Discovery of the First Environment-Sensitive Near-Infrared (NIR

Jan 28, 2016 - (18, 19) Compared with other members of AR, the characterization of α1-AR ...... the Program for Changjiang Scholars and Innovative Re...
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Discovery of the First Environment-Sensitive Near-Infrared (NIR) Fluorogenic Ligand for #-Adrenergic Receptors Imaging in vivo 1

Zhao Ma, Yuxing Lin, Yanna Cheng, Wenxiao Wu, Rong Cai, Shouzhen Chen, Benkang Shi, Bo Han, Xiaodong Shi, Yubin Zhou, Lupei Du, and Minyong Li J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01843 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on January 31, 2016

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Discovery of the First Environment-Sensitive NearInfrared (NIR) Fluorogenic Ligand for α1-Adrenergic Receptors Imaging in vivo Zhao Ma1, Yuxing Lin1, Yanna Cheng2, Wenxiao Wu1, Rong Cai3, Shouzhen Chen4, Benkang Shi4, Bo Han5, Xiaodong Shi3, Yubin Zhou6, Lupei Du1 and Minyong Li*1 1

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of

Pharmacy, Shandong University, Jinan, Shandong 250012, China; 2

Department of Pharmacology, School of Pharmacy, Shandong University, Jinan, Shandong 250012,

China; 3

C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506,

USA; 4

Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China;

5

Department of Pathology, School of Medicine, Shandong University, Jinan, Shandong 250012, China;

6

Center for Translational Cancer Research, Institute of Biosciences & Technology, Texas A&M

University Health Science Center, Houston, TX 77030, USA KEYWORDS. GPCR, fluorescent ligand, α1-adrenergic receptors, environment-sensitive, near-infrared, in vivo imaging, prostate sections (This article is dedicated to Professor Lin Xia on the occasion of her 80th birthday) ACS Paragon Plus Environment

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ABSTRACT

Fluorescent ligands are gaining popularity as tools to aid GPCR research. Nonetheless, in vivo application of such tools is hampered due to their short excitation wavelengths in the visible range and lack of fluorogenic switch. Here we report the discovery of fluorescent ligands (3a-f) for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore, cyane 5 (Cy5), with the quinazoline pharmacophore. Among them, the conjugated compound 3a, with acylated piperazine and the shortest carbon chain spacer, exhibited potent binding and remarkable changes in fluorescence (10fold) upon binding to α1-AR. Furthermore, it could be employed to selectively and specifically label α1ARs with no washing procedures in single cells, prostate tissue slices, intact tumor xenografts and organs in living mice. Especially, the slice imaging results gave direct and visual evidences that there is a close relationship between α1-ARs and pathological prostate. It is anticipated that our fluorescent tools will find broad applications in study of α1-ARs pharmacology and physiology to aid development of novel therapeutics.

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INTRODUCTION Fluorescent ligands are becoming attractive tools to study G-protein-coupled receptor (GPCR) since they can be employed at different experimental setups to afford insights into GPCR structure and function in native, live cells or tissues.1-3 During the past decade, we have witnessed remarkable advances in the wide application of fluorescently tagged drug molecules to visualize and interrogate GPCR targets.4,

5

Generally, fluorescently-tagged ligands can provide knowledge of receptor

localization that can be used to track GPCR’s dynamic processes, such as internalization and trafficking.6 Furthermore, the study of simple binding assays, ligand-receptor interactions and receptor– receptor interactions can be achieved.7 However, some obstacles remain that limit the applications of these fluorescent ligands. Although a lot of existing fluorescent ligands for GPCRs show very excellent receptor-oriented properties in vitro, in vivo imaging of GPCRs using them is not feasible because of the short excitation wavelengths, overmuch non-specific binding and difficulty in extensive washing, the latter step of which cannot be performed at animals.8 To overcome these obstacles, near-infrared (NIR) probes with fluorescence switches for GPCRs are drawing more and more attention from scientists. Though many fluorescent turn-on probes are designed upon enzymatic reactions, currently the design of fluorescent turn-on probes for proteins like GPCRs still remains challenging.9 One possible strategy is using environment-sensitive fluorophores because of their favorable spectroscopic behavior, which is dependent on the physicochemical properties of the surrounding environment. In GPCRs, canonical ligand-binding sites always belong to hydrophobic domains with low polarity and high viscosity. Once the environment-sensitive fluorescent ligands enter these regions, fluorescence switch is turned on, and then GPCRs are visualized. Taking advantage of these characteristics, several fluorogenic probes for adenosine receptor10, 5-hydroxytryptamine receptor11, 12, and oxytocin receptor13 have been designed.

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Adrenergic receptors (α1-, α2- and β-ARs) belonging to the superfamily of GPCR, mediate the actions of the endogenous catecholamines, and play a pivotal role in the modulation of sympathetic nervous system activity.14 Comprised of the three subtypes α1A, α1B and α1D, α1-ARs are the prime mediators of smooth muscle contraction, myocardial inotropy and chronotropy, and hepatic glucose metabolism.15 They also act as the therapeutic targets of hypertension16, benign prostatic hyperplasia (BPH)17 and lower urinary tract symptoms (LUTS)17. Additionally, some studies have suggested that α1-AR is abundant in prostate cancer cells.18, 19 Compared with other members of AR, the characterization of α1AR in molecular pharmacology lags behind greatly due to the lack of useful tools.20 Despite the powerful fluorescent technologies, to date, only a handful of fluorescent tools for α1-ARs are reported. Commercial antibodies widely used in quantifying and localizing the α1-AR subtypes are not specific for these receptors.21 With the shortcomings mentioned above, the commercial available 1-(4-(4-amino6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-3-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2c:2',1'-f][1,3,2]diazaborinin-3-yl)propan-1-one (BODIPY® FL prazosin)22,

23

and other fluorescent

prazosin derivatives, such as coumarin24, fluorescein24 and quantum dot (QD) conjugates25, are employed at best on cell-based studies always with the complicated washing work, which limits the study of α1-AR pharmacology in vivo. Thus, it is highly desired to develop puissant fluorescent ligands for α1-ARs. Herein we firstly discover that the common cyanine 5 (Cy5) could serve as an environmentsensitive fluorophore, and extend the “on-off” strategy into the design of fluorescent ligands for α1-AR (Figure 1). Then we synthesized and characterized six prazosin-triazole-Cy5 conjugates (3a-f) with nanomolar affinities and excellent fluorescence. Fluorescence turn-on can be achieved upon the binding of the fluorescent ligands to hydrophobic ligand-binding domain of α1-ARs. Using 3a, visualization, localization and expression level of α1-AR in living cells, mice tissue, and human tissue sections were conveniently investigated conveniently without cumbersome steps. Therefore, these environmentsensitive fluorescent probes beyond the traditional methods provide a powerful tool to study α1-AR pharmacology.

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RESULTS AND DISCUSSION Design and synthesis. As reported previously, prazosin derivatives with 4-amino-6,7-dimethoxy-2(piperazin-l-yl)quinazoline core always endure antagonism to α1-ARs.24,

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One major issue from

previous fluorescent ligands is the poor fluorescence properties. Hence, to generate fluorescent turn-on conjugates, the most concern is the choice of fluorophore. Cyanine dyes are broadly used in ultrasensitive imaging and spectroscopy in biological systems due to their long excitation wavelength.29 Furthermore, symmetrical cyanines accumulate in aqueous solution with a high degree of fluorescence quenching.30 Some new reports also describes that cyanine derivatives serve as environment-sensitive probes for their surrounding-dependent lifetimes.31, 32 To desgin environment-sensitive probes for α1-AR, as depicted in Figure 1, the key pharmacophore (4-amino-6,7-dimethoxy-2-(piperazin-l-yl)quinazoline moiety) of α1-AR antagonist prazosin and the environment-sensitive fluorophore, Cy5, were conjugated with the triazole linker, considering the powerful and modularized reactions generated by the Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC) chemistry. As environment-sensitive fluorescent probes, the conjugates emit very weak fluorescence themselves, and when bind to α1-AR, they enter the hydrophobic domains with low polarity and high viscosity and then release bright fluorescence. For prazosin, the furoyl or furan group was pruned away in order to introduce the azido group and to provide the key intermediate azides 1a-f. The symmetrical Cy5 was synthesized from 2,3,3-trimethylindole, and then 2-propynylamine was used synthesized another key intermediate 2 with the terminal alkyne by an amide condensation reaction. The end products 3a-f came into being from the CuAAC click reactions between the above azides (1) and acetylenic compound (2). More synthetic details can be found in Scheme 1 and the experimental section. In the previous research, we have gotten some preliminary results that acylation of 4-amino-6,7dimethoxy-2-(piperazin-l-yl)quinazoline may be beneficial to improve the binding affinity of α1-AR antagonist.24 To further demonstrate this conclusion and investigate the influence of carbonyl group on

