Synthesis and Preliminary Bioevaluation of 99m Tc(CO) 3 -17α-Triazolylandrost-4-Ene-3-One Derivative Prepared via Click Chemistry Route

Abstract

Azolyl steroids are known to manifest antiprostate cancer and antiandrogenic activities. These azolyl steroids have been shown to express affinity toward androgen receptors (ARs) overexpressed on LNCaP (human prostate adenocarcinoma) cell line. Hence, suitably derivatized azolyl steroids can be envisaged as potential vectors for targeting overexpression of ARs in prostate cancer. In the present study, testosterone has been derivatized to 17α-azidoandrost-4-ene-3-one using microwave-mediated azidation of the mesylate. Subsequently, a facile one-pot Cu(I)-catalyzed Click reaction was carried out to synthesize ^99mTc(CO)₃-labeled 17α-triazolylandrost-4-ene-3-one, which was characterized by HPLC. The chemical characterization of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one was carried out by preparing its corresponding rhenium complex using [NEt₄]₂[Re(CO)₃Br₃] precursor. The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one complex in LNCaP cell lines overexpressing ARs showed binding of 4.95%±1.2%, with inhibition of 8%±0.9%. In vivo biodistribution studies in male Wistar rats have shown uptake in the prostate to the extent of 0.48%±0.19% injected dose/g at 1 hpi and retention therein till 3 hpi. The present study demonstrates a novel and facile one-pot reaction for preparation of ^99mTc-labeled 17α-triazolylandrost-4-ene-3-one complex using Click chemistry. The corresponding Re-analog has been prepared for purpose of comparative characterization with the ^99mTc-labeled complex. The radiosynthetic strategy described in this article can be further extended toward preparation of radiolabeled complexes of other triazolyl steroidal derivatives.

Introduction

Prostate cancer is one of the most frequently diagnosed cancers among men.^1,2 Currently used methods for diagnosis of prostate cancer involve invasive procedures, and therefore, patients with prostatic cancer progress to an advanced stage of the disease at the time of diagnosis.^3

–6 To help in early diagnosis and monitoring disease progression, the need for additional means to complement and improve upon the existing approaches is therefore relevant. Noninvasive receptor imaging using radiolabeled molecules can provide an early indication of the disease and help in determining the therapeutic response. Androgen receptors (ARs) are upregulated in prostate cancer cells when compared with normal prostate cells, making them potential biological targets for developing radiotracers for diagnosis and therapy of prostate cancer.^7,8 In search of an ideal AR-based prostatic imaging agent,^8

–13 various androgens have been labeled with several γ-emitting radionuclides, such as ⁷⁷Br, ⁸²Br, ¹²⁵I, and ⁷⁵Se, as well as with the positron emitter ¹⁸F. Because of high cost of production, short half-life of positron (β+)-emitting isotopes, and the need for PET imaging instrumentation, PET has restricted clinical applications. However, the lower cost, easy availability, and longer half-lives of SPECT radioisotopes are the features that contribute toward development of new SPECT-based radiotracers that can be routinely used in hospitals. Technetium-99m is the most suitable SPECT radionuclide for imaging, having ideal nuclear and physical properties (t _½=6 hours, E _γ=140 keV).

The major impediments toward the use of radiolabeled androgen-based receptor imaging agents are rapid metabolic cleavage, low receptor binding affinity, and inadequate specific activity. Therefore, the need for a more effective androgen imaging agents still exists. The problem of inadequate specific activity can be circumvented by preparing radiotracers using organometallic technetium tricarbonyl precursor, [^99mTc(CO)₃(H₂O)₃]⁺, which leads to formation of high-specific-activity complexes required for the uptake in low-capacity targets such as ARs. [^99mTc(CO)₃(H₂O)₃]⁺ requires tridentate bifunctional chelating agents (BFCAs) for formation of stable complexes and this has been well studied and evaluated for radiolabeling of various molecules.^14

–18 However, the drawback of these BFCAs is that they require multistep synthesis involving protection of certain groups, which may interfere while incorporation of the targeting molecule. In this respect, it was felt pertinent to explore the versatile click chemistry route in designing a derivative for facile labeling with [^99mTc(CO)₃(H₂O)₃]⁺.^19
–21 The Click reaction, a Cu(I)-catalyzed cycloaddition reaction between a terminal azide and propargyl glycine, leads to formation of a 1,2,3-triazole derivative that provides tridentate coordination via N₃ of the triazole ring, which is required for forming stable complex with [Tc(CO)₃]⁺. In Click reaction, the formation of a chelating system in a biologically avid molecule derivatized as an azide and its complexation with ^99mTc can be achieved in a single step, making it an attractive strategy to be followed for preparation of Tc(I)-complexes.^22,23

