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European Journal of Medicinal Chemistry 58 (2012) 50e63

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

99mTc/Re complexes bearing bisnitroimidazole or mononitroimidazole aspotential bioreductive markers for tumor: Synthesis, physicochemicalcharacterization and biological evaluation

Lei Mei, Yue Wang, Taiwei Chu*

Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, College of Chemistry and MolecularEngineering, Peking University, Beijing 100871, PR China

a r t i c l e i n f o

Article history:Received 21 June 2012Received in revised form25 September 2012Accepted 27 September 2012Available online 4 October 2012

Keywords:BisnitroimidazoleTechnetiumRheniumTumor hypoxiaBiodistribution

* Corresponding author. Tel./fax: þ86 10 62754319E-mail address: twchu@pku.edu.cn (T. Chu).

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.09.042

a b s t r a c t

Four monoamine-monoamide dithiol (MAMA) ligands containing two or one nitroimidazole moietieswere synthesized and labeled with 99mTc (labeling yield > 95%). The proposed structures of 99mTc-complexes are identified by comparison with analogous Re-MAMA complexes. 99mTc-MAMA complexesshow better physicochemical characters than 99mTcO-(PnAO-1-(2-nitroimidazole)). Reduction potentialsof nitro groups of the rhenium complexes are within the range for bioreductive compounds. As expected,biodistribution studies demonstrate that the 2-nitroimidazole complex shows better tumor-to-tissueratios than 4-nitroimidazole analog for mononitroimidazole complexes, but not for MAMA-bisnitroimidazoles due to higher lipophilicity. Both the bisnitroimidazole compounds show rapiderexcretion, lower background activity in liver and higher tumor-to-tissue ratios than the mono-nitroimidazoles. Better biodistribution characteristic makes both the MAMA-bisnitroimidazolecomplexes, especially 99mTc-15, be potential tumor hypoxia marker.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Tumor hypoxia is associated with deterioration of diffusionconditions and subsequent structurally and functionally disturbedmicrocirculation [1,2]. The existence of hypoxic regions has draw-backs in clinic therapy for their resistance toward radiation damageand chemotherapy [3,4] and is related with malignant progressionandmetastasis of cancer [5,6]. Thedetectionof tumorhypoxia canbeuseful to evaluate tumoroxygenationanddesign treatmentplans forcontrol of cancer. Hypoxia markers, which can be selectivelyreduced and trapped in hypoxic condition, havebeen developed [7e16]. Radiopharmaceuticals are mostly applied for their sensitivityandpenetrability. Inmost of the radiopharmaceuticals, bioreductivepharmacophores such as nitroimidazole groups [10,11,17e20] havebeen employed as bioactive moieties for hypoxia imaging.

For a nitroimidazole-based hypoxia marker, the nitroimidazolegroup as the bioreductive pharmacophore plays a vital role inhypoxia selectivity. Only one nitroimidazole group was included inmost of nitroimidazole-based hypoxia markers. A recent work byMukai et al. reported a radio-labeled hypoxia marker 67Ga-DOTA-

.

son SAS. All rights reserved.

MN2 containing two metronidazole moieties, which displayedsignificant tumor uptake and rapid blood clearance in NSFa tumor-bearing mice [21]. But direct comparison with the correspondingmarker with one metronidazole moiety was lacking. In ourprevious study, three propylene amine oxime (PnAO) derivativescontaining two nitroimidazole groups were synthesized and radi-olabeled with 99mTc, and comparison of the accumulations of thesecomplexes in hypoxic cells with those of the corresponding mon-onitroimidazole complexes was carried out [22]. In vitro uptakeexperiments indicated that introduction of 2-nitroimidazoleenhanced greatly the hypoxic accumulation, but not for 4-nitroimidazole. Initial results shed light on the way to improvehypoxia selectivity through cooperation of two bioreductivegroups. However, there are still some drawbackswith 99mTcO-PnAOmarkers. Their stabilities are not favorable just similar to theiranalog 99mTcO-(PnAO-1-(2-nitroimidazole)) (BMS181321), anestablished hypoxia imaging marker [22,23]. They undergo chem-ical decomposition a few hours after preparation. Moreover, theircharacter of high lipophilicity makes them suboptimal for imaginghypoxia due to high hepatobiliary background [23]. A novel seriesof complexes bearing more than one bioreductive moiety but withbetter stability and lower lipophilicity are needed. Therefore, weemploy a monoamine-monoamide dithiol (MAMA) ligand whichforms a stable and slightly lipophilic complex with 99mTc (Fig. 1a).

a

b

Fig. 1. a) Different 99mTc-labeled complexes: 99mTc-PnAO and 99mTc-MAMA derivatives. b) Structures of designed 99mTcO/ReO labeled MAMA-bisnitroimidazole and mono-nitroimidazole complexes.

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 51

99mTc-labeled radiotracers have been used widely due to favorablecharacteristics of Tc-99m radioisotope such as optimal g-ray energy(140 keV), moderate half life (6.0 h) and widespread availability of99Mo/99mTc generator. The 99mTc-MAMA complex has been usedextensively to radiolabel a variety of functional molecules [24e26].Conjugation of this complex to bioreductive groups has potential toimprove physicochemical properties of hypoxia markers.

Herein two novel MAMA-conjugated bisnitroimidazole ligandsas well as the correspondingmononitroimidazole compoundsweredesigned and synthesized (Fig. 1b). The [99mTc(V)O] complexes andthe corresponding [Re(V)O] analogs were prepared and theirphysicochemical and electrochemical characteristics were investi-gated. The [99mTc(V)O] complexes as hypoxia imaging agents wereevaluated in vivo.

2. Results and discussion

2.1. Synthesis

The designed MAMA-bisnitroimidazole and MAMA-mononitroimidazole compounds were prepared by conjugation ofterminal amino-functional bisnitroimidazole compounds withMAMA carboxylic acid derivatives. Compounds 6 and 7 were

synthesized from L-lysinemethyl ester hydrochloride as depicted inScheme 1A. Reaction of L-lysine methyl ester hydrochloride withacryloyl chloride gave the acryloyl derivatives 1. The resultingacryloyl derivative reacted with 4-nitroimidazole to obtain bisni-troimidazole derivatives 2 along with a certain amount of mono-addition product 4. The corresponding 2-nitroimidazole deriva-tives 3 and 5 were synthesized by the same procedure from 2-nitroimidazole and compound 1. Interestingly, 1H NMR of themono-addition compound 5 shows that there are two region-isomers, which are not found in compound 4, the mono-additionproduct of 4-nitroimidazole analog. Further aminolysis of 2 and 3by 1,3-diaminopropane gave terminal amino-functionalizedcompounds 6 and 7. The terminal amino-functional mono-nitroimidazole compounds 10 and 11 were synthesized accordingto the procedure reported previously [27] (Scheme 1B). Reaction of2-bromoethylphthalimide with 4-nitroimidazole or 2-nitroimidazole followed by hydrazinolysis and acidification of theresulting intermediate products 8 or 9 gave compounds 10 and 11.

MAMA(12)waspreparedas reportedpreviously [28] (Scheme2).Further reaction of compound 12with ethyl bromoacetate affordedethyl ester 13. MAMA-AA (14) was obtained by saponification of 13and subsequent acidification. The syntheses of MAMA-AA-B4NIL(15), MAMA-AA-B2NIL (16), MAMA-AA-4NI (17) and MAMA-AA-

a Reagents: (a) TEA, CH2Cl2; (b) for compound 2 and 4: 4-nitroimidazole, TEA, CH3OH; for

compound 3 and 5: 2-nitroimidazole, TEA, CH3OH; (c) 1, 3-diaminopropane, CH3OH.

b Reagents: (a) for compound 8: 4-nitroimidazole, K2CO3, DMF; for compound 9: 2-nitroimidazole,

K2CO3, DMF; (b) NH2NH2, alcohol.

A

B

Scheme 1. Synthesis of (A) bisnitroimidazole compounds 6 and 7a; (B) mononitroimidazole compounds 10 and 11b.

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6352

2NI (18) were performed by coupling MAMA-AA with the corre-sponding amine using BOP as condensation agent in DMF or aceto-nitrile solvent (Scheme 3). MAMA-AA-Pr (19) was also preparedsimilarly from 1-propylamine.

2.2. 99mTc-labeling

For 99mTc-labeling, the thiol groups of MAMA-nitroimidazolecompounds 15 and 16 were deprotected [26] in a mixture of HCl

a Reagents: (a) BrCH2COOCH2CH3, DIPEA, CH3CN; (b) i. NaOH, CH3OH; ii. HCl.

Scheme 2. Synthesis of MAMA-AAa.

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 53

and ethanol at 100 �C for 15 min and then reacted with 99mTc-GHintermediate (which was formed through reaction of 99mTcO4

� withglucoheptonate (GH) in the presence of stannous chloride asreducing agent) at 100 �C for another 15 min to give 99mTc-15 and99mTc-16. Similarly, 99mTc-17, 99mTc-18 and 99mTc-19 wereprepared using the same method. The radiochemical purities ofthese complexes were analyzed by radio-HPLC. The radio-HPLCchromatograms are shown in Fig. 2. The retention times of 99mTc-GH and 99mTcO4

� are 1.8 and 3.2 min, while those of 99mTc-15,99mTc-16, 99mTc-17, 99mTc-18 and 99mTc-19 are found to be 13.3,13.5, 13.3, 13.4 and 14.7 min, respectively. The peak of 99mTc-

a Reagents: (a) for compound 15: compound 6, BOP,

BOP, DIPEA, DMF; (b) for compound 17: compoun

compound 11, BOP, DIPEA, CH3CN; for compound 199mTc labeling: i. 0.5 M HCl, EtOH; ii. 99mTc-GH; fo

(Ph3P)2Re(=O)Cl3, CH3COONa, CH3OH.

Scheme 3. Synthesis of MAMA-AA-B4NIL, MAMA-AA-B2NIL, MAMA-AA-4NI, MAM

MAMA-AA, which appears at 9.4 min, is not found in all the aboveHPLC chromatograms and these results demonstrate the thermalstability of these complexes during the process of labeling. Theradiochemical purities of all the 99mTc-labeled complexes are over95% after preparation.

