Structure–Activity Relationship of the Linker Moiety in Photoinduced Electron Transfer-Driven Nitric Oxide Releasers

Abstract

Nitric oxide (NO) is involved in numerous physiological activities including vasodilation, neurotransmission, and immune system regulation. NO-releasing small compounds are used to investigate the physiological activity of NO and to treat circulatory diseases, such as hypertension and angina pectoris. Among them, light-controllable NO releasers (caged NOs) enable spatiotemporal control of NO’s bioactivities. We previously reported NORD-1, a photoinduced electron transfer (PeT)-driven NO releaser that responds to red light. In the PeT-driven NO releasers, the NO release is triggered by photoinduced electron transfer from the N-nitrosoaminophenol to the light-harvesting dye. However, additional functionalization of PeT-driven NO releasers is required to enable introduction of tissue targeting groups or novel release triggers. As such, structure–activity relationship studies are needed to identify a suitable site for modification so as not to affect the NO-releasing efficiency of the PeT. Here, we investigated the functional impact of introducing substituents into the linker region connecting the light-harvesting antenna and NO releasing moiety. Although introduction of various substituents elicited only minor changes in NO-releasing efficiency and vasodilation activity, dialkylamino groups induced pH-dependent changes in NO-releasing reactivity. The structure–activity relationship of the linker moiety could provide fruitful information in further functionalizing PeT-driven NO releasers for biological applications.

Introduction

Nitric oxide (NO) is biosynthesized from arginine in vivo and is known to be an important molecule related to vasodilation, neurotransmission, and immune response.^1–3) Under ambient conditions NO is a gaseous compound that readily reacts with biomolecules.⁴⁾ Thus, NO-releasing molecules (NO releasers) are used in NO biological applications, such as the treatment of hypertension and angina pectoris.^5,6) Some of the NO releasers are those in which NO release is controlled by light (caged NOs). Due to the spatiotemporal tunability of NO release, these caged NOs are valuable research tools as well as novel therapeutic agents for the treatment of cardiovascular diseases.^2,3) Typically, bond cleavage in photo-responsive compounds is induced by UV light. However, the application of UV light in biological systems is limited because short wavelength radiation can readily damage cells and tissues.^7,8) To overcome the drawback, we have developed caged NOs that can release NO upon exposure to even low-energy visible light via photoinduced electron transfer (PeT).^9–15) The PeT-driven NO releasers comprise an antenna moiety, which absorbs light, an NO-releasing moiety, and a linker that connects them together. Upon photoexcitation of the antenna moiety, PeT is induced from the NO-releasing moiety to the antenna site to generate an unstable phenoxyl radical that liberates NO (Fig. 1a). A previous in vivo study demonstrated NORD-1, a red light-responsive NO releaser, successfully photoregulated intracavernosal pressure in the rat penis.¹³⁾ To achieve more precise spatiotemporal control of pharmacological activity, functionalization studies (e.g., introduction of a tissue-targeting group) have been conducted involving clinically utilized drugs.^16,17) For PeT-driven NO releasers, we reported that introduction of various functional groups to the NO-releasing moiety decreases their NO-releasing efficiency.¹⁵⁾ Here, we aimed to identify a suitable site for the introduction of functional groups on PeT-driven NO releasers. Specifically, we focused on the linker region connecting the antenna and NO-releasing moiety (Fig. 1b).

Fig. 1. (a) A Plausible Mechanism of Photoinduced NO Release from PeT-Driven Caged NOs; (b) Structure of NORD-1 Derivatives with Various Functional Groups Substituted on the Linker Moiety

Results and Discussion

It was previously reported that an aryl group substituted at the 9-position of the xanthene ring can cause PeT.¹⁸⁾ Thus, it was considered that a substituent on the linker moiety at the 9-position of the PeT-driven NO releaser might alter the NO-releasing efficiency by modifying the PeT process. Thus, we designed a series of PeT-driven NO releasers by incorporating various electron donating/withdrawing groups on the linker moiety to identify substituents that affect the NO release reaction (Fig. 1b, 2–8). These derivatives were synthesized according to Chart 1. Briefly, a formyl group of 2-bromobenzaldehyde derivatives (10–16) was converted to a dimethyl acetal group. After lithiation of bromobenzene derivatives 17–23, compound 24 was added to form a Si-rhodamine scaffold followed by deprotection of the dimethyl acetal group under acidic conditions to obtain 25–31.¹⁹⁾ After reductive amination with 32 and N-nitrosation to introduce the NO-releasing moiety, the tert-butyldimethylsilyl (TBS) protection was removed using NaF/HF buffer to obtain 2–8.¹³⁾