Very few compounds have been labeled with ^99mTc for use as androgen-based receptor imaging agents.^24

–27 However, rapid metabolic cleavage, low receptor binding affinity, and inadequate specific activity are the major impediments toward use of these radiolabeled conjugates. Therefore, a need for more effective androgen imaging agents still exists. Ling et al., Njar et al., and Zhu et al. have reported the synthesis of novel 17-imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives as potential agents for the treatment of prostatic cancer.^28
–30 These novel 17-azolyl steroids were found to inhibit P450_17α, the key enzyme of androgen biosynthesis. Nnane et al. and Grigoryev et al. have shown that these 17-azolyl steroids manifest antiandrogenic activity in cultures of human prostate cancer cell lines (LNCaP) by preventing the labeled synthetic androgen R1881 from binding to the ARs.^31,32 In the present work, 17-(5′-isoxazolyl)androsta-4,16-dien-3-one (L-39,)³¹ and VN/109-1 (Scheme 1)³² were used as lead compounds, to design and synthesize its 17-triazolylandrost-4-ene-3-one analog labeled with ^99mTc, in a one-pot procedure from the precursor 17α-azido-androst-4-ene-3-one, utilizing the novel Click chemistry route for targeting ARs upregulated in prostate cancer.

Materials and Methods

Materials

Carbonyl kit (Isolink^® kit) for the synthesis of [^99mTc(CO)₃(H₂O)₃]⁺ precursor was obtained as a gift from Mallinckrodt Medical. All the animal experiments were carried out in compliance with the relevant national laws as approved by the local committee on the conduct and ethics of animal experimentation. LNCaP cells (human prostate adenocarcinoma cell line) were procured from National Centre for Cell Sciences (Pune), the cell repository of India.

General methods

^99mTcO₄ ⁻ was eluted from an in-house ⁹⁹Mo/^99mTc column generator using 0.9% saline. HPLC analyses was performed on a Jasco PU 1580 system and a Jasco 1575 tunable absorption detector and a radiometric detector system having C-18 reversed-phase HiQ Sil (5 μm, 250×4 mm) column. The gradient system consisting of eluting solvents H₂O (solvent A) and acetonitrile (solvent B) with 0.1% trifluoroacetic acid was used for HPLC analyses (0–28 minutes, 90% A–10% A; 28–30 minutes, 10% A; 30–32 minutes, 10% A–90% A).

Chemical synthesis

Detailed chemical synthesis, characterization data, and NMR or ESI-MS spectra (Supplementary figures S1–S7) of all newly reported compounds are provided in the Supplementary Data (available online at www.liebertonline.com/cbr).

Radiochemistry

[^99mTc(CO)₃(H₂O)₃]⁺ precursor

In brief, ^99mTcO₄ ⁻ in 0.9% NaCl from ⁹⁹Mo/^99mTc generator (1 mL, 20–30 mCi) was added to the Isolink kit via syringe. The vial was kept in a boiling water bath for 15 minutes. After cooling the vial to room temperature, 1 N HCl was added to neutralize the solution to pH 7 and decompose any residual boranocarbonate. Radiochemical purity of the precursor was checked by reverse-phase HPLC (Fig. 1A).

FIG. 1.

HPLC profile of (A) [^99mTc(CO)₃(H₂O)₃]⁺ core, (B) ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one and (C) [Re(CO)₃-17α-triazolylandrost-4-ene-3-one].

^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5)

To 0.2 mLsolution (t-butanol/H₂O 1:1) of compound 4 (1.36 mg, 3.2 μmol), 1 mL of [^99mTc(CO)₃(H₂O)₃]⁺ (20 mCi) was added, followed by heating at 80°C for 1 hour. The complex prepared was characterized by HPLC (Fig. 1B). In the one-pot procedure, compound 3 (1.36 mg, 3.2 μmol), propargyl glycine (361 μg, 3.2 μmol), 0.2 mL (t-butanol/H₂O 1:1), [^99mTc(CO)₃(H₂O)₃]⁺ (1 mL), and catalytic amounts of Cu(II)acetate and Na-ascorbate were incubated at 80°C for 1 hour.

