Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

Accepted Manuscript Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands Mads Corvinius Nielsen, Anders Foller Larsen, Faisal Hussein Abdikadir, Trond Ulven PII:

S0223-5234(13)00769-1

DOI:

10.1016/j.ejmech.2013.11.027

Reference:

EJMECH 6573

To appear in:

European Journal of Medicinal Chemistry

Received Date: 6 August 2013 Revised Date:

17 November 2013

Accepted Date: 25 November 2013

Please cite this article as: M.C. Nielsen, A.F. Larsen, F.H. Abdikadir, T. Ulven, Phenanthroline-2,9bistriazoles as selective G-quadruplex ligands, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2013.11.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands†

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Mads Corvinius Nielsen, Anders Foller Larsen, Faisal Hussein Abdikadir and Trond Ulven*a

Dr. M. C. Nielsen, A. F. Larsen, F. H. Abdikadir, Prof. T. Ulven, Department of Physics, Chemistry and

Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

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Fax: (+45) 6615 8780; Tel: (+45) 6550 2568; E-mail: [email protected]

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† Electronic supplementary material available: methods, compound characterizations, figures and tables.

Abstract

G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and

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evaluated as G4 stabilizers. The compounds were efficiently assembled by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA. The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric

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sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands were thus

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identified as potent G4 ligands with high selectivity over duplex DNA, and preliminary results indicate that the scaffold may form basis for the development of subtype-specific G4 ligands.

Keywords G-quadruplex, phenanthroline, G-quadruplex ligands, anticancer, click reaction

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1

Introduction

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It is well established that nucleic acid sequences rich in guanosine (G) residues can form secondary structures known as G-quadruplexes (G4s) [1-3]. Putative G4 forming DNA sequences have been located throughout the human genome [4, 5]. The most studied sequences are human telomeric DNA [6-13] and promoter regions of proto-oncogenes [14, 15] such as c-MYC [16-18], c-KIT [19-23], VEGF [24-26] and RET [27, 28]. Promising approaches have recently emerged that target telomeric DNA or specific proto-

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oncogene regions with small molecules and may lead to the development of new and selective chemotherapeutic compounds [29-34].

The extended aromatic structure of a 1,10-phenanthroline scaffold provides a basis for efficient π-π stacking

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interactions with the terminal G-tetrads of G4s. Introducing appendages with positive charges or H-bond donating abilities around this scaffold provides the possibility for G4 groove and/or loop interactions. Indeed, several ligands based on phenanthrolines have been investigated as G4 stabilizers (Fig. 1). The platinum phenanthroline complex (A) was demonstrated to efficiently stabilize telomeric G4 [35, 36]. The importance of the central platinum atom was stressed by the observation that the metal free ligand showed lower potency. Furthermore, the highly potent and selective bisquinolinium amide PhenDC3 (B) [37] was

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initially evaluated by a fluorescence resonance energy transfer (FRET) Tm assay [38, 39]. The selectivity of this ligand was also demonstrated by a G-quadruplex fluorescent intercalator displacement (G4-FID) assay [40]. A series of 4,7-diamino-substituted phenanthrolines (C) has been described where the substituents were shown to have beneficial effect on the quadruplex stabilizing effect of phenanthroline, with positively charged side chains required for significant G4 stabilization [41]. Recently, hybrids of B and C, resulting in

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4,7-DPDC (D), were reported to show improved G4 potency and aqueous solubility [42, 43]. Additional phenanthroline G4 ligands have also been described [44-58].

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Triazole linked compounds have been reported as potent and selective G4 ligands. The acridine based ligand E showed selectivity for human telomeric quadruplex (Fig. 1) [59]. Similarly, F and analogues hereof also stabilized telomeric G4 structures [60, 61], as did triazole ligands based on a diarylurea scaffold [62, 63]. Other triazole based G4 ligands include C3-symmetric ligands [64, 65]. We speculated that a combination of the phenanthroline scaffold with triazole-linked positively charged groups would result in potent and selective G4 ligands. We here report the synthesis of a series of phenanthroline-2,9-bistriazoles and the characterization of their interaction with two G4 structures and duplex DNA, resulting in the identification of potent and selective G4 ligands.

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2

Results and Discussion

By combining previously described G4 ligands (Fig. 1), phenanthroline-2,9-bistriazole appears as an interesting scaffold carrying aromatic nitrogens with lone pairs that can be arranged to mimic half of telomestatin and other potent macrocyclic G4 ligands inspired from this compound [66]. Molecular modeling

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of these ligands with published G4 structures indicated that phenanthroline-2,9-bistriazole has a shape and size that fits well on top of a G4 tetrad and that positively charged end groups can be positioned on close proximity to phosphate residues of the backbone by 2-4 carbon tethers to the triazole units.

The triazole-linked phenanthroline ligands were obtained by conversion of 1,10-phenanthroline-2,9-

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dicarbaldehyde (1) to bis-alkyne 2 using the Ohira-Bestmann protocol (Scheme 1) [67, 68]. Phenanthroline 2 was further subjected to Cu-catalyzed azide-alkyne cycloaddition (CuAAC) [69, 70] with azides 3a-c [7173] to provide phenanthrolines 4a-c. Initial attempts to perform the cycloaddition reactions with Sharpless’

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standard procedure using Cu(II) and sodium ascorbate in THF, MeCN, MeOH or DMSO with water as cosolvent resulted in very poor conversions and complex reaction mixtures. A change from colorless to red was observed when adding the copper source to the reaction mixtures, indicating coordination of phenanthroline 2 to the copper ions and possibly quenching of the reaction. Therefore, the ligand tris[(1-benzyl-1H-1,2,3triazol-4-yl)methyl]amine (TBTA) was added to bind the copper ions and catalyze CuAAC reactions [74]. Surprisingly, no conversion was obtained when using THF or MeCN as solvents, even when N,Ndiisopropylethylamine (DIPEA) was added to the reaction mixtures. Full conversions of the CuAAC

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reactions were only obtained with a two-phase system of CH2Cl2 and water as reaction medium using Cu(II)TBTA complex (2 mol%) and sodium ascorbate, ultimately providing 4a, 4b and 4c in yields of 71%, 79% and 77%, respectively [75].

