Dibenzocyclooctynes: Effect of Aryl Substitution on Their Reactivity

Article Cite This: J. Org. Chem. 2019, 84, 8542−8551

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Dibenzocyclooctynes: Effect of Aryl Substitution on Their Reactivity toward Strain-Promoted Alkyne−Azide Cycloaddition Vida Terzic,† Guillaume Pousse,† Rachel Meá llet-Renault,‡ Philippe Grellier,§ and Joëlle Dubois*,† †

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Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Univ. Paris-Sud, Univ. Paris-Saclay, 1, av. de la Terrasse, Gif-sur-Yvette 91198, France ‡ Institut des Sciences Moléculaires d’Orsay, CNRS UMR 8214, Univ. Paris-Sud, Univ. Paris-Saclay, Orsay 91405, France § UMR 7245 CNRS MCAM, Muséum National d’Histoire Naturelle, CP52, 57 Rue Cuvier, Paris 75005, France S Supporting Information *

ABSTRACT: Five new dibenzocyclooctynes bearing different substituents on their aryl moieties were synthesized and evaluated for their reactivity toward strain-promoted alkyne− azide cycloaddition (SPAAC). The dinaphthylcyclooctynes proved to be poorly reactive with azides, and the formation of triazole required many days compared to a few hours for the other cyclooctynes. Fluoride atoms and methoxy groups were also introduced to the aryl rings, leading to more active compounds. Oxidation of the alcohol on the cyclooctyne ring also increased the reaction rates by 3.5- to 6-fold. 3,9-Difluoro4,8-dimethoxy-dibenzocyclooctyne-1-one thus displayed a SPAAC kinetic rate of 3.5 M−1 s−1, which is one of the highest rates ever described. Furthermore, the dibenzocyclooctyn-1-one displayed fluorescence properties that have allowed their detection in the protozoan parasites Plasmodium falciparum and Trypanosoma brucei by microscopy imaging, proving that they can cross cell membranes and that they are stable enough in biological media.

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INTRODUCTION Cyclooctynes have recently met renewed interest in the context of bioconjugation because of their ability to react with azides in a copper-free Huisgen 1,3-dipolar cycloaddition named strain-promoted alkyne−azide cycloaddition (SPAAC).1,2 Many articles have reported the use of cyclooctynes in SPAAC to label proteins, glycans, or lipids.3−6 To compete with other chemical ligation methods, cyclooctynes should be highly reactive toward azides and sufficiently inert toward other biomolecular functionality like thiols or amines. The first description of a biocompatible cycloaddition between a cyclooctyne and azides was realized with a 3-(benzyloxy)cyclooct-1-yne derivative, which displayed a low reactivity.1 Many modifications have been realized on cyclooctyne to increase their reaction rate constants (Figure 1).7 It rapidly appears that the introduction of fluorine atoms on propargylic carbon was beneficial to the reactivity.8−10 Actually, an increase of the ring strain was the most efficient way to improve SPAAC kinetics, as observed with 3,3,6,6-tetramethylthiaheptyne (TMTH),11 bicyclononyne (BCN),12 and dibenzoannulated derivatives like DIBO.13 Modulations around the DIBO structure have been then realized like aryl ring substitution (TMDIBO),14 introduction in the eight-membered ring of an heteroatom (DIBAC15,16 and ODIBO17), of a carbonyl (BARAC18 and keto-DIBO19) or of a cyclopropenone (FlDIBO20 and MC-DIBOD21). More recently, Popik’s group © 2019 American Chemical Society

published the synthesis and properties of the monotriazole derivative of Sondheimer diyne22 (Triazol-DIBO), the most reactive cyclooctyne ever described today.21 This overview shows that a few modifications have been realized on the aryl rings of DIBO. Only one example of substitution by fluorine atoms has been reported (difluoro-DIBAC) leading to a moderate enhancement of the kinetic rate (Figure 1).23 Therefore, the design of new substituted dibenzocyclooctynes appeared propitious to bring out new SPAAC substrates with an improved reactivity like with DIBAC analogues.24 The aim of this work was to explore the effect of fluorine substituents and of a more conjugated aromatic system on cyclooctyne properties. Furthermore, since oxidation of DIBO to keto-DIBO has led to enhanced reaction rate constants, the synthesis of keto-derivatives of TMDIBO and of the newly designed molecules was also scheduled. This article describes the synthesis and properties of five new dibenzocyclooctynes: difluorodimethoxydibenzocyclooctynol (FMDIBO, 1a); dinaphthyldibenzocyclooctynol (DINO, 1b); and the corresponding dibenzocyclooctynones, keto-FMDIBO 2a, ketoDINO 2b, and keto-TMDIBO 2c (Figure 2). Received: April 1, 2019 Published: June 3, 2019 8542

DOI: 10.1021/acs.joc.9b00895 J. Org. Chem. 2019, 84, 8542−8551

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The Journal of Organic Chemistry

Figure 1. Overview of cyclooctynes designed for bioconjugation and their reaction rate constants.

Figure 2. Structure of the new cyclooctynes.

Scheme 1. Retrosynthesis of Dibenzocyclooctynols 1

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RESULTS AND DISCUSSION FMDIBO and DINO were synthesized according to the method described by Leeper for the synthesis of TMDIBO.14 The triple bond could be installed by dibromination followed by didehydrobromation of cyclooctene 3 coming from the cleavage of the cyclic ether 4, the product of phenylacetaldehyde 5 dimerization (Scheme 1). The synthesis of compounds 1a and 1b is presented in Scheme 2. As the desired aldehydes 5a (2-(4-fluoro-3methoxyphenyl)acetaldehyde) and 5b (2-(naphthalen-1-yl)-

acetaldehyde) were not commercially available, they have been prepared by aldehyde homologation and by oxidation of the corresponding alcohol, respectively. Aldehyde homologation was achieved with a modest yield despite many modifications of the experimental conditions to improve it. Dimerization of the acetaldehydes 5 was easy and afforded the cyclic ethers 4 in excellent yields. The ether cleavage was not straightforward especially for 4a. Many variations of the experimental conditions have been tried: base amount, base nature (nBuLi, tBuLi, LDA, phosphazene base), temperature, 8543


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The Journal of Organic Chemistry Scheme 2. Synthesis of Dibenzocyclooctynols 1a and 1b

and reaction time. The best result was obtained with 1.8 equiv of nBuLi at room temperature for 2 h, whereas the conditions employed for TMDIBO (2.4 equiv of nBuLi at −78 °C, 4 h) have led to the formation of 3b in a very good yield. Protection of the alcohol by a tert-butyldimethysilyl group was improved by replacing pyridine by imidazole, and crystallization of 6b allowed its structure assignment by X-ray diffraction. Again a noticeable reactivity difference was observed between the two series since the fluoromethoxy derivative 3a was stirred at room temperature for 18 h and the naphthyl compound 3b was heated for 3 h at 60 °C to afford 6a and 6b in similar yields. The most difficult step in these syntheses was the formation of the triple bond. The dibrominated products were easily obtained but were too unstable to be isolated. It should be noted that carrying out the reaction at −30 °C instead of −78 °C, as described for TMDIBO synthesis, reduced the time required for complete conversion and therefore avoided many degradation, such as TBS group removal. Application of the previously defined conditions for dehydrobromination afforded cyclooctynols 7a and 7b in a modest yield but no improvement could be made for this step. Compound 7b purification was troublesome due to its low solubility and to the presence of a side product hardly separable from the expected compound. Classical removal of the TBS group by TBAF afforded fluoromethoxy derivative 1a in a quantitative yield when the reaction was carried out at a low temperature. However, these conditions afforded 7b in a modest yield because of the formation of numerous side products. Therefore, another less nucleophilic fluoride source was tried, and TBS removal was more effective with TASF at room temperature but required a longer reaction time. Dibenzocyclooctynols 1a and 1b were synthesized in 7 steps from their commercially available precursors, 4-fluoro-3-methoxybenzaldehyde and 2-(naphthalen-1-yl)ethan-1-ol, in 5.6% and 15.3% overall yields, respectively. All alcohols 1 were oxidized to ketones 2 by using either Dess−Martin periodinane (1a and 1b) or MnO2 (1c). The reactivity of all new derivatives was evaluated in a model SPAAC-reaction using an equimolar amount of benzyl azide in methanol at room temperature (Scheme 3). The

Scheme 3. Synthesis of the SPAAC-Based Probes 2 and of Their Triazole Adducts

reaction required less than 3 h to be completed for 1a, 2a, and 2c but took a longer reaction time for the dinaphthyl series. Indeed, a total conversion was observed after 3 days for 2b. Only 80% of 1b was converted into triazole adduct 8b after more than 4 days. Both triazoles were generally obtained with a large predominance of one isomer named 1,4-triazole represented in Scheme 3, except for DINO 1b. The 1,4:1,5 isomer ratio was determined by NMR 1D and 2D experiments based on chemical shifts of protons and carbons, mainly for those in the β-position to the triazole ring. A ROESY experiment on compound 9c showed a NOE crosspeak with the benzyl methylene only for the major isomer, thus confirming the structure of the 1,4-triazole. The estimated 8544


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Figure 3. Absorption (dashed lines) and fluorescence emission (solid lines) spectra of (a) DINO 1b (20 μM, λ exc = 340 nm); (b) keto-FMDIBO 2a (40 μM, λ exc = 375 nm); (c) keto-DINO 2b (30 μM, λ exc = 375 nm); (d) keto-TMDIBO 2c (50 μM, λ exc = 375 nm).

