Synthesis and evaluation of radiolabeled analogs of the antidepressant drug zimelidine as potential SPECT-ligands for the serotonin transporter

Nuclear Medicine and Biology 31 (2004) 563–569

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Synthesis and evaluation of radiolabeled analogs of the antidepressant drug zimelidine as potential SPECT-ligands for the serotonin transporter Jos L.H. Eerselsa,*, Rob P. Kloka, Joost Verbeeka, Allard J. Jonkerb, Jacobus D.M. Herscheida a

b

Radionuclide Center, Vrije Universiteit, de Boelelaan 1085c, 1081 HV Amsterdam, The Netherlands VU University Medical Center, Department Anatomy, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands

Abstract Z-3-(4-bromophenyl)-N,N-dimethyl-3-(3-pyridinyl)-2-propen-1-amine or zimelidine (ZIM) and its first metabolite nor-zimelidine, were radioiodinated via a nonisotopic exchange, using the Cu(I)-assisted nucleophilic labeling method. To evaluate their potential as SPECT ligands for the serotonin transporter (SERT), the biodistribution of both ligands was determined and pretreatment “blocking” studies performed. Both radioligands demonstrated a good brain penetration of 0.8-1% ID/g, stable after 60 min., p.i., and a brain/blood ratio of up to 3. In vivo brain distribution did not reveal specific binding. Blocking studies by pretreatment with a known SERT ligand, had minor influence on the uptake of [123I]I-ZIM, between the several isolated brain regions. It may therefore be concluded that [123I]I-ZIM and [123I]I-nor-ZIM do not appear to be promising SPECT ligands for the SERT. © 2004 Elsevier Inc. All rights reserved. Keywords: Zimelidine; SSRI; SPECT; [123I] labeling; SERT

1. Introduction In the modern pharmacological treatment of mental disorders, particularly affective and anxiety disorders, selective serotonin reuptake inhibitors (SSRIs) play a predominant role [1]. The higher affinity and specificity of these drugs toward the SERT, and therefore comparatively fewer pharmacological side effects, confers a higher safety and tolerability in therapeutic doses than the older tricyclic antidepressants (e.g., imipramine, amitriptyline) or MAOIs (monoamine oxidase inhibitors). They are also clinically effective. These findings, together with alterations in functional SERT levels in neuropsychiatric disorders [2], and dose-related decrease in SERT in MDMA users (methylenedioxymethamphetamine or Ecstasy) [3,4], suggest that nuclear imaging techniques, like single photon emission computed tomography (SPECT) and positron emission tomography (PET), could provide useful medical data for the assessment of the biochemical status and availability of the

* Corresponding author. Tel.: ⫹31-20-4449712; fax: ⫹31-204449121. E-mail address: [email protected] (J.L.H. Eersels). 0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2004.01.006

SERT in human brain, and thus provide insight into the pathology of these disorders. In this context, extensive efforts have led to the development of several radioligands for SPECT and PET applications, albeit with different and diverse chemical base structure. This is in part due to the absence of a single pharmacophore model for the SERT. Until now, most-promising SERT radioligands have been derived from chemical base structures, such as; NO2-quipazine, diphenylsulfide, and to a lesser extent tropane-congeners. [123I]-NO2-quipazine showed appropriate binding characteristics. However, in vivo dehalogenation obscured the recorded images [5]. There has been more success with diphenylsulfide-based structures; small modifications on the accessible, chemical base structure of diphenylsulfide, with the absence of an asymmetric center, have given rise to different radioligands for SPECT and PET applications. Preliminary studies in healthy volunteers, with [123I]ADAM [6], [11C]-DASB [7], and [11C]-DADAM [8], all show a regional distribution of radioactivity, corresponding to known SERT-rich regions, whereas pretreatment with citalopram leads to significantly lower binding in these regions (e.g., [123I]-ADAM, [11C]-DASB). Despite numerous substitutions on the tropane-structure (Fig. 1); on posi-

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Fig. 1. 3-Aryltropane.