2.3. Preparation of rhenium complexes

The rhenium complexes were prepared by substitution ofdeprotected ligands with the [Re(V)O] precursor tri-chlorobis(triphenylphophine)oxorhenium(V) as shown in Scheme

DIPEA, DMF; for compound 16: compound 7,

d 10, BOP, DIPEA, CH3CN; for compound 18:

9: 1-propylamine, BOP, DIPEA, CH3CN; (c) for

r rhenium complexes: i. TFA, triethylsilane; ii.

A-AA-2NI and MAMA-AA-Pr and their rhenium and technetium compoundsa.

Fig. 2. HPLC chromatograms of 99mTc-labeled complexes: (A) 99mTcO4�, 3.2 min; (B) 99mTc-GH, 1.8 min; (C) 99mTc-15, 13.3 min; (D) 99mTc-16, 13.5 min; (E) 99mTc-17, 13.3 min; (F)

99mTc-18, 13.4 min; (G) 99mTc-19, 14.7 min.

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6354

3. Structures of these complexes were characterized by IR, 1H NMR,13C NMR and HR-MS. IR spectra show the expected [Re]O]stretching band in the region of 950e960 cm�1 together withintense bands around 1340 and 1500 cm�1 arising from nitrogroups. The disappearance of d at 7.20e7.40 ppm in 1H NMR cor-responding to protons signals on trityl groups and the change ofsignals on the MAMA ligands indicate the formation of ReO-MAMAcomplexes. The signals on the skeleton of ReO-MAMA complexes,which form square pyramidal configuration, are diastereotopic.Moreover, chemical shifts of carbon atoms belonging to MAMA

ligand move toward lower field in 13C NMR. It is another charac-teristic of ReO-MAMA complexes. All signals are assigned accordingto the literature data [28]. The accurate molecular formulas ob-tained by High-resolution mass spectra are consistent with theproposed structures of ReO-MAMA complexes.

HPLC analysis (shown in Figure S1) was performed with thesame solvent systems as those for 99mTc-labeled complexes. Each ofthe rhenium complex reveals only one major peak, i.e. only onechemical species in these complexes. The similarity of retentiontimes between the rhenium complexes and the corresponding

Table 2Radiochemical purity of four complexes in rat serum in vitro at different timeintervals.

1 h 2 h 4 h99mTc-15 92 e 8999mTc-16 98 e 8699mTc-17 93 90 8899mTc-18 99 93 87

A

B

C

D

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 55

99mTc-labeled complexes suggests their identical structures foreach of these five complexes.

2.4. Physicochemical studies

2.4.1. Partition coefficientsThe partition coefficients were determined by distributing the

99mTc-labeling complex in a mixture of 1-octanol and phosphatebuffer solution (0.1 M, pH 7.4) [27]. The results are shown in Table 1.All the complexes are less lipophilic than BMS181321 (log P(o/w):1.59) [22] and 99mTc-15 shows less lipophilicity than the othercomplexes.

2.4.2. In vitro stabilityAll the 99mTc-MAMA complexes of nitroimidazole derivatives

show no decomposition in PBS (0.1 M, pH 7.4) at room tem-perature during 4 h. The results indicate that 99mTc-MAMAcomplexes are more stable than the series of 99mTc-labeled PnAOcomplexes [22]. The in vitro stability in rat plasma was alsoevaluated [29]. Table 2 shows the radiochemical purity of thesefour complexes in rat serum in vitro. The major decompositionproduct is 99mTcO4

� (retention time: 3.2 min). The radiochemicalpurities are all over 85% after 4 h. High stability is observed for allthe compounds.

2.4.3. ElectrophoresisIn order to determine the charges of the labeled complexes,

paper electrophoresis was performed in phosphate buffer (0.05 M,pH 7.4). Pertechnetate (99mTcO4

�) was used as the reference in thesame electrophoretic conditions. The paper electrophoresispatterns of the complexes are shown in Supporting Information(Figure S2). Pertechnetate shows a trend of migration toward theanode, while the radioactivity of 99mTc-15 or 99mTc-16was found toremain at the origin in the same electrophoretic condition. Itsuggests that the complex is neutral, which is consistent with theproposed structures of the 99mTc-MAMA and Re-MAMA complexes.99mTc-17 and 99mTc-18 show similar paper electrophoresispatterns.

2.5. Electrochemical study

It is accepted that the hypoxia-selective accumulation ofhypoxia markers is initiated by enzyme-mediated one-electronreduction of bioreductive group within the cells [30,31]. The one-electron reduction is crucial to the hypoxia-selective tumoruptake. So the redox behaviors of the bisnitroimidazole derivatives(MAMA-bisnitroimidazole ligands and their rhenium complexes)were studied by cyclic voltammetry to evaluate their bioreductivecapacities.

Fig. 3 shows the cyclic voltammograms of the bisnitroimidazolecompounds 2, 3 and the corresponding mononitroimidazolecompounds 4, 5 in anhydrous DMF. The 4-nitroimidazole-derivedmononitroimidazole compound 4 displays a quasi-reversible redoxprocess with a cathodic peak potential, Epc(SCE) ¼ �1.456 V, whilethe corresponding bisnitroimidazole compound 2 displays anincrease of peak-to-peak separation and peak current, where

Table 1Partition coefficients of 99mTc-15, 99mTc-16, 99mTc-17 and 99mTc-18.

99mTc-15 99mTc-16 99mTc-17 99mTc-18

P(o/w) 1.32 � 0.07 1.90 � 0.12 4.09 � 0.13 4.86 � 0.13log P(o/w) 0.12 � 0.02 0.28 � 0.03 0.61 � 0.01 0.69 � 0.01

Epc(SCE) appears at a slightly more positive potential,�1.423 V. Theminor change of bisnitroimidazole compound with more positivereduction potential may result from the addition of the second 4-nitroimidazole group. Similarly, reduction potential (�1.136 V) ofthe 2-nitroimidazole compound 3 is slightly more positive thanthat (�1.161 V) of the mononitroimidazole 5. Though the intro-duction of a second nitroimidazole group leads to a slight increaseof reduction potential, the difference between them is negligible.

The redox behaviors of MAMA-derived ligands, MAMA-AA-B4NIL (15) and MAMA-AA-B2NIL (16), and their rheniumcomplexes were studied as well as those of MAMA-AA-4NI (17),MAMA-AA-2NI (18). Fig. 4 shows typical voltammograms forselected complex Re-18 and the corresponding ligand. Voltam-mograms for the other three rhenium complexes and the corre-sponding ligands are given in Supporting Information (Figures S3eS6). The voltammograms show that the reduction processes ofligands follow simple patterns which involve a redox couple cor-responding to an oxidationereduction process of nitro group. Thereduction potentials of 2-nitroimidazole derivatives are compa-rable to that of PnAO-1-(2-nitroimidazole), while 4-nitroimidazolederivatives are about 200e300 mV lower. Comparison between the4-nitroimidazole ligands shows that the MAMA-bisnitroimidazole15 possesses a more positive reduction potential thanMAMA-mononitroimidazole 17. On the contrary, the reductionpotential of MAMA ligands with two 2-nitroimidazole, i.e.compound 16, is lower than that of 18. Further comparisonbetween the reduction potentials of MAMA ligands 15e18 andcompounds 2e5 indicate that the reduction potentials for both ofthe 2-nitroimidazole and 4-nitroimidazole derivatives fluctuate

-1.8 -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

Potential, E(SCE) / V

Fig. 3. Cyclic voltammograms of bisnitroimidazole compounds 2 (A), 3 (C) and mon-onitroimidazole compounds 4 (B), 5 (D) recorded at 50 mV s�1 scanning in a negativedirection in anhydrous DMF. All potential are reported relative to the SCE (Ferrocenewas used as a reference).

-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0-80

-60

-40

-20

0

20

40

60

80

100

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0

-100

-80

-60

-40

-20

0

20

40

60

80

100

Cu

rre

nt /

A

Potential, E(SCE) / V

Cu

rre

nt /

A

Potential, E(SCE) / V

µ

µFig. 4. Voltammograms for selected complex Re-18 and the corresponding ligand (inset).

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6356

within small ranges and keep relatively constant (for 2-nitroimidazole: �1.04 to �1.17 V; for 4-nitroimidazole: �1.31to �1.45 V). It is consistent with the result obtained above thatnegligible difference is found between reduction potentials ofcompounds 2 and 4 or between those of 3 and 5.

The rhenium complexes display two or three reductionwaves. Itis found that the reduction of oxo Re(V) core is involved in thevoltammograms of Re-17 and Re-18, but not in those of Re-15 andRe-16 through comparison with the voltammogram of Re-19,a rhenium complex without nitro group. It may be related with themore complicated redox process of MAMA-bisnitroimidazoleligands. Table 3 gives the results of the cathodic peaks of nitrogroup and/or oxo Re(V) core for these compounds. The reduction ofnitro groups occurs between �1.04 and �1.51 V, while that of oxoRe(V) cores is between �1.84 and �2.01 V. All the rheniumcomplexes showmore negative reduction potentials of nitro groupsthan the corresponding free ligands, which is similar to the resultsreported by Rey et al. [32]. It indicates that the reduction of nitrogroup become more difficult after formation of the rheniumcomplex. The reduction potentials of the rhenium complexes Re-17

Table 3Reduction potential of MAMA ligands and their rhenium complexes recorded inDMF versus SCE (scan rate: 0.05 V s�1, concentration: 5 mM).

Epca (V) for NO2 reduction Epc (V) for Re(V) reduction

PnAO-2NIb �1.101 e

15 �1.314 e

16 �1.173 e

17 �1.382 e

18 �1.049 e

Re-15 �1.426 e

Re-16 �1.352 e

Re-17 �1.513 �1.943Re-18 �1.267 �1.845Re-19 e �2.010

a Epc: potential of cathodic peak.b PnAO-2NI: PnAO-1-(2-nitroimidazole).

and Re-18 are more positive than that of Re-19, which can beattributed to the stronger induction effect of nitroimidazole groupin Re-17 and Re-18.