In vitro stability assessment

The stability of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) was assayed by monitoring the HPLC elution profile and determining the radiochemical purity after incubation at room temperature for 6 hours. To determine the in vitro serum stability, 200 μL of radiolabeled complex was incubated with 1 mL human serum at 37°C for 6 hours. Acetonitrile (1 mL) was added to the above solution. The precipitate was separated by centrifugation. The supernatant was injected in HPLC to determine the stability of the complex.

To study the stability of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) toward exchange with histidine and cysteine, challenge experiments were performed. The complex (100 μL) was incubated with freshly prepared histidine and cysteine solutions (10 μL, 0.5 M) in separate test tubes in a water bath at 37°C for 1 hour. The solution was then analyzed by HPLC to determine the extent of exchange, if any.

In vitro cell uptake studies

Cell uptake studies were carried out using LNCaP cells (human prostate adenocarcinoma cell line). The cells were maintained at 37°C in humidified 5% carbon dioxide atmosphere in RPMI 1640 medium with 2 mM l-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate containing 10% fetal bovine serum. The adherent cells from confluent flasks were isolated by trypsinization. The tumor cells were washed twice, and finally, a suspension of 7×10⁶ cells/mL was prepared in 50 mM of Tris-buffer (pH 8) containing 0.2% bovine serum albumin. To determine the percentage of in vitro cell binding of the complex, 0.1 mL cell suspension was incubated with 50 μL of the ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) (2 nmol) complex in triplicate at 37°C for 60 minutes. The specificity of the cellular uptake was studied by incubation in the presence of 100 μL of 10⁻⁷ to 10⁻⁴ M solution of testosterone. After incubation, cells were centrifuged at 2000 rpm for 5 minutes for pellet formation. The cell pellets were then washed twice with ice-cold assay buffer to remove any unbound radioactivity and centrifuged to collect supernatants. After the removal of the supernatant, radioactivity associated with the cell pellet was measured in an NaI(Tl) detector. Similar studies were carried out using 50 μL (0.1 μCi) of [^99mTc(CO)₃(H₂O)₃]⁺.

Biodistribution studies

The biodistribution studies were carried out according to the relevant national regulation using mature (5–6 weeks old) male Wistar rats weighing between 180 and 200 g. The ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) was reconstituted in saline and injected via the tail vein. The dose employed was 80 μCi per animal (1.03 μg), and one set of animals was coinjected with radiotracer, together with a blocking dose (25 μg) of testosterone. At 1 and 3 hours after injection of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one, groups of 3 animals each were sacrificed; tissues of interest were removed and weighed, and radioactivity was counted. The injected dose (ID) was calculated by comparison with dose standards prepared from the injected solution of appropriate counting rates, and the data were expressed as percentage of ID per gram of tissue (% ID/g of tissue). For each animal group, the data were averaged and standard deviation was calculated.

Results and Discussion

Synthesis

The structure of L-39 and VN/109-1 (Scheme 1) essentially consists of an androst-4-ene-3-one skeleton substituted with an azolyl group at the 17 position. Hence, any analog based on this molecule should possess these basic structural features. From the retrosynthetic perspective, it was envisaged that the incorporation of the triazole at the 17 position of the androst-4-ene-3-one skeleton (i) could be accomplished as shown in Scheme 1. It was possible to synthesize i easily via the 3+2 alkyne azide cycloaddition reaction. Struthers et al. and Mindt et al. have demonstrated that using the click-to-chelate approach, the triazole group and the radioactive atom can be incorporated in a biomolecule (derivatized as an azide) in a one-pot reaction.^22,23 Using this approach as shown in Scheme 1, incorporation of the triazole group and ^99mTc in the androst-4-ene-3-one skeleton could be achieved in a single step. The azide ii should be obtainable from iii through nucleophilic azidation. Hence, it was envisaged that testosterone (1, Scheme 2) would be an ideal synthon for the targeted compound. The Rhenium analog could also be synthesized following a similar route from testosterone.

SCHEME 1.

Retrosynthetic analysis of the ^99mTc-labeled triazole analog of L-39. M=^99mTc, Re.

SCHEME 2.