tion 2 (carbomethoxy-group [9]), on position 3 (phenyl-ring substitution [10], and replacement by –naphthyl [11]), and on position 8 (substitution of -N; carba-; and oxo-analogs [12]); it has proved difficult to isolate an ideal SERT ligand for use in vivo. Recently, even the constrained tricyclic-tropane structures with promising affinity and specificity data [13], failed in the in vivo evaluation, either due to poor brain penetration, high nonspecific binding, or in vivo deiodination [14]. In order to develop a new SPECT tracer, we started from the SSRI drug zimelidine. This drug, discovered like fluoxetine in the late 1970s, was inspired by the 5-HT reuptake blocking properties of certain H1-receptor antagonist antihistamines, addition to the knowledge of the pharmacological mechanisms of action of tricyclic-antidepressants (e.g., imipramine). The structure of H1 antagonists, like pheniramine and diphenhydramine, were the template for zimelidine and fluoxetine respectively (Fig. 2), [15]. Zimelidine or Z-3-(4-bromophenyl)-N,N-dimethyl-3-(3pyridinyl)-2-propen-1-amine, has a moderate affinity for the SERT, Ki⫽39 nM, and is almost devoid of action at other receptors (beta-adrenergic, opiate, GABA, or benzodiaz-

epine receptors) [16], whereas the selectivity ratios toward other monoamine transporters such as norepinephrine (NET) and dopamine (DAT) are 10 (NET/SERT) and 22.5 (DAT/SERT), respectively [17]. Moreover, the analogs have a significantly higher affinity for SERT (I-ZIM, Ki⫽19, and nor-Br-ZIM, Ki⫽3.3 nM, [18]). Therefore, [123I]I-ZIM and [123I]I-nor-ZIM were screened for in vivo evaluation in rats, as potential SPECT ligands for the SERT.

2. Materials and methods 2.1. Reagents Zimelidine or Z-3-(4-bromophenyl)-N,N-dimethyl-3-(3pyridinyl)-2-propen-1-amine dihydrochloride was bought from Sigma, and its iodinated analog (in oxalate-form), and the desmethyl brominated analog (in dihydrochloride-form) were donated by Astra-Zeneca (So¨ derta¨ lje, Sweden). Reagents used for labeling such as 2,5-dihydroxybenzoic acid (Janssen Chimica), CuSO4.5H2O (Merck), SnSO4 (Janssen Chimica), citric acid-1-aqua (Merck), sodium dihydrogenphosphate-1-aqua (Merck), sodium acetate-3-aqua (Merck), and phosphoric acid (85 %, Baker) were analytical grade. Solvents for HPLC were HPLC-grade (Baker). Ion-pair reagents used for optimum HPLC separations were 1-heptanesulfonic acid sodium salt (Eastman) and N,N-dimethyloctylamine (DMOA), technical grade (Aldrich). -N.C.A. Na123I (10-2 M NaOH; 8.7ⴱ103 TBq/mmol) was purchased from the B.V. Cyclotron, Vrije Universiteit Amsterdam. The selective DA uptake blocker GBR-12909 was obtained from Research Biochemicals International (RBI, Natick, MA, USA) and fluvoxamine was donated by Solvay Pharmaceuticals (Weesp, The Netherlands). 2.2. HPLC equipment

Fig. 2. SSRI-drugs derived from H1-antagonists.

Semi-preparative: Rheodyne injector (2 ml loop), a LKB 2405 pump with a LKB VWM-2141 variable wavelength UV monitor at 230 nm, and a flow-through NaI(Tl)-radioactivity detector (Ortec electronics) was used. Both semi preparative separations for [123I]I-ZIM and [123I]I-nor-ZIM were done with a RP Select B, LiChrosorb (Merck) column, 250⫻10 mm, 10 ␮ with a H2O/MeCN/MeOH eluent; 0.08 M sodium dihydrogenphosphate solution 65/20/15, containing 5.5 mM heptanesulfonic acid, pH 5.0, at a flow rate of 5.5 ml/min. Analytical 1: Rheodyne injector (0.2 ml loop), and LKB pump, with UV and radioactivity detection, as described previously, was used. Quality control, of synthesized radioligands [123I]I-ZIM and [123I]I-nor-ZIM, was performed on a RP Select B, LiChrospher (Merck) column, 125⫻4 mm, 5 ␮ with the same eluent as for semipreparative. Flow rate was 1 ml/min. Analytical 2: Rheodyne injector (0.2 ml loop), and LKB