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α1-AR affinity, the acylated compounds 3a-c and non-acylated compounds 3d-f were designed for a comparison (Scheme 1). Additionally, in order to provide the ideal conjugated compound with optimal balance in pharmacologic and spectroscopic properties, the linkers of different length, two carbons to four carbons, were employed. Pharmacologic and spectroscopic properties. Receptor binding affinity. Affinity values were determined by competition binding assays on membrane proteins from CHO cells stabley expressing the human α1A -, α1B- or α1D-ARs, using [3H]prazosin as radioligand. As depicted in Table 1 and Figure S1, all compounds 3a-f exhibit high affinities to α1-ARs in the nanomolar range. In general, a fluorescent ligand designed by conjugating a large fluorophore to a prototype ligand almost displays decreased binding affinity at receptors.2 Here we found the similar results that the addition of Cy5 to the N4 position of 2-piperazine of prazosin decreases the affinity by 20-400 fold (Table 1). Nonetheless, these conjugates were almost equal to phentolamine in affinity to α1-AR, except for 3e and 3f. The acylated compounds 3a-c showed higher affinity at α1-AR than non-acylated compounds 3d-f, which well supports our previous viewpoint that acylation of 4-amino-6,7-dimethoxy-2-(piperazin-l-yl)quinazoline will be conducive to increase affinity of antagonists.24 In the acylated compounds, compound 3b with the spacer comprised of three carbon atoms showed lowest affinity, while the other two compounds 3a and 3c, which have a linker of two or four carbon atoms, respectively, displayed higher and similar affinity to α1-AR. The non-acylated compounds were identified in the binding affinity at α1-AR with the rank order of 3d > 3e > 3f, which demonstrates that the non-acylated compound with a shorter carbon chain as the spacer will possess higher receptor affinity. Cytotoxicity. The cytotoxicity of 3a-f using a 48 h MTT cytotoxicity assay and doxazosin as a comparator was investigated to confirm they were be less toxic and less harmful to cells and animals. Four types of cells were chosen, including normal HEK293A cells, α1A- and α1D-AR transfected HEK293A cells, and PC-3 cells. As a result (Table S1), all these conjugates just showed a low,

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micromolar cytotoxicity range, which was similar to doxazosin. This indicated that there was no significant increase in cytotoxicity after conjugating Cy5 to the quinazoline core. 3a-c with higher affinity were less toxic than 3b-f, and among all of them, 3a gave the minimal toxicity. Spectra. To test the environment-sensitive property, the fluorescence spectra and quantum yields of compounds were measured firstly. In Table 1, spectral analysis showed the absorption and emission spectra of conjugates were located in the near infrared region (λex = 640nm, λem = 665nm). Although the quantum yields in DMSO and PBS were low and similar, the influence of solvents on absorption and fluorescence spectra was obvious (Figure S2). Comparing with organic solvents, the aqueous PBS solution quenched the fluorescence gravely. When the polarity of solvent decreases, the absorbance and fluorescence intensity increases dramatically. For example, a ~7-fold increase of fluorescence from PBS to DMSO was found on 3a (Figure 2A and 2B). Additionally, we investigated the fluorescence lifetime of 3a in glycerol-water solutions. The fluorescence lifetime increases from 0.56 ns to 1.88 ns with an increasing glycerol volume fraction from 0% to 100% (viscosity increase), and meanwhile the fluorescence intensity improved correspondingly (Figure 2C). The lifetime displayed non-correlation with the concentration of 3a (Figure 2D). These results revealed that these fluorescent conjugates could serve as environment sensors because their fluorescence intensities were dependant on the polarity and viscosity of surrounding environment. Specific interaction of 3a with human α1-AR. With the highest receptor affinity, strongest sensitivity to the surrounding solvents and lowest cytotoxicity, 3a stood out from the six conjugates. To confirm whether its fluorescence switch could be specially recognized and turned on by human α1-ARs, 3a (20 nM) was incubated with various proteins for 2 h on 96-well plates and the fluorescence intensity was recorded (Figure 3A and 3B). While the fluorophore compound 2 was unselective to all proteins, three membrane proteins of α1-ARs subtypes led 3a to emit about 10-time stronger fluorescence than 10-fold excess of BSA, α-chymotrypsin and pepsin. The nonspecific fluorescence was determined in presence

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of a 100-fold excess of doxazosin. Obvious decrease of fluorescence intensity was observed when doxazosin was added (Figure 3C). Binding of 3a to entire cells expressing α1A- and α1D-AR stably was measured as well. Similar results were given in Figure 3D. The 2 h co-incubation of 3a with transfected HEK293 cells produced strong fluorescence, which could be quenched largely by excessive doxazosin as well. In situ visualizing human α1-ARs on cells. Transfected HEK293 cells. To visualize the specific binding of 3a to α1-ARs on transfected living cells in situ, the imaging study of 3a on α1-ARs transfected HEK293A cells were conducted. It was worth mentioning that HEK293 cells transiently transfected with α1B-AR/GFP was also established. After 10-min incubation at 37 °C, the cells were tested immediately without washing. In Figure 3E, the α1A-, α1B- and α1D-AR transfected HEK293A cells were “highlighted” clearly by 3a, which could be prevented by a 10-fold excess of doxazosin while the un-transfected HEK293A cells showed only slight fluorescence. Through this approach, the awareness about subcellular localization of three α1-AR subtypes in HEK293A cells was readily acquired. The fluorescence signals existed in both cytomembrane and cytoplasm of α1A-AR cells, while the α1D-AR transfected cells were mainly stained intracellularly. In the α1B-AR/GFP cells, the red fluorescence (3a) overlapped with GFP (green), which was observed mainly on the cell surface. These results effectually supports our previous study24 and is highly consistent with Piascik’s conclusion33. Cancer cell imaging. To investigate if 3a can specifically bind to α1-ARs on cancer cells, the fluorescent imaging was further evaluated by using PC-3, DU145, HepG2, ES-2, Hela, MCF-7 and A549 cells. Figure 4A shows the labeling results of 3a (300 nM) in these cells. Compared with the feeble fluorescence in HepG2, ES-2, Hela, MCF-7 and A549 cells, bright labeling can be detected intracellularly in prostate cancer PC-3 and DU145 cells, and the intracellular binding in both cells can be markedly inhibited by doxazosin. The cell imaging with compound 2 in HEK293, PC-3, and ES-2 cells were also performed, and the whole cells were stained without selectivity (Figure S4). Thus,

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through 3a, we could draw a conclusion that α1-ARs are highly expressed in prostate cancer PC-3 and DU145 cells, and most of them are distributed in the cytoplasm. Using radioligand and EGFP-tagged α1AR, Shi et. al reached the same conclusion that the intracellular abundance of α1-ARs is determined in PC-3 and DU145 cells.18 The authors also found that the prolonged pre-incubation with an antagonist of α1-AR would provoke α1-AR/EGFP to be translocated to cell surface. As a fluorescent antagonist, 3a could stimulate and trace this activity simultaneously by itself. As shown in Figure 4B, fluorescence in both PC-3 and DU145 cells was congregated at 1h. After stimulation with 3a for 24 h and 48 h, the fluorescence region became annular, and especially, some cell-surface fluorescence was detected in DU145 cells, which signified the cytomembrane-oriented movement was happening. Flow cytometry analysis. Subsequently, the binding of 3a to the living cells was analyzed by flow cytometry (FCM). The total binding of 3a to the α1A-AR cell was significantly higher than the nonspecific binding to α1A-AR cells treated by 3a and doxazosin and untransfected cells treated by 3a (Figure 4C). Besides, after the same incubation with 3a, the peak of PC-3 was higher than that of the Hela cells, which meant PC-3 cells expressed more α1-ARs than Hela cells (Figure 4C). These results from FCM are in accordance with the conclusion above. In vivo optical imaging of α1-ARs. Tumor xenograft mouse model. To study the ability of 3a in α1AR-targeted imaging in vivo, the subcutaneous PC-3, ES-2 and Hela xenograft mouse models were established firstly. The nude mice with tumors were dosed by a tail intravenous injection of 3a (50 µM). 12 h later, when the mice and dissected tumors were imaged in a small animal living imaging system, strong fluorescence in PC-3 tumors, slight fluorescence in Hela tumors and little fluorescence in ES-2 tumors were detected (Figure 5). Within the tumor xenograft model and quantitative analysis, it was confirmed that 3a could be applied to label selectively tumors expressing α1-ARs and could distinguish expression level of α1-ARs in different tumors according to fluorescence intensity. Here α1-ARs were identified in the three kinds of tumors with a rank order of PC-3 > Hela > ES-2.