2.6. Biodistribution studies

In order to assess the potentiality of 99mTc-labeled MAMA-bisnitroimidazole complexes for targeting tumor hypoxia, bio-distribution studies were performed inmale Kunmingmice bearingmurine sarcoma (S180), which is a typical hypoxia model of solidtumors [22,33e36]. The 99mTc-labeled MAMA-mononitroimidazolecomplexes, 99mTc-17 and 99mTc-18, were also evaluated in the samemode. The complete biodistribution results of the 99mTc-labeledMAMA complexes are summarized as Supplementary Material(Tables S1e4). All the complexes show high initial blood, kidneyand liver activity at 30 min. Blood clearance is relatively fast, whilekidney and liver activity remain high at 2 h. The activity is excretedmainly through the heptatobiliary tract as demonstrated by thehigh intestinal activity and part of renal tract.

Biological behaviors of the MAMA complexes are comparedwith that of BMS181321. At 2 h post-injection, the MAMA-mononitroimidazole complex 99mTc-18 shows higher tumor-to-blood ratio (0.51 � 0.11) than BMS181321 (tumor-to-blood ratio:0.31, tumor-to-muscle ratio: 2.63, in KHT tumor [23]), but tumor-to-muscle ratio (1.63 � 0.24) is not better. The 4-nitroimidazoleanalog 99mTc-17 shows similar results (tumor-to-blood ratio:0.58 � 0.06, tumor-to-muscle ratio: 1.32 � 0.23). However, theMAMA-bisnitroimidazole complex 99mTc-16 as well as its 4-nitroimidazole analog 99mTc-15 show higher tumor specificity(99mTc-16: tumor-to-blood ratio, 0.64 � 0.16, tumor-to-muscleratio, 2.73 � 0.47; 99mTc-15: tumor-to-blood ratio, 0.75 � 0.09,tumor-to-muscle ratio, 2.68 � 0.45) than BMS181321.

Tables 4 and 5 show tumor uptakes of the developed complexesat different time points post-injection. Figure S7 shows bio-distribution patterns of the MAMA-bisnitroimidazole complexes,99mTc-15 and 99mTc-16, in Kunming mice bearing murine sarcomatumor. Though both of them display similar distribution pattern,

Table 4Tissue or organ uptakes of 99mTc-15 and 99mTc-17 in Kunming mice bearing murinesarcoma tumor (% ID/g).

Time post injection

30 min 60 min 120 min 240 min99mTc-15Blood 9.16 � 0.83 5.69 � 0.47 3.71 � 0.37 2.51 � 0.11Muscle 2.05 � 0.16 1.39 � 0.30 1.04 � 0.04 0.57 � 0.05Tumor 4.83 � 0.07 3.72 � 0.53 2.77 � 0.40 1.83 � 0.26T/B 0.56 � 0.06 0.64 � 0.05 0.75 � 0.09 0.73 � 0.10T/M 2.37 � 0.13 2.95 � 0.09 2.68 � 0.45 3.23 � 0.46

99mTc-17Blood 6.23 � 0.81 4.12 � 0.23 3.85 � 0.32 2.78 � 0.37Muscle 2.91 � 0.81 1.19 � 0.10 1.69 � 0.23 0.92 � 0.09Tumor 2.48 � 0.33 1.90 � 0.41 2.21 � 0.14 2.01 � 0.35T/B 0.40 � 0.07 0.48 � 0.10 0.58 � 0.06 0.77 � 0.09T/M 0.88 � 0.18 1.47 � 0.28 1.32 � 0.23 2.17 � 0.39

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 57

activities in all the organs for 99mTc-16 are significantly lower than99mTc-15. As for 99mTc-15 and 99mTc-16, because of lower reductionpotentials of 4-nitroimidazole derivatives (i.e. relatively inefficientenzymatic reduction for 4-nitroimidazole [27,37,38]) than the cor-responding 2-nitroimidazoles, it is supposed that the tumor-to-tissue ratios of 4-nitroimidazole tracer, 99mTc-15, are not as goodas those of its 2-nitroimidazole analog. In fact, this is not the case inthe present study. Tumor-to-muscle and tumor-to-blood ratios for99mTc-15 (tumor-to-muscle and tumor-to-blood ratios at 240 min:3.23 � 0.46 and 0.73 � 0.10) are slightly better. It is probably due torelatively lower lipophilicity of 99mTc-15 (log P: 0.12 � 0.02) thanthat of 99mTc-16 (log P: 0.28 � 0.03), resulting in faster plasmaclearance. Therefore, electrochemical behavior of the selectedpharmacophore is important for the biological results, but lip-ophilicity is of greater importance here. On the contrary, MAMA-mononitroimidazole complexes, 99mTc-17 and 99mTc-18, displaythe expected distribution patterns that better tumor-to-tissueratios are obtained for the 2-nitroimidazole analog 99mTc-18. It isbecause that they have similar lipophilicity (log P: 0.61 � 0.01 and0.69 � 0.01) and, hence, the bioreductive activity plays a vital role.

Biological behavior of 99mTc-15 is better than that of the corre-sponding mononitroimidazole compound, 99mTc-17. The bio-distribution results show that tumor uptake of 99mTc-15 is higherthan that of 99mTc-17 and the activity in muscle is lower. Therefore,higher tumor-to-muscle ratios are achieved for the bisni-troimidazole complex 99mTc-15. Moreover, though more activity inblood is observed for 99mTc-15, tumor-to-blood ratios are also

Table 5Tissue or organ uptakes of 99mTc-16 and 99mTc-18 in Kunming mice bearing murinesarcoma tumor (% ID/g).

Time post injection

30 min 60 min 120 min 240 min99mTc-16Blood 2.93 � 0.28 2.62 � 0.34 1.32 � 0.15 0.92 � 0.17Muscle 0.59 � 0.06 0.52 � 0.12 0.31 � 0.05 0.24 � 0.03Tumor 1.49 � 0.15 1.24 � 0.21 0.83 � 0.16 0.64 � 0.09T/B 0.51 � 0.07 0.48 � 0.11 0.64 � 0.16 0.70 � 0.09T/M 2.54 � 0.37 2.43 � 0.42 2.73 � 0.47 2.68 � 0.50

99mTc-18Blood 7.40 � 0.16 6.50 � 1.24 3.03 � 0.46 2.32 � 0.44Muscle 2.20 � 0.40 1.63 � 0.16 0.95 � 0.16 0.66 � 0.08Tumor 3.09 � 0.41 3.13 � 0.85 1.56 � 0.41 1.86 � 0.31T/B 0.42 � 0.06 0.48 � 0.10 0.51 � 0.11 0.81 � 0.03T/M 1.42 � 0.18 1.91 � 0.32 1.63 � 0.24 2.83 � 0.45

higher than that of 99mTc-17 and show an increase with time due tofast clearance from blood. Unlike the 4-nitroimidazole analogs,99mTc-16 displays a lower tumor uptake than the corresponding99mTc-18. But relatively higher tumor-to-blood and tumor-to-muscle ratios are also achieved due to low activity in blood andmuscle. 99mTc-18, on the other hand, shows higher uptake in tumorbut blood and muscle activities are also significantly higher.

The biodistribution results demonstrate that MAMA-bisnitroimidazole complexes show better biodistribution charac-teristics (lower background activity in liver and fast clearance fromblood) compared with MAMA-mononitroimidazole complexes.Higher tumor-to-blood and tumor-to-muscle ratios are alsoobserved. These features make them more beneficial for tumortargeting and imaging. One of the factors that lead to the improvedresults for MAMA-bisnitroimidazole complexes is their lower lip-ophilicity (log P: 0.12 � 0.02 and 0.28 � 0.03 for MAMA-bisnitroimidazole complexes; log P: 0.61 � 0.01 and 0.69 � 0.01for MAMA-mononitroimidazole). Relatively moderate lipophilicitypromotes rapid excretion and provides better tumor-to-backgroundratios. Moreover, the number of bioreductive pharmacophores maycorrelate with the improved results. After increase of the bio-reductive pharmacophores in a molecule, cooperation of bioactiv-ities of the initial pharmacophore and the latteronehas contributionto relatively higher uptake and better retention in tumor asdemonstrated above, such as 99mTc-15. In addition, the reductionpotential may also affect the accumulation of the complex in tumor.But there are no obvious differences between the reduction poten-tials of the MAMA-bisnitroimidazole and MAMA-mononitroimidazole compounds. Therefore, although the reduc-tionpotential is important for the cellular uptake, other factors are ofgreater importance here.

3. Conclusions

Four designed MAMA-nitroimidazole compounds weresynthesized. The corresponding [99mTc(V)O] and [Re(V)O]complexes were prepared and evaluated as potential bioreductivemarkers for tumor.

1. Physicochemical studies show that the [99mTc(V)O] complexesof MAMA-nitroimidazole display more favorable charactersthan 99mTcO (PnAO-1-(2-nitroimidazole)) (BMS181321) due totheir lower lipophilicity and better stability.

2. The results of cyclic voltammetry indicate that the reductionpotentials for bothof the2-nitroimidazole and4-nitroimidazolederivatives fluctuate within small ranges and keep relativelyconstant. It is found that although the reductionofnitro group inthe [Re(V)O] complexes become more difficult after ligationwith oxo-rhenium core, reduction potentials of nitro groups inthe ReO-MAMA complexes are still in the range for potentiallybioreductive compounds [39e42] (i.e. tirapazamine, �0.90 V[41]; PnAO-1-(2-nitroimidazole), �1.101 V as shown above).

3. Biodistribution studies demonstrated that each MAMA-mononitroimidazole complex with either single 2-nitroimidazole or 4-nitroimidazole moiety shows highertumor-to-blood ratio but lower tumor-to-muscle ratio thanBMS181321, while both of the MAMA-bisnitroimidazolecomplexes show higher tumor specificity (higher tumor-to-blood ratios and tumor-to-muscle ratios) compared withBMS181321.