N-Boc groups of 4a-c were cleaved using TFA in CH2Cl2 to provide primary amines 5a-c in quantitative

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yields. Finally, the primary amines were converted to the corresponding primary guanidine (6a–c) or diisopropylguanidine (7a-c) analogues by microwave assisted reactions using 1H-pyrazole-1-carboxamidine

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or N,N′-diisopropylcarbodiimide, respectively (Scheme 1). Compounds 6a-c and 7a-c were obtained in 5772% yield after purification by preparative HPLC. FRET Tm assays were employed to evaluate the effect of ligand binding on the thermal stability of various DNA structures [38, 39]. Each ligand was examined at a range of concentrations against human telomeric (htelo) and c-KIT G4-forming oligonucleotides and against a hairpin (hairpin) structure as a double stranded DNA (dsDNA) reference (Table 1; see also Table S1 in the ESI). The change in thermal stability (∆Tm) produced by ligand binding to DNA was measured by recording the emission of 6-carboxyfluorescein (FAM) with increasing temperature. The results of these experiments showed that all ligands were capable of G4 stabilization. Ligands with longer linkers between the tetrazoles and the positive charges (b and c series;

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n = 2 or 3) were generally more potent stabilizers of htelo and c-KIT than the corresponding ethylene ligands (a series, n = 1). No significant stabilization of dsDNA was observed for ligands 5a-c and 7a-c. The guanidinium

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functionalized ligands 6a-c were generally more potent stabilizers of both htelo and c-KIT, but was also found to stabilize dsDNA to a small extent, presumably due to unspecific ligand-DNA interactions of the unsubstituted guanidinium groups in these compounds. The most potent G4 stabilizers were 6b (∆Tm(htelo) = 18.0 °C; ∆Tm(c-KIT) = 16.1 °C) and 6c (∆Tm(htelo) = 17.3 °C; ∆Tm(c-KIT) = 16.8 °C). The G4 stabilizing abilities of these compounds are lower than that of TMPyP4 [76] (∆Tm(htelo) = 23.1 °C; ∆Tm(c-KIT) > 25

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°C), but they discriminate better against dsDNA. Additional tests of ligands 5b, 6b and 7b with htelo in Na+containing buffer revealed that these ligands stabilize the telomeric G4 structure significantly better in K+-

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buffer than in Na+-buffer.

Surface plasmon resonance (SPR) studies (Table 1) using biotinylated DNA attached to a streptavidin-coated sensor chip were used to evaluate the kinetics of the ligand-DNA interactions. In agreement with the FRET Tm studies, all phenanthroline-2,9-bistriazole ligands showed highly selective binding towards htelo and cKIT compared to hairpin dsDNA, in contrast to TMPyP4, which has high affinity for both G4 and dsDNA structures (KD = 33-200 nM and 200 nM, respectively) [77]. For ligands 5a-c and 6a-c only concentrations from 250 nM gave significant binding to dsDNA, whereas the binding of 7a-c to dsDNA was too low to

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determine KD-values. All ligands exhibit KD-values for G4 binding that are 2 to 3-fold lower for c-KIT compared to htelo, indicating an inherent preference of all ligands for c-KIT over htelo. The KD-values obtained from c-KIT (KD ≤ 50 nM) indicates a very strong binding to this G4 type. Binding to htelo is also significant for most of the ligands (KD-values from 57 to 176 nM), with the exception of 7a and 7b (KD = 567 nM and 440 nM respectively) that both displayed a 13-fold selectivity for c-KIT over htelo. The

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guanidinium functionalized ligands with the longer tethers 6b-c displayed the lowest KD-values on both cKIT and htelo, which agrees well with the findings from the FRET Tm assay.

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The G4-FID assay is a method aiming at evaluating quadruplex-DNA binding affinity and quadruplex- over duplex-DNA selectivity based on the displacement of the fluorescent probe thiazole orange (TO) [78-80]. G4-FID curves can be obtained by plotting percentage displacement of TO against ligand concentration. From this the

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DC50- and

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DC50-values, representing the concentration required to decrease the

fluorescence by 50% upon binding to quadruplex or duplex, respectively, can be determined. In the cases where 50% displacement was not obtained,

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DC(2.5 µM)- and dsDC(2.5 µM)-values are reported, indicating TO-

displacement induced by the presence of 2.5 µM ligand. Ligand 6b exhibited the highest TO displacement ability from c-KIT with G4DC50 = 0.25 µM (Fig. 2). In case of 22AG (htelo) G4DC50 = 0.94 µM was obtained in K+-solution and G4DC50 > 2.5 µM in Na+-solution, in the latter case displacing 47% TO at 2.5 µM ligand

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6b. A TO displacement of only 21% from dsDNA at 2.5 µM ligand 6b confirms the selectivity over dsDNA. These results coincide very well with the observations from FRET Tm and SPR. Circular dichroism (CD) spectroscopy can be used to examine the structures and effects of ligand binding on

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quadruplex DNA.[81] In K+-solution, c-KIT adopts a parallel stranded conformation [23, 82], which is indicated in the spectrum by a major positive band at 260 nm and a negative band at 240 nm, and htelo adopts a mixture of antiparallel and parallel conformations, characterized by a maximum at around 290 nm with a shoulder at 270 nm, a positive band at 245 nm and a minimum at 235 nm [83]. We studied the c-KIT and htelo G4 structures in K+-solution by CD, containing different concentrations of ligand 6b, and found

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that the overall G4 folding in each case was preserved in the presence of ligand (see the ESI).

Conclusion

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We have designed and synthesized a series of G4 stabilizers based on a phenanthroline-2,9-bistriazole scaffold. The ligands were shown by SPR and FRET Tm to have high affinity towards G4 DNA and selectively stabilize this over dsDNA. The primary guanidinium functionalized ligands 6b and 6c were identified as the most potent G4 stabilizers, with a moderate selectivity for c-KIT over htelo, which was confirmed by a G4-FID assay on ligand 6b. However, the diisopropylguanidine ligands 7a and 7b exhibited a 13-fold higher selectivity for c-KIT over htelo in the SPR assay. Interestingly, FRET Tm data did not reflect

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the high selectivity of 7a and 7b for c-KIT over htelo and 7c showed opposite selectivity in the two assays. A possible explanation of this may be the differences in buffer systems and ion concentration, which can have significant implications on G4 stability. The fluorescent tags, the high temperature, and the cacodylate buffer required by the FRET assay makes this system highly artificial, whereas the SPR assay is closer to the intracellular situation in terms of buffer system, temperature and potassium concentration. Altogether, we

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have established phenanthroline-2,9-bistriazole as a G4 interacting scaffold with high selectivity over duplex DNA, and preliminary results indicate that the scaffold can form basis for development of ligands with

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selectivity for specific G4 subtypes.