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Distribution of α1-ARs in mouse tissues. The results in transplanted tumor experiments above clearly indicated that the in vivo visualization of α1-ARs was available using probe 3a. Therewith, the tissuespecific distribution of α1-ARs was studied by living animal imaging on male and female nude mice treated by 3a. As shown in Figure 6A, 3a mainly “lighted up” the neck, abdomen and dorsal kidney of mice. To further study the tissue distribution of 3a, we dissected the mice and acquired fluorescence images of their internal organs (Figure 6B and S5). For female mice, fluorescence signals were visible in the liver, kidney, salivary glands, lung, and heart, and for the male, fluorescence was found in the same parts plus the bladder and prostate. In order to verify whether the fluorescence signal is localized on prostate or bladder in the male, the ex vivo imaging of separated prostate and bladder was supplemented. As shown in Figure S7, the prostate displays much brighter fluorescence than bladder, which indicates that the prostate tissue could be stained by 3a obviously. An essential difference between genders is the prostate, in which α1-AR is abundant. The male has a prostate and this region is colored, while the female has no prostate and no dyeing, which further indicates 3a could visualize α1AR specifically. No fluorescence was detected in the brain perhaps because of impermeability of 3a to the blood brain barrier. As a control, the fluorophore 2 was employed in the animal imaging as well. In Figure 6A, very strong fluorescence signals were found in the whole bodies of mice treated with 5. We also dissected the mice and found that the fluorescence signals were visible in most tissues and their distribution was associated with blood flow (Figure 6B and S5), which demonstrated that the fluorophore 2 has no selectivity to different tissues. It is undeniable that any fluorophores injected will stay in the organs such as liver and kidneys for a certain period of time, so the fluorescence signals in organs may not be associated with the existence of α1-AR. To make it clear whether fluorescence signals of 3a could reflect the existence of α1-AR, the competitive binding experiments using an excess of prazosin were carried out on mice. As shown in Figure 6C, the tissues that treated by compound 3a together with prazosin, such as liver, kidneys, salivary glands, lung and prostate, showed subdued fluorescence compared to these organs treated by 3a only (Figure 6C). In quantitative analysis (Figure

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S6), the large decrease was found in liver, kidney, and salivary glands, the moderate in lung and prostate, and the slight in heart. Therefore, according the results above, we could draw a conclusion that α1-ARs in mice are distributed primarily in the liver, kidney, and salivary glands, moderately in lung and prostate, slightly in heart, little in the spleen. This is roughly identical to the Michel’s conclusion from [3H]prazosin binding studies to murine tissues homogenates.34 Here, 3a provides a simple and efficient method to detect the tissue-specific distribution of α1-AR in vivo to displace the reverse transcriptionpolymerase chain reaction (RT-PCR), northern blotting and radioligand binding, which consumed huge amounts of time and resources or required high-security protection. Imaging of prostate sections. To analyze the expression of α1-AR in human prostate, 3a was applied in the imaging on human prostate sections. Upon a 24 h-incubation with 300 nM 3a, human prostate cancer (Figure 7C) and BPH sections (Figure 7B) showed stronger fluorescence than normal prostate slices (Figure 7A and 7A’). In contrast to the transmission image, fluorescence image of the normal tissue revealed localization of fluorescence on glandular epithelium (G1, Figure 7A) and stromal smooth muscle (G2, Figure 7A) in prostate tissues. Some smooth muscle tissues with weak fluorescence were observed as well (G’, Figure 7A’). Additionally, though incubated for 30 min only, the low-level cancer tissues could be dyed and showed a striking contrast to the surrounding tissues (White box, Figure 7D). In histochemical experiments, comparing to normal tissues, the stronger fluorescence in BPH and lowor high-level prostate cancer indicated there was a significant increase for α1-AR expression when cancer or hyperplasia happened in prostate. For normal prostate tissues, smooth muscle and glandular epithelium had strong fluorescent signal, showing α1-AR was mainly localized on these regions. It keeps into correspondence with the research reported, in which by immunohistochemistry and radioligand binding assay, α1A- and α1D-ARs were detected in the stroma and α1B-AR was localized predominantly in the epithelium.35, 36 It was very positive that we found not all of smooth muscle displayed strong fluorescence. It may be caused by another subtype α1L-AR, which distributes in prostatic smooth muscle and showed low affnity to normal α1-AR antagonist as prazosin.17, 37 ACS Paragon Plus Environment

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CONCLUSION In conclusion, based on the environment-sensitive Cy5 fluorophore, we have created a series of coveted “off-on” NIR ligands for α1-ARs. By conjugation with quinazoline, this fluorescence property of Cy5 was caged, which released strong fluorescence when bound to α1-ARs. Among these fluorescent ligands, 3a exhibited potent binding and remarkable changes in fluorescence (10-fold) upon binding to α1-AR. This compound 3a can be further employed as a powerful fluorogenic tool to selectively and specifically interrogate α1-ARs in vitro and in vivo, for example, it can “off-on” label α1-AR in single cells, prostate tissue slices, intact tumor xenografts and organs in living mice. This fluorescent ligand also gave us direct and visual evidences that there is a very close relationship between α1-ARs and pathological prostate. It’s anticipated that such a Cy5-caged strategy could be similarly extended to other targets to provide novel method to portray GPCRs.

EXPERIMENTAL SECTION General materials and instruments. General chemicals used in the synthesis were purchased from J&K, Accela, and Aladdin. All solvents were used after appropriate distillation or purification. The twice-distilled water was used throughout all chemical experiments. Buffer reagents purchased from Aldrich and Acros were used without purification. Water used in activity studies was doubly distilled and further purified with a Mill-Q filtration system. HEK293 A, PC-3, DU145, HepG2, ES-2, Hela, MCF-7 and A549 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). α1A and α1D-AR stably transfected HEK293A cells were given by Professor Youyi Zhang from Peking University. BSA, α-chymotrypsin and papain were supplied by Sigma. The prostate tissue sections, including normal tissue sections, BHP, and prostate cancer pathological sections were provided by the Department of Urology and the Department of Pathology, Qilu Hospital of Shandong University.

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Melting points were determined on an electrothermal melting point apparatus and were uncorrected. 1

H NMR and 13C NMR were recorded on a Bruker 300 or 400 MHz NMR spectrometer. Mass spectra

were recorded in ESI mode and performed by the analytical and the mass spectrometry facilities at Shandong University. HRMS spectra were performed in the Shandong Analysis and Test Center. The purity of all final compounds (>95%) was determined by RP-HPLC analysis. Analytical HPLC was carried out on Agilent Technologies 1260 Series high-performance liquid chromatography using a C8 reversed-phase column (5 micron, Phenomenex). UV and fluorescence spectra were obtained with Thermo Varioskan microplate reader. Quantum yields were measured using Shimadzu UV-2401PC UVvisible spectrometer, Hitachi F-2500 Fluorescent spectrometer and WAY-2S Abbe refractometer. The excited-state lifetime was measured on a Horiba Fluorolog-3 spectrofluorometer. Fluorescence intensity of proteins and cells treated by 3a and 2 was determined with a BMG Omega microplate reader. Fluorescence images of cells (except for α1B-eGFP HEK293 cells) and tissue sections were performed on a Zeiss Axio Observer A1. Fluorescence images of α1B-EGFP HEK293 cells were recorded on Zeiss LSM700. Fluorescence imaging of living animals were recorded on an IVIS Kinetic (Caliper Life Sciences, USA) equipped with a cooled CCD camera. 1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-azidoethanone (1a). To a solution of compound 10a (1.0 g, 2.73 mmol) in 10 mL DMSO, sodium azide (0.89 g, 13.65 mmol) was added carefully. The mixture was stirred at 100 °C for 6 h, and then allowed to cool to room temperature. The solution was diluted with 200 mL dichloromethane, washed with saturated sodium bicarbonate solution, water, and brine. After dried by MgSO4, the crude product was purified by silica gel column chromatography with dichloromethane and methanol to afford white powder as desired product 1a. Yield: 0.66 g, 65%. M.p. 231-233 °C. 1HNMR (400MHz, DMSO-d6): δ 7.42 (s, 1 H), 7.15 (s, 2 H), 6.74 (s, 1 H), 4.20 (s, 2 H), 3.83 (s, 3 H), 3.79 (s, 3 H), 3.74 (m, 4 H), 3.53 (t, 2 H), 3.37 (t, 2 H); 13CNMR (100 MHz, DMSO-d6): δ 166.5, 161.6, 158.7, 154.7, 149.2, 145.5, 105.7, 104.1, 103.4, 56.3, 55.9, 50.2, 44.6, 44.0, 43.8, 42.0; ESI-MS: m/z [M+H]+ calcd for C16H21N8O3+ 373.1731, found 373.1723. ACS Paragon Plus Environment

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1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-3-azidopropan-1-one (1b). Compound 1b was synthesized as described for 1a, except for the use of compound 10b. Yield: 440 mg, 35%. M.p. 192-195 °C. 1HNMR (400 MHz, DMSO-d6): δ 7.43 (s, 1 H), 7.16 (s, 2 H), 6.75 (s, 1 H), 3.83 (s, 3 H), 3.79 (s, 3 H), 3.75 (t, 2 H, J = 5.2 Hz), 3.71 (t, 2 H, J = 5.6 Hz), 3.56 (m, 6 H), 2.70 (t, 2 H, J = 6.4 Hz); 13

CNMR (100 MHz, DMSO-d6): δ 168.9, 161.6, 158.7, 154.7, 149.2, 145.5, 105.6, 104.2, 103.4, 56.3,