Table 1. Photophysical Properties of Novel Cyclooctynes λabs (nm) λem (nm) Stokes shift (cm−1) Φfa ελexc (M−1 s−1) Bb

keto-DIBO

1a

1b

2a

2b

2c

294 470 5390 0.26 155 40

257−283−307−320

267−286−298−340−357 387 3572 0.18 700 126

269−309 510 7059 0.13 1000 130

288 490 6260 0.11 300 33

275−308−320 528 7727 0.04 3915 157

Fluorescence quantum yield; quinine sulfate (0.1 M) in H2SO4 was used as a standard. bB: brillance, B = ελexc × Φf.

a

does not absorb anymore above 370 nm, which might be a limitation for further optical microscopy use. Its emission is also blue-shifted compared to the reference compound (ketoDIBO). Interestingly the emission wavelengths of 2a−c are located in the blue-green region (490−528 nm). The presence of electron-donating groups such as methoxy groups (2a−2c) and the extension of the system conjugated in 2b are likely responsible for such emission wavelength shift (from 470 nm for keto-DIBO to 528 nm for 2c). The replacement of two methoxy groups with fluorine atoms (2a) resulted in a slight hypsochromic effect of the emission relative to 2c. For these three keto dibenzocyclooctynes, the Stokes shift was thus improved, compared to keto-DIBO (a gain of more 900 cm−1 is observed). Absorption at 375 nm was significantly increased for 2a and 2c thanks to n → π* transitions (methoxy groups conjugated to the system). For compound 2b, this increase is lower certainly because the

isomeric ratios for 1a, 1b, 2a, 2b, and 2c were 70:30, 100:0, 83:17, 73:27, and 90:10 respectively. The lower reactivity of the triple bond in DINO and ketoDINO may be due not only to steric hindrance but also to a more delocalized electronic system, owing to a higher conjugation with the naphthalenyl moiety. Among the 5 novel cyclooctynes, only FMDIBO 1a was not fluorescent. As observed with keto-DIBO,19 no triazole adduct displayed fluorescent properties. Absorption spectra were recorded in methanol for the 5 new cyclooctynes, and fluorescent spectra were also measured in methanol for 1b and 2a−c (Figure 3). Keto-DIBO was synthesized according to Mbua et al.,19 and its photophysical properties were recorded under the same conditions as for the new cyclooctynes. The photophysical properties are presented in Table 1. Compounds 2a−c have a similar absorption range between 260 and 400 nm. Compound 1b (DINO) has a specific behavior since it 8545


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The Journal of Organic Chemistry involved transitions lie on lower energy (π → π*). Although the luminescence efficiency (quantum yield, see Table 1) is decreased compared to keto-DIBO, the brilliance is increased (except for 2b). Since the brilliance is a key parameter for optical imaging, we envision that such luminescent compounds might be used for bioimaging (see Figure 4).

chlorine or bromine atoms.22,24 The combination of a carbonyl and fluorine atoms on the dibenzocyclooctyne structure greatly improved the kinetic reaction rate, leading to keto-FMDIBO 2a as the second most reactive cyclooctyne described to date, with reactivity comparable to TMTH, a thiacycloheptyne derivative. We then envisioned that the fluorescent properties of our probes could be useful to label molecules inside cells. In the course of our search for new antiparasitic agents, we were interested in verifying whether compounds 2a−c could penetrate into two protozoan parasites, Plasmodium falciparum, responsible for malaria, and Trypanosoma brucei, responsible for the African sleeping sickness, our main targets. The fluorescent probes were incubated with the intraerythrocytic form of P. falciparum and the bloodstream form of T. brucei, and images were recorded after 30 min of incubation by fluorescence microscopy (Figure 4). These images showed that fluorescence was only observed inside parasites proving that they are able to cross membranes and suggesting that there was a concentration effect. It should be noted that, for the intraerythrocytic form of P. falciparum, no fluorescence was detected in the erythrocyte, the host cell of the protozoan parasite. This is very promising for a possible use of this kind of probe for intracellular imaging because of the absence of fluorescence quenching by biological media and of their accumulation inside cells.

Figure 4. Incorporation of 2a−c by the free bloodstream form of T. brucei (A−C) and the intraerythrocytic stage of P. falciparum (D−F). Fluorescence (B, C and E, F) was observed under 352−402 nm excitation wavelengths and emission wavelengths > 412 nm. Parasite autofluorescence (control) was captured after 30 min of incubation in PBS. (B) Autofluorescence of the T. brucei bloodstream form. (C) Corresponding phase contrast image. (E) Autofluorescence of the P. falciparum intraerythrocytic form. (D) Corresponding phase contrast image. The intracellular parasite is indicated by an arrow. (C) Fluorescence of T. brucei bloodstream forms incubated 30 min with 10 μM 2a−c. (F) Fluorescence of the intraerythrocytic P. falciparum incubated 30 min with 50 μM 2a−c. Bar scale = 5 μm.

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CONCLUSION Since the discovery of dibenzocyclooctyne, few modifications have been made on the aryl part of the molecules. In this context, we have synthesized 5 new cyclooctynes to examine the influence of substituents like naphthalene, methoxy, or fluoro groups. Replacement of the phenyl by a 1-naphthyl group was detrimental to alkyne reactivity unlike the introduction of methoxy and fluoro groups. The weak reactivity of naphthyl derivatives may be due to steric hindrance and electronic delocalization. The presence of a ketone on the cyclooctyne ring greatly enhanced the alkyne reactivity, leading to 2a, one of the most reactive cyclooctyne toward SPAAC described today. An additional benefit of the cyclooctynone is the appearance of fluorescence as was observed with keto-DIBO. The introduction of methoxy or fluoro groups led to a red shift of the emission wavelength and therefore to an increased Stokes shift. These fluorescent properties allowed us to visualize their entry into two protozoan parasites, P. falciparum and T. brucei, showing that they were able to cross membranes and were stable enough in biological media during the time required for microscopy imaging. Therefore, such compounds could serve as fluorogenic probes to visualize biological events inside cells, providing that other substituted dibenzocyclooctynes with improved fluorescent properties were designed. However, the main restraint to go further in this study resides in the dibenzocyclooctyne synthesis. Indeed, carrying out the synthesis according to Leeper’s method generally leads to unsymmetrical aryl substituents relative to the triple bond. Other methods should be tried like ring expansion of cycloheptenone19 or double Friedel−Crafts alkylation of 1,2diphenylethane,20 which is under current investigation. However, none of these synthetic methods could be widespread to any substitution pattern, and new innovative methods are sorely needed.

To evaluate the substituent effects on dibenzocyclooctyne reactivity in SPAAC, we have measured the second-order rate constants for the most reactive derivatives 1a, 2a, and 2c. The determination of the rate constants has been realized by UV measurements in pseudo-first-order conditions with an excess of benzyl azide at different concentrations. Because of the moderate solubility of our derivatives in methanol, SPAAC has been conducted in acetonitrile at 25 °C. The progress of the reaction was followed by measuring the absorbance decay of the cyclooctyne triple bond at a specific wavelength (309−320 nm according to the derivative). The pseudo first-order rate constants derived from these experiments were then plotted as a function of benzyl azide concentration, and the second-order rate constants presented in Table 2 were calculated by leastsquares fitting of the data to a linear equation. Table 2. SPAAC Kinetic Rate Constants for Dibenzocyclooctynes compound

keto-DIBOa

TMDIBOb

1a

2a

2c

wavelength k2 (M−1 s−1)

317 0.26

0.094

320 1.01

309 3.50

320 0.63

a From ref 19, measured in methanol. bFrom ref 13, measured by 1H NMR in CDCl3.