Experimental section Synthesis

4.1.1 General methods. Commercially available chemicals and solvents were used without further purification, unless otherwise stated. CH2Cl2 was freshly distilled from CaH2 and MeOH from Mg and iodine. Microwave reactions were

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performed in an Emrys Creator microwave reactor. Reactions were monitored by TLC (SiO2-60, F254, Merck). Purification by column chromatography was carried out using silica gel 60 (0.040-0.063 mm, Merck). NMR spectra were recorded on a Bruker Avance III 400 (400 MHz) spectrometer or a Varian Inova 500 (500 MHz) at 55 °C. Chemical shifts are reported in ppm relative to TMS (1H; internal standard, δH 0.00

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ppm) or the residual solvent peaks (1H: 2.50 ppm for DMSO-d6; 13C: 77.16 ppm for CDCl3; 39.52 ppm for DMSO-d6). ESI-HRMS was recorded on a Q-Star Pulsar hybrid QqTOF instrument. Ligands 5a-7c were purified by RP-HPLC (Dionex UltiMate 3000) using a Phenomenex Luna 5µ C18(2) 100A 50x21.20 mm column; 20 mL/min flow; gradients of MeCN in water containing 0.05% TFA as modifier with gradient I being 10-20% MeCN (0-7 min), gradient II 10-25% MeCN (0-7 min) and gradient III 10-40% MeCN (0-12

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min). Stock solutions of ligands 5a-7c were prepared as 1 mM in distilled water and stored at -20 °C. Purity of stock solutions was analyzed by RP-HPLC (Dionex UltiMate 3000) using a Dionex Acclaim 5 µm C18 120A 4.6x150 mm column; 1 mL/min flow; a gradient of 10% MeCN in water (0-1 min), then 10-100%

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MeCN in water (1-11 min) followed by 100% MeCN (11-15 min) with 0.05% TFA as modifier. Oligonucleotides were purchased from Sigma Aldrich and concentrations are expressed in strand molarity, using the nearest-neighbor approximation for the absorption coefficients of the unfolded species at 260 nm [84].

4.1.2 General procedure A: Cu-catalyzed azide-alkyne cycloaddition. 2,9-Diethynyl-1,10-phenanthroline (2, 46 mg, 0.2 mmol) and the corresponding azide (3a, 3b or 3c; 0.44

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mmol) were dissolved in a mixture of CH2Cl2 (4 mL, 0.05 M) and H2O (3.6 mL). Sodium ascorbate (79 mg, 0.4 mmol), Cu(II)-TBTA complex (2 mol%; 800 µL of a 5 mM stock solution in H2O/DMSO (1:1 v/v) containing equimolar amounts of CuSO4x5H2O and TBTA) and DIPEA (175 µL, 1.0 mmol) was added. The mixture was stirred vigorously at RT for 24 h and diluted with CH2Cl2 (30 mL). The organic phase was washed with water (25 mL) and brine (25 mL), dried (MgSO4) and concentrated. Products were crystallized

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from crude reaction mixtures.

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4.1.2.1 Di-tert-butyl ((4,4'-(1,10-phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1diyl))bis(ethane-2,1-diyl))dicarbamate (4a). Following general procedure A: 3a (0.082 g) provided 4a (0.085 g, 71%) as a colorless amorphous solid after crystallization from CH2Cl2/PE. Mp 175-6 °C; 1H NMR (400 MHz, CDCl3) δ 9.33 (s, 2H), 8.58 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 8.0 Hz, 2H), 7.88 (s, 2H), 4.79 (br. s, 2H), 3.84 (m, 4H), 3.06 (m, 4H), 1.37 (s, 18H); 13C NMR (100 MHz, CDCl3) δ 155.7, 150.9, 148.7, 145.3, 137.6, 128.6, 126.6, 124.9, 120.6, 80.0, 50.2, 40.2, 28.4; ESI-HRMS calcd for C30H36N10NaO4 [M + Na+] 623.2813, found 623.2810.

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4.1.2.2 Di-tert-butyl ((4,4'-(1,10-phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1diyl))bis(propane-3,1-diyl))dicarbamate (4b). Following general procedure A: 3b (0.088 g) provided 4b (0.100 g, 79%) as a colorless amorphous solid after crystallization from CH2Cl2/PE. Mp 183-5 °C; 1H NMR (400 MHz, CDCl3) δ 9.61 (s, 2H), 8.61 (d, J =

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8.0 Hz, 2H), 8.46 (d, J = 8.0 Hz, 2H), 7.96 (s, 2H), 4.49 (br. s, 2H), 3.47 (m, 4H), 2.58 (m, 4H), 1.41 (s, 18H), 1.09 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 155.8, 151.3, 148.6, 145.3, 137.7, 128.7, 126.7, 124.8, 120.6, 79.6, 47.2, 37.1, 30.2, 28.5; ESI-HRMS calcd for C32H40N10NaO4 [M + Na+] 651.3126, found 651.3125.

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4.1.2.3 Di-tert-butyl ((4,4'-(1,10-phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1diyl))bis(butane-4,1-diyl))dicarbamate (4c).

Following general procedure A: 3c (0.094 g) provided 4c (0.101 g, 77%) as a colorless amorphous solid after crystallization from EtOAc/PE. Mp 191-3 °C; 1H NMR (400 MHz, CDCl3) δ 9.66 (s, 2H), 8.64 (d, J = 8.0

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Hz, 2H), 8.52 (d, J = 8.0 Hz, 2H), 7.96 (s, 2H), 4.38 (br. s, 2H), 3.31 (m, 4H), 2.76 (m, 4H), 1.46 (s, 18H), 0.98 (m, 4H), 0.80 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 155.9, 151.5, 148.3, 145.3, 137.7, 128.6, 126.6, 125.0, 120.6, 79.4, 49.6, 39.8, 28.6, 27.7, 26.9; ESI-HRMS calcd for C34H44N10NaO4 [M + Na+] 679.3439, found 679.3437.

4.1.3 General procedure B: synthesis of ligands 5a-c (cleavage of N-Boc group). The corresponding N-Boc protected compound (4a, 4b or 4c; 0.1 mmol) was dissolved in CH2Cl2 (1.6 mL)

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and TFA (0.4 mL). The mixture was stirred at RT for 2h and concentrated to give crude TFA salt. A portion hereof (~25%) was purified by RP-HPLC and isolated as the TFA salt for analyses.

4.1.3.1 2,2'-(4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diethanamine (5a). Following general procedure B: 4a (0.060 g) provided the TFA salt of 5a quantitatively as a white

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hygroscopic solid. Purification by RP-HPLC (gradient I, Rt 4.2 min). Analytical purity by RP-HPLC: 100%. 1

H NMR (500 MHz, 55 °C, DMSO-d6) δ 9.01 (s, 2H), 8.61 (d, J = 8.0 Hz, 2H), 8.44 (d, J = 8.0 Hz, 2H), 8.27

(br. s, 4H), 8.01 (s, 2H), 4.85 (t, J = 6.0 Hz, 4H), 3.55 (t, J = 6.0 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ

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149.6, 147.8, 145.1, 137.6, 128.3, 126.5, 125.1, 119.7, 47.5, 38.6; ESI-HRMS calcd for C20H21N10 [M + H+] 401.1945, found 401.1929.

4.1.3.2 3,3'-(4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(propan-1amine) (5b). Following general procedure B: 4b (0.063 g) provided the TFA salt of 5b quantitatively as a white hygroscopic solid. Purification by RP-HPLC (gradient II, Rt 4.2 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C, DMSO-d6) δ 9.00 (s, 2H), 8.60 (d, J = 8.0 Hz, 2H), 8.45 (d, J = 8.0 Hz, 2H), 8.01 (s, 2H), 7.95 (br. s, 4H), 4.63 (t, J = 7.0 Hz, 4H), 2.93 (t, J = 7.5 Hz, 4H), 2.27 (quintet, J = 7.0 Hz,

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J = 7.5 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ 149.8, 147.6, 145.1, 137.5, 128.3, 126.4, 124.7, 119.7, 46.9, 36.3, 27.8; ESI-HRMS calcd for C22H25N10 [M + H+] 429.2258, found 429.2248.

