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Nuclear Medicine and Biology 37 (2010) 997–1004www.elsevier.com/locate/nucmedbio

Preparation and preliminary bioevaluation of99mTc(CO)3-11β-progesterone derivative prepared via

click chemistry routeManish V. Dhyania, Drishty Satpatia, Aruna Kordea, Haladhar Dev Sarmab,

Chandan Kumara, Sharmila Banerjeea,⁎aRadiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

bRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India

Received 28 January 2010; received in revised form 23 April 2010; accepted 6 May 2010

Abstract

Introduction: Progesterone receptors (PRs) overexpressed in breast cancers serve as potential targets for developing radiotracers for use innuclear medicine. Hence, suitably derivatized progesterone can be envisaged as a potential vector for targeting overexpression of receptors inbreast cancer. In the present article, we report the preparation of a 99mTc(CO)3-progesterone triazole using the Cu(I)-catalyzed novel clickchemistry route. Preliminary evaluation of the radiolabeled derivative has been carried out in binding studies with MCF 7 cell lines.Methods: 11-Hydroxyprogesterone has been synthetically derivatized to 11-azidoprogesterone. Subsequently, the cycloaddition reactionbetween progesterone azide and propargyl glycine was carried out to prepare 1,4-bifunctionalized progesterone triazole analogue. The clickedprogesterone triazole derivative was radiolabeled with 99mTc and characterized by HPLC. The chemical characterization of 99mTc(CO)3-progesterone triazole has been carried out by preparing its corresponding rhenium complex using the [NEt4]2[Re(CO)3Br3] precursor. Whilein vitro studies were carried out in MCF7 cell lines, in vivo distribution studies were performed in female Swiss mice.Results: The radiolabeled complex could be prepared in N95% radiochemical yield as determined by HPLC. In vitro studies of 99mTc(CO)3-progesterone complex in MCF7 cell lines overexpressing receptors for breast cancer showed binding up to 30%. In vivo distribution studiesin female Swiss mice have shown uterine uptake of 0.41 (0.06) % ID/g at 3 h postinjection (pi) and retention therein till 24 h pi.Conclusion: The present study demonstrates a novel and facile route for preparation of 99mTc-labeled progesterone complex using clickchemistry. This strategy can be further extended towards preparation of radiolabeled complexes of other steroidal derivatives.© 2010 Elsevier Inc. All rights reserved.

Keywords: Click chemistry; Progesterone; [99mTc(CO)3(H2O)3]+

1. Introduction

Progesterone receptors (PRs) belong to the class of steroidreceptors which are overexpressed in breast [1] andendometrial [2] cancers, making them favorable targets fordevelopment of radiotracers for imaging as well as fortherapy [3–5]. This feature can be utilized in designingprogesterone-based radiotracers as target-specific vectors totarget tumors overexpressing these receptors. Radiolabeledprogestins have been studied to a limited extent [6], the

⁎ Corresponding author. Tel.: +91 22 25593910; fax: +91 22 25505151.E-mail addresses: sharmila@barc.gov.in,

banerjeesharmila1@rediffmail.com (S. Banerjee).

0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nucmedbio.2010.05.005

reason being probably attributed to the lower number ofPR-positive breast tumors as compared to those that showoverexpression of estrogen receptors [7,8]. However, aprogesterone-based ligand has the significant advantage infollow-up of patients undergoing hormone replacementtherapy over that of estrogen-based ones since estrogenreceptors get saturated owing to the use of hormones.Tamoxifen used in the treatment of breast and endometrialcancer also leads to increase in the levels of PRs providing animpetus to identify suitable PR-based ligands [9,10]. More-over, in some estrogen receptor-positive tumors, progester-one levels are higher than estrogen levels, where radiolabeledprogestins may prove efficacious [11]. The aforementionedadvantages provided the rationale for the present study to

998 M.V. Dhyani et al. / Nuclear Medicine and Biology 37 (2010) 997–1004

prepare a progesterone derivative for possible diagnosticapplication in targeting tumors expressing PRs.