4. The 2-nitroimidazole complex shows better tumor-to-tissueratios than the corresponding 4-nitroimidazole analog forMAMA-mononitroimidazole complexes, whereas the oppositeresults are obtained forMAMA-bisnitroimidazole complexes dueto higher lipophilicity of 2-nitroimidazole analog. Moreover,

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6358

relatively rapider excretion, lower background activity in liverand higher tumor-to-blood and tumor-to-muscle ratios are ach-ieved for both of MAMA-bisnitroimidazole complexes than thecorresponding MAMA-mononitroimidazole complexes.

4. Experimental procedures

L-Lysine methyl ester hydrochloride (>99%) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophos-phate (BOP) (>98%) were obtained from GL Biochem (Shanghai,China). 4-Nitroimidazole (98%), 2-nitroimidazole (98%) and1,3-diaminopropane were purchased from ACROS OrganicsCompany (NJ, USA). Trichlorobis(triphenylphophine)oxo-rhenium(V) ((Ph3P)2Re(]O)Cl3) were purchased from Aldrich (MO,USA). Methylene chloride, acrylonitrile and N,N0-dimethyl form-amide were purified by distillation. All other reagents were of A. R.Grade and used as received. 3,3,9,9-Tetramethyl-1-(2-nitro-lH-imidazol-L-yl)-4,8-diazaundecane-2,10-didone dioxime wassynthesized as reported [43]. 99mTcO4

� was obtained fromcommercial 99Moe99mTc generator (China Institute of Atom Energy,Beijing). Murine sarcoma (S180) cell line and male Kunming mice(20e25 g, male) were supplied by Department of LaboratoryAnimal Science, Peking University Health Science Center.

NMR spectrawere recorded on a VarianMercury Plus (300MHz,Varian, USA) and a Bruker AVANCE III (500 MHz, Bruker,Switzerland) with deuterated dimethyl sulfoxide (DMSO-d6),chloroform (CDCl3) or deuterium oxide as a solvent, and with tet-ramethylsilane (TMS) as an internal standard. ESI-MS spectra(including high resolutionmass spectra, HRMS)were obtainedwitha Bruker APEX IV Fourier transform ion cyclotron resonance massspectrometer (Bruker, USA). Melting points were determined ina Tech XTL-20 melting point apparatus (Tech Instrument Co. Ltd,Beijing, China) and were uncorrected. IR spectra were recorded atIR Affinity-1 FT-IR instrument (Shimadzu, Japan). Cyclic voltam-mograms were recorded on a CHI600C electrochemical worksta-tion (Chen Hua Instrument Co. Ltd, Shanghai, China) employinga glassy carbon working electrode, a platinum wire counter elec-trode and an Ag/Agþ reference electrode. Radioactivity in organs ortissues of mice was assayed using aWizard II 2470 series AutomaticGamma Counter (Perkin Elmer, USA). Radio-HPLC analysis wasperformed on a reversed-phase column (Agilent HC-C18,4.6 � 150 mm, size 5 m) and a Waters 2487 dual wavelengthabsorbance detector (Waters 600E series) and a radiometricdetector (Packard 500TR series) system with the following solventsystems: A, 0.1 M ammonium acetate; B, CH3CN. Gradient: 0e2min90%A; 2e15 min 90%e50%A; 15e20 min 50e20%A; 20e22 min 20%A; 22e24 min 20%e90%A; flow rate 1.0 mL/min.

4.1. Synthesis

4.1.1. Synthesis of methyl 2,6-diacrylamidohexanoate (1)Acrylyl chloride (2.34 g, 25.9 mmol) in dichloromethane (DCM)

(20 mL) was added dropwise to a suspension of L-lysine methylester hydrochloride (2.33 g, 10.0 mmol) and triethylamine (TEA)(5.20 g, 51.4 mmol) in DCM (20 mL) in ice-water bath. After anadditional 1 h at 0 �C, the reaction mixture was warmed to roomtemperature and placed in darkness overnight. The mixture waswashed with 20 mL of 1 M aqueous HCl, 20 mL of a saturatedaqueous solution of NaHCO3 and 20 mL of brine. The organic phasewas dried over magnesium sulfate and evaporated under reducedpressure. The crude residue was purified by chromatography(MeOH:DCM 1:50) to give compound 1 (0.71 g, 26.5%), mp 84e85 �C. IR (KBr): 2951, 2862, 1734, 1654, 1624, 1249, 1215, 1177,995, 952, 810 cm�1. 1H NMR (300MHz, CDCl3, d ppm): 6.51 (d,1H,e

CONHCHCOOCH3); 6.26e6.37 (m, 2H, CH2(trans)]CHe); 6.07e6.25(m, 2H, CH2]CHe); 6.00 (s, 1H, eCONHCH2CH2e); 5.62e5.72 (m,2H, CH2(cis)]CHe); 4.66 (m,eCHCOOCH3); 3.76 (s, 3H,eCOOCH3);3.34 (m, 2H, eCH2CH2NHCOe); 1.78e1.89 (m, 2H, eCH2CH(NHCO)COOCH3); 1.59 (m, 2H, eCH2CH2NHCOe); 1.40 (m, 2H, e

CH2CH2CH2NHCOe). 13C NMR (500 MHz, CDCl3, d ppm): 172.85(COOCH3); 166.09 and 165.68 (CONH); 130.99 and 130.40 (CH]CH2); 127.11 and 126.17 (CH]CH2); 52.42 (CH); 51.97 (CH3); 38.64(CH2); 31.59 (CH2); 28.69 (CH2); 22.28 (CH2). HRMS (ESI): masscalculated for C13H21N2O4 (M þ Hþ), 269.14958; m/z found,269.14901.

4.1.2. Synthesis of methyl 2,6-bis(3-(4-nitro-1H-imidazol-1-yl)propanamido)hexanoate (2) and methyl 6-acrylamido-2-(3-(4-nitro-1H-imidazol-1-yl)propanamido)hexanoate (4)

4-Nitroimidazole (0.23 g, 2.0 mmol), compound 1 (0.27 g,1.0 mmol) was added to methanoleTEA mixed solvents (10 mL, v/v¼ 1/1). The suspensionwas heated to reflux for 7 d. After removingthe solvent under reduced pressure, the residue was chromato-graphed with a gradient (1:50e1:20) of methanol/DCM to givecompound 2 (0.06 g,12.2%),mp 94e95 �C. Rf 0.32 (MeOH:DCM1:10).IR (KBr): 2951, 2862, 1734, 1653, 1560, 1332, 1288, 983, 823 cm�1. 1HNMR (300 MHz, DMSO-d6, d ppm): 8.41 (d, 1H, eCONHCHCOOCH3);8.35 (m, 2H, imidazole C2eH); 7.97 (t, 1H, eCONHCH2CH2e); 7.79(m,2H, imidazoleC5eH);4.28 (m, 4H,eCH2CH2eimidazole); 4.15 (m,1H, eCHCOOCH3); 3.58 (s, 3H, eCOOCH3); 2.96 (m, 2H, e

CH2CH2NHCOe); 2.73 (t, 2H, eCH2CH2eimidazole); 2.63 (t, 2H, eCH2CH2eimidazole); 1.47e1.62 (m, 2H, eCH2CH(NHCO)COOCH3);1.25 (m, 2H, eCH2CH2NHCOe); 1.13 (m, 2H, eCH2CH2CH2NHCOe).13C NMR (500 MHz, DMSO-d6, d ppm): 172.38 (COOCH3); 169.40and 168.80 (CONH); 146.77 (imidazole C4); 137.47 (imidazole C2);121.53 (imidazole C5); 51.83 (CH); 51.76 (CH3); 43.88 and 43.76(imidazoleeCH2CH2); 38.08 (CH2); 35.85 and 35.50 (imidazoleeCH2CH2); 30.34 (CH2); 28.46 (CH2); 22.54 (CH2). MS (ESI): masscalculated for C19H26N8O8, 494.2; m/z found, 495.2 (M þ Hþ); 517.2(M þ Naþ); 533.1 (M þ Kþ). HRMS (ESI): mass calculated forC19H27N8O8 (Mþ Hþ), 495.19464;m/z found, 495.19404.

Mono-addition product 4 was also isolated (0.09 g), mp 56e57 �C. Rf 0.54 (MeOH:DCM 1:10). IR (KBr): 2954, 2864, 1734,1683, 1655, 1560, 1541, 1332, 1288, 823 cm�1. 1H NMR (500 MHz,DMSO-d6, d ppm): 8.40 (d, 1H, eCONHCHCOOCH3); 8.32 (m, 1H,imidazole C2eH); 8.06 (t, 1H, eCONHCH2CH2e); 7.79 (m, 1H,imidazole C5eH); 6.17e6.22 (dd, 1H, CH2]CHe); 6.04e6.07 (dd,1H, CH2(trans)]CHe); 5.55e5.57 (dd, 1H, CH2(cis)]CHe); 4.29 (t,2H,eCH2CH2eimidazole); 4.19 (m,1H,eCHCOOCH3); 3.59 (s, 3H,eCOOCH3); 3.07 (m, 2H, eCH2CH2NHCOe); 2.72 (t, 2H, eCH2CH2e

imidazole); 1.51e1.67 (m, 2H, eCH2CH(NHCO)COOCH3); 1.38 (m,2H,eCH2CH2NHCOe); 1.21 (m, 2H,eCH2CH2CH2NHCOe). MS (ESI):mass calculated for C16H23N5O6, 381.2; m/z found, 382.2 (M þ Hþ),404.2 (M þ Naþ), 420.1 (M þ Kþ). HRMS (ESI): mass calculated forC16H24N5O6 (M þ Hþ), 382.17211; m/z found, 382.17163.