55.9, 47.2, 45.2, 44.2, 43.8, 41.6, 32.3; ESI-MS: m/z [M+H]+ calcd for C17H23N8O3+ 387.1888, found 387.1893. 1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-4-azidobutan-1-one (1c). Compound 1c was synthesized as described for 1a, except for the use of compound 10c. Yield: 1.1 g, 88%. M.p. 199-202 °C. 1HNMR (400MHz, DMSO-d6): δ 7.43 (s, 1 H), 7.16 (s, 2 H), 6.75 (s, 1 H), 3.83 (s, 3 H), 3.79 (s, 3 H), 3.74 (t, 2 H, J = 4.8 Hz), 3.69 (t, 2 H, J = 5.2 Hz), 3.50 (m, 4 H), 3.39 (t, 2 H, J = 6.8 Hz), 2.46 (t, 2 H, J=7.2 Hz), 1.82-1.75 (m, 2 H);

13

CNMR (100 MHz, DMSO-d6): δ 170.2, 161.2, 158.7,

154.7, 149.5, 145.5, 105.6, 104.2, 103.4, 56.3, 55.9, 50.8, 45.3, 44.3, 43.9, 41.6, 29.7, 24.6; ESI-MS: m/z [M+H]+ calcd for C18H25N8O3+ 401.2044, found 401.2045. 2-(4-(2-Azidoethyl)piperazin-1-yl)-6,7-dimethoxyquinazolin-4-amine (1d). To the solution of compound 7 (1 g, 3.5 mmol) and 13a (920 mg, 3.8 mmol) in 60mL MeCN was added potassium carbonate (1.43 g, 10.5 mmol). The reaction mixture was refluxed at 90°C for 24h, and filtered immediately. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with dichloromethane and methane to afford white powder as desired product 1d. Yield: 0.86g, 69%. M.p. 119-121°C. 1HNMR (300 MHz, CDCl3): δ 6.94 (s, 1H), 6.80 (s, 1H), 5.13 (s, 2 H), 3.97 (s, 3 H) 3.93 (s, 3 H), 3.89 (t, 4 H, J = 4.8 Hz), 3.42 (t, 2 H, J = 6.0 Hz), 2.66 (t, 2 H, J = 6.0 Hz), 2.59 (t, 4 H, J = 5.1 Hz);

13

CNMR (100 MHz, DMSO-d6): δ 166.5, 158.8, 154.7,

149.0, 145.4, 105.5, 104.2, 103.3, 57.3, 56.3, 55.5, 47.6, 44.1, 44.0; ESI-MS: m/z [M+H]+ calcd for C16H23N8O2+ 359.2, found 359.6.

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Journal of Medicinal Chemistry

2-(4-(3-Azidopropyl)piperazin-1-yl)-6,7-dimethoxyquinazolin-4-amine (1e). Compound 1e

was

synthesized as described for 1d, except for the use of 13b (893 mg, 3.5 mmol). Yield: 0.8 g, 68%. mp. 117-120 °C. 1HNMR (300 MHz, CDCl3): δ 7.08 (s, 1 H), 6.98 (s, 1 H), 5.12 (s, 2 H), 3.96 (s, 3 H) 3.95 (s, 3 H), 3.88 (t, 4 H, J = 3.9 Hz), 3.40 (t, 2 H, J = 6.9 Hz), 2.53-2.44 (m, 6 H), 1.86-1.76 (m, 2 H); 13

CNMR (100 MHz, DMSO-d6): δ 161.5, 158.9, 154.7, 149.2, 145.4, 105.6, 104.2, 103.3, 56.3, 55.8,

55.3, 53.3, 49.4, 44.0, 26.1; ESI-MS: m/z [M+H]+ calcd for C17H25N8O2+ 373.2, found 373.4. 2-(4-(4-Azidobutyl)piperazin-1-yl)-6,7-dimethoxyquinazolin-4-amine (1f). Compound 1f

was

synthesized as described for 1d, except for the use of 13c (1.2 g, 4.2 mmol). Yield: 0.85g, 64%. M.p. 140-141 °C. 1HNMR (300MHz, CDCl3): δ 6.93 (s, 1 H), 6.79 (s, 1 H), 5.10 (s, 2 H), 3.97 (s, 3 H) 3.93 (s, 3 H), 3.88 (t, 4 H, J=4.8 Hz), 3.34 (t, 4 H, J = 6.0 Hz), 2.53 (t, 4 H, J = 5.1 Hz), 2.43 (t, 2H, J=6.9 Hz) 1.66-1.62 (m, 2 H);

13

CNMR (100 MHz, DMSO-d6): δ 161.5, 160.0, 154.6, 149.3, 145.3,

105.7, 104.2, 103.3, 57.8, 56.3, 55.8, 53.4, 51.1, 44.1, 26.8, 23.9; ESI-MS: m/z [M+H]+ calcd for C18H27N8O2+ 387.2, found 387.5. 3,3-Dimethyl-1-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (2). To the solution of compound 7 (427 mg, 0.82 mmol), 2-Propynylamine (158 µL, 2.46 mmol), DMAP (150 mg, 1.23 mmol) in 20 mL dichloromethane, EDCI (173 mg, 0.9 mmol) were added slowly. The mixture was stirred at room temperature for 6 hours. The organic solution was diluted with 100 mL dichloromethane, and washed with saturated citric acid solution, saturated sodium bicarbonate solution, water, and brine. After dried by MgSO4, the solvent was removed under reduced pressure to afford the blue powder as desired product 2. Yield: 243 mg, 53%. Decomposition point: 175-176 °C. 1HNMR (400 MHz, DMSO-d6): δ 8.53 (t, 1H, J=5.2Hz), 8.39-8.27 (q, 2H, J = 12.8 Hz), 7.64 (d, 1 H, J = 7.2 Hz) 7.61 (d, 1 H, J = 7.6 Hz), 7.43-7.20 (m, 6 H), 6.58 (t, 1 H, J = 12.4 Hz), 6.36 (d, 1 H, J = 14.0 Hz), 6.22 (d, 1 H, J = 13.6 Hz), 4.32 (t, 2 H, J = 6.0 Hz) 3.81 (dd, 2 H, J2 = 5.2 Hz, J2 = 2.4 Hz) 3.63 (s, 3 H), 3.11 (t, 2 H, J=2.4 Hz)

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2.60 (t, 2 H, J=6.4 Hz), 1.67 (s, 6 H), 1.66 (s, 6 H).

13

Page 16 of 41

CNMR (100 MHz, DMSO-d6): δ 174.2, 172.5,

169.5, 154.8, 154.2, 143.2, 142.3, 141.6, 141.4, 128.9, 128.8, 126.0 125.4, 124.9, 122.8, 111.7, 111.4, 104.4, 103.4, 81.2, 73.8, 49.5, 49.2, 40.4 (covered with DMSO) 33.3, 31.7, 28.5, 27.7, 27.4.ESI-MS: m/z [M-I-] calcd for C32H36N3O+ 478.3, found 478.5. Conjugation of azido and acetylenic compounds (3a-f). To the solution of alkyne 2 (22.0 mg, 0.05 mmol) and azides 1 (0.05 mmol) in 3.9 mL t-BuOH: H2O (2:1), 0.1 M sodium ascorbate (1.8 mL) and 0.1 M CuSO4 (0.5 mL) were added. The reaction mixture was warmed to 50°C for 1 h in the dark, cooled to room temperature and the solvent was removed by rotary evaporation. To the crude reaction mixture, 2M NH4OH (5 mL) was added and extracted with DCM (3 x 25 mL). The organic layer was dried with MgSO4 and evaporated, and the residue was purified by silica gel column chromatography with dichloromethane and methane to afford the corresponding 1,4-triazole oxime. 1-(3-(((1-(2-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-oxoethyl)-1H-1,2,3triazol-4-yl)methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2ylidene) penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (3a). Compound 1a (20.4 mg,0.05 mmol) was used to synthesize 3a by affording the blue solid. Yield: 23.9 mg, 48.9%. 1HNMR (300MHz, CD3OD): δ 8.30-8.18 (m, 2 H), 7.68 (s, 1H), 7.50-7.20 (m, 9 H), 6.93 (s, 1 H), 6.50 (t, 1 H, J = 12.3 Hz), 6.356.25 (m, 2 H), 5.46 (s, 2 H), 4.40-4.34 (m, 4 H), 3.92-3.83 (m, 10 H), 3.69 (t, 4 H, J = 3.3 Hz), 2.72 (t, 2 H, J = 6.9 Hz), 1.72 (s, 6 H), 1.68 (s, 6 H); ESI-HRMS: m/z [M-I-] calcd for C48H56N11O4+ 850.4511, found 850.4512. HPLC purity 99.1%, tR = 10.459 min, 250 × 10.00 mm, CH3OH (0.05% triethylamine): H2O=95:5, 2 mL/min. 1-(3-(((1-(3-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-3-oxopropyl)-1H-1,2,3-triaz ol-4-yl)methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-yliden e)penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (3b). Compound 1b (21.1 mg,0.05 mmol) was used to synthesize 3b by affording the blue solid. Yield: 33.4 mg, 67.3%. 1HNMR (300MHz, CD3OD): δ 8.22ACS Paragon Plus Environment