The radiolabeled progesterone derivatives can be poten-tial agents for imaging of PR-positive tumors using bothpositron emission tomography and single photon emissioncomputed tomography (SPECT). In this respect, various 18F-[12–14], 99mTc- [4,15] and 123I-labeled [16–18] receptorligands have been reported to be selective towards PRs. Thelower cost associated with SPECT radioisotopes, easyavailability and longer half-lives are the features thatcontribute in maintaining interest in research towardsdevelopment of new SPECT-based radiotracers. Techne-tium-99m is the most suitable SPECT radionuclide forimaging, having ideal nuclear and physical properties (t½=6h, Eγ=140 keV). The technetium tricarbonyl precursor[99mTc(CO)3(H2O)3]

+ leads to the formation of high specificactivity complexes that are required for high uptake intargeted tissue, a feature that is especially important forradiopharmaceuticals with uptake mediated by low-capacitytargets such as receptors. [99mTc(CO)3(H2O)3]

+ requirestridentate bifunctional chelating agents (BFCAs) for forma-tion of stable complexes and this has been well studied andevaluated for radiolabeling of various molecules [19–23].However, the drawback of these BFCAs is that they requiremultistep synthesis involving protection of certain groupswhich may interfere with incorporation of the targetingmolecule. In this respect, it was felt pertinent to explore theversatile click chemistry route [24–26] in designing aprogesterone precursor for facile labeling with [99mTc(CO)3(H2O)3]

+. The click reaction, a Cu(I)-catalyzedcycloaddition reaction between a terminal azide andpropargyl glycine leads to the formation of a 1,2,3-triazolederivative that provides tridentate coordination via N3 of thetriazole ring which is required for forming a stable complexwith [Tc(CO)3]

+. In click reaction, the formation of achelating system in a biologically avid molecule forradiolabeling can be achieved in a single step, making it anattractive strategy to be followed for preparation of Tc(I)complexes [27,28].

The present work demonstrates the application of clickchemistry in the preparation of an organometallic 99mTccomplex of a progesterone derivative. 11-Hydroxyproges-terone has been derivatized to 11-azidoprogesterone whichundergoes [3+2] cycloaddition reaction with propargylglycine, and the 1,2,3-triazole derivative thus formed hasbeen radiolabeled with [99mTc(CO)3(H2O)3]

+ core. Theaffinity of radiolabeled complex towards PRs has beentested by carrying out suitable in vitro and in vivo studies.

2. Materials and methods

2.1. Materials

All solvents were distilled and dried prior to use.Reagents were obtained from Sigma Aldrich Chemicals(USA) and were used without further purification. Carbonyl

kit for the synthesis of [99mTc(CO)3(H2O)3]+ precursor was

obtained as a gift from Mallinckrodt Medical (Netherlands).[NEt4][Re(CO)3Br3] was prepared from Re(CO)5Cl accord-ing to the published procedure [29].

All the animal experiments were carried out in compli-ance with the relevant national laws as approved by the localcommittee on the conduct and ethics of animal experimen-tation. MCF7 and HT-29 cells were procured from theNational Centre of Cell Sciences (Pune, India).

2.2. General methods

99mTcO4− was eluted from an in-house 99Mo/99mTc column

generator using 0.9% saline. HPLC analyses were performedon a Jasco PU 1580 system and on a Jasco 1575 tunableabsorption detector and on a radiometric detector systemhaving C-18 reversed-phase HiQ Sil (5 μm, 250×4 mm)column. The gradient system consisting of eluting solventsH2O (solvent A) and acetonitrile (solvent B) with 0.1% tri-fluoroacetic acid was used for HPLC analyses (0–28 min,90%A–10%A; 28–30min, 10%A; 30–32min, 10%A–90%A). Melting points were determined using Fisher-Johns melt-ing point apparatus. IR spectra were recorded using a JascoFT IR 4100 spectrophotometer. Proton NMR spectra wererecorded on a 300-MHzVarianVXR 300S spectrophotometer.Mass spectra were recorded on a QTOFMicromass Instrumentusing electron spray ionization (ESI) in positive mode.