Reagents and conditions. (i) MsCl, Pyridine, 0°C, 30 minutes; (ii) DMSO, NaN₃, microwave (350 W), 2 minutes; (iii) Cu (II) acetate, Na-ascorbate, propargylglycine, t-BuOH+H₂O (1:1), room temperature, 12 hours; (iv) for 5: [^99mTc(CO)₃(H₂O)₃]⁺, pH 7, 80°C, 1 hour, M=^99mTc, for 6: [NEt₄]₂[Re(CO)₃Br₃], t -BuOH+H₂O (1:1), 100°C, 1 hour, M=Re. (v) for 5: Cu (II) acetate, Na-ascorbate, propargylglycine, [^99mTc(CO)₃(H₂O)₃]⁺, pH 7, 80°C, 1 hour, M=^99mTc, for 6: Cu(II)acetate, Na-ascorbate, propargylglycine, [NEt₄]₂[Re(CO)₃Br₃], t-BuOH+H₂O (1:1), 100°C, 1 hour, M=Re.

The present synthesis commenced with mesylation of testosterone (1) to afford the mesylate (2, Scheme 2). Mesylation led to a downfield shift in the position of the C-17 proton from δ 3.64 ppm in testosterone (1) to δ 4.49 ppm in 2. The S_N2 azidation led to the inversion of stereochemistry, leading to the formation of the 17α-azidoandrost-4-ene-3-one (3). Azidation led to an upfield shift of the C-17 proton from δ 4.49 ppm in 2 to δ 3.54 ppm in case of 3. Azidation was also confirmed by the presence of a sharp prominent peak at 2098 cm⁻¹ in the IR spectrum of 3. Azidation in DMF afforded negligible yields (2%–5%) even on heating the reaction mixture at 110°C for 5 hours. However, using DMSO under similar conditions afforded better yield (50%–60%). Under microwave conditions using DMSO as solvent, the reaction could be completed in 2 minutes with a yield of 80%. No formation of the elimination product was observed, which is commonly formed in S_N2 reactions involving azidation of the steroidal skeletons especially in the B and C rings. Compound 3 has been functionalized to 1,2,3-triazole 4 by Cu(I)-catalyzed [3+2] cycloaddition reaction with propargyl glycine. Toward this, 3 was reacted with propargyl glycine in the presence of Cu(I) as catalyst, generated in situ from Cu(II)acetate by reduction with Na-ascorbate in t-butanol/H₂O (1:1), at room temperature, overnight, to provide 1,2,3-triazole derivative. The formation of compound 4 has been confirmed by mass spectral analyses of the reaction mixture. The presence of a peak corresponding to compound 4 at [M - H]⁻=425.2 (calc. for C₂₄H₃₄N₄O₃: 426.552) and the absence of peaks corresponding to the molecular ion peak of starting azide and alkyne confirmed the completion of the reaction and formation of 4.

Radiolabeling

Triazole 4 could be radiolabeled by incubation with the [^99mTc(CO)₃(H₂O)₃]⁺ (1 mL, 20 mCi) precursor for 1 hour at 80°C at pH 7 (Scheme 2) to afford the nonpurified radioligand 5 (specific activity: 6.25 Ci/mmol). The ^99mTc(CO)₃ complex 5 was characterized using HPLC, wherein the complex (Fig. 1B) having retention time of 26.1 minutes was well separated from the ^99mTc-tricarbonyl precursor (Fig. 1A) eluting out at 16 minutes. Propargyl glycine being a bidentate ligand having amino and carboxyl groups for coordination was also complexed with ^99mTc-tricarbonyl precursor. The retention time of ^99mTc(CO)₃-propargyl glycine was 12 minutes in HPLC, and hence, it could be readily distinguished from ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5). However, such a complex was unstable and dissociated with time. The HPLC chromatogram indicated >95% (Fig. 1B) radiochemical purity of the complex. The radiolabeled complex was purified by RP-HPLC with gradient mixtures of water/acetonitrile at a flow rate of 1 mL/min and collected in a vial. The solution was evaporated to dryness to obtain the radioligand in a specific activity of 42 Ci/mmol. The radioligand was suitably reconstituted and diluted with saline for animal and cell studies. HPLC pattern revealed no degradation of the reconstituted complex.