4.1.3. Synthesis of methyl 2,6-bis(3-(2-nitro-1H-imidazol-1-yl)propanamido)hexanoate (3) and methyl 6-acrylamido-2-(3-(2-nitro-1H-imidazol-1-yl)propanamido)hexanoate (5)

Compound 3 was obtained in a similar procedure from 2-nitroimidazole and compound 1. Yield: 0.10 g, 20.4%, mp 87e88 �C. Rf 0.20 (MeOH:ethyl acetate 1:20). IR (KBr): 2928, 2888,1734,1653,1560,1541,1358,1198, 835, 744 cm�1. 1HNMR (300MHz,DMSO-d6, d ppm): 8.35 (d, 1H, CONHCHCOOCH3); 7.93 (m, 1H, eCONHCH2CH2e); 7.52 (m, 2H, imidazole C5eH); 7.14 (m, 2H, imid-azole C4eH); 4.59 (m, 4H, eCH2CH2eimidazole); 4.15 (m, 1H, eCHCOOCH3); 3.60 (s, 3H,eCOOCH3); 2.95 (m, 2H,eCH2CH2NHCOe);2.73 (t, 2H,eCH2CH2eimidazole); 2.64 (t, 2H,eCH2CH2eimidazole);1.46e1.60 (m, 2H, eCH2CH(NHCO)COOCH3); 1.25 (m, 2H, e

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 59

CH2CH2CH2NHCOe); 1.11 (m, 2H, eCH2CH2CH2NHCOe). 13C NMR(500 MHz, DMSO-d6, d ppm): 172.35 (COOCH3); 169.33 and 168.77(CONH); 144.60 (imidazoleC2); 127.91 and127.56 (imidazoleC4 andC5); 51.79 (CH); 46.02 and 45.84 (imidazoleeCH2CH2); 38.13 (CH2);36.43 (CH3); 35.65 and 35.23 (imidazoleeCH2CH2); 30.46 (CH2);28.45 (CH2); 22.54 (CH2). MS (ESI): mass calculated for C19H26N8O8,494.2;m/z found, 495.2 (Mþ Hþ); 517.2 (Mþ Naþ); 533.1 (Mþ Kþ).HRMS (ESI): mass calculated for C19H27N8O8 (M þ Hþ), 495.19464;m/z found, 495.19389.

Similarly, mono-addition product 5 was isolated (0.06 g), mp59e60 �C. Rf 0.38 (MeOH:ethyl acetate 1:20). IR (KBr): 2961, 2864,1734, 1655, 1560, 1541, 1363, 1261, 1198, 1097, 987, 804 cm�1. 1HNMR (500 MHz, DMSO-d6, d ppm): As a mixture of two isomers.Isomer 1 (65% of total): 8.35 (d, 1H, eCONHCHCOOCH3); 8.05 (m,1H, eCONHCH2CH2e); 7.50 (s, 1H, imidazole C5eH); 7.15 (s, 1H,imidazole C4eH); 6.17e6.22 (dd, 1H, CH2]CHe); 6.04e6.07 (dd,1H, CH2(trans)]CHe); 5.55e5.57 (dd, 1H, CH2(cis)]CHe); 4.57e4.59 (m, 2H, eCH2CH2eimidazole); 4.15e4.19 (m, 1H, e

CHCOOCH3); 3.60 (s, 3H, eCOOCH3); 3.09 (m, 2H, eCH2CH2NHCOe); 2.73 (m, 2H, eCH2CH2eimidazole); 1.51e1.67 (m, 2H, e

CH2CH(NHCO)COOCH3); 1.17e1.40 (m, 2H, eCH2CH2NHCOe and e

CH2CH2CH2NHCOe). Isomer 2 (35% of total): 8.44 (d, 1H, eCON-HCHCOOCH3); 7.91 (m,1H,eCONHCH2CH2e); 7.50 (s, 1H, imidazoleC5eH); 7.13 (s, 1H, imidazole C4eH); 6.28e6.33 (dd, 1H, CH2]CHe); 6.09e6.12 (dd, 1H, CH2(trans)]CHe); 5.62e5.64 (dd, 1H,CH2(cis)]CHe); 4.57e4.59 (m, 2H, eCH2CH2emidazole); 4.25e4.29 (m, 1H, eCHCOOCH3); 3.63 (s, 3H, eCOOCH3); 2.97 (m, 2H, eCH2CH2NHCOe); 2.63 (m, 2H, eCH2CH2eimidazole); 1.51e1.67(m, 2H, eCH2CH(NHCO)COOCH3); 1.17e1.40 (m, 2H, e

CH2CH2NHCOe and eCH2CH2CH2NHCOe). MS (ESI): mass calcu-lated for C16H23N5O6, 381.2; m/z found, 382.2 (M þ Hþ), 404.2(M þ Naþ), 420.1 (M þ Kþ). HRMS (ESI): mass calculated forC16H24N5O6 (M þ Hþ), 382.17211; m/z found, 382.17166.

4.1.4. Synthesis of compound 6 and 7A mixture of compound 2 (0.20 g, 0.40 mmol) and 1,3-

diaminopropane (0.74 g, 10.0 mmol) in methanol was reacted atroom temperature for 40 h. After evaporatingmethanol, the residuewas pumped in high vacuum to remove the residual 1,3-diaminopropane as much as possible. Chromatography of theresidue on silica gel with MeOH:DCM (1:1) as eluents gavecompound 6 (0.12 g, 55.3%). IR (KBr): 2928, 2858, 1655, 1541, 1491,1363, 1132, 1261, 1161, 835 cm�1. 1H NMR (300 MHz, DMSO-d6,d ppm): 8.35 (m, 2H, imidazole C2eH); 8.16 (d, 1H, e

CHNHCOCH2CH2eimidazole); 7.98 (t, 1H, eCH2NHCOCH2CH2e

imidazole); 7.80 (m, 2H, imidazole C5eH); 4.28 (t, 4H, eCH2CH2e

imidazole); 4.12 (m, 1H, eCHCONHCH2CH2CH2NH2); 3.16 (s, 2H, eCONHCH2CH2CH2NH2); 3.06 (m, 2H, eCH2NHCOCH2CH2e

imidazole); 2.94 (t, 2H, eCONHCH2CH2CH2NH2); 2.72 (m, 2H, eCH2CH2eimidazole); 2.64 (t, 2H, eCH2CH2eimidazole); 2.48 (t,2H, eCONHCH2CH2CH2NH2); 1.42 (m, 4H, eCH2CH(NHCO)CONHeand CONHCH2CH2CH2NH2); 1.24 (m, 2H, eCH2CH2CH2NHCOe);1.07 (m, 2H, eCH2CH2CH2NHCOe). 13C NMR (500 MHz, DMSO-d6,d ppm): 171.22 (CONH); 168.95 (CONH); 168.77 (CONH); 146.76(imidazole C4); 137.46 (imidazole C2); 121.55 (imidazole C5); 52.46(CH); 43.88 (imidazoleeCH2CH2); 38.91 (CH2); 38.26 (CH2); 36.17(CH2); 35.85 and 35.63 (imidazoleeCH2CH2); 32.66 (CH2); 31.77(CH2); 28.62 (CH2); 22.60 (CH2). MS (ESI): mass calculated forC21H32N10O7, 536.2; m/z found, 537.3 (M þ Hþ). HRMS (ESI): masscalculated for C21H33N10O7 (M þ Hþ), 537.25282; m/z found,537.25190.

The preparation of compound 7 was similar from compound 3(0.04 g, 13.8%). mp 105e106 �C. IR (KBr): 2927, 1654, 1570, 1541,1490, 1363, 1261, 1161, 1132, 810 cm�1. 1H NMR (300 MHz, DMSO-d6, d ppm): 8.13 (m,1H,eCHNHCOCH2CH2eimidazole); 7.98 (m,1H,

eCH2NHCOCH2CH2eimidazole); 7.53 (m, 2H, imidazole C5eH);7.15 (m, 2H, imidazole C4eH); 4.58 (t, 4H, eCH2CH2eimidazole);4.11 (m, 1H, eCHCONHCH2CH2CH2NH2); 3.17 (s, 2H, e

CONHCH2CH2CH2NH2); 3.08 (m, 2H, eCH2NHCOCH2CH2e

imidazole); 2.95 (m, 2H, eCONHCH2CH2CH2NH2); 2.72 (m, 2H, eCH2CH2eimidazole); 2.64 (m, 4H, eCH2CH2eimidazole and e

CONHCH2CH2CH2NH2); 1.53 (m, 4H, eCH2CH(NHCO)CONHe andCONHCH2CH2CH2NH2); 1.24 (m, 2H,eCH2CH2CH2NHCOe); 1.07 (m,2H, eCH2CH2CH2NHCOe). 13C NMR (500 MHz, DMSO-d6, d ppm):171.90 (CONH); 169.16 (CONH); 168.80 (CONH); 144.63 (imidazoleC2); 127.88 (imidazole C5); 127.59 (imidazole C4); 52.61 (CH); 45.98and 45.90 (imidazoleeCH2CH2); 38.28 (CH2); 36.76 (CH2); 35.65(CH2); 35.49 and 35.40 (imidazoleeCH2CH2); 31.51 (CH2); 28.63(CH2); 27.37 (CH2); 22.64 (CH2). MS (ESI): mass calculated forC21H32N10O7, 536.2; m/z found, 537.3 (M þ Hþ); 559.2 (M þ Naþ).