2.3. Chemical synthesis

2.3.1. 11α-(Methylsulphonyl)oxyprogesterone 2Compound 1, 11α-hydroxyprogesterone (100 mg, 0.3

mmol), was dissolved in 3 ml of CH2Cl2, and Et3N (84 μl,0.6 mmol) was added to it. After cooling the solution to 0°C,methanesulphonyl chloride (35 μl, 0.45 mmol) was addedand stirring continued at the same temperature for 30 min.Water was added to the reaction mixture and the aqueousphase was extracted with CHCl3 (2×10 ml). The organicextracts were washed with water and brine. The pooledorganic extract was dried over anhydrous sodium sulphateand solvent was removed under reduced pressure. Theproduct was purified on a silica-gel column using 2%MeOH/CHCl3 as the eluting solvent. Yield: 95%; whitesolid; mp: 158°C; IR (nujol, ν cm−1): 2964, 1705, 1666,1610; 1H NMR (CDCl3): δ 0.74 (s, 3H, 18-CH3), 1.36 (s,3H, 19-CH3), 2.13 (s, 3H, 21-CH3), 3.04 (s, 3H, SO2-CH3),5.12 (m, 1H, 11-H), 5.75 (s, 1H, 4-H); ESI MS m/z: 409.19[M+H]+, (calc for C22H32O5S: 408.187).

2.3.2. 11β-Azidoprogesterone 3To a stirred solution of compound 2 (100 mg, 0.24 mmol)

in 2.5 ml dry DMF, sodium azide (48 mg, 0.72 mmol) wasadded and the stirring was continued at 100°C for 5 h. Waterwas then added to the reaction mixture and the aqueousphase extracted with Et2O (2×10 ml). The organic extractswere washed with water, brine and dried over Na2SO4. Thesolvent was removed under vacuum and the product waspurified by silica gel column using 1% MeOH/CHCl3 as the

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eluting solvent. Yield: 25%; yellow solid; mp: 109°C; IR(nujol, ν cm−1): 2955, 2942, 2857, 2095, 1707, 1677, 1621; 1HNMR (CDCl3): δ 0.91 (s, 3H, 18-CH3), 1.42 (s, 3H, 19-CH3),2.15 (s, 3H, 21-CH3), 4.17 (m, 1H, 11-H), 5.68 (s, 1H, 4-H);ESIMSm/z: 356.20 [M+H]+, (calc for C21H29N3O2: 355.226).

2.3.3. Elimination product 4The above reaction also yielded the elimination product

3b. Yield: 55%; yellow solid; mp: 112 °C; IR (nujol, νcm−1): 1697,1652, 2918, 2848; 1H NMR (CDCl3): δ 0.60 (s,3H, 18-CH3), 1.33 (s, 3H, 19-CH3), 2.14 (s, 3H, 21-CH3),5.51 (s, 1H, 11-H), 5.74 (s, 1H, 4-H); ESI MS m/z: 313.14[M+H]+, (calc for C21H28O2: 312.209).

2.3.4. 11β-Progesterone triazole 5An aliquot of compound 3 (5 mg, 14 μmol), propargyl

glycine (1.5 mg, 13 μmol), copper sulphate (100 μg, 0.4μmol) and sodium ascorbate (100 μg, 0.5 μmol) wasdissolved in CH2Cl2/H2O (1:1, 2 ml). The reaction mixturewas stirred at room temperature overnight. ESI MS m/z:507.20 [M+K]+ (calc for C26H36N4O4: 468.274).

2.3.5. Re(CO)3-Progesterone triazole 7Compound 5, 11β-progesterone triazole (5 mg, 14 μmol),

was dissolved in methanol, and [NEt4]2[ReBr3(CO)3] (11mg, 14 μmol) was added followed by incubation in a sealedvial at 100°C for 5 h. The complex prepared wascharacterized by UV-HPLC (retention time 24.20 min). IR(KBr, ν cm−1): 2016, 1888; ESI MS m/z: 777.70 [M+K]+

(calc for C29H35N4O7Re: 738.206).