The ^99mTc(CO)₃-triazole complex 5 has been characterized by comparison with the corresponding rhenium complex 6, which has been synthesized using [NEt₄][Re(CO)₃Br₃] precursor using conditions similar to ^99mTc labeling. This precursor was prepared from rhenium pentacarbonyl chloride, [Re(CO)₅Cl], using a published procedure.³³ Formation of the rhenium complex has been confirmed by mass spectral analysis of the reaction mixture. The peak corresponding to compound 6 [M - H]⁻=695.4 (calc. for C₂₇H₃₃N₄O₆Re: 695.78) was present in the negative ion mode, whereas the pseudomolecular ion peak corresponding to the [M+Na]⁺=719.1 was obtained in the positive ion mode, confirming formation of the Re(CO)₃-triazole complex 6. Absence of molecular ion peak corresponding to the precursor triazole 4 in both the positive as well as negative ion mode confirmed the completion of the reaction. The technetium-99m complex prepared in tracer level was characterized by comparing the retention time of γ-trace of ^99mTc(CO)₃-triazole 5 in HPLC with that of UV trace of corresponding rhenium complex 6. The UV-HPLC chromatogram retention time of rhenium complex (Fig. 1C) was observed to be 26.6 minutes, which matches well with 26.1 minutes γ-HPLC chromatogram retention time of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) (Fig. 1B). A one-pot reaction of azide 4, Cu(II)acetate, and Na-ascorbate with [^99mTc(CO)₃(H₂O)₃]⁺ and [NEt₄][Re(CO)₃Br₃] gave the products 5 and 6, respectively, which were characterized using mass spectral and HPLC analysis as described earlier.

The lipophilicity of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5) as determined by octanol/saline partitioning was inferable from the log P value of 1.21±0.30.

In vitro stability and cell uptake studies

The affinity of the radiolabeled complex toward ARs has been tested by carrying out in vitro and in vivo studies. In vitro stability of the complex was studied in phosphate-buffered saline at pH 7.0 as well as in serum after 6 hours of incubation at 37°C. The complex was found to be stable even after 6 hours of preparation. In serum stability studies, nearly 50% of the activity was associated with the precipitate obtained after acetonitrile addition, indicating high binding of complex with serum proteins. The acetonitrile fraction was characterized by HPLC, wherein a single peak was observed at the same retention time (26.1 minutes, >95%) as that of the complex, indicating no decomposition of the complex and hence its stability under 37°C incubation.

To assess the kinetic inertness of the complex under physiological condition, the challenge studies were carried out by incubation of the radiolabeled complex with excess of histidine and cysteine. The HPLC pattern of the complex (retention time: 26.1 minutes) remained unaltered with no indication of the appearance of a new peak corresponding to that of either ^99mTc(CO)₃-histidine or ^99mTc(CO)₃-cysteine complex. This demonstrates the kinetic inertness of the complex, indicating no transchelation with the ligands, histidine, or cysteine.

To determine the affinity of the ^99mTc(CO)₃ triazole complex (5) toward binding with ARs, in vitro cell studies were carried out in LNCaP, human prostate carcinoma cells. The percentage of binding of the complex in cells was found to be 4.95%±1.2% when 1 μg of the tracer was added. The binding study of the tricarbonyl core has been carried out with LNCaP cells and the binding was found to be 0.69%±0.07%. The binding in inhibition studies in the presence of 5 μM cold testosterone is 4.55%±1.5% (8% inhibition).

In vivo studies

^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (80 μCi/animal, 1.03 μg) was injected into mature male Wistar rats via the tail vein. Animals were sacrificed at 1 and 3 hours postinjection. Table 1 presents the uptake of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one in tissues of mature male Wistar rats at 1 hpi, 1 hpi with coinjected testosterone to block AR, and 3 hpi. At 1 hpi, uptake of 0.48%±0.19% ID/g was observed in the prostate, which was found to be retained till 3 hpi. Uptake in the blood and liver was significantly less at 1 hpi, being 0.03%±0.01% and 0.54%±0.10% ID/g, respectively. Low blood uptake indicated lack of in vivo binding of the complex to proteins present in the blood. Most of the activity was concentrated in the intestine (7.4±4.8), indicating a hepatobiliary route of excretion as expected of the lipophilic complexes. The prostate/muscle and prostate/blood ratios were 7.10±3.15 and 19.15±13.19, respectively. These ratios are significantly higher compared with that reported for other radiolabeled AR imaging derivatives.^26,34,35

Table 1.