4.1.5. Synthesis of 2-(4-nitro-1H-imidazolyl)ethylamine (10) and 2-(2-nitro-1H-imidazol-1-yl)ethylamine (11)

Compounds 10 and 11were prepared as reported by Rauth et al.[27] with minor modifications. Yield of 2-(4-nitro-1H-imidazolyl)ethylamine 10, 1.64 g (61.7%). 1H NMR (300 MHz, D2O, d ppm): 8.29(s, 1H, imidazole C2eH); 7.85 (s, 1H, imidazole C5eH); 4.51 (t, 2H,eCH2CH2eimidazole); 3.54 (t, 2H, eCH2CH2eimidazole). 13C NMR(500 MHz, D2O, d ppm): 147.05 (imidazole C4); 137.91 (imidazoleC2); 121.50 (imidazole C5); 45.26 (CH2); 39.26 (CH2). MS (ESI): masscalculated for C5H8N4O2, 156.1; m/z found, 157.1(M þ Hþ). Yield of2-(2-nitro-1H-imidazol-1-yl)ethylamine 11, 0.12 g (43.4%). 1H NMR(300 MHz, D2O, d ppm): 7.41 (s, 1H, imidazole C5eH); 7.13 (s, 1H,imidazole C4eH); 4.69 (t, 2H, eCH2CH2eimidazole); 3.45 (t, 2H, eCH2CH2eimidazole). 13C NMR (500 MHz, D2O, d ppm): 144.72(imidazole C2); 128.43 (imidazole C5); 128.01 (imidazole C4); 46.72(CH2); 39.26 (CH2). MS (ESI): mass calculated for C5H8N4O2, 156.1;m/z found, 157.1(M þ Hþ).

4.1.6. Synthesis of MAMA-AA (14)MAMA-AA (14) was prepared as reported in the literature [29].

Ethyl 2-bromoacetate (0.21 g, 1.2 mmol) in 8 mL of acetonitrile wasgradually added to a mixed solution of MAMA (12) [28] (0.68 g,1.0 mmol) and diisopropylethylamine (DIPEA) (0.39 g, 3.00 mmol)in 2mL of acetonitrile. The reaction mixture was refluxed overnightand the solvent was removed in vacuum. The oily residue waspurified by silica gel chromatography using ethyl acetateepetroleum (1:3) as the eluent to provide compound 13 (0.69 g,90.2%) as a yellow oil. 1H NMR (300 MHz, CDCl3, d ppm): 7.41e7.36(m, 12H, Ph H); 7.17e7.27 (m, 18H, Ph H); 4.06 (q, 2H, eCH2CH3);3.16 (s, 2H, eNCH2COOe); 3.07 (s, 2H, eNCH2CONHe); 3.03 (td, 2H,eCH2NHCOe); 2.55 (t, 2H, eCH2NCH2COe); 2.36 (t, 2H, eCH2Se);2.25 (t, 2H, eCH2Se); 1.20 (t, 3H, eCH2CH3). MS (ESI): masscalculated for C48H48N2O3S2, 764.3; m/z found, 764.9 (M þ Hþ),787.3 (M þ Naþ). Compound 14 was obtained by hydrolysis of theethyl ester 13 in a mixture of NaOH and methanol followed byacidification with HCl (1 M) and exaction by chloroform. Yield,0.62 g (93.9%). 1H NMR (300 MHz, CDCl3, d ppm): 7.37e7.40 (m,12H, Ph H); 7.21e7.27 (m, 18H, Ph H); 3.21 (s, 2H, eNCH2COOe);3.12 (s, 2H, eNCH2CONHe); 3.02 (td, 2H, eCH2NHCOe); 2.55 (t,2H,eCH2NCH2COe); 2.37 (t, 2H,eCH2Se); 2.28 (t, 2H,eCH2Se). 13CNMR (500 MHz, CDCl3, d ppm): 172.49 and 170.45 (CO); 144.64,129.55, 127.97 and 126.75 (Ph); 67.04 and 66.80 (CPh3); 57.32, 54.54and 53.70 (CH2); 38.34 (CH2NH); 31.64 and 29.65 (CH2S). MS (ESI):mass calculated for C46H44N2O3S2, 736.3; m/z found, 736.7(M þ Hþ), 759.3 (M þ Naþ).

4.1.7. Synthesis of MAMA-AA-B4NIL (15), MAMA-AA-B2NIL (16)MAMA-AA-B4NIL (15) BOP (0.029 g, 0.066mmol), DIPEA (0.021 g,

0.17mmol)andcompound14 (0.048g,0.065mmol)weredissolved in

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6360

DMF (2 mL) and stirred for 30 min. To the solution was addedcompound 6 (0.032 g, 0.060mmol). The mixture was stirred at roomtemperature overnight. The solvent was evaporated under reducedpressure and the crude residuewas dissolved in ethyl acetate (10mL)and the organic phases were washed with 1 M HCl (10 mL), water(10 mL), saturated NaHCO3 aqueous solution (10 mL), and brine(10 mL). The combined organic layers were dried over magnesiumsulfate. The solvent was evaporated under reduced pressure and thecrude residue was purified by chromatography on silica gel(MeOH:DCM, 1:20e1:10) to give compound 15 (0.053 g, 70.8%), mp94e95 �C. IR (KBr): 2930, 2860, 1647, 1541, 1522, 1491, 1442, 1335,1288, 1128, 823, 744, 700 cm�1. 1H NMR (500 MHz, CDCl3, d ppm):7.84e7.86 (m, 2H, imidazole C2eH); 7.48e7.54 (m, 2H, imidazole C5eH); 7.34e7.36 (m,12H, PhH); 7.22e7.26 (m,18H, PhH); 4.36 (m, 6H,eCH2CH2eimidazole andeCHCONHe); 3.02 (m,12H,eCH2NHe ande

CH2Ne); 2.82 (m, 2H,eCH2CH2eimidazole); 2.72 (m, 2H,eCH2CH2e

imidazole); 2.54 (t, 2H,eCH2NCH2COe); 2.40 (t, 2H,eCH2Se); 2.33 (t,2H, eCH2Se); 1.47e1.55 (m, 4H, eCH2CH(NHCO)CONHe andCONHCH2CH2CH2NH); 1.26 (m, 2H,eCH2CH2CH2NHCOe); 0.95e0.97(m, 2H,eCH2CH2CH2NHCOe). 13CNMR (500MHz, DMSO-d6, dppm):171.37,169.80,169.72,169.07 and 168.76 (CO); 146.78 (imidazole C4);144.43 and 144.39 (Ph); 137.45 (imidazole C2); 129.07, 129.02,127.97and 126.67 (Ph); 121.53 (imidazoleC5); 66.24 and 65.88 (CPh3); 57.67,57.58, 54.87 and 53.79 (CH2); 52.57 (CH); 43.88 and 43.85(imidazoleeCH2); 38.29, 37.30, 35.99, 35.87, 35.78, 35.65, 31.69, 31.35,29.16, 28.96, 28.67 and 22.69 (CH2). MS (ESI): mass calculated forC67H74N12O9S2, 1254.51; m/z found, 1255.15 (M þ Hþ), 1277.37(MþNaþ). HRMS (ESI):mass calculated for C67H75N12O9S2 (MþHþ),1255.52159;m/z found, 1255.52232.

MAMA-AA-B2NIL (16): The preparation of compound 16 wassimilar to that of compound 15 from compound 7. Yield: 0.027 g(33.0%), mp 89e90 �C. IR (KBr): 2928, 2858, 1647, 1533, 1489, 1442,1361, 1159, 1130, 1033, 835, 744, 702 cm�1. 1H NMR (500 MHz,CDCl3, d ppm): 7.48e7.49 (m, 2H, imidazole C5eH); 7.06e7.12 (m2H, imidazole C4eH); 7.36e7.38 (m, 12H, Ph H); 7.24e7.27 (m, 18H,Ph H); 6.76 (br, 1H, eCONHe); 4.73 (m, 4H, eCH2CH2eimidazole)4.31 (m, 1H, eCHCONHe); 3.02e3.10 (m, 12H, eCH2NHe and e

CH2Ne); 2.83e2.89 (m, 4H, eCH2CH2eimidazole); 2.58 (t, 2H, eCH2NCH2COe); 2.42 (t, 2H, eCH2Se); 2.36 (m, 2H, eCH2Se);1.65e1.70 (m, 2H, CONHCH2CH2CH2NH); 1.52 (m, 2H, e

CH2CH(NHCO)CONHe); 1.27 (m, 2H, eCH2CH2CH2NHCOe); 0.89(m, 2H, eCH2CH2CH2NHCOe). 13C NMR (500 MHz, DMSO-d6,d ppm): 171.37, 169.84, 169.74, 169.10 and 168.77 (CO); 144.60(imidazole C2); 144.42, 144.38, 129.07, 129.01 and 127.96 (Ph);127.60 and 127.52 (imidazole C4 and C5); 126.68 (Ph); 66.24 and65.87 (CPh3); 57.58, 54.86 and 53.79 (CH2); 52.59 (CH); 46.02 and45.92 (imidazoleeCH2); 38.32, 37.30, 35.94, 35.75, 35.66, 35.39,31.66, 31.33, 29.16, 28.93, 28.65 and 22.68 (CH2). MS (ESI): masscalculated for C67H74N12O9S2,1254.51;m/z found,1255.11 (MþHþ),1277.46 (M þ Naþ). HRMS (ESI): mass calculated for C67H75N12O9S2(M þ Hþ), 1255.52159; m/z found, 1255.52041.