4.1.3.3 4,4'-(4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(butan-1-amine) (5c).

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Following general procedure B: 4c (0.066 g) provided the TFA salt of 5c quantitatively as a white hygroscopic solid. Purification by RP-HPLC (gradient II, Rt 4.7 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C, DMSO-d6) δ 9.17 (s, 2H), 8.63 (d, J = 8.0 Hz, 2H), 8.47 (d, J = 8.0 Hz, 2H), 8.05 (s, 2H), 7.79 (br. s, 4H), 4.25 (m, 4H), 2.77 (m, 4H), 1.74 (m, 4H), 1.45 (m, 4H); 13C NMR (100 HRMS calcd for C24H29N10 [M + H+] 457.2571, found 457.2562.

4.1.4 General procedure C: synthesis of ligands 6a-c.

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MHz, DMSO-d6,) δ 150.0, 147.6, 144.8, 137.8, 128.3, 126.5, 124.5, 119.7, 48.9, 38.1, 26.7, 23.9; ESI-

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The crude TFA salt of primary amine (5a, 5b or 5c; 25 µmol) was mixed with 1H-pyrazole-1-carboxamidine hydrochloride (88 mg, 60 µmol) in a microwave vial and added abs. EtOH (400 µL) and Et3N (35 µL, 250 µmol). The mixture was heated at 120 °C for 30 min in a microwave reactor and afterwards concentrated. The crude mixture was dissolved in water and purified by RP-HPLC. The products were isolated as TFA salts.

4.1.4.1 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1diyl))di-guanidine (6a).

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Following general procedure C: 5a (10.0 mg) provided 6a (6.9 mg, 57%) as a clear film after purification by RP-HPLC (gradient I, Rt 5.6 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C, DMSO-d6) δ 8.96 (s, 2H), 8.60 (d, J = 8.0 Hz, 2H), 8.46 (d, J = 8.0 Hz, 2H), 8.01 (s, 2H), 7.73 (t, J = 6.0 Hz, 2H), 7.29 (br. s, 6H), 4.71 (t, J = 6.0 Hz, 4H), 3.85 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 157.0, 149.7,

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147.7, 145.1, 137.5, 128.3, 126.4, 124.8, 119.7, 49.0, 40.6; ESI-HRMS calcd for C22H25N14 [M + H+] 485.2381, found 485.2379.

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4.1.4.2 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(propane-3,1diyl))-diguanidine (6b). Following general procedure C: 5b (10.7 mg) provided 6b (9.2 mg, 72%) as a clear film after purification by RP-HPLC (gradient II, Rt 5.4 min). Analytical purity by RP-HPLC: 97%. 1H NMR (500 MHz, 55 °C, DMSO-d6) δ 9.06 (s, 2H), 8.61 (d, J = 8.0 Hz, 2H), 8.46 (d, J = 8.0 Hz, 2H), 8.02 (s, 2H), 7.80 (s, 2H), 7.22 (br. s, 6H), 4.46 (m, 4H), 4.06 (m, 4H) 2.09 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 156.9, 149.9, 147.7, 145.0, 137.6, 128.3, 126.4, 124.6, 119.7, 47.0, 37.9, 29.0; ESI-HRMS calcd for C24H29N14 [M + H+] 513.2694, found 513.2687.

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4.1.4.3 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(butane-4,1diyl))-diguanidine (6c). Following general procedure C: 5c (11.4 mg) provided 6c (8.9 mg, 65%) as a clear film after purification by RP-HPLC (gradient II, Rt 6.0 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C,

(br. s, 6H), 3.97 (m, 4H), 3.00 (m, 4H), 1.52 (m, 4H), 1.17 (m, 4H);

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DMSO-d6) δ 9.33 (s, 2H), 8.66 (d, J = 8.0 Hz, 2H), 8.49 (d, J = 8.0 Hz, 2H), 8.08 (s, 2H), 7.63 (s, 2H), 7.14 C NMR (100 MHz, DMSO-d6) δ

156.7, 150.2, 147.5, 144.7, 138.0, 128.4, 126.5, 124.6, 119.8, 48.8, 39.5(merged with DMSO), 27.1, 25.3; ESI-HRMS calcd for C26H33N14 [M + H+] 541.3007, found 541.3002.

4.1.5 General procedure D: synthesis of ligands 7a-c.

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The crude TFA salt of the corresponding primary amine (5a, 5b or 5c; 25 µmol) was mixed with abs. EtOH (300 µL) and Et3N (70 µL, 500 µmol) in a microwave vial. After addition of N,N′-diisopropylcarbodiimide

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(78 µL, 500 µmol) the mixture was heated at 120 °C for 30 min in a microwave reactor and afterwards concentrated. The crude mixture was dissolved in water, filtered and purified by RP-HPLC. The products were isolated as TFA salts.

4.1.5.1 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1diyl))-bis(2',3-diisopropylguanidine) (7a). Following general procedure D: 5a (10.0 mg) provided 7a (11.4 mg, 70%) as a white hygroscopic solid after purification by RP-HPLC (gradient III, Rt 8.3 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500

TE D

MHz, 55 °C, DMSO-d6) δ 8.95 (s, 2H), 8.59 (d, J = 8.0 Hz, 2H), 8.44 (d, J = 8.0 Hz, 2H), 8.00 (s, 2H), 7.50 (t, J = 6.0 Hz, 2H), 7.10 (d, J = 8.5 Hz, 4H), 4.74 (t, J = 6.0 Hz, 2H), 3.91-3.87 (m, 4H), 3.83-3.73 (m, 4H), 1.13 (d, J = 6.0 Hz, 24H); 13C NMR (100 MHz, DMSO-d6) δ 152.6, 149.8, 147.8, 145.2, 137.5, 128.3, 126.4, 124.8, 119.6, 48.7, 43.7, 41.2, 22.2; ESI-HRMS calcd for C34H49N14 [M + H+] 653.4259, found 653.4259.