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Journal of Medicinal Chemistry

8.07 (m, 2 H), 7.63 (s, 1H), 7.43-7.12 (m, 9 H), 6.83 (s, 1 H), 6.53 (t, 1 H, J = 12.3 Hz), 6.27 (d, 1 H, J = 14.1 Hz), 6.16 (d, 1 H, J = 12.3 Hz), 4.60 (t, 2 H, J = 6.3 Hz), 4.30-4.22 (m, 4 H), 3.82 (s, 3 H), 3.79 (s, 3 H), 3.69 (t, 4 H, J = 5.4 Hz), 3.57 (m, 5 H), 3.48 (t, 2 H, J = 5.4 Hz), 3.03 (t, 2 H, J = 6.3 Hz), 2.61 (t, 2 H, J = 6.9 Hz), 1.64 (s, 6 H), 1.57 (s, 6 H); ESI-HRMS: m/z [M-I-] calcd for C49H58N11O4+ 864.4668, found 864.4656. HPLC purity 99.9%, tR = 10.664 min, 250 × 10.00 mm, CH3OH (0.05% triethylamine): H2O=95:5, 2 mL/min. 1-(3-(((1-(4-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-4-oxobutyl)-1H-1,2,3triazol -4-yl)methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2ylidene) penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (3c). Compound 1c (21.8 mg,0.05 mmol) was used to synthesize 3c by affording the blue solid. Yield: 30.4 mg, 60.4%. 1HNMR (300MHz, CD3OD): δ 8.20-8.09 (m, 2 H), 7.57 (s, 1H), 7.43-7.13 (m, 9 H), 6.91 (s, 1 H), 6.54 (t, 1 H, J = 9.3 Hz), 6.26 (d, 1 H, J = 10.5 Hz), 6.19 (d, 1 H, J = 10.2 Hz), 4.35-4.28 (m, 6 H), 3.85 (s, 3 H), 3.81 (s, 3 H), 3.79 (t, 2 H, J = 3.6 Hz), 3.75 (t, 2 H, J = 3.3 Hz), 3.60 (t, 2 H, J = 4.5 Hz), 3.57 (s, 3H), 3.52 (t, 2 H, J = 3.9 Hz), 2.65 (t, 2 H, J = 5.1 Hz), 2.39 (t, 2 H, J = 5.1 Hz), 2.12 (m, 2 H), 1.64 (s, 6 H), 1.58 (s, 6 H); ESIHRMS: m/z [M-I-] calcd for C50H60N11O4+ 878.4824, found 878.4823. HPLC purity 99.1%, tR = 9.733 min, 250 × 10.00 mm, CH3OH (0.05% triethylamine): H2O=95:5, 2 mL/min. 1-(3-(((1-(2-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl) methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta -1,3-dien-1-yl)-3H-indol-1-ium iodide (3d). Compound 1a (17.9 mg , 0.05 mmol) was used to synthesize 3d by affording the blue solid. Yield: 26.8 mg, 55.6%. 1HNMR (400MHz, CD3OD): δ 8.208.10 (m, 2 H), 7.71 (s, 1H), 7.42-7.15 (m, 9 H), 6.91 (s, 1 H), 6.55 (t, 1 H, J = 12.4 Hz), 6.25 (d, 1 H, J = 13.6 Hz), 6.20 (d, 1 H, J = 13.6 Hz), 4.45 (t, 2 H, J = 6.0 Hz), 4.29 (m, 4 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.72 (t, 4 H, J = 4.4 Hz), 3.56 (s, 3 H), 2.80 (t, 2 H, J = 6.0 Hz), 2.65 (t, 2 H, J = 6.8 Hz), 2.52 (t, 2 H, J = 4.8 Hz), 1.64 (s, 6 H), 1.59 (s, 6 H); ESI-HRMS: m/z [M-I-] calcd for C48H58N11O3+ 836.4719, found

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Page 18 of 41

836.4714. HPLC purity 100.0%, tR = 13.981 min, 250 × 4.60 mm, CH3OH (0.05% triethylamine): H2O=9:1, 1 mL/min. 1-(3-(((1-(3-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)propyl)-1H-1,2,3-triazol-4yl)methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2ylidene)penta -1,3-dien-1-yl)-3H-indol-1-ium iodide (3e). Compound 1b (18.6 mg,0.05 mmol) was used to synthesize 3e by affording the blue solid. Yield: 27.0 mg, 55.2%. 1HNMR (400MHz, CD3OD): δ 8.20-8.10 (m, 2 H), 7.61 (s, 1H), 7.43-7.13 (m, 9 H), 6.91 (s, 1 H), 6.55 (t, 1 H, J = 12.4 Hz), 6.26 (d, 1 H, J = 14.0 Hz), 6.19 (d, 1 H, J = 13.6 Hz), 4.37 (t, 2 H, J = 6.8 Hz), 4.30-4.27 (m, 4 H), 3.85 (s, 3 H), 3.81 (s, 3 H), 3.74 (t, 4 H, J = 4.8 Hz), 3.56 (s, 3 H), 2.64 (t, 2 H, J = 6.8 Hz), 2.45 (t, 4 H, J = 7.2 Hz), 2.33 (t, 2 H, J = 6.8 Hz), 2.05-1.98 (m, 2 H), 1.64 (s, 6 H), 1.59 (s, 6 H); ESI-HRMS: m/z [M-I-] calcd for C48H58N11O3+ 836.4719, found 836.4714. ESI-HRMS: m/z [M-I-] calcd for C49H60N11O3+ 850.4875, found 850.4873. HPLC purity 96.2%, tR = 14.110 min, 250 × 4.60 mm, CH3OH (0.05% triethylamine): H2O=9:1, 1 mL/min. 1-(3-(((1-(4-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)butyl)-1H-1,2,3-triazol-4-yl) methyl)amino)-3-oxopropyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta -1,3-dien-1-yl)-3H-indol-1-ium iodide (3f). Compound 1c (19.3 mg , 0.05 mmol) was used to synthesize 3f by affording the blue solid. Yield: 36.0 mg, 72.5%. 1HNMR (400MHz, CD3OD): δ 8.218.10 (m, 2 H), 7.60 (s, 1H), 7.41-7.13 (m, 9 H), 6.90 (s, 1 H), 6.56 (t, 1 H, J = 12.4 Hz), 6.26 (d, 1 H, J = 14.0 Hz), 6.21 (d, 1 H, J = 13.6 Hz), 4.32-4.29 (m, 6 H), 3.85 (s, 3 H), 3.81 (s, 3 H), 3.74 (t, 4 H, J = 6.4 Hz), 3.57 (s, 3 H), 2.66 (t, 2 H, J = 6.8 Hz), 2.49 (t, 4 H, J = 4.4 Hz), 2.40 (t, 2 H, J = 7.2 Hz), 1.88-1.80 (m, 2 H), 1.64 (s, 6 H), 1.60 (s, 6 H), 1.51-1.48 (m, 2 H); ESI-HRMS: m/z [M-I-] calcd for C50H62N11O3+ 864.5032, found 864.5028. HPLC purity 95.4%, tR = 14.251 min, 250 × 4.60 mm, CH3OH (0.05% triethylamine): H2O=9:1, 1 mL/min.

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Journal of Medicinal Chemistry

1-(2-Carboxyethyl)-2,3,3-trimethyl-3H-indol-1-ium iodide (5). The mixture of 2,3,3-trimethyl-3Hindole (1.0 g, 6.23 mmol) and 3-iodopropionic acid (1.86 g, 9.3 mmol) dissolved in acetonitrile (15 mL) was refluxed for 48 hours, then allowed to cool to room temperature. The solvent was removed under reduced pressure, and 100 mL ethyl acetate was added slowly while stirring or ultrasonically shaking. The precipitation was collected by filtration and washed with ethyl acetate to afford the white powder as desired product 5. Yield: 1.8 g, 81%. M.p.181-183 °C. 1HNMR (400 MHz, DMSO-d6): δ 12.72 (s, 1 H), 8.01-7.98 (m, 1 H), 7.85-7.83 (m, 1 H), 7.62-7.60 (m, 2H), 4.67 (t, 2H, J = 7.0 Hz), 3.00 (t, 2H, J = 7.0 Hz), 2.86 (s, 3 H), 1.53 (s, 6 H). 13CNMR (100 MHz, DMSO-d6): δ 198.4, 172.0, 142.2, 141.3, 129.8, 129.4, 124.0, 116.0, 54.7, 44.0, 31.6, 22.4, 14.9. ESI-MS: m/z [M-I-] calcd for C14H18N2O2+ 232.1, found 232.4. 1,2,3,3-Tetramethyl-3H-indol-1-ium iodide (6). The mixture of 2,3,3-trimethyl-3H-indole (2.0 g, 12.5 mmol) and methyl iodide (2.3 g, 16.3 mmol) dissolved in acetonitrile (20 mL) was refluxed for 12 hours, then allowed to cool to room temperature. The precipitation was collected by filtration, and washed with acetonitrile to afford the white powder as desired product 6. Yield: 3.2g, 86%. Decomposition point: 244-245°C. 1HNMR (400 MHz, DMSO-d6): δ 7.93-7.91 (m, 1 H), 7.85-7.82 (m, 1 H), 7.65-7.60 (m, 2 H), 3.98 (s, 3 H), 2.78 (s, 3 H), 1.54 (s, 6 H). 13CNMR (100 MHz, DMSO-d6): δ 196.5, 142.6, 142.1, 129.8, 129.3, 123.8, 115.6, 54.4, 35.2, 22.2, 14.7. ESI-MS: m/z [M-I-] calcd for C12H14N+ 174.1, found 174.4. 1-(2-Carboxyethyl)-3,3-dimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3dien-1-yl)-3H-indol-1-ium iodide (7). Compound 5 (684.6 mg, 2.2 mmol) and malonaldehydedianilide hydrochloride (517.5 mg, 2 mmol) was dissolved in 4 mL AcOH/ 4 mL AC2O, and the mixture was refluxed under the argon atmosphere for 4 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 4 mL pyridine/4 mL AcOH. Compound 6 (602.5 mg, 2 mmol) was added to the solution, and the mixture was refluxed for 2 hours. The solvent was removed under reduced