4.1.8. Synthesis of MAMA-AA-4NI (17), MAMA-AA-2NI (18) andMAMA-AA-Pr (19)

MAMA-AA-4NI (17): To a suspension of compound 14 (0.74 g,1.0 mmol) in acetonitrile (10 mL) were added DIPEA (0.31 g,2.4 mmol), BOP (0.45 g, 1.0 mmol). After stirring for 30 min,compound 10 (0.19 g, 1.0 mmol) was added to the solution. Themixture was stirred at room temperature overnight. The solventwas evaporated under reduced pressure and the crude residue wasdissolved in ethyl acetate (25 mL) and the organic phases werewashed with 1 M HCl (20 mL), water (20 mL), saturated NaHCO3aqueous solution (20mL), and brine (20mL). The combined organiclayers were dried over magnesium sulfate. The solvent was evap-orated under reduced pressure and the crude residue was purified

by chromatography on silica gel (ethyl acetate) to give compound17 (0.53 g, 60.6%), mp 77e78 �C. IR (KBr): 2926, 1654, 1541, 1490,1442, 1336, 1290, 1132, 823, 744, 700 cm�1. 1H NMR (500 MHz,CDCl3, d ppm): 7.95 (br, 1H, eCONHe); 7.63 (d, 1H, imidazole C5eH); 7.35e7.40 (m, 13H, Ph H and imidazole C4eH); 7.22e7.31 (m,18H, Ph H); 6.39 (br, 1H, eCONHe); 3.95 (t, 2H, eCH2CH2e

imidazole); 3.41 (m, 2H, eCH2CH2eimidazole); 3.04 (m, 4H, e

NCH2COe and eCH2NHCOe); 2.93 (s, 2H, eCH2NHCOe); 2.47 (m,4H, eCH2NCH2COe and CH2Se); 2.35 (t, 2H, eCH2Se). 13C NMR(500MHz, CDCl3, d ppm): 170.83 and 170.08 (CO); 147.12 (imidazoleC4); 143.35 (Ph); 135.15 (imidazole C2); 128.48, 127.09, 127.06 and125.97 (Ph); 118.40 (imidazole C5); 66.34 and 65.98 (CPh3); 57.72,56.81 and 53.29 (CH2N); 45.92 (imidazoleeCH2); 38.25(imidazoleeCH2CH2), 36.95 (CH2NH), 31.08 and 29.28 (CH2S). MS(ESI): mass calculated for C51H50N6O4S2, 874.33; m/z found, 874.66(M þ Hþ), 897.18 (M þ Naþ), 913.10 (M þ Kþ). HRMS (ESI): masscalculated for C51H51N6O4S2 (M þ Hþ), 875.34077; m/z found,875.33915.

MAMA-AA-2NI (18): The preparation of compound 18 wassimilar from compound 11. Yield: 0.34 g (78.1%), mp 75e77 �C. IR(KBr): 2926,1654,1541,1489,1443, 1364,1273,1244,1163, 833, 744,700 cm�1. 1H NMR (500 MHz, CDCl3, d ppm): 7.61 (br, 1H, eCONHe); 7.37e7.39 (m, 12H, Ph H); 7.20e7.29 (m, 18H, Ph H); 7.05 (s, 1H,imidazole C5eH); 7.00 (s, 1H, imidazole C4eH); 6.50 (br, 1H, eCONHe); 4.37 (t, 2H, eCH2CH2eimidazole); 3.50 (eCH2CH2e

imidazole); 3.00e3.03 (m, 4H, eNCH2COe and eCH2NHCOe);2.92 (s, 2H, eCH2NHCOe); 2.48 (m, 4H, eCH2NCH2COe andCH2Se); 2.31 (t, 2H, eCH2Se). 13C NMR (500 MHz, CDCl3, d ppm):170.98 and 169.62 (CO); 146.89 (imidazole C2); 144.37 (Ph); 129.50,128.23, 128.03, 127.92, 127.00 and 126.91 (Ph and imidazole C2 andC5); 67.28 and 66.99 (CPh3); 58.55, 57.92 and 54.14 (CH2N); 48.82(imidazoleeCH2); 39.01 (imidazoleeCH2CH2), 37.94 (CH2NH), 32.09and 30.07 (CH2S). MS (ESI): mass calculated for C51H50N6O4S2,874.33; m/z found, 897.31 (M þ Naþ). HRMS (ESI): mass calculatedfor C51H51N6O4S2 (M þ Hþ), 875.34077; m/z found, 875.33840.

MAMA-AA-Pr (19): The preparation of compound 19was similarfrom propylamine. Yield: 0.67 g, 85.9%, mp 53e55 �C. IR (KBr):2927, 2872, 1655, 1525, 1490, 1442, 1240, 842, 743, 700 cm�1. 1HNMR (500 MHz, CDCl3, d ppm): 7.35e7.38 (m, 12H, Ph H); 7.19e7.26(m, 18H, Ph H); 3.08 (m, 2H, eCH2NHCOe); 2.95e3.00 (m, 6H, eNCH2COe and eCONHCH2CH2CH3); 2.50 (t, 2H, e

SCH2CH2NCH2COe); 2.40 (t, 2H, eCH2Se); 2.31 (t, 2H, eCH2Se);1.39 (m, 2H, eCH2CH2CH3); 0.78 (t, 3H, CH2CH2CH3). 13C NMR(500 MHz, CDCl3, d ppm): 171.16 and 169.57 (CO); 144.56, 129.46,127.98,126.95 and 126.82 (Ph); 67.28 and 66.99 (CPh3); 58.63, 58.07and 54.05 (CH2N); 40.98 and 38.08 (CH2NH); 32.02 and 30.13(CH2S); 22.70 (CH2CH3); 11.50 (CH3). MS (ESI): mass calculated forC49H51N3O2S2, 777.34; m/z found, 777.87 (M þ Hþ). HRMS (ESI):mass calculated for C49H52N3O2S2 (M þ Hþ), 778.34955;m/z found,778.34981.

4.2. 99mTc-labeling

The technetium complexes were prepared by thiol groupdeprotection and labeling by means of glucoheptonate as thetransfer ligand [26]. To a solution of sodium glucoheptonate (GH) inwater was added 10 mL of SnCl2$H2O solution (1 mg/mL, dissolvedin 10 mM HCl). The mixture was adjusted to neutral by addition of0.1 M NaOH and 1e2 mCi of Na99mTcO4 was added. After vortexedfor 5 min, the mixture was then kept at room temperature for10 min to obtain 99mTc-GH. 0.5 mg of ligand was dissolved inethanol (0.5 mL) followed by addition of 0.5 M HCl (0.5 mL). Thesolution was vortexed for 5 min and heated at 100 �C for 15 min99mTc-GH prepared as above was added to the deprotected ligandand the mixture was incubated at 100 �C for another 15 min. After

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e63 61

cooled to room temperature, the labeled solution was analyzed byradio-HPLC.

4.3. Preparation of rhenium complexes

The compound was dissolved in 5 mL TFA and the resultingbright yellow solution was stirred for 5 min and cooled to 5 �C. Thereaction mixture was then titrated with triethylsilane until thedisappearance of the yellow color. The solution was evaporated todryness by rotary evaporation at room temperature. The residuewas dissolved in methanol (10 mL) followed by addition of sodiumacetate solution in methanol (5 mL) and (Ph3P)2Re(]O)Cl3. Themixture was refluxed for 2 h. After cooling to room temperature,the suspension was diluted with 20 mL of ethyl acetate and thenfiltered. The filtration was concentrated and the crude residue waspurified by chromatography on silica gel to give a brown solid.

Re-15: Gradient of chromatography: MeOH:DCM, 1:50 to 1:20.Yield: 45.8%. IR (KBr): 2927, 1676, 1541, 1442, 1340, 1294, 1211, 1134,968, 842, 802 cm�1. 1H NMR (500 MHz, CD3OD, d ppm): 7.42 (d, 2H,imidazole C5eH); 7.11 (m, 2H, imidazole C4eH); 5.33 (m, 1H,NCH2CONHCH2CH2CH3); 4.73 (m, 4H, eCH2CH2eimidazole); 4.41e4.61 (m, 5H); 4.12 (m, 1H, eCHCOOCH3); 4.04 (m, 1H, e

SCH2CH2NCOe); 3.66 (m, 1H, eSCH2CH2NCH2e); 3.46 (m, 2H, eSCH2CH2NCH2e and eSCH2CH2NCOe); 3.22 (m, 3H, eCH2NHCOeand eSCH2CH2NCOe); 3.09 (m, 4H, eCH2NHCOe); 2.95 (m, 1H, eSCH2CH2NCH2e); 2.89 (m, 2H, eCH2CH2eimidazole); 2.77 (t, 2H,eCH2CH2eimidazole); 1.69 (m, 4H, eCH2CH(NHCO)CONHe andCONHCH2CH2CH2NH2); 1.54 (m, 1H, eSCH2CH2NCH2e); 1.39 (m,2H, eCH2CH2CH2NHCOe); 0.91 (m, 2H, eCH2CH2CH2NHCOe). 13CNMR (500 MHz, DMSO-d6, d ppm): 188.74, 171.51, 169.25, 168.93and 165.56 (CO); 146.72, 137.46 and 121.50 (imidazole C4, C2 andC5); 67.73, 64.94, 62.65 and 59.21 (CH2); 52.76 (CH); 48.47 (CH2);46.69 (CH2); 43.87 and 43.82 (imidazoleeCH2); 38.22, 38.14, 36.11,36.04, 35.85, 35.65, 31.46, 28.53 and 22.61 (CH2). HRMS (ESI): masscalculated for C29H43N12NaO10ReS2 (M þ Naþ), 993.21162; m/zfound, 993.20965.

Re-16: Gradient of chromatography: MeOH:DCM, 1:50 to 1:10.Yield: 44.8%. IR (KBr): 2929, 1647, 1541, 1489, 1361, 1211, 1136, 966,835, 800 cm�1. 1H NMR (500 MHz, CD3OD, d ppm): 8.14 (m, 2H,imidazole C2eH); 7.72 (m, 2H, imidazole C5eH); 5.35 (m, 1H,NCH2CONHCH2CH2CH3); 4.46e4.59 (m, 4H); 4.41 (m, 4H, e

CH2CH2eimidazole); 4.14 (m, 1H, eCHCOOCH3); 4.04 (m, 1H, eSCH2CH2NCOe); 3.68 (m, 1H, eSCH2CH2NCH2e); 3.45 (m, 2H, eSCH2CH2NCH2e and eSCH2CH2NCOe); 3.22 (m, 4H, eCH2NHCOe);3.18 (m, 3H, eCH2NHCOe and eSCH2CH2NCOe); 2.95 (m, 1H, eSCH2CH2NCH2e); 2.84 (m, 2H, eCH2CH2eimidazole); 2.73 (t, 2H,eCH2CH2eimidazole); 1.70 (m, 4H, eCH2CH(NHCO)CONHe andCONHCH2CH2CH2NH2); 1.54 (m, 1H, eSCH2CH2NCH2e); 1.38 (m,2H, eCH2CH2CH2NHCOe); 0.90 (m, 2H, eCH2CH2CH2NHCOe). 13CNMR (500 MHz, DMSO-d6, d ppm): 188.66, 171.31, 169.05, 168.76and 165.52 (CO); 144.61,127.90,127.59 and 127.54 (imidazole C4, C2and C5); 67.73, 64.86, 62.62 and 59.24 (CH2); 54.87 (CH2); 52.53(CH); 46.74 (CH2); 46.01 and 45.94 (imidazoleeCH2); 38.30, 38.16,36.19, 36.08, 35.65, 35.41, 31.68, 28.64 and 22.63 (CH2). HRMS (ESI):mass calculated for C29H43N12NaO10ReS2 (MþNaþ), 993.21162;m/zfound, 993.21213.