2.4. Radiochemistry

2.4.1. [99mTc(CO)3(H2O)3]+ precursor

The precursor [99mTc(H2O)3(CO)3]+ was prepared

according to the reported procedure [20]. In brief, 99mTcO4−

in 0.9% NaCl from the 99Mo/99mTc generator was added tothe Isolink kit via a syringe. The vial was kept in a boilingwater bath for 15 min. After cooling the vial to roomtemperature, 175 μl of 1N HCl was added to neutralize thesolution and decompose any residual boranocarbonate.Radiochemical purity of the precursor was checked byreversed-phase HPLC.

2.4.2. 99mTc(CO)3-Progesterone triazole 6To 0.1 ml methanolic solution of compound 5 (200 μg,

4 mM), 0.4 ml of [99mTc(CO)3(H2O)3]+ was added and

the pH of the resultant mixture was adjusted to 7 followedby heating at 80°C for 1 h. The complex prepared wascharacterized by HPLC.

2.5. In vitro stability assessment

The stability of 99mTc(CO)3-progesterone triazole wasassayed by monitoring the HPLC elution profile anddetermining the radiochemical purity after incubation atroom temperature for 6 h. To determine the in vitro serumstability, 200 μl of radiolabeled complex was incubated with1 ml human serum at 37°C for 1 h. Acetonitrile (1 ml) was

added to the above solution. The precipitate was separatedby centrifugation. The supernatant was injected in HPLC todetermine the stability of the complex.

In order to study the stability of 99mTc(CO)3-progesteronetriazole towards exchange with histidine and cysteine,challenge experiments were performed. The complex (100μl) was incubated with freshly prepared histidine andcysteine solutions (10 μl, 0.5 M) in separate test tubes in awater bath at 37°C for 1 h. The solution was then analyzedby HPLC to determine the extent of exchange, if any.

2.6. In vitro cell uptake studies

Cell uptake studies were carried out by using MCF7breast cancer cells as well as HT-29 colon cancer cells. Thecells were cultured in Dulbecco's Modified Eagles Mediumcontaining 10% fetal bovine serum, 100 IU/ml penicillin, 50μg/ml streptomycin and 10 mM glutamine in humidifiedatmosphere under 5% CO2 at 37°C. Trypsin-EDTA solutionwas used for subculturing and isolation of cells fromconfluence cultures. The tumor cells were washed twiceand, finally, a suspension of 106 cells/ml was prepared in 50mM of Tris-buffer (pH 8) containing 0.2% bovine serumalbumin. To determine the percentage of in vitro cell bindingof the complex, 0.1 ml of cell suspension (105 cells) wasincubated with different amounts of the 99mTc(CO)3-progesterone triazole complex in triplicate at 37°C for 60min. After incubation, cells were centrifuged at 2000 rpm for5 min for pellet formation. The cell pellets were then washedtwice with ice-cold assay buffer to remove any unboundradioactivity and centrifuged to collect supernatants. Afterthe removal of the supernatant, radioactivity associated withthe cell pellet was measured in a NaI(Tl) detector. Inhibitionstudies were carried out by using a similar procedure whichinvolved incubation of 105 cells with 100-fold excess ofprogesterone along with the radiolabeled compound. Similarstudies were carried out using HT-29 colon cancer cell line.

2.7. Biodistribution studies

Biodistribution studies were carried out in normal Swissmice. The radiolabeled product (0.1 ml, 3–7 MBq) wasinjected via the tail vein. The studies were carried out at 1, 3and 24 h postinjection (pi) using four mice for each timepoint. All major organs were excised, weighed and countedin a NaI(Tl) flat geometry detector in order to estimate thepercent of injected dose per gram of the tissue. Blood,muscle and bone weights were taken as 7%, 40% and 10% oftotal body weight, respectively.

3. Results and discussion

3.1. Synthesis

It is reported in the literature that synthetic manipulationat the C-11 position of progesterone and introduction of abulky 11β group do not alter its affinity towards PRs.