The three new cyclooctynes displayed a higher SPAAC reactivity than the model compounds keto-DIBO and TMDIBO. The SPAAC rates for cyclooctynones were 3.5- to 6.5-fold greater than their corresponding alcohols, confirming that alkyne reactivity was dependent on ring constraints. Fluoro or methoxy substituents on the aryl rings were also beneficial to the SPAAC rate with a more pronounced effect with fluorine atoms. This result could be compared to the SPAAC rate enhancement when DIBAC was substituted with 8546


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temperature for 2 h, quenched carefully with water, and extracted twice with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under a vacuum. General Procedure E: Dibenzocyclooctyn-1-one 2 Synthesis. Dess−Martin periodinane (2.0 equiv) was added to a solution of dibenzocyclooctyn-1-ol 1 in dry CH2Cl2. The mixture was stirred at room temperature sheltered from light for 1 h. The reaction was quenched by saturated sodium thiosulfate and then extracted three times with CH2Cl2. The combined organic layers were washed with water and brine, dried over magnesium sulfate, and concentrated under a vacuum. General Procedure F: Triazole Formation. Benzyle azide (1.0 equiv) was added to a solution of dibenzocyclooctyne in methanol (1 mL/0.01 mmol). The reaction was stirred at room temperature sheltered from light until complete conversion. The solution was concentrated under a vacuum. 2-(4-Fluoro-3-methoxyphenyl)acetaldehyde (5a). (E)- and (Z)-1Fluoro-2-methoxy-4-(2-methoxyvinyl)benzene. To a solution of (methoxymethyl)triphenylphosphonium chloride (16 g, 46.7 mmol, 1.2 equiv) in dry THF (45 mL) was added sodium hydride (60% in oil, 1.9 g, 46.7 mmol, 1.2 equiv) slowly under argon. After stirring under reflux for 1 h, the reaction mixture was cooled to 0 °C before adding 4-fluoro-3-methoxybenzaldehyde (6.0 g, 38.9 mmol). After stirring for 5 h at room temperature, the solution was poured on a NH4Cl solution and the aqueous layer was extracted 3 times by ethyl acetate. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under a vacuum to afford 20 g of crude enol ether that was used in the next reaction step without any purification. To characterize the enol ether formed, a portion of the crude product (1 g) was purified by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 95:5 (v/v) in 40 min) to afford 1-fluoro-2-methoxy-4-(2-methoxyvinyl)benzene (264 mg, 74%) in an E/Z (41:59) ratio as a colorless oil. 1H NMR (300, MHz, CDCl3): 7.22 (dd, J = 8.4, 1.7 Hz, 1H), 6.95 (dd, J = 8.4, 5.0 Hz, 1H), 6.93 (d, J = 1.7 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H′), 6.88 (d, J = 13.1 Hz, 1H), 6.74 (dd, J = 8.4, 2.2 Hz, 1H′), 6.67 (ddd, J = 8.4, 4.3, 2.2 Hz, 1H′), 6.04 (d, J = 7.2 Hz, 1H′), 5.69 (d, J = 13.1 Hz, 1H), 5.09 (d, J = 7.2 Hz, 1H′), 3.81 (s, 6H), 3.72 (s, 3H), 3.61 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3): 150.9 (d, J13CF = 244.0 Hz, 2C), 148.8, 148.7, 147.6, 147.6, 120.8 (d, J13CF = 6.4 Hz), 117.4 (d, J13CF = 6.6 Hz), 116.2, 115.9, 115.7, 115.4, 113.3 (d, J13CF = 1.6 Hz), 110.2 (d, J13CF = 1.4 Hz), 104.9, 104.3, 60.8, 56.6, 56.2, 56.1. 19F NMR (300 MHz, CDCl3): δ (ppm) = −138.4 (d, JHF = 5.0 Hz, 1F), −139.3 (dd, JHF = 4.3 Hz, 1F). IR (neat): 3006, 2939, 2835, 1643, 1602, 1464, 1414, 1277, 1262, 1188, 1209, 1148, 1093, 1033. MS (APPI, MeCN + CH2Cl2): m/z = 182.1 [M•]+. HRMS: m/z calcd for C10H11FO2•+ [M•+], 182.0743; found, 182.0733. 2-(4-Fluoro-3-methoxyphenyl)acetaldehyde (5a). To a solution of the crude enol ether (3.6 g) in acetone (18 mL) placed in a pressure tube was added sulfuric acid (1 M, 140 μL, 0.14 mmol, 0.05 equiv), and the reaction was stirred at 80 °C for 6 h. After removal of the solvent, the crude mixture was purified by flash chromatography on silica gel (gradient heptane/CH2Cl2 7:3 (v/v) to heptane/CH2Cl2 3:7 (v/v) in 12 min) to afford pure 5a (436 mg, 37% for two steps) as a yellow oil. 1H NMR (300 MHz, CDCl3): 9.72 (t, J = 2.1 Hz, 1H), 7.04 (dd, J = 11.2; 8.1 Hz, 1H), 6.79 (dd, J = 8.0; 1.6 Hz, 1H), 6.73 (ddd, J = 8.1; 4.0; 1.6 Hz, 1H), 3.86 (s, 3H), 3.65 (d, J = 2.1 Hz, 2H). 13 C{1H} NMR (75 MHz, CDCl3): δ (ppm) = 198.9, 153.3 (d, J13CF = 20.6 Hz), 151.8 (d, J13CF = 245.6 Hz), 128.0 (d, J13CF = 3.6 Hz), 121.9 (d, J13CF = 7.1 Hz), 116.3 (d, J13CF = 18.2 Hz), 114.6 (d, J13CF = 2.2 Hz), 56.2, 50.1. 19F NMR (300 MHz, CDCl3): −137.0 (ddd, JHF = 11.2; 8.0; 4.0 Hz, 1F). IR (neat): 3440, 2925, 2853, 1723, 1465, 1420, 1270, 1153, 1121, 1032. MS (APPI, MeCN + CH2Cl2): m/z 168.1 [M•]+. HRMS: m/z calcd for C9H9FO2•+ [M•+], 168.0587; found, 168.0577. 2-(Naphthalene-1-yl)acetaldehyde (5b). 2-Iodoxybenzoic acid (IBX) (4.9 g, 17.4 mmol, 1.5 equiv) was added to a solution of 2(naphthalen-1-yl) ethanol (2.0 g, 11.6 mmol) in acetonitrile (110 mL). The mixture was stirred at 70 °C for 90 min. Then, the mixture was cooled down and stirred for 1 h at 0 °C. The solid was filtered off,

EXPERIMENTAL SECTION

General Information. All commercial reagents were used without any further purification. Where necessary, organic solvents were routinely dried and/or distilled prior to use and stored over molecular sieves under argon. All reactions were conducted in oven-dried glassware under an argon atmosphere, unless stated otherwise. Analytical thin-layer chromatography was carried out on precoated silica gel aluminum plates (SDS TLC plates, silica gel 60F254). Column chromatography was performed on prepacked Redisep columns. Preparative TLC (PLC) was performed on Merck TLC with silica gel 60F254. Supercritical fluid chromatography (SFC) was realized on an Investigator II SFC System (Waters, USA). NMR spectra, including 1H, 19F, 13C (HMQC and HMBC) experiments, were recorded on Bruker Avance 300 (300 MHz) and Avance 500 (500 MHz) spectrometers. Chemical shifts (δ) are given in ppm relative to CDCl3 (7.26 ppm; 77.2 ppm), acetone-d6 (2.05 ppm; 30.5 ppm), or DMSO-d6 (2.50 ppm; 39.5 ppm). 19F chemical shifts were correlated with a deuterium chemical shift of the corresponding solvent. Splitting patterns are designed as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet and combinations thereof. Coupling constants (J) are reported in hertz (Hz). IR spectra were recorded on a PerkinElmer Spectrum BX. ESI mass spectra were recorded on Thermoquest AQA Navigator with a TOF detection (ESI-HRMS) or on a Waters LCT premier XE using electrospray ionization with a TOF detection (ESI-HRMS) coupled with Waters Acquity UPLC. MALDI mass spectra were recorded on a Voyager DE-STR (AB Sciex) or on a MALDI-TOF UltrafleXtreme (Bruker) unit for HRMS using the DCTB (2-[3-(4-tert-butylphenyl)-2-methyl2-propenylidene]malononitrile) matrix. APPI mass spectra were recorded on a Q-ToF 6540 (Agilent) instrument with positive ionization. Melting points were measured on a Büchi b-450 apparatus and are uncorrected. UV spectra were recorded on a Cary 5000 spectrophotometer at END Cachan (Varian), and kinetic measurements were realized at ICSN on a Cary 500 (Varian) spectrophotometer. Fluorescence measurements were realized on a Fluoromax-4 spectrofluorometer, Horiba Jobin Yvon. General Procedure A: Compound 4 Synthesis. To a solution of aldehyde in dry dichloromethane (10 mL/mmol) at −78 °C under argon was slowly added with vigorous stirring trimethylsilyl iodide (1.5 equiv). The solution was stirred at −78 °C for 5 h and stirred at room temperature. The reaction was quenched by a saturated solution of sodium thiosulfate and extracted three times with CH2Cl2. The combined organic layers were washed with saturated NaHCO3 and brine, dried over magnesium sulfate, and concentrated under a vacuum. General Procedure B: Dibenzocycloocten-1-ol 3 Synthesis. n-Butyllithium (1.6 M in THF) was slowly added to a solution of compound 4 in dry THF (10 mL/mmol). The reaction was stirred for 2 h at room temperature. The reaction was carefully quenched with water, and the aqueous layer was extracted three times with CH2Cl2. The combined organic phases were washed with brine, dried over MgSO4, and concentrated under a vacuum. General Procedure C: Silyl Protecting Group Introduction. tert-Butyldimethylsilyl chloride (3.0 equiv) was added to a stirred solution of alcohol 3 and imidazole (5.0 equiv) in dichloromethane in a pressure tube. The reaction mixture was stirred at 60 °C in an oil bath for 3 h. The reaction was diluted with CH2Cl2 and quenched with HCl (1 M). The aqueous layer was extracted three times with CH2Cl2. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under a vacuum. General Procedure D: Dibenzocyclooctyne 7 Synthesis. Bromine (1.1 equiv) was added dropwise, with stirring, to protected dibenzocyclooctene 6 in dichloromethane at −30 °C. The reaction mixture was stirred for 1.5 to 3 h. The reaction was quenched by saturated sodium thiosulfate, and the aqueous layer was extracted twice with CH2Cl2. The combined organic layers were dried over MgSO4 and concentrated under a vacuum. The residue was dissolved in dry THF and N-methylpiperazine (10 equiv) then potassium tertbutoxide (5.0 equiv) were added. The suspension was stirred at room 8547