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4.1.5.2 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(propane-3,1diyl))-bis(2',3-diisopropylguanidine) (7b). Following general procedure D: 5b (10.7 mg) provided 7b (10.9 mg, 64%) as a clear film after purification

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by RP-HPLC (gradient III, Rt 9.6 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C, DMSO-d6) δ 9.09 (s, 2H), 8.59 (d, J = 8.0 Hz, 2H), 8.45 (d, J = 8.0 Hz, 2H), 8.00 (s, 2H), 7.43 (m, 2H), 7.01 (d, J = 8.5 Hz, 4H), 4.62 (m, 4H), 3.92-3.85 (m, 4H), 3.35 (m, 4H), 2.25 (m, 4H), 1.18 (d, J = 6.5 Hz, 24H); 13

C NMR (100 MHz, DMSO-d6) δ 152.6, 149.8, 147.7, 145.0, 137.5, 128.2, 126.4, 124.7, 119.6, 47.2, 43.6,

38.5, 29.0, 22.3; ESI-HRMS calcd for C36H54N14 [M + 2H+] 341.2323, found 341.2327.

4.1.5.3 1,1'-((4,4'-(1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(butane-4,1diyl))-bis(2',3-diisopropylguanidine) (7c). Following general procedure D: 5c (11.4 mg) provided 7c (12.0 mg, 68%) as a clear film after purification by RP-HPLC (gradient III, Rt 9.7 min). Analytical purity by RP-HPLC: 100%. 1H NMR (500 MHz, 55 °C,

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DMSO-d6) δ 9.17 (s, 2H), 8.64 (d, J = 8.0 Hz, 2H), 8.47 (d, J = 8.0 Hz, 2H), 8.04 (s, 2H), 7.28 (m, 2H), 6.92 (d, J = 8.0 Hz, 4H), 4.28 (m, 4H), 3.86-3.80 (m, 4H), 1.71 (m, 4H), 1.37 (m, 4H), 1.15 (d, J = 6.5 Hz, 24H); 13

C NMR (100 MHz, DMSO-d6) δ 152.4, 150.1, 147.5, 144.8, 137.9, 128.3, 126.5, 124.6, 119.7, 48.9, 43.5,

39.5(merged with DMSO), 26.9, 25.1, 22.2; ESI-HRMS calcd for C38H57N14 [M + H+] 709.4885, found

4.2

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709.4884.

Evaluation of G-quadruplex interaction

4.2.1 FRET Tm

The ability of the ligands 5a-7c to stabilize G-quadruplex DNA was investigated using a fluorescence

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resonance energy transfer (FRET) assay in a 96-well format using an Mx3000P real time PCR machine (Stratagene). DNA was initially dissolved as 100 µM stock solutions in double distilled water, further dilutions were carried out in the relevant buffer. The labeled oligonucleotides used as the FRET probes were

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telomeric G-quadruplex (F21T: 5'-FAM-d(G3[T2AG3]3)-TAMRA-3'), c-KIT G-quadruplex (5'-FAMG3CG3CGCGAG3AG4-TAMRA-3') and hairpin DNA (5'-FAM-TATAGCTATAT7ATAGCTATA-TAMRA3'), with donor fluorophore FAM (6-carboxyfluorescein) and acceptor fluorophore TAMRA (6carboxytetramethylrhodamine). The FRET probes were diluted from stock to the correct concentration (400 nM) in the relevant buffer and then annealed by heating to 85 °C for 10 min, followed by cooling to room temperature in the heating block. Afterwards 12.5 µL was pipetted into the relevant wells. Ligand solutions

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were diluted from stock to 5 µM using buffer and added to the relevant wells. Buffer was used as a negative control. All experimental values were determined in triplicate with a total volume of 25 µL in each well. The FAM fluorophore was excited at 492 nm and the emission collected at 516 nm in 0.5 °C intervals as the plate was heated from 25 to 100 °C, with a constant temperature being maintained for 30 seconds prior to each reading to ensure a stable value. Final analysis of the data was carried out using Excel where Tm values were

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determined from normalized melting curves.

4.2.2 Surface plasmon resonance (SPR) Inc.)

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SPR measurements were performed with a four-channel BIAcore 3000 optical biosensor system (Biacore equipped

with

a

streptavidin-coated

sensor

chip.

The

hairpin

DNA

5'-biotin-

d[T10ATAGCTATAT7ATAGCTATA]-3' was heated at 95 °C for 5 min and annealed by slow cooling to form a hairpin structure in filtered and degassed buffer 20 mM Tris–HCl, 200 mM KCl, pH 7.4. The DNA was then immobilized (~400 RU) on flow cell 2. Similarly, telomeric G-quadruplex 5'-biotin-d[T7(T2AG3)4]3'

was

immobilized

(~400

RU)

on

flow

cell

3

and

c-KIT

G-quadruplex

5'-biotin-

d[(T9G3CG3CGCGAG3AG4]-3' was immobilized (~400 RU) on flow cell 4, leaving flow cell 1 as blank. DNA binding experiments were carried out in running buffer (filtered and degassed 20 mM KH2PO4, 150 mM KCl, 0.005% surfactant P20, 0.5 mM EDTA, pH 7.4) at a flow rate of 20 µL/min. Ligand solutions at different concentrations (10, 25, 50, 100, 250, 500 and 1000 nM) were prepared in degassed running buffer

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by serial dilutions from stock solution. These solutions were injected (80 µL/min for 120 s) in random series to avoid any systematic error. Chip regeneration was performed using 1 M KCl, 50 mM KOH solution. Data was analyzed using BIAevaluation 4.0.1. Data from the blank flow cell were subtracted from the sample flow cells (2, 3 and 4) to remove bulk responses caused by different refractive indexes of sample and running

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buffer. The baselines were adjusted to zero on the y (SPR response) axis and aligned to the injection time on the x (time) axis. The equilibrium dissociation constants (KD-values) were determined in BIAevaluation 4.0.1 from plots of Req (5 seconds before injection stop) as function of ligand concentration using a steady state affinity fit.

4.2.3 G-quadruplex fluorescent intercalator displacement (G4-FID)

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Measurements were performed using a LS-55 Luminescence spectrometer (Perkin Elmer) at a constant temperature of 20 °C. Each experiment was performed on a total volume of 1.25 mL, 10 mm cell length, in

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10 mM sodium cacodylate buffer pH 7.3 with 100 mM KCl or 100 mM NaCl depending on the experiments. In the G4-FID protocol 0.25 µM pre-folded DNA target was mixed with thiazole orange (0.50 µM for 22AG (5'-AG3(T2AG3)3-3') and c-KIT (5'-G3CG3CGCGAG3AG4-3'), and 0.75 µM for ds26 (self-complementary 5'CA2TCG2ATCGA2T2CGATC2GAT2G-3')). Each ligand addition step of ligand 6b (from 0.2 to 10 equivalents) was followed by a 3-min equilibration period after which the fluorescence spectrum was recorded. The percentage of displacement is calculated from the fluorescence area (FA, 510-750 nm, λex = 501 nm), using: percentage of displacement = 100 - [(FA/FA0) × 100], FA0 being the fluorescence of thiazole

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Acknowledgements

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orange bound to DNA without added ligand.

This study was supported by the Danish Council for Independent Research, Technology and Production

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(grants 274-08-056 and 09-070364).