1000 M.V. Dhyani et al. / Nuclear Medicine and Biology 37 (2010) 997–1004

Therefore, 11α-hydroxyprogesterone was chosen as theparent molecule for derivatization to an azide in preparing aclicked 1,2,3-triazole of progesterone. 11α-Hydroxyproges-terone 1 has been converted to 11β-azidoprogesterone 3 in atwo-step reaction as shown in Scheme 1. All the inter-mediates and final product have been characterized by IR,NMR and ESI-MS. In 1H NMR, a downfield shift has beenobserved for C-11 proton from δ 4 ppm corresponding tocompound 1 to δ 5.12 ppm indicating the desired mesylationof compound 1. The SN2 azidation reaction led to inversionof stereochemistry leading to the formation of the 11β-azidogroup, a desirable and preconceived feature since the 11β-substituted progesterone has been reported to show higherreceptor binding affinity. In IR spectra the stretching band at2095 cm−1 characteristic of N3 functionality indicated thedesired derivatization. This has been further confirmed by ashift in 1H NMR signal corresponding to C-11 proton from δ5.12 to δ 4.17 ppm after azidation. The elimination product 4is also formed in 55% yield. Elimination reactions arecommonly encountered in SN2 reactions. The formation ofthe elimination product 4 in greater yield as compared to theazido product 3 could be possibly explained by the stericcrowding at the reaction center, thereby making elimination

Scheme 1. Reagents and conditions: (i) MsCl, CH2Cl2, Et3N, 0°C, 30 min; (ii) DMH2O, RT=12 h; (iv) for 6, [99mTc(CO)3(H2O)3]

+, pH 7, 80°C, 1 h, M=99mTc; for

a preferred pathway with respect to substitution. Compound4 was characterized by the appearance of C-11 alkene protonin 1H NMR spectra at δ 5.51 ppm in addition to the C-4alkene proton at δ 5.74 ppm. The crude product could bepurified by silica gel column chromatography to yield thedesired compound 3 in pure form. Compound 3 has beenfunctionalized to 1,2,3-triazole 5 by Cu(I)-catalyzed [3+2]cycloaddition reaction with propargyl glycine [30]. Towardsthis, 11β-azidoprogesterone was reacted with propargylglycine in the presence of Cu(I) as catalyst, generated in situin DCM/H2O (1:1), at room temperature, overnight, toprovide 1,2,3-triazole derivative. The formation of com-pound 5 has been confirmed by mass spectral analysis of thereaction mixture. The peak corresponding to the [M+H]+=469.274 (calc. for C26H36N4O4: 468.274) was notpresent; however, the pseudomolecular ion peakcorresponding to the [M+K]+=507.2 was observed therebyconfirming the formation of 5.

3.2. Radiolabeling

Progesterone triazole could be radiolabeled by incubationwith the [99mTc(CO)3(H2O)3]

+ precursor for 1 h at 80°C at pH

F, NaN3, 100°C, 5 h; (iii) CuSO4, Na-ascorbate, propargylglycine, DCM,7, [NEt4] 2[Re(CO) 3Br3], MeOH, 100°C, 5 h, M=Re.

Fig. 1. HPLC profile of (A) [99mTc(CO)3(H2O)3]+ core (B) 99mTc(CO)3-

progesterone triazole and (C) Re(CO)3-progesterone triazole.

Table 1In vitro cell binding and inhibition studies in MCF7 and HT-29 cell lines

Cell line Tracer (ng) Progesterone (μg) Cell uptake (%)

MCF-7 200 – 35±2.3MCF-7 200 20 34±1.8MCF-7 50 – 30±1.9MCF-7 50 5 28±1.4HT-29 200 – 6±0.8HT-29 50 – 5±0.6

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7 (Scheme 1). The 99mTc(CO)3 complex 6 was characterizedusing HPLC where the complex (Fig. 1B) having a retentiontime of 24.1 min was well separated from the 99mTc-tricarbonyl precursor (Fig. 1A) eluting out at 16 min.Propargyl glycine being a bidentate ligand having aminoand carboxyl groups for coordination was also complexedwith 99mTc-tricarbonyl precursor. The retention time of 99mTc(CO)3-propargyl glycine was 12 min in HPLC and hence itcould be readily distinguished from 99mTc(CO)3-progesteronetriazole. The HPLC chromatogram indicated N95% (Fig. 1B)radiochemical purity of the complex.