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pump, with UV and radioactivity detection, as described previously was used. An efficient mutual separation of [123I]I-ZIM and [123I]I-nor-ZIM, needed for the metabolic studies, was performed on a RP Select B, LiChrospher (Merck) column, 125⫻4mm, 5 ␮ with a H2O/MeCN; 0.05 M sodium dihydrogenphosphate solution 85/15 eluent, acidified with phosphoric acid (pH 3⫺3.2), containing 4⫻10⫺4 M DMOA, at a flow rate of 1 ml/min. Chromatographic data were captured and analyzed using Gina Star software, version 14.0. 2.3. Radiosynthesis of [123I]Iodo-ZIM and its [123I]Iodonor-compound Pretreatment of precursors: Both precursors, ZIM and nor-ZIM were supplied in their dihydrochoride salt form. Modification to the acetate form was done by means of a home-made anion-exchange column. A 1 ml syringe, filled with Bio-Rad AG 1-X8 anion-exchanger Cl⫺ resin (0.4 g), was rinsed successively with 2 ml of a 1M NaOAc solution, and 2 ml of water. 2.4 mg (6.1 ␮mol) of the zimelidine compound was dissolved in 0.4 ml of water, and passed through the column till dry. Retention of the drug compound on the column was controlled by UV-HPLC; the concentration decrease of the eluate, versus start solution, was negligible and lower than 5 % for both precursors. Radioiodination: In a conical V-vial; 5 mg 2,5-dihydroxybenzoic acid, 8 mg citric acid and 0.2 mg SnSO4 were dissolved in a 0.2 ml eluate solution of Br-ZIM or nor-BrZIM, containing 1.2 mg of the drug compound in acetate form (vide supra). 30 ␮l of a 13 mM copper(II)-sulphate solution and NCA Na[123I] (up to 1000 MBq) were added, and the final solution was made oxygen free, with a gentle N2 stream, during 20 min. at room temperature. The vial, placed in a copper safety container, was heated in a thermoblock at 165°C, for 50 min. Thereafter, the content of the vial was diluted and rinsed with 1 ml of a 10% methanolic solution, filtered through a 0.45 ␮-filter, and injected for semi preparative HPLC separation. The collected RP-HPLC fraction was diluted 1.5 times its collected volume, with water, and the resulting solution was passed through a C18 Sep-Pak Classic. It was subsequently rinsed with 10 ml of a sodium hydroxide solution (pH 9) and 10 ml water. The radioactive compound was recovered from the cartridge, by reverse-elution with 0.3-0.4 ml acidified ethanol [0.6% (v/v) concentrated sulfuric acid]. For both radioligands [123I]IZIM and [123I]I-nor-ZIM, the overall radiochemical yield, after purification and preconcentration, was higher than 60%, and the radiochemical purity was more than 99%. The amount of starting bromocompound in the ethanolic concentrate was checked by the analytical 1 UV-HPLC system, operated at high UV sensitivity (␭⫽230 nm, i.e., ␭max of Br-ZIM or nor-Br-ZIM in the eluent). For both radioligands, in at least four separate runs each (labeling, collection of eluent and concentration), no peak corresponding to their bromoprecursor could been observed in the ethanolic concentrate.

