ESI-MS spectra of compounds 5a−5c, 6a−6d, 15a− 15c, 16a−16c, 27, methyl DOTAM-ω-aminocaproate, methyl DOTAM-3-amino-3-methylbutyrate, and methyl DOTAM-p-aminomethylbenzoate; HPLC chromatogram of compound 27; plots of concentration vs T2 for 4a−d, 5a−c, and 6a−d and general procedure for measuring exchange rates and Ω-plots for the data listed in Table 2 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +1 519-661-2111 ext 86349. E-mail: rhhudson@uwo. ca. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of this work was provided by the Ontario Institute for Cancer Research (OICR), Canadian Health Research Institute (CIHR), and National Science and Engineering Research Council of Canada (NSERC). We thank Elyse Hudson for assistance with graphic designs.

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ABBREVIATIONS USED Boc, tert-butyloxycarbonyl; BSA, bovine serum albumin; CA, contrast agent; CEST, chemical exchange saturation transfer; DCC, dicyclohexylcarbodiimide; DMF, dimethylformamide; DOTAM, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid amide; EI, electron impact; ESI, electron spray; FCC, flash column chromatography; FISP, fast imaging with steady state precession; FLASH, fast low angle shot; FLEX, frequency labeled exchange transfer; HBTU, 1-benzotriazolyl-N,N,N′,N′tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPDO3A, 10-(2-hydroxypropyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane; HPLC, high pressure liquid chromatography; HRMS, high resolution mass spectrometry; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; MT, magnetization transfer; NHS, N-hydroxysuccinimide; NMR, nuclear magnetic resonance; OPARACHEE, on resonance paramagnetic chemical exchange effects; PARACEST, paramagnetic chemical exchange saturation transfer; RT, room temperature; SAP, square antiprism; SEC, size exclusion chromatography; SPIO, super paramagnetic iron oxide; T3P, propanephosphonic acid anhydride; TES, triethylsilane; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; 6530



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