The 99mTc(CO)3-progesterone complex has been char-acterized by comparison with the corresponding rheniumcomplex 7, which has been synthesized using the [NEt4][Re(CO)3Br3] precursor. This precursor was prepared fromrhenium pentacarbonyl chloride [Re(CO)5Cl] using thepublished procedure [29]. Formation of the rhenium

complex has been confirmed by IR and mass spectrasince the complex was highly insoluble in the commonsolvents used for recording NMR spectra. The infraredspectra of the rhenium complex showed carbonyl stretch-ing at 2016 and 1888 cm−1 which are characteristic of fac-[Re(CO)3] unit. The peak corresponding to the [M+H]+=739.206 (calc. for C29H35N4O7Re: 738.206) wasnot present; however, the pseudomolecular ion peakcorresponding to the [M+K]+=777.7 was obtained con-firming the formation of 7. The technetium-99m complexprepared in tracer level was characterized by comparingthe retention time of the γ-trace of 99mTc in HPLC withthat of the UV trace of the corresponding rheniumcomplex. The UV-HPLC chromatogram retention time ofrhenium complex (Fig. 1C) was observed to be 24.2 minwhich matches well with the γ-HPLC chromatogramretention time of 24.1 min for 99mTc(CO)3-progesteronetriazole (Fig. 1B).

The lipophilicity of 99mTc(CO)3-progesterone triazole asdetermined by octanol/saline partitioning was inferable fromthe log P value of .85±.03.

3.3. In vitro stability and cell uptake studies

The affinity of the radiolabeled complex towards PRs hasbeen tested by carrying out in vitro and in vivo studies. Invitro stability of the complex was studied in phosphatebuffered saline at pH 7.0 as well as in serum after 1 h ofincubation at 37°C. The complex was found to be stable evenafter 6 h of preparation. In serum stability studies, nearly50% of the activity was associated with the precipitateobtained after acetonitrile addition, indicating the binding ofcomplex with serum proteins. The acetonitrile fraction wascharacterized by HPLC where a single peak was observed atthe same retention time (24.1 min, N95%) as that of thecomplex, indicating no decomposition of the complex andhence its stability under 37°C of incubation.

To assess the kinetic inertness of the complex underphysiological condition, the challenge studies were carriedout by incubation of the radiolabeled complex with excess ofhistidine and cysteine. The HPLC pattern of the complex(retention time 24.1 min) remained unaltered with noindication of the appearance of a new peak correspondingto that of either 99mTc(CO)3-histidine or 99mTc(CO)3-cysteine complex. This demonstrates the kinetic inertness

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of the complex indicating no transchelation with the ligands,histidine or cysteine.

In vitro cell studies were carried out in MCF7 breastcancer cell lines to determine the affinity of the 99mTc(CO)3-progesterone triazole complex towards binding with PRs.The percentage binding of the complex in cells was found tobe 30±1.9% when 50 ng of the tracer was added. Blankexperiments were carried out by incubating cells with the[99mTc(CO)3(H2O)3]

+ core as well as with 99mTcO4−.

Uptakes of 3±0.8% and 1±0.2% were observed, respectively.Inhibition studies were carried out by incubating 105 MCF7breast cancer cells with 100-fold excess of progesteronealong with the radiolabeled progesterone complex. Althoughthe binding experiments showed nearly 30% binding of the99mTc(CO)3-progesterone triazole complex, no inhibitionwas observed in blocking studies with unlabeled progester-one. The results are shown in Table 1. To investigate thespecificity of the radiolabeled complex towards PR over-expression, cell binding studies were carried out in a similarmanner using a nonspecific human colon carcinoma cell line,HT-29. The extent of binding was found to be 5±0.6%,which is significantly less compared to the binding observedwith MCF7 cells.