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Page 20 of 41

pressure, and the crude product was purified by silica gel column chromatography with dichloromethane and methanol to afford blue powder as desired product 7. Yield: 341 mg, 30%. Decomposition point: 171-172 °C. 1HNMR (400 MHz, DMSO-d6): δ 8.35 (q, 2 H, J = 12.0 Hz), 7.61 (t, 2 H, J = 7.6 Hz), 7.43-7.33 (m, 4 H), 7.26-7.20 (m, 2 H), 6.57 (t, 1H, J = 12.4 Hz), 6.40 (d, 1H, J = 14.0 Hz), 6.22 (d, 1 H, J = 13.6 Hz), 4.26 (t, 2 H, J = 7.2 Hz) 3.56 (s, 3 H), 2.26 (t, 2 H, J = 7.2 Hz), 1.67 (s, 6 H), 1.66 (s, 6 H). ESI-MS: m/z [M-I-] calcd for C29H33N2O2+ 441.2, found 441.6. 6,7-Dimethoxy-2-(piperazin-1-yl)quinazolin-4-amine (9). The mixture of compound 8 (9.96 g, 41.56 mmol) and piperazine (35.8 g, 41.56 mmol) in 50 mL water was stirred at 100 °C for 6 h, and then allowed to cool to room temperature. After slow addition of 50 mL 1.7 N KOH, the mixture was stirred for 1 h at room temperature. The precipitation was filtered, washed with water and recrystallized from methanol to produce the white solid as desired product 9. Yield: 9.7 g, 81%. M.p. 219-220°C. 1HNMR (400 MHz, DMSO-d6): δ 7.40 (s, 1 H), 7.03 (s, 2 H), 6.70 (s, 1 H), 3.82 (s, 3 H), 3.78 (s, 3 H), 3.64 (t, 4 H, J=4.8 Hz), 2.71 (t, 4 H, J = 4.4 Hz); 13CNMR (100 MHz, DMSO-d6): δ 161.4, 159.1, 154.6, 149.4, 145.2, 105.6, 104.2, 103.2, 56.3, 55.8, 46.2, 45.3; ESI-MS: m/z [M+H]+ calcd for C14H20N5O2+ 290.2, found 290.4. 1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-2-chloroethanone (10a). Compound 9 (1 g, 3.46 mmol) and triethylamine (1 mL, 6.9 mmol) was heated to be dissolved in 100mL acetonitrile, and then stirred at 0 °C. 2-chloroacetyl chloride (286.3 µL, 3.8 mmol) was dropwise added into the above mixture. After stirred for 1 h 0 °C, the solvent was removed under reduced pressure. The residue was stirred in 5 mL methanol for 20 min, filtered and washed with methanol to afford the white solid as desired product 10a. Yield: 950 mg, 75%. Decomposition point: 218-219 °C. 1HNMR (400 MHz, DMSO-d6): δ 7.43 (s, 1 H), 7.16 (s, 2 H), 6.75 (s, 1 H), 4.43 (s, 2 H), 3.83 (s, 3 H), 3.79-3.72 (m, 7 H), 3.52 (t, 4 H, J = 2.7 Hz);

13

CNMR (100 MHz, DMSO-d6): δ 165.2, 161.6, 158.7, 154.7, 149.1, 145.6,

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Journal of Medicinal Chemistry

105.7, 104.2, 103.5, 56.3, 55.9, 49.1 (Methanol), 45.8, 44.2, 43.8, 42.4, 42.2; ESI-MS: m/z [M+H]+ calcd for C16H21ClN5O3+ 366.1327, found 366.1334. 1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-3-chloropropan-1-one

(10b).

Compound 10b was synthesized as described for 10a, except for the use of 3-chloropropionyl chloride. Yield: 920 mg, 70%. M.p. 212-215 °C.1HNMR (400MHz, DMSO-d6): δ 7.42 (s, 1 H), 7.15 (s, 2 H), 6.74 (s, 1 H), 3.83-3.78 (m, 8 H), 3.74 (t, 2 H, J = 4.4 Hz), 3.69 (t, 2 H, J = 5.6 Hz), 3.52-3.51 (m, 4 H), 2.92 (t, 2 H, J = 6.8Hz);

13

CNMR (100 MHz, DMSO-d6): δ 168.3, 161.6, 158.7, 154.7, 149.2, 145.5,

105.7, 104.2, 103.4, 56.3, 55.9, 45.3, 44.2, 43.9, 41.7, 41.1, 35.8; ESI-MS: m/z [M+H]+ calcd for C17H23ClN5O3+ 380.1, found 380.5. 1-(4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)-4-chlorobutan-1-one

(10c).

Compound 10c was synthesized as described for 10a, except for the use of 4-chlorobutyl chloride. Yield: 850 mg, 62%. M.p. 186-189 °C. 1HNMR (400 MHz, DMSO-d6): δ 7.42 (s, 1 H), 7.16 (s, 2 H), 6.74 (s, 1 H), 3.83 (s, 3 H), 3.78 (s, 3 H), 3.75 (t, 2 H, J = 4.0 Hz) 3.70 (t, 4 H, J = 6.4 Hz), 3.52 (t, 4 H, J = 6.4Hz), 2.51-2.50 (m, 2 H), 1.99 (m, 2 H); 13CNMR (100 MHz, DMSO-d6): δ 170.2, 161.2, 158.7, 154.7, 149.5, 145.5, 105.6, 104.2, 103.4, 56.3, 55.9, 45.5, 45.3, 44.2, 43.9, 41.6, 29.9, 28.5, ; ESI-MS: m/z [M+H]+ calcd for C18H25ClN5O3+ 394.2, 2-Azidoethanol (12a). To a solution of 2-bromoethanol (2 g, 16 mmol) in 40 mL H2O/ 10 mL CH3CN, NaN3 (3.12 g, 48 mmol) was added, and the solution was stirred vigorously at 80 °C for 20 h. The solution was cool to room temperature, then extracted with dichloromethane (3 × 50 mL). The combined organic layers were dried over anhydrous MgSO4, filtered, and the solvent was removed under reduced pressure to afford 12a as a clear oil. Yield: 0.7 g, 50%. 1HNMR (300 MHz, CDCl3): δ 3.80 (td, 2H, J1 = 4.8 Hz, J2 = 3.0 Hz), 3.27 (t, 2 H, J = 5.4 Hz), 1.96 (s, 1 H).