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The Journal of Organic Chemistry and the filtrate was reduced under a vacuum to afford 5b as a yellow oil (1.95 g, 99%). 1H NMR (300 MHz, CDCl3): 9.80 (t, J = 2.4 Hz, 1H), 7.95−7.83 (m, 3H), 7.61−7.41 (m, 4H), 4.12 (d, J = 2.4 Hz, 2H). 13C{1H} NMR (75 MHz, CDCl3): 199.5, 134.0, 133.4, 132.3, 128.9, 128.5, 128.4, 126.7, 126.1, 125.6, 123.5, 48.4. IR (neat): 3047, 2824, 2725, 1722, 1597, 1511, 1395, 1217, 1165, 1078, 1037. MS (MALDI, MeCN + CH2Cl2): m/z 170.1 [M•]+. HRMS: m/z calcd for C12H10O•+ [M•+], 170.0732; found, 170.0721. 3,9-Difluoro-2,8-dimethoxy-5,6,11,12-tetrahydro-5,11epoxydibenzo[a,e](8)annulene (4a). The compound was prepared according to general procedure A from compound 5a (222 mg, 1.3 mmol). The reaction was carried out at room temperature and stirred for 20 h. The crude product was recrystallized in MeOH/CH2Cl2 95:5 (v/v) to afford pure 4a (231 mg, 96%) as a white solid. Mp: 222−224 °C. 1H NMR (300 MHz, CDCl3): 6.80 (d, J = 11.4 Hz, 2H), 6.57 (d, J = 8.4 Hz, 2H), 5.19 (d, J = 5.7 Hz, 2H), 3.81 (s, 6H), 3.45 (dd, J = 16.1, 5.7 Hz, 2H), 2.65 (d, J = 16.1 Hz, 2H). 13C{1H} NMR (75 MHz, CDCl3): 151.0 (d, J13CF = 245.0 Hz, 2C), 146.7 (d, J13CF = 10.8 Hz, 2C), 129.6 (d, J13CF = 5.2 Hz, 2C), 127.1 (d, J13CF = 3.7 Hz, 2C), 113.5 (d, J13CF = 1.7 Hz, 2C), 112.4 (d, J13CF = 18.3 Hz, 2C), 68.7 (d, J13CF = 1.4 Hz, 2C), 56.1 (2C), 35.6 (2 C). 19F NMR (300 MHz, CDCl3): −137.0 (dd, JHF = 11.4; 8.4 Hz, 2 F). IR (neat): 2973, 2924, 2850, 1463, 1447, 1344, 1312, 1262, 1228, 1195, 1109, 1077, 1017. MS (MALDI, THF): m/z 318.1 [M•]+. HRMS: m/z calcd for C18H16F2O3•+ [M•+], 318.11; found, 318.10. 7,8,15,16-Tetrahydro-7,15-epoxycycloocta[1,2-a:5,6-a′]dinaphthalene (4b). The compound was prepared according to general procedure A from compound 5b (1.97 g, 11.6 mmol) and stirred for 3 days at room temperature. The crude product was purified by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 4:1 (v/v) in 25 min) to afford pure 4b (1.55g, 83%) as a white solid. Mp: 208−211 °C. 1H NMR (300 MHz, CDCl3): 7.89 (d, J = 8.3 Hz, 2H), 7.75 (d, J = 7.0 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.49 (dt, J = 7.0 Hz, 2H), 7.42 (dt, J = 7.0 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 5.64 (d, J = 6.3 Hz, 2H), 3.82 (dd, J = 16.5, 6.3 Hz, 2H), 3.39 (d, J = 16.5 Hz, 2H). 13C{1H} NMR (75 MHz, CDCl3): δ (ppm) = 135.0 (2C), 132.6 (2C), 132.0 (2C), 128.5 (2C), 126.7 (2C), 126.5 (2C), 126.2 (2C), 125.4 (2C), 123.6 (2C), 122.5 (2C), 70.0 (2C), 32.2 (2 C). IR (neat): 3053, 2928, 1926, 1717, 1599, 1509, 1396, 1073, 812, 745. MS (MALDI, MeCN + CH2Cl2): m/z 322.1 [M•]+. HRMS: m/z calcd for C24H18O•+ [M•+], 322.1352; found, 322.1359. (Z)-3,9-Difluoro-2,8-dimethoxy-5,6-dihydrodibenzo[a,e](8)annulen-5-ol (3a). The compound was prepared according to general protocol B from compound 4a (1.0 g, 3.1 mmol) with BuLi (1.6 M, 3.5 mL, 1.8 equiv) added at room temperature. Purification by flash chromatography on silica gel heptane to heptane/EtOAc 4:1 (v/v) in 35 min afforded pure 3a (467 mg, 47%) as an off-white solid. Mp = 96−98 °C. 1H NMR (300 MHz, CDCl3): 7.13 (d, J = 12.1 Hz, 1H), 6.76 (d, J = 11.6 Hz, 1H), 6.71 (d, J = 8.5 Hz, 1H), 6.68 (d, J = 12.0 Hz, 1H), 6.62 (d, J = 12.0 Hz, 1H), 6.57 (d, J = 8.5 Hz, 1H), 5.12 (dd, J = 10.0, 5.1 Hz, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.30 (dd, J = 14.0, 5.7 Hz, 1H), 3.12 (dd, J = 14.0, 10.2 Hz, 1H). 13C{1H} NMR (75 MHz, CDCl3): 151.4 (d, J13CF = 247.3 Hz), 150.5 (d, J13CF = 246.1 Hz), 146.6 (d, J13CF = 10.8 Hz), 146.3 (d, J13CF = 10.8 Hz), 133.9 (d, J13CF = 4.8 Hz), 132.3 (d, J13CF = 3.6 Hz), 130.8, 130.6, 130.4 (d, J13CF = 3.6 Hz), 129.2 (d, J13CF = 6.6 Hz), 116.6 (d, J13CF = 18.0 Hz), 116.0 (d, J13CF = 18.0 Hz), 114.5 (d, J13CF = 10.8 Hz), 114.5 (d, J13CF = 10.8 Hz), 73.3, 56.2, 56.2, 42.2. 19F NMR (300 MHz, CDCl3): −136.0 (dd, JHF = 12.1, 8.5 Hz, 1F), −138.9 (dd, JHF = 11.6, 8.5 Hz, 1F). IR (neat): 3380, 2938, 2845, 1446, 1333, 1263, 1193, 1247, 1193, 1091, 1005. MS (MALDI, THF): m/z 318.1 [M•]+. HRMS: m/z calcd for C18H16F2O3•+ [M•+], 318.11; found, 318.10. (Z)-7,8-Dihydrocycloocta[1,2-a:5,6-a′]dinaphthalen-7-ol (3b). The compound was prepared according to general procedure B from compound 4b (1.56 g, 4.8 mmol) with BuLi (1.6M, 7.3 mL, 2.4 equiv) added at −78 °C, and then the mixture was stirred for 4 h at room temperature. Purification by recrystallization in heptane/ EtOAC 95:5 (v/v) followed by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 9:1 (v/v) in 30 min) afforded