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The detection limit was determined, for both precursors at 1.2⫻10⫺11 mol. The specific activity of the 123I-labeled zimelidine analogs can be estimated to be ⬎4 TBq/␮mol. 2.4. Biodistribution in rats For both radioligands, [123I]I-ZIM and [123I]I-nor-ZIM, we carried out methodologically identical biodistribution experiments. Male Wistar rats (250⫺280 g) were injected via the tail vein with approximately 3.7 MBq of the radioligand (⬍1 nmol) in a volume of 0.2 ml in saline (acidified with 1 mM NaHSO4, pH 3), and sacrificed by cervical dislocation at 5, 30, 60, and 120 min. postinjection. Four rats per group were used, and samples of blood, thyroid, heart, lung, liver, kidney and muscle were removed, weighed and counted for radioactivity. Regional brain distribution was measured by quickly dissecting the cerebellum, cerebral cortex, striatum, hippocampus and hypothalamus, which were immediately weighed and counted. Radioactivity was measured in an automated gamma counter, and percentage dose/g was calculated by comparison of the samples to standard dilutions (100 times diluted) of the injected dose (4-5 %). To reveal the specificity of the in vivo brain uptake; blocking studies were performed for [123I]I-ZIM, with GBR-12909 and fluvoxamine, DAT and SERT ligands, respectively. Drug compounds were dissolved in acidified saline and a dose of 4 mg/kg was ip injected, 60 min. before tracer administration, and animals were sacrificed at 60 min. post-tracer administration. All animal experiments were performed according to the principles of laboratory animal care (NIH publication 85-23, revised 1985) and the Dutch national law “Wet op de Dierproeven” (Stb 1985, 336). 2.5. Metabolite analysis of rat plasma and brain samples One hour after an iv injection of ⬃10 MBq [123I]I-ZIM (in three rats), a blood sample was taken before the brain was removed. The blood samples were centrifuged (5 min., 3000 g) and plasma was separated from the blood cells. Plasma specimen (0.5 ml) was treated with a TCA solution, mixed and centrifuged (5 min., 3000 g ) [19]. Supernatant of last centrifugation was analyzed with HPLC, according the setup procedure of Analytical 2 (vide supra). Brain samples were treated individually, frozen in liquid nitrogen, homogenized with a stamper, and extracted with ethyl acetate (2⫻1 ml). The extracted layers of ethyl acetate were evaporated, till nearly dry, diluted with eluent and analyzed with HPLC, following procedure Analytical 2 (vide supra). 3. Results and Discussions 3.1. Radiochemistry As described previously, both radioligands [123I]I-ZIM and [123I]I-nor-ZIM, were labeled according an identical

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Fig. 3. Radiosynthesis of [123I]I-ZIM and [123I]I-nor-ZIM.

procedure, with the Cu1⫹-assisted nucleophilic labeling method [20], via a nonisotopic exchange and under the same labeling conditions (Fig. 3). The absence of electron-donating groups in the precursor molecule implies that nucleophilic labeling is the method of choice [21]. The availability of the precursor as a dihydrochloride salt, however, suggested to us that it would be beneficial to modify its salt form to acetate. Indeed, using the acetate form we were able to obtain a reproducible labeling yield of more than 90% (notwithstanding the small amount of 1.2 mg precursor in the reaction mixture, favorable for a good semipreparative separation), whereas under the same labeling conditions, the dihydrochloride salt gave a significant lower labeling yield of 50⫺60 %, due to a competing chloro-exchange [22]. Concerning, the HPLC conditions, the presence of the ion-pairing agent heptanesulfonic acid was essential to obtain an optimum resolution between the bromo-precursor and the (radio)iodinated compound. For the semipreparative separation, we obtained a resolution factor of 2.7 and the retention times of precursor and radioligand, were 30 and 44 min., respectively. Concerning, the separation of the ZIM compound and its nor-

analog, as well as their radioligands, co-elution (by peakbroadening) was noticed and DMOA as an ion-pairing reagent was needed to obtain a optimum separation. After radiosynthesis for both radioligands, [123I]I-ZIM and [123I]I-nor-ZIM, minor side-products (cold and radioactive), 1-2 %, could be seen. As expected, analytical HPLC control of the reaction mixture could not reveal geometrical isomer conversion (Z to E-isomer) due to the regiospecific interaction mechanism of the Cu1⫹ labeling method. As published, both isomers Z and E-ZIM and their nor-analogs have distinct k⬘ values [23]. We achieved an overall radiochemical yield of more than 60%, and the quality control revealed a radiochemical purity of more than 99% and was still the same after 24 h, at 0-4°C in acidified ethanol. 3.2. Biodistribution in rats After in vivo injection in rats with [123I]I-ZIM (Table 1), as well as [123I]I-nor-ZIM (Table 2), there was good brain penetration of the tracers. The initial uptake at 5 min. postinjection, was for [123I]I-ZIM (1.27⫺2.3 % ID/g). This was more favorable

Table 1 Biodistribution of [123I]I-ZIM in rats Organ

5 min.