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3-Azidopropan-1-ol (12b). Compound 12b was synthesized as described for 12a, except for the use of 3-bromo-1-propanol. Yield: 1.2 g, 74%. 1HNMR (300 MHz, CDCl3): δ 3.77 (t, 2 H, J = 6.0 Hz), 3.48 (t, 2 H, J = 6.6 Hz), 1.91 (s, 1 H), 1.88-1.79 (m, 2 H). 4-Azidobutan-1-ol (12c). Compound 12c was synthesized as described for 12a, except for the use of 4chloro-1-butanol. Yield: 0.8 g, 43%. 1HNMR (300 MHz, CDCl3): δ 3.68 (td, 2 H, J1 = 6.0 Hz, J2 = 2.1 Hz), 3.48 (t, 3 H, J = 5.4 Hz), 1.75-1.59 (m, 4 H). 2-Azidoethyl methanesulfonate (13a). The solution of 12a (1.3 g, 15 mmol), TsCl (5.7 g, 30 mmol) and triethylamine (3 g, 30 mmol) in 20 mL dichloromethane was stirred at room temperature for 24 h. The solution was diluted with 200mL dichloromethane, washed with saturated sodium carbonate solution, water and brine. After dried by anhydrous MgSO4, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether and ethyl acetate to afford a yellow oil as desired product 13a. Yield: 2.3 g, 65%. 1HNMR (300 MHz, CDCl3): δ 7.83 (d, 2 H, J = 8.4 Hz), 7.38 (d, 2 H, J = 8.1 Hz), 4.18 (t, 2H, J = 5.1 Hz), 3.50 (t, 2 H, J = 5.1 Hz), 2.46 (s, 3 H). ESI-MS: m/z [M+NH4]+ calcd for C9H15N4O3S+ 259.1, found 259.3. 3-Azidopropyl methanesulfonate (13b). Compound 13b was synthesized as described for 13a, except for the use of 12b (1.12 g, 11 mmol). Yield: 2.5 g, 88%. 1HNMR (300MHz, CDCl3) δ 7.81 (d, 2 H, J = 8.1 Hz), 7.38 (d, 2 H, J = 8.1 Hz), 4.13 (t, 2 H, J = 6.0 Hz), 3.40 (t, 2 H, J = 6.6 Hz), 2.46 (s, 3 H), 1.931.85 (m, 2 H). ESI-MS: m/z [M+NH4]+ calcd for C10H17N4O3S+ 273.1, found 273.4. 4-Azidobutyl methanesulfonate (13c). Compound 12c was synthesized as described for 13a, except for the use of 12c (0.6 g, 5.2 mmol). Yield: 1.2 g, 86%. 1HNMR (300 MHz, CDCl3) δ 7.80 (d, 2 H, J = 8.4 Hz), 7.37 (d, 2 H, J = 8.1 Hz), 4.07 (t, 2 H, J = 6.0 Hz), 3.28 (t, 2 H, J = 6.6 Hz), 2.46 (s, 3 H), 1.821.57 (m, 4 H); ESI-MS: m/z [M+NH4]+ calcd for C11H19N4O3S+ 287.1, found 287.3.

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Measurement of photophysical properties. The absorption and fluorescence spectra (Ex=600nm) of six compounds and 5 (30 µM) were measured in water, methanol, ethanol, acetonitrile, DMSO, PBS solution with Thermo Varioskan microplate reader. The influence of the polarity of the medium on the fluorescence of 3a and 2 was investigated by addition of DMSO (indicated in % v/v) in HEPES buffer (50 mM, pH 7.4) measured at 600 nm. The excited-state lifetime of 3a in water-glycerol with different percentages (0, 25%, 50%, 75%, 100%), or at a concentration range from 0.03~30 µM in water, were measured on a Horiba Fluorolog-3 spectrofluorometer with a NanoLED-590nm as the light source. For measurement of fluorescent quantum yields of compounds in different kinds of solution, fluorescence, absorption and refractivity measurements were carried out at room temperature with same instrument parameters through Shimadzu UV-2401PC UV-visible spectrometer, Hitachi F-2500 Fluorescent spectrometer and WAY-2S Abbe refractometer, respectively. The relative quantum yield for each sample was calculated according the equation below. Φx = Φs (As /Ax) (FAx /FAs) (ηx /ηs)2 Where Φ is the quantum yield, A is the absorbance at the excitation wavelength, FA is the area under the corrected emission curve, n is the refractive index of the solvent. X refers to the sample, and S refers to the standard. Fluorescein was chosen as the standard, which has the fluorescence quantum yield of 0.92 in 1 M NaOH solution. Assays to determine binding affinities. Membrane preparation: The membrane proteins were provided by GenScript USA Inc (Nanjing, China). Adrenergic receptor (AdrA1A, AdrA1B and AdrA1D) overexpressing CHO-K1 cells were thawed and grown in F-12 medium supplemented with 10% FBS, 400 µg/ml geneticin and 1% penicillin-streptomycin at 37°C/5% CO2. Cell pellets were scrapped off the dish, washed with PBS and centrifuged at 200 g for 10 min at 4 °C. The pellet was resuspended in 20 ml ice-cold Buffer I (10 mM HEPES +10 mM EDTA, pH7.4) and homogenized for 50-60 sec followed by centrifugation at 39000 g for 45 min at 4 °C. After resuspended and centrifuged again as above, the

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resulting pellet was resuspended in 5 ml ice-cold storage buffer and stored at -80 °C. Membrane protein concentration was determined by Bradford protein assay (Coomassie Plus-The better Bradford Assaytm kit, Prod#23236, Thermo). [3H]-prazosin competition binding to α1-AR membrane proteins was performed using 3a-f, according to the previous method. The competition curves were shown in Figure S1. Turn on fluorescence switch by α1-AR. These experiments were carried out in tris-HCl solution (50 mM, pH=7.4) with 2 h incubation at 37 °C on 96-well black flat plates. Data are from three independent experiments. (1) Protein selectivity. To 10 µL of α1-AR membrane proteins (1 mg/mL), and BSA (10 mg/mL), α-chymotrypsin (10 mg/mL), papain (10 mg/mL), 3a (20 nM, 90 µL) was added, respectively. After incubation, the fluorescent intensity was measured. Data were obtained by deducting the values of those wells containing 3a only. (2) Specific binding on proteins. Auto-fluorescence: α1-AR proteins were incubated alone. Total fluorescence: α1-AR proteins were incubated with 20 nM 3a. Nonspecific fluorescence: α1-AR proteins were incubated with 20 nM 3a together with 2 µM doxazosin. (3) Specific binding on cells. Transfected HEK293 cells were plated at 40,000 cells per well. After 24h, the medium was discarded, then compounds were added. Total fluorescence: α1-AR cells were incubated with 20 nM 3a. Nonspecific fluorescence: α1-AR cells were incubated with 20 nM 3a together with 2 µM doxazosin. Cell culture and animal feeding. Cells were grown in the corresponding medium supplemented with 10% (v/v) fetal bovine serum in a 37 °C humidified 5% CO2:95% air atmosphere. The medium for untransfected and transfected HEK293A cells, HepG2 and Hela cells is DMEM, and for PC-3, DU145, ES-2, MCF-7 and A549 cells is RPMI-1640. α1B-AR-green fluorescent protein (α1B-AR/GFP) plasmid were constructed by ligating the human α1B-AR full-length gene (ADRA1B) into the basic pEGFP-N1 protein fusion vector as described previously by Youyi Zhang (See Figure S3). The recombinant plasmid were transfected into HEK293A cells mediated by Lipofectamine® 2000 reagent

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Journal of Medicinal Chemistry

(Invitrogen) according to the manufacturer’s instructions, and cells were assayed 48-72 h after transfection. All animal studies were approved by the Ethics Committee and IACUC of Qilu Health Science Center, Shandong University, and were conducted in compliance with European guidelines for the care and use of laboratory animals. Balb/c-nu male of female mice, 4 weeks of age, were purchased from the Animal Center of China Academy of Medical Sciences (Beijing, China). Mice were single or grouphoused on a 12:12 light-dark cycle at 22 ° C with free access to food and water. Cytotoxicity assay. The cell viability of α1A and α1D-AR transfected stably HEK293A cells, untransfected HEK293A cells and PC-3 cells exposed to 3a-f and doxazosin was evaluated using a 48 h MTT assay on 96-well plates (Table S1). The absorbance values were recorded at 490 nm to avoid the interference from absorption of 3a-f. Fluorescence imaging in living cells and tissue sections. Cells were grown in the confocal dish for at least 24 h and washed with 10 mM PBS twice carefully. After incubated for 10 min in HEK293 cells or 30 min in cancer cells with 300 nM compound 3a with or without 3 µM doxazosin, the cell imaging was performed without further washing on a Zeiss Observer A1 or LSM700 confocal microscopy. All images was adjusted by ImageJ software. The imaging of cells treated with 30 nM compound 2 was also conducted as the above, and results were displayed in Figure S4. For prolonged pre-incubation, 300 nM 3a was prepared using cell-culture medium. After incubated for 1 h, 24 h and 48 h, PC-3 and DU145 cells were imaged at the aseptic condition, respectively. For imaging of prostate tissue sections, the paraffin sections were dewaxed to water at first, and then exposed to Krebs solution with 300 nM 3a and incubated for 24 h at 4 °C. Tissue samples were washed for 5 min in Krebs solution, and observed using 20× objective.