pure 3b (1.56 g, quant.) as a white solid. Mp = 172−175 °C. 1H NMR (300 MHz, CDCl3): 8.23 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.6 Hz, 1H), 7.61− 7.51 (m, 4H), 7.51−7.21 (m, 5H), 5.84 (dd, J = 10.0, 7.4 Hz, 1H), 3.95 (d, J = 7.4 Hz, 1H), 3.94 (d, J = 10.0 Hz, 1H). 13C{1H} NMR (75 MHz, CDCl3): 138.2, 134.2, 134.0, 132.6, 132.2, 132.3, 131.9, 131.4, 130.2, 128.6, 128.5, 128.3, 128.1, 126.6, 126.6, 126.5, 126.2, 126.1, 125.8, 125.5, 125.0, 124.1, 73.8, 39.5. IR (neat): 3351, 3054, 2886, 2308, 1599, 1480, 1078, 1025, 807. MS (MALDI, MeCN + CH2Cl2): m/z 322.1 [M•]+. HRMS: m/z calcd for C24H18O+ [M•+], 322.1358; found, 322.1375. (Z)-tert-Butyl(dimethyl)((3,9-difluoro-2,8-dimethoxy-5,6dihydrodibenzo[a,e][8]annulen-5-yl)oxy)silane (6a). The compound was prepared according general protocol C from compound 3a (236 mg, 0.74 mmol) with stirring at room temperature for 18 h. Purification by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 4:1 (v/v) in 40 min) afforded compound 6a (274 mg, 86%) as an off-white solid. Mp = 98−100 °C. 1H NMR (300 MHz, CDCl3): 7.23 (d, J = 12.2 Hz, 1H), 6.77−6.68 (m, 2H), 6.65− 6.55 (m, 3H), 5.35 (dd, J = 9.9, 5.7 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.40 (dd, J = 15.4 5.7 Hz, 1H), 3.01 (dd, J = 15.4; 9.9 Hz, 1H), 0.88 (s, 9H), 0.00 (s, 3H), −0.07 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3): 151.8 (d, J13CF = 246.3 Hz), 150.0 (d, J13CF = 246.3 Hz), 146.1 (d, J13CF = 12.7 Hz), 145.8 (d, J13CF = 11.3 Hz), 136.5 (d, J = 5.9 Hz), 132.9, 132.6 (d, J = 3.6 Hz), 130.1 (d, J = 3.7 Hz), 129.1, 128.4 (d, J = 6.2 Hz), 117.1 (d, J13CF = 17.7 Hz), 115.3 (d, J = 1.8 Hz), 113.5 (d, J13CF = 19.2 Hz), 112.5 (d, J = 1.9 Hz), 70.6, 56.1, 56.0, 46.9, 29.7, 25.8 (3 C), −4.79, −4.86. 19F NMR (300 MHz, CDCl3): −136.1 (dd, JHF = 12.2, 8.1 Hz, 1F), −140.0 (dd, JHF = 11.8, 8.5 Hz, 1F). IR (neat): ν (cm−1) 2955, 2930, 2856, 1464, 1342, 1274, 1260, 1102, 1072. MS (MALDI, THF): m/z 432.2 [M•]+. HRMS: m/z calcd for C24H30F2O3Si•+ [M•+], 432.1932; found, 432.1898. (Z)-tert-Butyl((7,8-dihydrocycloocta[1,2-a:5,6-a′]dinaphthalen7-yl)oxy)dimethylsilane (6b). The compound was prepared according to general procedure C on compound 3b (1.08 g, 3.3 mmol). Purification by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 95:5 (v/v) in 30 min) afforded pure 6b (1.36 g, 93%) as a colorless solid. Mp = 143−145 °C. 1H NMR (300 MHz, CDCl3): 8.17 (d, J = 8.6 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.78−7.68 (m, 3H), 7.66 (d, J = 8.5 Hz, 1H), 7.53−7.32 (m, 5H), 7.29 (d, J = 12.0 Hz, 1H), 7.23 (d, J = 12.0 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.13 (dd, J = 10.8, 5.1 Hz, 1H), 3.96 (dd, J = 16.2, 10.8 Hz, 1H), 3.75 (dd, J = 16.2, 5.1 Hz, 1H), 0.96 (s, 9H), 0.10 (s, 3H), 0.01 (s, 3H). 13 C{1H} NMR (75 MHz, CDCl3): 141.1, 135.7, 133.0, 132.7, 132.4, 132.2, 131.1, 131.0, 129.7, 128.3, 128.2, 128.1, 127.9, 127.0, 126.3, 125.9 (2C), 125.5, 125.4, 124.7, 124.3 (2C), 71.1, 43.5, 26.0 (3 C), 18.4, −4.63, −4.76. IR (neat): 3052, 2928, 2855, 1724, 1508, 1470, 1251, 1082, 1066. MS (MALDI, THF): m/z 436.2 [M•]+. HRMS: m/ z calcd for C30H32OSi+ [M•+], 436.2217; found, 436.2210. tert-Butyl(dimethyl)((3,9-difluoro-2,8-dimethoxy-11,12-didehydro-5,6- dihydrodibenzo[a,e][8] annulen-5-yl)oxy)silane (7a). The compound was prepared according to general protocol D on compound 6a (320 mg, 0.74 mmol) with the dibromination step carried out for 1.5 h. Purification by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 98:2 (v/v) in 40 min) afforded pure compound 7a (123 mg, 39%) as a white solid. Mp = 61−63 °C. 1 H NMR (300 MHz, CDCl3): 7.42 (d, J = 12.6 Hz, 1H), 7.01 (d, J = 11.2 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 4.40 (bs, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.00 (dd, J = 14.7, 1.7 Hz, 1H), 2.82 (dd, J = 14.7, 3.0 Hz, 1H), 0.93 (s, 9H), −0.08 (s, 3H), −0.09 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3): 152.5 (d, J13CF = 248.7 Hz), 150.8 (d, J13CF = 245.2 Hz), 150.1 (d, J13CF = 5.6 Hz), 148.5, 147.3 (d, J13CF = 10.8 Hz), 146.1 (d, J13CF = 11.6 Hz), 116.3 (d, J13CF = 3.5 Hz), 115.6, 115.5, 113.8 (d, J13CF = 7.1 Hz), 113.5 (d, J13CF = 6.0 Hz), 112.0, 110.7, 109.6, 75.4, 56.5, 56.5, 50.4, 25.9 (s, 3C), 18.2, −4.53, −4.95. 19F NMR (300 MHz, CDCl3): −133.5 (dd, JHF = 12.6, 8.1 Hz, 1F), −137.3 (dd, JHF = 11.2, 8.4 Hz, 1F). IR (neat): 2957, 2930, 2855, 1731, 1638, 1613, 1512, 1492, 1333, 1275, 1254, 1156, 1143, 1066. MS (APPI, MeCN + CH2Cl2): m/z 430.2 [M•]+. HRMS: m/z calcd for C24H28F2O3Si•+ [M•+], 430.1776; found, 430.1765. 8548