The progesterone molecule was modified keeping in mindthat the 11β-position has higher tolerance for bulkysubstituents without affecting the receptor affinity. Hence,the strategy was to choose 11α-hydroxy progesterone andcarry out azidation reaction to invert the stereochemistry sothat the desired 11β derivative could be obtained. However,although this was achieved, introduction of a triazole moietyalong with the 99mTc-tricarbonyl core probably led to anincrease in the steric bulk, creating interference between theligand and the receptor ligand-binding pocket, thus affectingthe receptor affinity of the radiolabeled progesteronecomplex. Additionally, there exists a possibility arisingfrom the introduction of the bulky organometallic complexwhich now exceeds the volume of the PR ligand-bindingpocket, thereby rendering it unsuitable for fitting into thedesired volume of the receptor pocket. The present results arein similar line with observations reported by Dallagi et al.

Table 2Biodistribution studies of 99mTc(CO)3-progesterone triazole in Swiss mice(%ID/g) (n=4)

Organ 1 h pi 3 h pi 24 h pi

Blood 5.2±0.35 4.1±0.85 1.4±0.5Liver 28.6±0.6 28.8±0.3 25.7±3.1Kidney 10.5±0.7 10.4±0.15 8.8±1.9Intestine 22.4±1.7 23.6±4.3 31.9±1.4Stomach 1.3±0.4 1.5±0.15 1.8±0.2Heart 0.4±0.04 0.34±0.04 0.26±0.03Lungs 1.5±0.4 2.5±0.3 2.2±0.46Muscles 0.27±0.07 0.18±0.04 0.14±0.01Uterus 0.75±0.4 0.41±0.06 0.4±0.17Uterus/muscle 2.8 2.2 2.8Uterus/blood 0.15 0.1 0.28

[31] and He et al. [32], while designing androgen receptor-specific 99mTc-labeled agents.

3.4. In vivo studies

Tissue uptake was determined by carrying out biodis-tribution studies in female Swiss mice (Table 2). High uptakein the uterus was found at 1 and at 3 h pi. There was a slowwashout of radioactivity from the uterus even after 24 h.While the uterus/muscle ratio was observed to be 2.2 at 3h pi, it was 2.8 at 24 h. However, uterus/blood ratio waslower and there was high uptake in the liver and intestine.This may be attributed to the presence of metabolites as it isknown that the liver is the main site of steroid metabolism[33]. Labeling of small molecules with 99mTc requiresintroduction of chelating agents that lead to an increase in theoverall molecular size of the parent molecule altering itsbinding properties and also the physical properties that affectthe absorption, metabolism and clearance characteristics ofthe radiopharmaceutical rendering it unsuitable for imagingof these receptors.

The present strategy demonstrated a high-yielding routefor the preparation of a progesterone triazole chelate whichcould be radiolabeled with the 99mTc-tricarbonyl synthon, ata low ligand concentration and therefore resulting in higherspecific activity, a desirable feature in designing receptor-based agents. Current studies reveal the ease of incorporationof triazole moiety in steroids possessing azide functionality.The clicked product thus obtained provides an efficientmethodology for the preparation of 99mTc(CO)3- or

188Re(CO)3-labeled steroids for the diagnosis or therapy of variouscancers. This strategy can be utilized to synthesize similarsteroidal derivatives for targeting overexpression of steroidreceptors in cancer.

4. Conclusion

The progesterone-triazole derivative could be synthesizedand radiolabeled with the [99mTc(CO)3(H2O)3]

+ precursor inN95% radiochemical yield. The 99mTc complex wascharacterized by comparison of its HPLC profile with thatof the analogous rhenium complex. While in vitro studiescarried out in MCF7 cells indicate binding to the extent of30%, insignificant binding of the 99mTc(CO)3-progesteronetriazole complex was observed in case of binding studieswith nonspecific cell lines such as HT-29. Preliminarybiodistribution studies in female Swiss mice have shownfavorable uptake and retention in the uterus but high uptakein blood. These observations provide insight towardsenvisaging alternate synthetic modification of the progester-one molecule in order to achieve the desired receptorspecificity. This first attempt at applying the click chemistryapproach to prepare a radiolabeled progesterone tracer can befurther extended to prepare clicked derivatives of othersteroids in high radiochemical yield.

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Acknowledgments

The authors are thankful to Dr. V. Venugopal, Director,Radiochemistry and Isotope Group, for his constantencouragement and support towards this work. Manish V.Dhyani is thankful to the Department of Atomic Energy,Government of India, for the research fellowship.

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