References

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[66] M.C. Nielsen, T. Ulven, Macrocyclic G-Quadruplex Ligands, Curr. Med. Chem., 17 (2010) 3438-3448. [67] S. Müller, B. Liepold, G.J. Roth, H.J. Bestmann, An Improved One-pot Procedure for the Synthesis of Alkynes from Aldehydes Synlett, (1996) 521-522. [68] G.J. Roth, B. Liepold, S.G. Müller, H.J. Bestmann, Further Improvements of the Synthesis of Alkynes from Aldehydes, Synthesis, (2004) 59. [69] C.W. Tornoe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides, J. Org. Chem., 67 (2002) 3057-3064. [70] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes, Angew Chem Int Ed Engl, 41 (2002) 2596-2599. [71] J. Wang, M. Uttamchandani, J. Li, M. Hu, S.Q. Yao, "Click" synthesis of small molecule probes for activity-based fingerprinting of matrix metalloproteases, Chem. Commun., (2006) 3783-3785. [72] T.L. Mindt, C. Muller, M. Melis, M. de Jong, R. Schibli, "Click-to-chelate": in vitro and in vivo comparison of a 99mTc(CO)3-labeled N(tau)-histidine folate derivative with its isostructural, clicked 1,2,3triazole analogue, Bioconjug Chem, 19 (2008) 1689-1695. [73] S. Xiao, N. Fu, K. Peckham, B.D. Smith, Efficient synthesis of fluorescent squaraine rotaxane dendrimers, Org. Lett., 12 (2010) 140-143. [74] M. Meldal, C.W. Tornoe, Cu-catalyzed azide-alkyne cycloaddition, Chem. Rev., 108 (2008) 2952-3015. [75] B.Y. Lee, S.R. Park, H.B. Jeon, K.S. Kim, A new solvent system for efficient synthesis of 1,2,3triazoles, Tetrahedron Lett., 47 (2006) 5105–5109. [76] R.T. Wheelhouse, D.K. Sun, H.Y. Han, F.X.G. Han, L.H. Hurley, Cationic porphyrins as telomerase inhibitors: the interaction of tetra-(N-methyl-4-pyridyl)porphine with quadruplex DNA, J. Am. Chem. Soc., 120 (1998) 3261-3262. [77] A. Arora, S. Maiti, Effect of loop orientation on quadruplex-TMPyP4 interaction, J. Phys. Chem. B, 112 (2008) 8151-8159. [78] D. Monchaud, C. Allain, M.P. Teulade-Fichou, Development of a fluorescent intercalator displacement assay (G4-FID) for establishing quadruplex-DNA affinity and selectivity of putative ligands, Bioorg. Med. Chem. Lett., 16 (2006) 4842-4845. [79] D. Monchaud, C. Allain, H. Bertrand, N. Smargiasso, F. Rosu, V. Gabelica, A. De Cian, J.L. Mergny, M.P. Teulade-Fichou, Ligands playing musical chairs with G-quadruplex DNA: a rapid and simple displacement assay for identifying selective G-quadruplex binders, Biochimie, 90 (2008) 1207-1223. [80] D. Monchaud, M.P. Teulade-Fichou, G4-FID: a fluorescent DNA probe displacement assay for rapid evaluation of quadruplex ligands, Methods Mol Biol, 608 (2010) 257-271. [81] S. Paramasivan, I. Rujan, P.H. Bolton, Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding, Methods, 43 (2007) 324-331. [82] J. Dash, P.S. Shirude, S. Balasubramanian, G-quadruplex recognition by bis-indole carboxamides, Chem. Commun., (2008) 3055-3057. [83] A. Ambrus, D. Chen, J.X. Dai, T. Bialis, R.A. Jones, D.Z. Yang, Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution, Nucleic Acids Res., 34 (2006) 2723-2735. [84] C.R. Cantor, M.M. Warshaw, H. Shapiro, Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleolides, Biopolymers, 9 (1970) 1059-1077.

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Captions Table 1. FRET Tm and SPR data for ligands 5a-7c with htelo, c-KIT and hairpin DNA.

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Fig. 1 Examples of relevant G-quadruplex ligands.

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Scheme 1. Reagents and conditions: (i) dimethyl-1-diazo-2-oxopropylphosphonate (Ohira-Bestmann reagent), K2CO3, MeOH, 3h (57%); (ii) Cu(II)-TBTA complex (2 mol%), sodium ascorbate, CH2Cl2/H2O (1:1 v/v), 24h, RT, azide 3a, 3b or 3c (4a: 71%; 4b: 79%; 4c: 77%); (iii) TFA, CH2Cl2, 2h, RT (quantitative); (iv) 1H-pyrazole-1-carboxamidine hydrochloride, Et3N, EtOH, 120 °C (microwave), 30 min (6a: 57%; 6b: 72%; 6c: 65%); (v) N,N′-diisopropylcarbodiimide, Et3N, EtOH, 120 °C (microwave), 30 min (7a: 70%; 7b: 64%; 7c: 68%).

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Fig. 1 Examples of relevant G-quadruplex ligands.

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Fig. 2 G4-FID curves obtained for ligand 6b with c-KIT in K+-buffer (♦), 22AG in K+- (■) or Na+-buffer (▲) and duplex-DNA ds26 in K+-buffer (×). [Oligonucleotide]: 0.25 µM; [TO] = 0.5 µM for c-KIT and 22AG, 0.75 µM for ds26.

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Scheme 1. Reagents and conditions: (i) dimethyl-1-diazo-2-oxopropylphosphonate (Ohira-Bestmann reagent), K2CO3, MeOH, 3h (57%); (ii) Cu(II)-TBTA complex (2 mol%), sodium ascorbate, CH2Cl2/H2O (1:1 v/v), 24h, RT, azide 3a, 3b or 3c (4a: 71%; 4b: 79%; 4c: 77%); (iii) TFA, CH2Cl2, 2h, RT (quantitative); (iv) 1H-pyrazole-1-carboxamidine hydrochloride, Et3N, EtOH, 120 °C (microwave), 30 min (6a: 57%; 6b: 72%; 6c: 65%); (v) N,N′-diisopropylcarbodiimide, Et3N, EtOH, 120 °C (microwave), 30 min (7a: 70%; 7b: 64%; 7c: 68%).

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Table 1. FRET Tm and SPR data for ligands 5a-7c with htelo, c-KIT and hairpin DNA.