Article

The Journal of Organic Chemistry tert-Butyl((7,8-dihydrocycloocta[1,2-a:5,6-a′]dinaphthalen-7-yl)oxy)silane (7b). The compound was prepared according to general protocol D on compound 6b (240 mg, 0.55 mmol) with the dibromination step carried out for 3 h. Purification by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 95:5 (v/v) in 30 min) afforded compound 7b (82 mg, 34%) as a yellow solid. Mp = 57−59 °C. 1H NMR (300 MHz, CDCl3): 8.35 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.96−7.90 (m, 3H), 7.83 (d, J = 8.4 Hz, 1H), 7.65−7.50 (m, 5H), 4.65 (tl, J = 2.3 Hz, 1H), 4.23 (dd, J = 14.8 2.3 Hz, 1H), 2.83 (dd, J = 14.8, 2.9 Hz, 1H), 0.98 (s, 9H), −0.23 (s, 3H), −0.36 (s, 3H). 13 C{1H} NMR (75 MHz, CDCl3): 156.0, 150.2, 133.1, 132.5, 132.4, 130.4, 128.9, 128.0, 127.6, 127.1, 126.7, 126.5, 126.4, 126.1, 126.1, 124.5, 123.3, 123.4, 121.5, 119.6, 118.9, 111.3, 77.7, 45.2, 31.9, 25.9 (3C), −5.13,−5.27. IR (neat): 3054, 2953, 2927, 2855, 1923, 1507, 1471, 1359, 1252, 1090, 1070. MS (MALDI, THF): m/z 434.2 [M•]+. HRMS: m/z calcd for C30H30OSi + [M•+],: 434.2061; found, 434.2051. 3,9-Difluoro-2,8-dimethoxy-11,12-didehydrodihydrodibenzo[a,e][8]annulen-5(6H)-ol (1a). A solution of TBAF (1 M in THF) (500 μL, 0.50 mmol, 2.0 equiv) was added dropwise at −20 °C to compound 7a (109 mg, 0.25 mmol) in THF (4 mL) and stirred at this temperature for 1.5 h. The reaction was quenched by the addition of a saturated aqueous solution of NaHCO3. The reaction mixture was extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under a vacuum. The crude product was purified by recrystallization in heptane/CH2Cl2 95:5 (v/v) to afford pure 1a (80 mg, quant.) as a white solid. Mp = 230−235 °C. 1H NMR (500 MHz, DMSO-δ6): 7.44 (d, J = 12.7 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 11.5 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.89 (d, J = 5.0 Hz, 1H), 4.24 (ddd, J = 5.0, 3.0, 1.5 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.10 (dd, J = 14.5, 1.7 Hz, 1H), 2.63 (dd, J = 14.5, 3.0 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-δ6): 151.5 (d, J13CF = 249.8 Hz), 150.6 (d, J13CF = 1.7 Hz), 149.9 (d, J13CF = 243.0 Hz), 149.3 (d, J13CF = 2.9 Hz), 147.0 (d, J13CF = 10.5 Hz), 145.6 (d, J13CF = 11.9 Hz), 116.2 (d, J13CF = 3.4 Hz), 116.0, 114.4 (d, J13CF = 9.2 Hz), 113.1 (d, J13CF = 9.2 Hz), 113.0 (d, J13CF = 9.5 Hz), 111.7, 111.2, 109.5, 73.2, 56.3, 56.2, 48.7. 19F NMR (500 MHz, DMSO-δ6): −133.9 (dd, JHF = 12.7, 8.2 Hz, 1F), −137.5 (dd, JHF = 11.5, 8.6 Hz, 1F). IR (neat): 3228, 2964, 2917, 2146, 1724, 1610, 1572, 1495, 1449, 1330, 1312, 1221, 1178, 1066. MS (APPI, MeCN + CH2Cl2): m/z 316.1 [M•]+. HRMS: m/z calcd for C18H14F2O3•+ [M•+], 316.0911; found, 316.0885. Dinaphathalenyl Cyclooctyn-1-ol (1b). To a solution of compound 7b (133 mg, 0.31 mmol) in dry CH2Cl2 (5.3 mL) at room temperature under argon was added slowly tris(dimethylamino)sulfonium difluorotrimethylsilicate (137 mg, 0.50 mmol, 1.6 equiv). The reaction mixture was stirred for 44 h. The reaction was quenched by water, and the reaction mixture was extracted three times with CH2Cl2. The combined organic layers were washed with brine, dried over MgSO4, and concentrated under a vacuum. The crude mixture was purified by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 4:1 (v/v) in 25 min) to afford pure 1b (74.7 mg, 71%) as a white solid. Mp = 222−224 °C. 1 H NMR (300 MHz, DMSO-δ6): 8.39 (d, J = 8.6 Hz, 1H), 8.14−7.99 (m, 5H), 7.95 (d, J = 8.3 Hz, 1H), 7.73−7.55 (m, 5H), 6.11 (d, J = 5.7 Hz, 1H), 4.41 (m, 1H), 4.18 (dd, J = 14.3, 2.0 Hz, 1H), 2.66 (dd, J = 14.3, 2.5 Hz, 1H). 13C{1H} NMR (75 MHz, DMSO-δ6): 157.2, 150.2, 132.8, 132.0, 131.9, 129.6, 128.7, 128.1, 127.6, 127.1 (2C), 127.0, 126.4, 126.2, 125.7, 124.3, 123.3, 123.1, 120.7, 119.0, 117.7, 111.2, 75.5, 43.9. IR (neat): 3319, 3223, 2283, 2325, 1263, 1036, 1026. MS (APPI/MeCN + CH2Cl2): m/z 320.1 [M•]+. HRMS: m/z calcd for C24H16O+ [M•+], 320.1188; found, 320.1183. 3,9-Difluoro-2,8-dimethoxy-11,12-didehydrodihydrodibenzo[a,e][8]annulen-5(6H)-one (2a). The compound was prepared according to general protocol E from compound 1a (49.5 mg, 0.16 mmol). Purification by flash chromatography on silica gel (isocratic, heptane/EtOAc 95:5 (v/v)) afforded pure 2a (39.5 mg, 80%) as a white solid. Mp = 196−198 °C. 1H NMR (500 MHz, DMSO-d6): 7.50 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 11.5 Hz, 1H), 7.35 (d, J = 11.5

Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 3.96 (m, 1H), 3.92 (2s, 6H), 3.77 (m, 1H). 13C{1H} NMR (125 MHz, DMSO-d6): 197.1, 151.0 (d, J13CF = 250.0 Hz), 150.4 (d, J13CF = 229.3 Hz), 149.4 (d, J13CF = 3.9 Hz), 148.2 (d, J13CF = 11.0 Hz), 147.3 (d, J13CF = 4.9 Hz), 145.5 (d, J13CF = 3.1 Hz), 118.2 (d, J13CF = 4.2 Hz), 117.2 (d, J13CF = 1.9 Hz), 115.9 (d, J13CF = 20.3 Hz), 114.2 (d, J13CF = 9.0 Hz), 113.0 (d, J13CF = 20.3 Hz), 111.1 (d, J13CF = 1.8 Hz), 110.2, 108.3, 56.6, 56.4, 48.2. 19F NMR (300 MHz, DMSO-d6): −133.7 (dd, JHF = 10.2, 8.4 Hz, 1F), −136.1 (dd, JHF = 11.1, 9.8 Hz, 1F). IR (neat): 2941, 2160, 1686, 1605, 1571, 1495, 1446, 1329, 1223, 1179, 1072, 1029. MS (APPI, MeCN + CH2Cl2): m/z 314.1 [M•]+. HRMS: m/z calcd for C18H12F2O3•+ [M•+], 314.0755; found, 314.0729. Dinaphathalenyl Cyclooctyn-1-one (2b). The compound was prepared according to general protocol E from compound 1b (20 mg, 0.06 mmol). Purification by flash chromatography on silica gel (gradient heptane to heptane/EtOAc 95:5 (v/v) in 25 min) afforded pure 2b (17.4 mg, 87%). Mp = 156−159 °C. 1H NMR (500 MHz, DMSO-δ6): 8.52 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.12 (d, J = 8.6 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.81−7.71 (m, 4H), 7.69−7.62 (m, 2H), 4.91 (d, J = 13.3 Hz, 1H), 4.15 (d, J = 13.3 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-δ6): 198.7, 154.0, 146.4, 133.9, 133.5, 132.9, 128.9, 128.8, 128.5, 128.5, 128.4, 128.2 (2C), 127.5, 127.0, 126.4, 124.8, 124.6, 122.4, 120.6, 120.3, 116.9, 109.4, 43.5. IR (neat): 3056, 2926, 2147, 1678, 1589, 1433, 1396, 1336, 1231, 1054, 1024. MS (MALDI, THF): m/z 318.1 [M•]+. HRMS (ESI+, MeOH): m/z calcd for C24H15O+ [M + H+], 319.1123; found, 319.1108. 2,3,8,9-Tetramethoxy-11,12-didehydrodihydrodibenzo[a,e][8]annulen-5(6H)-one (2c). MnO2 (2.56 g, 29.4 mmol, 20 equiv) was added to a solution of TMDIBO13 1c (500 mg, 1.47 mmol) in CH2Cl2 (20 mL). The mixture was stirred at room temperature for 4 days. The mixture was filtered, and the filtrate was concentrated under a vacuum. The crude material was purified by flash chromatography (gradient heptane to heptane/EtOAc 1:1 (v/v) in 25 min) to afford pure 2c (250 mg, 50%) as a yellow solid. Mp = 175−178 °C. 1H NMR (500 MHz, CDCl3): 7.13 (s, 1H), 7.02 (s, 1H), 6.77 (s, 1H), 6.75 (s, 1H), 4.14−3.75 (m, 14H). 13C{1H} NMR (75 MHz, CDCl3): 197.8, 151.3, 150.0, 148.7, 148.3, 147.8, 141.1, 115.8, 115.7, 114.6, 111.7, 111.4, 109.5, 108.2, 108.1, 56.1 (4C), 49.3. IR (neat): 3053, 2928, 1663, 1595, 1509, 1465, 1426, 1396, 1336, 1307, 1260, 1218, 1179, 1093, 1074, 1051, 1026. MS (ESI+, MeCN + CH2Cl2): m/z 339.1 [M + H]+. HRMS: m/z calcd for C20H19O5+ [M + H+], 339.1232; found, 339.1227. 1- and 3-Benzyl-6,12-difluoro-5,11-dimethoxy-3,9-dihydro-8Hdibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-ol (8a). The compound was prepared according to general protocol F from 1a (6.1 mg, 0.019 mmol). After the mixture was stirred for 3.5 h at room temperature and after the solvent was removed, the crude product was purified by preparative TLC (heptane/EtOAc 3:7 (v/v)) then by SFC (on Viridis 2-ethylpyridine column, 12 mL/min, 150 bar, 40 °C, 20% EtOH) to afford a 70:30 mixture of 1,4- and 1,5-triazole 8a (4.4 mg, 51%) as a white solid. 1H NMR (300 MHz, CDCl3): 7.42 (d, J = 12.2 Hz, 0.7H), 7.36−7.26 (m, 4H), 7.15−7.05 (m, 2.3H), 6.88 (d, J = 8.4 Hz, 0.3H), 6.67 (d, J = 8.5 Hz, 0.7H), 6.56 (d, J = 7.6 Hz, 0.3H), 6.53 (d, J = 7.8 Hz, 0.7H), 5.78 (d, J = 15.1 Hz, 0.3H), 5.67 (d, J = 15.3 Hz, 0.7H), 5.65 (d, J = 15.1 Hz, 0.3H), 5.50 (d, J = 15.3 Hz, 0.7H), 4.76 (m, 1H), 3.91 (s, 1H), 3.85 (s, 2H), 3.65 (s, 1H), 3.61 (s, 2H), 3.51 (dd, J = 15.8, 5.1 Hz, 0.7H), 3.01 (m, 1H), 2.54 (dd, J = 13.6 Hz, 0.3H). 13C{1H} NMR (125 MHz, CDCl3): 153.6 (d, J13CF = 255.6 Hz), 150.9 (d, J13CF = 248.9 Hz), 147.7, 146.9, 137.5, 135.8, 133.8, 131.8, 129.1, 128.9, 128.6, 128.4, 127.5, 127.6, 126.7, 123.4, 121.2, 121.1, 119.2, 119.0, 115.8, 114.0, 113.6, 113.4, 113.0, 75.6, 68.1, 56.3, 56.2, 56.1, 53.1, 52.6, 45.6, 40.2. 19F NMR (300 MHz, CDCl3): −130.7 (dd, JHF = 11.9, 9.1 Hz, 0.7 F), −132.1 (bt, JHF = 9.6 Hz, 0.3 F), −136.9 (bt, JHF = 8.9 Hz, 0.3 F), −136.1 (bt, JHF = 10.1 Hz, 0.7 F). MS (ESI+, MeOH): m/z 450.1 [M + H]+. HRMS: m/z calcd for C25H22F2N3O3+ [M + H+], 450.1629; found, 450.1624. 15-Benzyl-8,15-dihydro-7H-dinaphtho[1′,2′:3,4;1″,2″:7,8]cycloocta[1,2-d][1,2,3]triazol-7-ol (8b). The compound was prepared according to general protocol F from 1b (15. 0 mg, 0.05 mmol). 8549