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Flow cytometry analysis. Cells were harvested and washed with cold 10 mM PBS. 3a with the final concentration of 60 nM was added solely or together with doxazosin, which had the final concentration of 600 nM, into 1 × 106 cells in 100 µL of 10 mM PBS. After incubated for 30 min at 37 °C, the samples were analyzed by flow cytometry. In vivo optical imaging. To generate tumor xenografts in mice, PC-3, Hela and ES-2 cells (1 × 107) were implanted subcutaneously in the right armpit region of each 5-week-old female nude mouse, respectively. Tumors were allowed to grow to 1cm3. To study the distribution of α1-AR in vivo, the female and male mice were employed. 12 h before optical imaging, the nude mice with or without tumors were dosed by a tail intravenous injection of 3a (50 µM, 20 µL/g). Then mice were anesthetized with isoflurane and imaging was performed immediately in a small animal living imaging system (IVIS Spectrum, λex=640 nm, Cy5.5 channel). ASSOCIATED CONTENT Supporting Information. Binding affinity curves, optical spectra in different solvents, cell imaging results of compound 2, plasmid information of EGFP-ADRA1B, quantitative data of Figure 5, ex vivo imaging of separated prostate and bladder, 1HNMR, 13CNMR, MS, HRMS and HPLC data. AUTHOR INFORMATION Corresponding Author * Tel./fax.: +86-531-8838-2076. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

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The present work was supported by grants from the National Natural Science Foundation of China (No. 21228204), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Fok Ying Tong Education Foundation (No. 122036), the China–Australia Centre for Health Sciences Research (CACHSR) (No. 2015GJ02), and the Fundamental Research Funds of Shandong University (No. 2014JC008). Our cell imaging work was performed at the Microscopy Characterization Facility, Shandong University. We also thank Professor Youyi Zhang from Peking University for her generous gift, the α1A-AR- and α1D-AR-transfected HEK293A cells. ABBREVIATIONS NIR, near-infrared; GPCR, G-protein-coupled receptor; 5-HT, 5-hydroxytryptamine; BPH, benign prostatic hyperplasia; LUTS, lower urinary tract symptoms; BODIPY® FL prazosin, 1-(4-(4-amino-6,7dimethoxyquinazolin-2-yl)piperazin-1-yl)-3-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'f][1,3,2]diazaborinin-3-yl)propan-1-one; QD, quantum dot; RT-PCR, reverse transcription-polymerase chain reaction. REFERENCES 1.

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Scheme 1. Synthesis of prazosin-trizole-Cy5 conjugates 3a-fa

a

Reagents and conditions: (a) 3-iodopropionic acid, acetonitrile, reflux, 48h, 81%; (b) methyl iodide,

acetonitrile, reflux, 12h, 86%; (c) malonaldehydedianilide hydrochloride, AcOH/AC2O, 120 °C, 4h; pyridine/AcOH, 120 °C, 4h; 30% for two steps; (d) propargylamine, EDCI, DMAP, dichloromethane, r.t., 6h, 53%; (e) piperazine, H2O, 100 °C, 81%; (f) acyl chloride, TEA, acetonitrile, 1h, 0 °C, 62-75%; (g) NaN3, DMSO, 100°C, 6h, 35-88%; (h) H2O/ acetonitrile, NaN3, 80 °C, 20h, 43-74%; (i) TsCl, triethylamine, dichloromethane, r.t., 24h, 65-88%; (j) K2CO3, acetonitrile, 90 °C, 64-69%; (k) CuSO4, Sodium ascorbate, t-BuOH/H2O=2:1, 50 °C, 1h, 48.9-72.5%.

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Table 1. Summary of spectroscopic and receptor binding affinity properties of compounds. λex a

λem a

(nm)

(nm)

PBSa

Atropine

ND

ND

Prazosin38

ND

Phentolamine

Compounds

Ki b(nM)

Φ DMSO

α1A

α1B

α1D

ND

ND

NA

NA

NA

ND

ND

ND

0.2

0.25 0.32

ND

ND

ND

ND

2.2

17.6 19.5

3a

640

665

0.0041

0.0050

8.2

4.8

14

3b

640

665

0.0037

0.0106

12.2

7.7

17.9

3c

640

665

0.0041

0.0076

8.4

5.6

14.4

3d

640

665

0.0032

0.0090

19.3 16.6 29.9

3e

640

665

0.0040

0.0087

37.2 26.1 54.8

3f

640

665

0.0045

0.0081

53.6 34.2

150

2

640

665

0.0063

0.0100

NA

NA

a

in 50 mM PBS solution; ND, not detection; NA, not available.

b

Ki was calculated from IC50 using the Cheng-Prusoff equation;

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NA

Journal of Medicinal Chemistry

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Figure 1. Design concept of the conjugates of prazosin and Cy5 as the environment-sensitive nearinfrared fluorescent ligands for α1-AR. (a) Illustration of the fluorescence turn-on strategy of prazosinCy5 conjugates used for detecting α1-AR selectively. (b) Design flow of chemical structure of the fluorescent ligands 3.

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Journal of Medicinal Chemistry

Figure 2. Emission spectra of 3a in different solvents (A) and in the mixture of DMSO and HEPES buffer (B); Fluorescence lifetime of 3a in mixture of glycerol and water (C), and at different concentrations (D).

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Journal of Medicinal Chemistry

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Figure 3. (A, B) Fluorescence response of 3a (A) and 2 (B) to various proteins. 3a or 2 (18 nM) were incubated with proteins for 2 h, and fluorescence intensity was recorded at 680 nm. The data were obtained by deducting the mean value intensity of those wells containing 3a without any protein; (C) Fluorescence measurement of 3a bound to three α1-AR proteins. Membrane proteins were incubated alone (autofluorescence), with 20 nM 3a, supplemented with (nonspecific binding) or without (total binding) 2 µM doxazosin; (D) Fluorescence measurement of 3a bound to α1A-and α1D-AR transfected stably HEK293A cells. Cells were incubated with 3a, supplemented (nonspecific binding) or not (total binding) with 2µM doxazosin. These assay above were conducted for 2 h in the Tris-HCl binding buffer (50 mM, pH 7.4), and the values are from three independent experiments; (E) Fluorescence labeling of transfected and untransfected HEK293A cells with 3a was observed without washing on a Zeiss fluorescence microscopy (a, b, c, d, e) or LSM700 (g, f). Scale bar = 20 µm. Cells were incubated for 5 min with 300 nM 3a, together with (b, d, g) or without (a, c, e, f) 3 µM doxazosin. (a, b) α1A-AR transfected stably HEK293A cell; (c, d) α1D-AR transfected stably HEK293A cell; (e) untransfected HEK293A cell; (f, g) α1B-AR/GFP transfected transiently HEK293A cell.

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Journal of Medicinal Chemistry

Figure 4. (A) Fluorescence imaging of cancer cells with 3a observed on a Zeiss fluorescence microscopy. Scale bar = 20 µm. Cells were incubated for 30 min with 300 nM 3a, without (a, b, c, d, e, f and g) or with (a’ and b’) 3 µM doxazosin , and then cells were observed immediately without any washing. PC-3 cells (a, a’); DU145 cells (b, b’); Hela cells (c); ES-2 cells (d); A549 cells (e); MCF-7 cells (f); and HepG2 cells (g); (B) Localization of the α1-AR in PC-3 cells (Aa) and DU145 cells; (Bb). 3a (300 nM) is exposed to PC-3 or DU145 cells for 1h (aa), 24h (bb) and 48h (cc); (C) Flow cytometry analysis of binding of 3a to the living cells. Scale bar = 20 µm; (upside) Binding to α1A-AR transfected and untransfected HEK293 cells. Untransfected HEK293 cells were treated with (nonspecific binding) or without 3a (negative); α1A-AR transfected HEK293 cells were treated without 3a (negative) or with 3a (total binding) or with doxazosin and 3a (nonspecific binding); Bottom, Binding to PC-3 and Hela cells. Cells were treated without 3a (negative) or with 3a (total binding).

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Journal of Medicinal Chemistry

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Figure 5. (A) In vivo and ex vivo imaging result of tumor-bearing mice at 12 h after intravenous injection of NS (a) and 3a (b). Upside of A: imaging of tumors (marked with the red loops) in the entire mice; Bottom of A: the fluorescence images of the tumors after anatomy. (B) The quantitative data from fluorescence imaging of three types of tumors injected by 3a.

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Journal of Medicinal Chemistry

Figure 6. In vivo and ex vivo imaging of mice at 12 h after intravenous injection. (A) ventral and dorsal view of mice treated with 3a or 2; (a) male, 3a, (b) male, 2, (c) female, 3a, (d) female, 2; (B) fluorescence imaging of the internal organs from the dissected mice in A. (1) Blood, (2) Salivary glands, (3) Brain, (4) Lung, (5) Heart, (6) Kidney, (7) Liver, (8) Bladder and Prostate, (9) Urethra, (10) Spleen, (11) Bladder; (C) The competition assay of prazosin in mouse organs with 3a. The mice were injected with 3a (50 µM) firstly, and after 9h, they were injected with NS (-) or with prazosin (+). And then the mice were dissected for imaging study.

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Journal of Medicinal Chemistry

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Figure 7. Microscopic views of human prostate sections, including representative transmission and red fluorescent images. (A, A’) Prostate normal tissues; (B) BPH tissues; (C) Prostate highlevel cancer tissues (Gleason score=8), and (D) Prostate low-level cancer tissues (Gleason score=7). G1 refers to the glandular epithelium with strong fluorescence; G2 and G’ refer the smooth muscle tissues with strong and weak fluorescence, respectively; D’ is a HE staining image; and the white box in D and D’ refers to the cancer tissue (Gleason score=7). D was gained after 30 min incubation of 3a at r.t. With the exception of D, other red fluorescent images were recorded after the 24 h incubation of 3a at 4 °C.

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