5c 6a 6b 6c 7a 7b 7c

hairpin

htelo

4.9 ± 0.3

0.6 ± 0.2

139 ± 31

11.8 ± 0.4

1.2 ± 0.5

75 ± 6

13.6 ± 0.4

1.3 ± 0.3

115 ± 13

15.0 ± 0.4 18.0 ± 0.5 (11.4 ± 0.3) 17.3 ± 0.4

13.9 ± 0.1

2.5 ± 0.1

75 ± 10

16.1 ± 0.4

2.8 ± 0.4

57 ± 6

28 ± 2

1060 ± 130

16.8 ± 0.1

3.7 ± 0.3

61 ± 7

29 ± 3

1020 ± 80

9.7 ± 0.7 9.5 ± 1.0 (4.3 ± 0.5) 15.1 ± 0.3

10.4 ± 0.1

1.1 ± 0.3

567 ± 20

40 ± 8

n.d.c

12.0 ± 0.5

1.3 ± 0.1

440 ± 19

33 ± 3

n.d.c

13.9 ± 0.2

1.3 ± 0.4

176 ± 21

35 ± 2

n.d.c

5.5 ± 0.3 13.3 ± 0.4 (8.4 ± 0.6) 12.9 ± 0.2

hairpin

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5b

htelo

KD [nM]b c-KIT 47 ± 7

1180 ± 120

45 ± 4

914 ± 180

50 ± 2

1060 ± 90

50 ± 2

1120 ± 110

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5a

∆Tm [°C]a c-KIT

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Ligand

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TMPyP4 23.1 ± 0.2 14.8 ± 1.0 >25 °C a ∆Tm data (°C) is reported at a 1 µM ligand concentration in 10 mM KCl, 100 mM LiCl, 10 mM cacodylate (pH 7.3), and for htelo with ligands 5b, 6b and 7b also in 100 mM NaCl, 10 mM LiCl, 10 mM cacodylate (pH 7.3) (data obtained for Na+-containing buffer is shown in brackets). Tm (htelo, K+) =54.2 ± 0.3 °C; Tm (htelo, Na+) =52.2 ± 0.2 °C; Tm (c-KIT, K+) = 64.5 ± 0.5 °C; Tm (hairpin, K+) = 63.0 °C. b SPR data was obtained in 100 mM K+-containing running buffer and is reported as KD-values (nM). c n.d. = not detected, e.i., no significant binding detected at 1 µM ligand concentration.

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Highlights

A series of G4 stabilizers based on phenanthroline-2,9-bistriazole was synthesized.

•

The compounds were evaluated by SPR, FRET Tm, G4-FID and CD spectroscopy.

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Primary guanidinium functionalized ligands were identified as the most potent G4 stabilizers.

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Diisopropylguanidine ligands exhibited a 13-fold binding selectivity for c-KIT over htelo.

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Supplementary Material

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Selective phenanthroline-2,9-bistriazole G-quadruplex ligands

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Mads Corvinius Nielsen, Anders Foller Larsen, Faisal Hussein Abdikadir and Trond Ulven*

Contents:

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Synthesis of compounds 2 and 3a-c

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Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, Fax: (+45) 6615 8780, E-mail: [email protected]

FRET Tm data

2 4 15

Circular dichroism

8

1

9

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CD data for Ligand 6b

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SPR sensorgrams and Scatchard plots for ligand 6b

H and 13C NMR of compounds 2-7c

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RP-HPLC profiles of ligand 5a-7c stock solutions

23

References

27

S1  

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Synthesis of compound 2 & 3a-c

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2,9-Diethynyl-1,10-phenanthroline (2). A solution of dimethyl-1-diazo-2-oxopropylphosphonate1 (Bestmann reagent, 0.342 g, 1.78 mmol) in MeOH (7 mL) was added dropwise to a suspension of 1,10-phenanthroline-2,9-dicarbaldehyde (1, 0.200 g, 0.85 mmol) and K2CO3 (0.351 g, 2.54 mmol)

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in MeOH (10 mL) under argon. The resulting mixture was stirred for 3 h at RT, diluted with CHCl3 (50 mL) and washed sequentially with 5% NaHCO3 solution and brine. The organic phase was

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dried (MgSO4) and concentrated. Purification by flash column chromatography (0-1% MeOH in CHCl3) provided 2 (0.110 g, 57%) as a light yellow solid. Mp > 210 C; NMR spectra were in accordance with previously reported data:2 1H NMR (400 MHz, CDCl3)  8.21 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H), 7.79 (s, 2H), 3.30 (s, 2H); 13C NMR (100 MHz, CDCl3)  145.8, 143.0,

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136.3, 128.3, 127.1, 127.0, 83.7, 79.0; EI-MS m/z (%) 228 (M+, 100).

General procedure A: Synthesis of azides. Azides were synthesized following literature procedures.3-4 Briefly: sodium azide (2.60 g, 40 mmol) was dissolved in a mixture of H2O (6.6 mL) and CH2Cl2 (11 mL) at 0 °C. Triflyl anhydride (1.35 mL, 8.0 mmol) was added dropwise to the

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solution and stirring was continued for 2 h. The organic layer was separated and washed once with satd. Na2CO3 solution and afterwards added to a solution containing the corresponding amine (4.0

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mmol), K2CO3 (0.829 g, 6.0 mmol), CuSO4x5H2O (10 mg, 40 μmol), H2O (13.2 ml) and MeOH (26.1 ml). The resulting mixture was stirred at RT overnight. Subsequently, the organic solvents were removed under reduced pressure and the aqueous slurry was diluted with H2O (75 ml) and acidified to pH 2 with 4 M hydrochloric acid solution. The resulting mixture was extracted with CH2Cl2 (3 x 50 mL) and the combined organic phase was dried (MgSO4), concentrated and the product purified by flash column chromatography (1% MeOH in CHCl3).

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tert-Butyl (2-azidoethyl)carbamate (3a). Following general procedure A: tert-butyl 2aminoethylcarbamate5 (0.641 g) provided 3a (0.737 g, 99%) as a light yellow oil. NMR spectra were in accordance with previously reported data:4 1H NMR (400 MHz, CDCl3) δ 4.84 (br s, 1H), 13

C NMR (100 MHz, CDCl3)  155.9, 79.9,

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3.43–3.40 (m, 2H), 3.32-3.28 (m, 2H), 1.45 (s, 9H); 51.4, 40.2, 28.5.

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tert-Butyl (3-azidopropyl)carbamate (3b). Following general procedure A: tert-butyl 3-

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aminopropylcarbamate6 (0.697 g) provided 3b (0.777 g, 97%) as a clear oil. NMR spectra were in accordance with previously reported data:6 1H NMR (400 MHz, CDCl3) δ 4.66 (br s, 1H), 3.36 (t, J = 6.8 Hz, 2H), 3.24–3.19 (m, 2H), 1.81–1.74 (m, 2H). 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3) 

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156.1, 79.6, 49.3, 38.2, 29.4, 28.5.

 

tert-Butyl (4-azidobutyl)carbamate (3c). Following general procedure A: tert-butyl 4aminobutylcarbamate6 (0.753 g) provided 3c (0.815 g, 95%) as a clear oil. NMR spectra were in

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accordance with previously reported data:5 1H NMR (400 MHz, CDCl3) δ 4.57 (br s, 1H), 3.30 (t, J = 6.8 Hz, 2H), 3.17–3.13 (m, 2H), 1.65–1.54 (m, 4H). 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3) 

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156.1, 79.4, 51.2, 40.1, 28.5, 27.6, 26.3.