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1- with 3-Benzyl-5,6,11,12-tetramethoxy-1,9-dihydro-8Hdibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-one (9c). The compound was prepared according to general protocol F from 2c (15.3 mg, 0.045 mmol). After the mixture was stirred for 2 h and 40 min at room temperature and after the solvent was removed, the crude product was purified by preparative TLC (heptane/EtOAc 3:7 (v/v)) to afford a 9:1 mixture of 1,4- and 1,5-triazole 9a (11.8 mg, 55%) as a yellow solid. Mp = 149−152 °C. 1H NMR (500 MHz, CDCl3): 7.78 (s, 0.9H), 7.72 (s, 0.1H), 7.47 (s, 0.9H), 7.30−7.22 (m, 3H), 7.17 (s, 0.1H), 7.12−7.09 (m, 2H), 6.90 (s, 0.9H), 6.78 (s, 0.1H), 6.47 (s, 0.9H), 6.40 (s, 0.1H), 5.78 (d, J = 15.8 Hz, 0.9H), 5.74 (d, J = 15.7 Hz, 0.1H), 5.47 (d, J = 15.7, 0.1H), 5.32 (d, J = 15.8 Hz, 0.9H), 3.96 (s, 2.7H), 3.87 (s, 2.7H), 3.86 (s, 0.3H), 3.85 (s, 2.7H), 3.84 (s, 0.3H), 3.81 (s, 0.3H), 3.67 (d, J = 13.1 Hz, 0.1H), 3.61 (d, J = 12.1 Hz, 0.9H), 3.57 (d, J = 12.1 Hz, 0.9H), 3.49 (d, J = 13.1 Hz, 0.1H), 3.41 (s, 2.7H), 3.34 (s, 0.3H). 13C{1H} NMR (125 MHz, CDCl3): 195.6, 194.2, 152.9, 152.5, 151.7, 151.3, 150.0, 149.1, 148.8, 148.2, 146.3, 135.9, 134.5, 134.0, 129.1, 128.6, 128.4, 128.3, 127.5, 126.7, 126.4, 126.0, 119.9, 116.8, 114.7, 113.6, 112.9, 112.2, 111.5, 110.8, 110.4, 56.4, 56.2, 56.0, 55.7, 55.6, 53.0, 52.1, 47.9, 46.9. IR (neat): 2941, 1655, 1609, 1509, 1456, 1359, 1343, 1263, 1212, 1179, 1093, 1032. MS (APPI, MeCN + DMSO): m/z 471.2 [M•]+. HRMS: m/z calcd for C27H25N3O5•+ [M•+], 471.1794; found, 471.1750. Kinetics of the 1,3-Dipolar Cycloaddition Reaction. The accurate rate measurements of benzyl azide addition to alkynes 1a, 2a, and 2c were performed in MeCN solution at 25.0 ± 0.1 °C under pseudofirst-order conditions using a 25 μM concentration of alkynes and a 10- to 100-fold excess of azide. The consumption of alkynes was followed by the decay of the absorbance band of the starting material: 320 nm for 1a and for 2c and 309 nm for 2a. The pseudo-first-order rate constants were obtained by least-squares fitting of the data and were plotted as a function of azide concentration (Figure S2). Leastsquares fitting of the data to a linear equation allowed the determination of the second-order rate constant. Absorption and Fluorescence Measurements. Absorption spectra were recorded on a Varian Cary 5000 spectrophotometer. The fluorescence spectra of keto-DIBO and compounds 1b, 2a, 2b, and 2c were recorded on a Fluoromax-4 from Horiba Jobin Yvon. The quantum yield was measured in MeOH using quinine sulfate as a standard. Fluorescence Microscopy. The chloroquine-resistant strain FcB1/ Colombia of P. falciparum was maintained in vitro on human erythrocytes in RPMI 1640 medium supplemented by 8% (v/v) heatinactivated human serum, at 37 °C, under an atmosphere of 3% CO2, 6% O2, and 91% N2. Bloodstream forms of Trypanosoma brucei brucei strain 93 were cultured in HMI9 medium supplemented with 10% fetal calf serum at 37 °C under an atmosphere of 5% CO2. Experiments were performed on log-phase T. brucei cultures. For fluorescence microscopy, protozoan cell cultures were washed twice in phosphate-buffered saline (PBS) and incubated with 0, 10, or 50 μM of compounds 2a and 2c in PBS for 30 min, at 37 °C. After two washes in PBS, parasites were mounted on slides and viewed using a Nikon Eclipse TE 300DV inverted microscope with a 100× oil objective. Fluorescence was recorded with filters at λexc 352−402 nm and λem > 412 nm using a back illuminated cooled detector (Roper Scientific, France). Images of controls and treated cultures were acquired using the Metamorph software (Roper Scientific) and processed using the ImageJ software (https://imagej.nih.gov/ij/) under the same conditions for comparison.