30 min.

60 min.

120 min.

Blood Thyroid Heart Lungs Liver Kidney Muscle Cerebellum Cer. Cortex Striatum Hippocampus Hypothalamus

0.13 ⫾ 0.02 0.80 ⫾ 0.17 0.82 ⫾ 0.11 6.06 ⫾ 1.00 1.49 ⫾ 0.59 2.54 ⫾ 0.77 0.15 ⫾ 0.05 1.27 ⫾ 0.12 2.30 ⫾ 0.34 2.05 ⫾ 0.51 2.04 ⫾ 0.36 1.64 ⫾ 0.27

0.19 ⫾ 0.04 0.61 ⫾ 0.08 0.67 ⫾ 0.06 4.28 ⫾ 0.38 1.79 ⫾ 0.22 1.92 ⫾ 0.17 0.27 ⫾ 0.04 1.07 ⫾ 0.13 1.57 ⫾ 0.17 1.46 ⫾ 0.18 1.49 ⫾ 0.21 1.22 ⫾ 0.13

0.23 ⫾ 0.02 0.54 ⫾ 0.04 0.53 ⫾ 0.03 3.93 ⫾ 0.20 1.65 ⫾ 0.10 1.66 ⫾ 0.08 0.23 ⫾ 0.01 0.65 ⫾ 0.04 0.86 ⫾ 0.05 0.75 ⫾ 0.04 0.84 ⫾ 0.06 0.75 ⫾ 0.04

0.36 ⫾ 0.03 0.61 ⫾ 0.03 0.58 ⫾ 0.03 3.72 ⫾ 0.51 1.82 ⫾ 0.10 1.72 ⫾ 0.10 0.25 ⫾ 0.01 0.59 ⫾ 0.03 0.75 ⫾ 0.03 0.69 ⫾ 0.04 0.71 ⫾ 0.04 0.65 ⫾ 0.03

Values represented as % ID/g (mean ⫾ standard error of the mean, n ⫽ 4).

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Table 2 Biodistribution of [123I]I-nor-ZIM in rats Organ

5 min.

30 min.

60 min.

120 min.

Blood Thyroid Heart Lungs Liver Kidney Muscle Cerebellum Cer. Cortex Striatum Hippocampus Hypothalamus

0.24 ⫾ 0.02 0.93 ⫾ 0.07 1.36 ⫾ 0.08 9.96 ⫾ 0.79 2.74 ⫾ 0.18 3.93 ⫾ 0.27 0.30 ⫾ 0.02 0.71 ⫾ 0.03 0.80 ⫾ 0.05 0.81 ⫾ 0.06 0.71 ⫾ 0.04 0.67 ⫾ 0.03

0.32 ⫾ 0.03 0.83 ⫾ 0.05 0.98 ⫾ 0.02 8.18 ⫾ 0.44 2.78 ⫾ 0.09 2.71 ⫾ 0.05 0.40 ⫾ 0.01 0.93 ⫾ 0.02 1.19 ⫾ 0.01 1.17 ⫾ 0.02 1.05 ⫾ 0.03 0.97 ⫾ 0.01

0.30 ⫾ 0.00 0.55 ⫾ 0.03 0.68 ⫾ 0.02 5.55 ⫾ 0.46 2.11 ⫾ 0.11 1.97 ⫾ 0.04 0.29 ⫾ 0.01 0.68 ⫾ 0.04 0.89 ⫾ 0.05 0.82 ⫾ 0.06 0.73 ⫾ 0.04 0.76 ⫾ 0.05

0.44 ⫾ 0.11 0.58 ⫾ 0.15 0.67 ⫾ 0.17 4.72 ⫾ 1.19 2.33 ⫾ 0.60 2.07 ⫾ 0.53 0.28 ⫾ 0.07 0.61 ⫾ 0.15 0.83 ⫾ 0.21 0.91 ⫾ 0.24 0.82 ⫾ 0.21 0.69 ⫾ 0.17

Values represented as % ID/g (mean ⫾ standard error of the mean, n ⫽ 4).