Biodistribution of ^99m Tc(CO) ₃-17α-Triazolylandrost-4-Ene-3-One in Mature Male Wistar Rats (Percentage of Injected Dose per Gram±Standard Deviation of Tissue, n=3)

Tissue	1 hour control	1 hour block	3 hours control
Blood	0.03±0.01	0.06±0.02	0.06±0.03
Liver	0.54±0.10	0.34±0.08	0.50±0.045
Int-Gb	7.45±4.87	5.51±.88	5.04±0.35
Stomach	0.17±0.13	0.23±0.09	0.16±0.09
Heart	0.06±0.00	0.04±0.02	0.04±0.01
Lung	0.18±0.03	0.15±0.05	0.12±0.04
Spleen	0.07±0.03	0.09±0.04	0.11±0.07
Bone	0.35±0.05	0.38±0.02	0.24±0.17
Muscle	0.07±0.00	0.15±0.04	0.02±0.01
Prostate	0.48±0.19	0.81±0.29	0.45±0.08
Kidneys	0.45±0.04	0.46±0.20	0.42±0.04
Testicles	0.04±0.03	0.02±0.01	0.02±0.01
Prostate/muscle	7.10±3.15	5.91±3.87	24.22±14.68
Prostate/blood	19.15±13.19	14.09±5.24	8.50±3.25

To determine whether the uptake was mediated by AR, a blocking study was carried out by coinjection of 80 μCi of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one with 25 μg testosterone to saturate the AR. The blocking studies did not show any decrease in the prostate uptake though inhibition was observed in in vitro cell studies. This may be attributed to the difference in physicochemical conditions while carrying out cell uptake studies in vitro and that present in vivo.

At 3 hours postinjection point, uptake by prostate was 0.45±0.08 similar to that at 1 hour postinjection. There was a significant decrease in muscle uptake to 0.02±0.01, thereby significantly increasing the prostate/muscle ratio to 24.22±14.68. Uptake in the blood was 0.06±0.03, causing the prostate/blood ratio to reduce to 8.50±3.25 compared with 1 hour postinjection. In vivo biodistribution studies revealed that 45%–50% of the total injected activity was associated with the carcass. The binding of steroidal hormones to the adipose tissue in rats has been reported.³⁶ The low activity in blood and liver may be probably attributed to binding of the radioligand to the adipose tissue associated with the carcass.

Conclusion

The testosterone molecule could be successfully derivatized at 17-position to introduce a triazole moiety radiolabeled with ^99mTc(CO)₃ core via click chemistry route. The radiolabeled complex could be obtained in >95% radiochemical yield as determined by radiochromatogram in HPLC. The retention time in HPLC-UV profile of Re(CO)₃-17α-triazolylandrost-4-ene-3-one was similar to that of the radioactive peak of ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one observed in HPLC, thereby confirming the identity of the complex. Preliminary biodistribution studies in normal male Wistar rats have shown favorable uptake in prostate with significantly less uptake in other organs. However, ^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one did not show specificity toward ARs in in vivo biodistribution studies, indicating the loss of affinity due to introduction of a chelating group and the lipophilic ^99mTc-tricarbonyl core.

Footnotes

Acknowledgments

The authors are thankful to Dr. V. Venugopal, Director, Radiochemistry and Isotope group, and Dr. Meera Venkatesh, Head Radiopharmaceuticals Division, for their constant encouragement and support toward this work. Manish V. Dhyani is thankful to Department of Atomic Energy, Government of India, for the research fellowship.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Synthesis and Preliminary Bioevaluation of 99m Tc(CO) 3 -17α-Triazolylandrost-4-Ene-3-One Derivative Prepared via Click Chemistry Route

Abstract

Introduction

Materials and Methods

Materials

General methods

Chemical synthesis

Radiochemistry

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

99mTc(CO)3-17α-triazolylandrost-4-ene-3-one (5)

In vitro stability assessment

In vitro cell uptake studies

Biodistribution studies

Results and Discussion

Synthesis

Radiolabeling

In vitro stability and cell uptake studies

In vivo studies

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

[^99mTc(CO)₃(H₂O)₃]⁺ precursor

^99mTc(CO)₃-17α-triazolylandrost-4-ene-3-one (5)