After the mixture was stirred for 4.5 days at room temperature and after the solvent was removed, the crude product was purified by preparative TLC (heptane/EtOAc 9:1 (v/v)) to afford pure 8b (8.6 mg, 41% with 80% conversion) as a white solid. Mp = 240−245 °C. 1 H NMR (300 MHz, CDCl3): 8.00−7.93 (m, 1H), 7.87−7.78 (m, 2H), 7.69−7.59 (m, 4H), 7.54−7.30 (m, 5H), 7.13−7.06 (m, 1H), 7.03−7.96 (m, 2H), 6.52 (bd, J = 7.1 Hz, 2H), 5.79 (d, J = 14.5 Hz, 1H), 5.06 (d, J = 14.5 Hz, 1H), 4.71 (dd, J = 11.2, 4.2 Hz, 1H), 3.70 (dd, J = 17.0, 11.2 Hz, 1H), 3.51 (dd, J = 17.0, 4.2 Hz, 1H). 13C{1H} NMR (75 MHz, CDCl3): 150.6, 143.2, 134.9, 133.4, 132.8, 132.7, 131.7, 131.1, 130.1, 129.7, 129.1, 128.9, 128.5 (2C), 128.3 (3 C), 127.9, 127.3, 126.7, 126.4, 126.2, 126.2, 124.5, 124.1, 122.0, 120.9, 119.8, 69.2, 54.0, 43.1. IR (neat): 3411, 2944, 1596, 1505, 1456, 1433, 1384, 1342, 1296, 1263, 1236, 1204, 1146, 1102, 1074, 1034. MS (ESI+, MeCN + CH2Cl2): m/z 454.2 [M + H]+. HRMS: m/z calcd for C31H24N3O+ [M + H+], 454.1919; found, 454.1917. 1- and 3-Benzyl-6,12-difluoro-5,11-dimethoxy-3,9-dihydro-8Hdibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8-one (9a). The compound was prepared according to general protocol F from 2a (16.4 mg, 0.05 mmol). After the mixture was stirred for 2.5 h at room temperature and after the solvent was removed, the crude product was purified by preparative TLC (heptane/EtOAc 1:1 (v/v)) to afford a 83:17 mixture of 1,4- and 1,5-triazole 9a (15.1 mg, 65%) as an orange solid. Mp = 198−200 °C. 1H NMR (500 MHz, CDCl3): 8.01 (d, J = 12.8 Hz, 0.83H), 7.94 (d, J = 12.3 Hz, 0.17H), 7.54 (d, J = 8.2 Hz, 0.83H), 7.43 (d, J = 11.1 Hz, 0.17H), 7.36−7.30 (m, 0.5H), 7.30− 7.27 (m, 2.5H), 7.16 (bd, J = 7.5 Hz, 0.34 H), 7.09 (m, 1.66H), 7.0 (d, J = 8.3 Hz, 0.83H), 6.92 (d, J = 11.1 Hz, 0.83H), 6.91 (d, J = 8.0 Hz, 0.17H), 6.54 (d, J = 7.8 Hz, 0.17H), 5.78 (d, J = 15.9 Hz, 0.17H), 5.67 (d, J = 15.3 Hz, 0.83H), 5.51 (d, J = 15.9 Hz, 0.17H), 5.50 (d, J = 15.3 Hz, 0.83H), 4.00 (s, 2.5H), 3.91 (s, 2.5H), 3.87 (s, 0.5H), 3.74 (d, J = 13.6 Hz, 0.17H), 3.62 (d, J = 12.1 Hz, 0.83H), 3.54 (d, J = 12.1 Hz, 0.83H), 3.54 (d, J = 13.6 Hz, 0.17H), 3.43 (s, 0.5H). 13 C{1H} NMR (125 MHz, CDCl3): 193.0 (2C), 152.0 (d, J13CF = 250.6 Hz), 151.6 (d, J13CF = 248.0 Hz), 151.2 (d, J13CF = 246.3 Hz), 151.0 (d, J13CF = 248.0 Hz), 151.0, 150.2, 150.0, 146.0, 135.4, 134.8, 134.6, 133.1, 131.9, 131.9, 129.3, 129.0, 128.6, 127.2, 126.7, 118.9, 117.9 ((d, J13CF = 19.9 Hz), 117.8, 116.8, 116.7, 115.6 (d, J13CF = 18.7 Hz), 115.4, 113.7 (d, J13CF = 2.2 Hz), 113.3, 56.5, 56.4, 56.3, 55.9, 53.1, 52.6, 48.3, 47.0. 19F NMR (300 MHz, CDCl3): −131.3 (dd, JHF = 12.3, 7.5 Hz, 0.17 F), −134.5 (dd, JHF = 12.8, 8.3 Hz, 0.83 F), −134.9 (dd, JHF = 11.1, 8.2 Hz, 0.83 F), −135.6 (dd, JHF = 11.1, 8.3 Hz, 0.17 F). IR (neat): 2948, 1655, 1609, 1511, 1457, 1360, 1342, 1264, 1212, 1180, 1093, 1033, 900, 891, 833, 735, 707. MS (APPI, MeCN + CH2Cl2): m/z 447.1 [M•]+. HRMS: m/z calcd for C25H19F2N3O3•+ [M•+], 447.1394; found, 447.1351. 15-Benzyl-8,15-dihydro-7H-dinaphtho[1′,2′:3,4;1″,2″:7,8]cycloocta[1,2-d][1,2,3]triazol-7-one (9b). The compound was prepared according to general protocol F from 2b (14.6 mg, 0.05 mmol). After the mixture was stirred for 3 days at room temperature and after the solvent was removed, the crude product was purified by preparative TLC (heptane/EtOAc 1:1 (v/v)) to afford a 73:27 mixture of 1,4- and 1,5-triazole 9b (12.0 mg, 58%) as a white solid. Mp = 230−234 °C. 1H NMR (300 MHz, CDCl3): 8.15 (d, J = 8.4 Hz, 0.73H), 8.02 (d, J = 8.4 Hz, 0.27H), 8.02−7.95 (m, 1.46H), 7.90 (d, J = 8.7 Hz, 0.27H), 7.81−7.67 (m, 4.1H), 7.60−7.37 (m, 4H), 7.28 (d, J = 8.5 Hz, 0.73H), 7.23−6.98 (m, 4.8H), 6.50 (d, J = 7.1 Hz, 0.27H), 5.76 (d, J = 14.6 Hz, 0.27H), 5.67 (s, 1.46H), 4.83 (d, J = 14.6 Hz, 0.27H), 4.36 (d, J = 15.2 Hz, 0.37H), 4.31 (d, J = 14.7 Hz, 0.73H), 3.57 (d, J = 15.2 Hz, 0.27H), 3.50 (d, J = 14.7 Hz, 0.73H). 13 C{1H} NMR (75 MHz, CDCl3): 198.4, 198.2, 147.9, 145.2, 136.2, 135.6, 135.0, 134.7, 134.3, 134.2, 134.0, 133.7, 132.9, 132.3, 132.1, 131.9, 131.1, 130.2, 130.1, 129.3, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 128.1, 127.9, 127.8, 127.6, 127.4, 127.3, 127.0, 125.9, 125.8, 125.5, 125.4, 125.0, 124.6, 124.2, 123.3, 54.1, 52.8, 46.6, 46.4. IR (neat): 3676, 3408, 2988, 1958, 1658, 1617, 1597, 1504, 1496, 1457, 1382, 1342, 1302, 1264, 1246, 1219, 1143, 1074, 1065, 1034. MS (ESI+, MeCN + CH2Cl2): m/z 452.2 [M + H]+. HRMS: m/z calcd for C31H22N3O+ [M + H+], 452.1763; found, 452.1752.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.9b00895. Crystallographic data for 6b, kinetics measurements of SPAAC rate constants, and 1H, 13C, and 19F NMR spectra for synthesized compounds (PDF) 8550


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(15) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition. Chem. Commun. 2010, 46, 97. (16) Kuzmin, A.; Poloukhtine, A.; Wolfert, M. A.; Popik, V. V. Surface Functionalization Using Catalyst-Free Azide-Alkyne Cycloaddition. Bioconjugate Chem. 2010, 21, 2076. (17) McNitt, C. D.; Popik, V. V. Photochemical generation of oxadibenzocyclooctyne (ODIBO) for metal-free click ligations. Org. Biomol. Chem. 2012, 10, 8200. (18) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. Rapid Cu-Free Click Chemistry with Readily Synthesized Biarylazacyclooctynones. J. Am. Chem. Soc. 2010, 132, 3688. (19) Mbua, N. E.; Guo, J.; Wolfert, M. A.; Steet, R.; Boons, G.-J. Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) Reveal New Features of Glycoconjugate Biosynthesis. ChemBioChem 2011, 12, 1912. (20) Friscourt, F.; Fahrni, C. J.; Boons, G.-J. A Fluorogenic Probe for the Catalyst-Free Detection of Azide-Tagged Molecules. J. Am. Chem. Soc. 2012, 134, 18809. (21) Sutton, D. A.; Popik, V. V. Sequential Photochemistry of Dibenzo[a,e]dicyclopropa[c,g][8]annulene-1,6-dione: Selective Formation of Didehydrodibenzo[a,e][8]annulenes with Ultrafast SPAAC Reactivity. J. Org. Chem. 2016, 81, 8850. (22) Wong, H. N. C.; Garratt, P. J.; Sondheimer, F. Unsaturated eight-membered ring compounds. XI. Synthesis of sym-dibenzo-1,5cyclooctadiene-3,7-diyne and sym-dibenzo-1,3,5-cyclooctatrien-7-yne, presumably planar conjugated eight-membered ring compounds. J. Am. Chem. Soc. 1974, 96, 5604. (23) Chadwick, R. C.; Van Gyzen, S.; Liogier, S.; Adronov, A. Scalable Synthesis of Strained Cyclooctyne Derivatives. Synthesis 2014, 46, 669. (24) Debets, M. F.; Prins, J. S.; Merkx, D.; van Berkel, S. S.; van Delft, F. L.; van Hest, J. C. M.; Rutjes, F. P. J. T. Synthesis of DIBAC analogues with excellent SPAAC rate constants. Org. Biomol. Chem. 2014, 12, 5031.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Rachel Méallet-Renault: 0000-0002-1083-6288 Joëlle Dubois: 0000-0002-5162-2438 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Fondation pour la Recherche Médicale, DCM20121225779, to J.D. including a Ph.D. grant for V.T. G.P. thanks the Region Ile de France for a postdoctoral fellowship (DIM-Malinf). The authors thank O. Thoison for SFC purification, J. F. Gallard for NMR spectra, N. Elie for APPI measurements, V. Guérineau for Maldi analyses, P. Retailleau for X-ray analysis, the CeMIM platform from MNHN (C. Willig and M. Gèze) for the fluorescence microscopy imaging, and ICSN and CNRS for financial support. Dr Gilles Clavier is greatly acknowledged for helpful discussions and advice.

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REFERENCES

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