Re-17: Gradient of chromatography: MeOH:ethyl acetate, 1:50to 1:20. Yield: 60.4%. IR (KBr): 2960, 1676, 1448, 1323, 1136, 966,847, 804 cm�1. 1H NMR (500 MHz, CD3OD, d ppm): 8.22 (m, 1H,imidazole C2eH); 7.76 (m, 1H, imidazole C5eH); 5.26 (d, J ¼ 17.5,1H, NCH2CONHCH2CH2CH3); 4.39e4.53 (m, 4H, eNCOCH2e,NCH2CONHCH2CH2CH3, eSCH2CH2NCOe and eNCOCH2e); 4.29(ddd, 1H, eCH2CH2eimidazole); 4.20 (ddd, 1H, eCH2CH2e

imidazole); 4.04 (dd, 1H, eSCH2CH2NCOe); 3.82 (ddd, 1H, e

CH2CH2eimidazole); 3.76 (ddd, 1H, eCH2CH2eimidazole); 3.57

(dd, 1H, J ¼ 13, 3.5, eSCH2CH2NCH2e); 3.33 (m, 1H, e

SCH2CH2NCH2e); 3.03e3.16 (m, 2H, eSCH2CH2NCOe and e

SCH2CH2NCOe); 2.93 (dd, J ¼ 14, 4.5, 1H, eSCH2CH2NCH2e); 1.65(td, J¼ 12.5, 4.5, 1H, eSCH2CH2NCH2e). 13C NMR (500 MHz, DMSO-d6, d ppm): 188.66 and 166.11 (CO); 147.03, 137.75 and 121.89(imidazole C4, C2 and C5); 67.66, 64.67, 62.23, 59.24, 46.98, 46.79,38.90 and 38.15 (CH2). HRMS (ESI): mass calculated forC13H19N6NaO5ReS2 (M þ Naþ), 613.03135; m/z found, 613.03084.

Re-18: Gradient of chromatography: MeOH:ethyl acetate, 1:50.Yield: 57.8%. IR (KBr): 2926, 1635, 1533, 1487, 1360, 1265, 1163, 966,835, 792 cm�1. 1H NMR (500 MHz, CD3OD, d ppm): 7.44 (m, 1H,imidazole C5eH), 7.16 (m,1H, imidazole C4eH); 5.16 (d, J¼ 17.5, 1H,NCH2CONHCH2CH2CH3); 4.66 (ddd, 1H, eCH2CH2eimidazole);4.45e4.54 (m, 4H, eCH2CH2eimidazole, eNCOCH2e,NCH2CONHCH2CH2CH3 and eSCH2CH2NCOe); 4.36 (d, J ¼ 15.5,1H, eNCOCH2e); 4.02 (dd, J ¼ 11, 4.5, 1H, eSCH2CH2NCOe), 3.68(ddd, 1H, eCH2CH2eimidazole), 3.62 (ddd, 1H, eCH2CH2e

imidazole), 3.52 (dd, 1H, J ¼ 13, 3.5, eSCH2CH2NCH2e); 3.35 (m,1H, eSCH2CH2NCH2e); 3.04e3.17 (m, 2H, eSCH2CH2NCOe and e

SCH2CH2NCOe); 2.93 (dd, J ¼ 14, 4.5, 1H, eSCH2CH2NCH2e); 1.68(td, J¼ 12.5, 4.5, 1H, eSCH2CH2NCH2e). 13C NMR (500 MHz, DMSO-d6, d ppm): 188.65 and 166.19 (CO); 144.68, 128.61 and 127.85(imidazole C4, C2 and C5); 67.63, 64.43, 62.04, 59.24, 49.19, 46.81,38.26 and 37.89 (CH2). HRMS (ESI): mass calculated forC13H20N6O5ReS2 (M þ Hþ), 591.04853; m/z found, 591.04858.

Re-19: Gradient of chromatography: ethyl acetate/petroleumether, 1:2 to 1:1. Yield: 84.2%. IR (KBr): 2960, 2931, 1641, 1541, 1436,962 cm�1. 1H NMR (500 MHz, CD3OD, d ppm): 5.30 (d, J ¼ 17.5, 1H,NCH2CONHCH2CH2CH3); 4.49e4.58 (m, 3H, eNCOCH2e,NCH2CONHCH2CH2CH3 and eSCH2CH2NCOe); 4.40 (d, J ¼ 15.5,1H, eNCOCH2e); 4.02 (dd, J ¼ 10.5, 4.5, 1H, eSCH2CH2NCOe); 3.65(dd, 1H, J ¼ 13, 3.5, eSCH2CH2NCH2e); 3.43 (td, J ¼ 14, 3.5, 1H, eSCH2CH2NCH2e); 3.18 (t, J ¼ 7, 2H, eCONHCH2CH2CH3); 3.09 (m,2H, eSCH2CH2NCOe and eSCH2CH2NCOe); 2.93 (dd, 1H, e

SCH2CH2NCH2e); 1.69 (ddd, 1H, eSCH2CH2NCH2e); 1.54 (m, 2H,eCH2CH2CH3); 0.94 (t, J ¼ 7, 3H, CH2CH2CH3). 13C NMR (500 MHz,DMSO-d6, d ppm): 189.19 and 165.90 (CO); 133.37, 132.55, 131.96,131.89, 129.28 and 129.18 (Ph of residual Ph3P]O); 68.25, 65.37,63.16, 59.72, 47.20, 40.73, 38.58 and 22.48 (CH2); 11.80 (CH3). HRMS(ESI): mass calculated for C11H21N3O3ReS2 (M þ Hþ), 494.05818;m/z found, 494.05837.

4.4. Physicochemical studies

4.4.1. Partition coefficientsPartition coefficients were determined in a similar method as

reported [27]. 1 mL of 1-octanol was added to a mixture of 0.9 mL ofphosphate buffer solution (0.1 M, pH 7.4) and 0.1 mL of the radio-labeled complex. The mixture was vortexed at room temperaturefor 5 min and centrifuged at 2000 rpm, for 5 min. Equal aliquots(100 mL) of sample were taken from each phase of water andoctanol and counted for radioactivity. The partition coefficient wascalculated by dividing the radioactivity of the octanol layer withthat of the water layer. This measurement was repeated threetimes.

4.4.2. StabilityTo evaluate the stability of these labeled complexes, they were

dissolved in phosphate buffer solution (0.1 M, pH 7.4) and incu-bated at room temperature after preparation. Certain aliquots ofsample were taken at different time points over a period of 4 h andthe radiochemical purity was analyzed by HPLC. Moreover, in vitroserum stability studies of these complexes in rat serum were alsoperformed using a method reported earlier [29]. About 50 mL of thelabeled compound was added to 0.5 mL of rat serum and this

L. Mei et al. / European Journal of Medicinal Chemistry 58 (2012) 50e6362

mixture was incubated at 37 �C for 4 h. At intervals of 1, 2 and 4 h,100 mL of aliquots were withdrawn and 200 mL of ethanol wasadded to precipitate the serum protein. The mixture was filteredwith a 0.45 mm Millipore filter and the filtrate was analyzed byHPLC to assess the radiochemical purity of the complex.

4.4.3. ElectrophoresisPaper electrophoresis was carried out using Whatman No. 3

chromatography paper. The solution of labeled complex wasspotted in the middle of strip with glass capillary and developedunder a potential gradient of 10 V/cm�1 in 0.05 M phosphate buffer(pH 7.4) for 4 h. Then the strip was dried and cut into 1 cmsegments and the radioactivity associated with these was deter-mined using a Wizard II 2470 series Automatic Gamma Counter(Perkin Elmer, USA).

4.5. Electrochemical studies

Electrochemical behavior was studied by cyclic voltammetry.Cyclic voltammograms were recorded on a CH Instruments(CHI600C) electrochemical workstation employing a glassy carbonworking electrode, a platinum wire counter electrode and an Ag/Agþ reference electrode. All solutions were 5 mM of analyte inanhydrous DMF (spectroscopic grade) with tetrabutylammoniumperchlorate (0.1 mol mL�1). The measurements were performed inglove boxes filled with argon at room temperature. Ferrocene wasused as a reference which the one-electron redox process occurs atE1/2 ¼ 0.47 V with 0.1 mol mL�1 of tetrabutylammonium perchlo-rate in DMF versus SCE [44].

4.6. Biodistribution

The murine sarcoma (S180) model was established by hypo-dermic injection of approximately 1.0 � 106 cells into the left frontleg of male Kunming mice. Tumor grew to diameters of 10e15 mm,corresponding to weights of 0.5e1.5 g during about a week’s time.The newly prepared complex (100 mL, 5 MBq, 0.04 mg of a totalamount of ligand) was injected into the mice bearing S180(weighing 20e25 g) via the tail vein. The mice were sacrificed ingroups of five at various time intervals after injection and theorgans or tissues of interest were removed and washed, whichwere then weighed in plastic tubes and counted in an auto gammacounting system. The results were calculated as percentages ofinjected dose per gram tissue (i.e., % ID/g), using a standardequivalent to 1% of the injected dose. Tumor-to-tissue ratios werecalculated from % ID/g for the tumor and relevant organs. The finalresults were expressed as mean � S.D. (standard deviation). Allexperiments were carried out following the principles of laboratoryanimal care and the China’s law on the protection of animals.

Acknowledgments

This work was supported by the National Science Foundation ofChina (Grant No. 21071010).

Appendix A. Supporting information

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2012.09.042.

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