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FRET Tm data Table S1. FRET Tm data obtained for ligands 5a-7c with htelo, c-KIT and hairpin DNA. ΔTm data (°C) is reported at a 0.5 and 2 µM ligand concentration in 10 mM KCl, 100 mM LiCl, 10 mM cacodylate (pH 7.3), and for htelo with ligands 5b, 6b and 7b also in 100 mM NaCl, 10 mM LiCl, determined. Tm [C]

Ligand

5a

c-KIT

0.5 µM

2 µM

0.5 µM

4.4 ± 0.4

6.8 ± 0.3

3.7 ± 0.4

2 µM

0.5 µM

6.2 ± 0.2

0.4 ± 0.2

9.8 ± 0.6

13.9 ± 0.5

1.1 ± 0.9

16.1 ± 0.4

(5.1 ± 0.4)

(11.9 ± 0.4)

5c

10.1 ± 0.4

15.8 ± 0.2

11.1 ± 0.2

15.2 ± 0.2

1.0 ± 0.1

6a

11.0 ± 0.3

18.9 ± 0.3

11.0 ± 0.4

16.4 ± 0.3

1.2 ± 0.5

11.4 ± 0.8

18.4 ± 0.2

1.6 ± 0.5

21.8 ± 0.3

13.8 ± 0.1

18.5 ± 0.4

1.7 ± 0.2

13.6 ± 0.6

7.4 ± 1.0

12.5 ± 0.2

0.7 ± 0.2

8.3 ± 0.6

14.6 ± 0.4

0.7 ± 0.2

12.2 ± 0.4

22.0 ± 0.3

6b

(6.6 ± 0.4)

(16.4 ± 0.6)

6c

12.2 ± 0.7

7a

4.2 ± 0.7

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10.3 ± 0.3 5b

3.2 ± 0.2

14.5 ± 0.5

7b

(2.8 ± 0.3)

7c

5.1 ± 0.6

18.1 ± 0.2

10.3 ± 0.9

15.9 ± 0.3

0.8 ± 0.3

TMPYP4

19.7 ± 0.5

> 35

> 25

n.d.

9.3 ± 0.4

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(8.8 ± 0.5)

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hairpin

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Htelo

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10 mM cacodylate (pH 7.3) (data obtained for Na+-buffer is shown in brackets). n.d.: not

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SPR sensorgrams and Scatchard plots for ligand 6b

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Htelo ‐ Ligand 6b 70

30

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Ligand concentrations: 10, 25, 50, 100, 250, 500 and 1000 nM.

Htelo ‐ Ligand 6b

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R² = 0,9409

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c‐KIT ‐ Ligand 6b 60

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c‐KIT ‐ Ligand 6b

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Req/Conc

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R² = 0,9097

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Hairpin ‐ Ligand 6b 20

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Ligand concentrations: 250, 500 and 1000 nM.

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Hairpin ‐ Ligand 6b

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Req/Conc

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4

6

Req

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Circular dichroism (CD) To confirm the conservation of G-quadruplex fold in presence of test compounds, CD measurements were performed with different concentrations of ligand 6b. Samples were prepared to provide a Tris–HCl buffer (100 mM KCl, 10 mM Tris–HCl, pH 7.4) and a 2 µM final concentration of telomeric G-quadruplex 5'-d[(G3(T2A3G3)3]-3' or c-KIT G-quadruplex 5'-G3CG3CGCGAG3AG4-

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3' in double distilled water. After annealing, by heating to 95 C for 10 min followed by slow cooling to room temperature, the ligand was added from aqueous stock solution in appropriate amounts to give the desired final concentrations. CD spectra were recorded on a J-815 CDSpectrometer (JASCO) using a quartz cell of 5 mm optical path length and 50 nm/min scanning

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speed with a response time of 1 s in the wavelength ranges 220–320 nm with a data pitch of 0.1 nm. The final CD spectra are results of five averaged scans at 25 C. Data were corrected for signal

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contributions due to the buffer.

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CD data for Ligand 6b cKIT & Ligand 6b 25

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cKIT

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2 µM 6b 4 µM 6b

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240

250

260

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280

290

300

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CD Signal [mdeg]

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6 µM 6b 8 µM 6b 10 mM 6b 320

Wavelength [nm]

 

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htelo & Ligand 6b

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CD Signal [mdeg]

4

230

240

htelo 2 µM 6b 4 µM 6b 6 µM 6b 8 µM 6b 10 µM 6b

250

260

270

280

‐2

‐4

Wavelength [nm]

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320

ACCEPTED MANUSCRIPT H and 13C NMR of compound 2

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 4c

N N N

N

N

N

N N

BocHN

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NHBoc

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 5a

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 5b

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 5c

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 6a

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 6b

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9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0 (ppm)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

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10.0

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 7a

9.5

180

170

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0 (ppm)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

20

10

0.0

AC C

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 7b

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0 (ppm)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

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ACCEPTED MANUSCRIPT H and 13C NMR of compound 7c

N N N

N

N

N

N

NH

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N NH

9.5

180

170

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0 4.5 (ppm)

4.0

3.5

3.0

2.5

2.0

1.5

1.0

30

20

0.5

0.0

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N

N

N

N

H2N

N N NH2

5a

N

5b

N N

NH2

AC C

H2N

N

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N N N

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RP-HPLC profiles of ligand 5a-7c stock solutions (254 nm)

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N

N N N

N

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H2N

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N N NH2

N

N

H N NH2

N N H N

6a

NH

H2N

AC C

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N

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HN

N

NH

6b

N H

N H

NH2

N

NH2

N

N

6c

H N

NH

H2N

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H N

N N

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S25

N

N

N

N N

H N

N

H N

7a

N NH

N

HN

N H

N

N

7b

N N

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N

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N H

NH

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N N N

RI PT

ACCEPTED MANUSCRIPT

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N N N

N

N

H N

N

N N H N

7c

HN

N NH

M AN U

SC

N

RI PT

ACCEPTED MANUSCRIPT

TE D

References 1

AC C

EP

Dirat, O.; Clipson, A.; Elliott, J. M.; Garrett, S.; Jones, A. B.; Reader, M.; Shaw, D. Tetrahedron Lett., 2006, 47, 1729-1731. 2 Ziessel, S.; Suffert, J.; Youinou, M.-T. J. Org. Chem. 1996, 61, 6535-6546. 3 Wang, J.; Uttamchandani, M.; Li, J.; Hu, M.; Yao, S. Q. Chem. Commun., 2006, 3783-3785. 4 Mindt, T. L.; Müller, C.; Melis, M.; de Jong, M.; Schibli, R. Bioconjugate Chem. 2008, 19, 16891695. 5 Krapcho, A. P.; Kuell, C. S. Synth. Commun., 1990, 20, 2559-2564. 6 Xiao, S.; Fu, N., K.; Smith, B. D. Org. Lett., 2010, 12, 140-143.

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