than for [123I]I-nor-ZIM (0.67⫺0.81 % ID/g) and is unlikely to be due to increased lipophilicity, both ligands have a similar structure. The uptake of [123I]I-ZIM decreased gradually with time, whilst the washout of [123I]I-nor-ZIM over the same period was negligible. Both radioligands displayed a stable level of brain uptake, (0.8⫺1 % ID/g), after about 60 min. The uptake, however, was homogeneous throughout the different brain regions studied. This is indicative of a nonspecific uptake. The regional serotonergic neuronal distribution, with highest concentration in the hypothalamus, followed by hippocampus, striatum, and cortex, and low in cerebellum was not reflected in the distribution of either radioligand. The cerebellum had nearly the same uptake profile as the SERT-rich regions, and after 60 min. only a slight lower uptake value, (10⫺20 %), was found. This is in contrast with other SERT-specific radioligands, like

[123I]-ADAM, where SERT-rich regions to cerebellum ratios, peak at values of 4⫺5 [6]. Blocking studies, by pretreatment with the specific SERT ligand fluvoxamine as well as DAT ligand GBR-12909, confirmed the nonspecific uptake of [123I]I-ZIM (Fig. 4). Concerning peripheral organs, the high lung uptake can be probably explained not only by the presence of SERT binding sites [24,25], but also due to binding sites of amine metabolic enzymes, like monoamine oxidase [26]. Interestingly, there was stable and low thyroid uptake during the time course, for both radioligands. This is indicative of the good in vivo stability of the radioligands. As described previously, due to co-elution of the ZIM compound and its nor-analog, the HPLC eluent had to be adapted with DMOA as ion-pairing reagent [23], and we obtained an efficient separation (Rs⬃4, analytical setup). Centrifugation of the blood samples (60 min. p.i.) gave a recovery of nearly 80% of the

Fig. 4. Blocking studies [123I]I-ZIM; effect of fluvoxamine and GBR-12909 on binding of [123I]I-ZIM.

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radioactivity in the plasma, and subsequently treatment with TCA and centrifugation, gave only 10% of the radioactivity in the protein free fraction. This indicates that [123I]I-ZIM (and or its metabolite) is intensely bound to proteins in the plasma. Radiochromatographic analysis of in vivo plasma and brain samples, from the animals sacrificed at 60 min. p.i., showed more than 92% of unchanged radioligand with only a few percent of the total activity representing free radioiodide. Previous metabolism studies [27] of the drug zimelidine, in rats, have demonstrated several metabolites (among them N-oxides of zimelidine, N-demethylation, and deamination). All of these metabolites, however, were measured from collected urine samples several days after different doses of the drug compound. It can be assumed that the difference between these results and the metabolite analysis we performed is due solely to pharmacokinetic factors.

4. Conclusions Starting with the lead structure of zimelidine, a selective compound for the SERT, [123I]I-ZIM and [123I]I-nor-ZIM could be easily synthesized in a good radiochemical yield and purity, via a nonisotopic, nucleophilic labeling. Some favorable aspects in the evaluation of the tracer were observed, such as a good brain penetration, high brain to blood ratios, and a high in vivo and in vitro stability. However, both radioligands failed to show specific binding in brain distribution and pretreatment receptor blocking experiments. In the development of new (neuro) receptor radioligands the chemical base structure of existing (antidepressant) drugs are often used as lead structure, unfortunately as we have also found, not always with success. Developments of SPECT and PET tracers, based on other tricyclic and selective antidepressants (e.g., citalopram, imipramine) have yielded negative results, probably because the lipophilic tricyclic aryl moiety promotes a high nonspecific binding in vivo [28]. On the other hand, modifications to the structure of the nonselective drug 403U76, have resulted in promising radioligands with a specific uptake, suitable for human applications (e.g., [123I]-ADAM [6] and [11C]-DASB [7]). So although, prerequisite criteria (e.g., affinity, lipophilicity, . . .), as expressed by their related parameter (e.g., Ki, log P, . . .), may ideally be fulfilled in vitro [29], as they were for zimelidine, these data do not have always a predictive value for the in vivo behavior of a receptor ligand.

Acknowledgments The authors would like to thank Dr. Mike Travis (London, Great Britain) for valuable comments on this manuscript.

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