Chart 1. Synthetic Scheme to Obtain 2–8

To explore the effect of the substituents on the photochemical properties of the PeT-driven releasers, the absorbance/fluorescence spectra of each compound were measured (Fig. 2). While compound 1–8 showed almost the same maximum absorbance wavelength (λ_max) and absorption coefficient (ε_max), fluorescence quantum yields (Ф_F) of 7 and 8 were lower than those of the other compounds. The difference in quantum yield was probably due to the dimethyl/diethylamino group having strong electron-donating properties, which likely increases the electron density of the linker site thereby promoting PeT from the linker to the antenna.²⁰⁾

Fig. 2. Absorbance (Solid Line) and Fluorescence (Dashed Line) Spectra of a Solution of 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), 6 (f), 7 (g), and 8 (h)

Each compound (10 μM) was dissolved in HEPES buffer (100 mM, pH 7.4, DMSO 0.1%). Maximum absorption wavelength (λ_max for absorbance), maximum extinction coefficient (ε_max), maximum fluorescence wavelength (λ_max for fluorescence), and fluorescence quantum yield (Ф_F) of each compound are given at the bottom of the table.

The influence of the amino groups for 7 and 8 were investigated by measuring their fluorescence properties when dissolved in various buffers at different pH. The results were then compared with those of 1, which does not possess an amino group on the linker (Fig. 3). While the absorbance spectra in various buffer solutions were similar for 1, 7, and 8 (Figs. 3a–3c, 3g), fluorescence intensity of 7 or 8 was significantly raised under acidic conditions (Fig. 3e, 3f, 3h). These findings are explicable because the amino groups of 7 and 8 are protonated under acidic conditions, which would inhibit PeT from the linker moiety. For 8, the fluorescence intensity decreased sharply at pH 4–6 (Figs. 3f, 3h), and the pK_a of protonated 8 was calculated to be 5.3.

Fig. 3. Absorbance (a–c) and Fluorescence (d–f) Spectra of a Solution of 1 (a, d), 7 (b, e), and 8 (c, f) under Different pH Conditions (3–9); The Absorbance at 655 nm (g) and Fluorescence Intensity at 680 nm (h) of Each Compound Was Plotted against pH

Next, we investigated the photoresponsive NO-releasing activity of each compound with the aid of an NO electrode²¹⁾ (Fig. 4). A solution of each compound (10 μM) in N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (100 mM, pH 7.3, 0.1% dimethyl sulfoxide (DMSO)) was irradiated with red light (660 nm, 144 mW cm⁻²) while measuring NO release using an NO electrode (ISO-NOP). The maximum NO concentrations of 1–6 were >1.5 μM (Figs. 4a–4f) while those of 7 and 8 were <0.5 μM (Figs. 4g, 4h). These findings suggest that PeT from the N,N-dialkylaminophenyl groups on the linker moiety of 7 and 8 compete with that of the NO-releasing moiety. To suppress PeT from the linker moiety of 8 by protonation of the diethylamino group, a solution of 8 in a weakly acidic solution (phosphate buffer, pH 5.0) was irradiated (Fig. 4i). Under these mildly acidic conditions the maximum NO concentration was 10-fold of that observed at pH 7.4.

Fig. 4. NO Release Detected by an NO Electrode from a 10 μM Solution of Each Compound (HEPES Buffer 100 mM, pH 7.3, DMSO 0.1%) of 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), 6 (f), 7 (g), 8 (h), (i) NO Release from a 10 μM Solution of 8 at pH 5.0 (Solid Line) and pH 7.4 (Dashed Line)

To investigate NO release efficiency, the NO release quantum yields (Φ_NO) were calculated based on the previously reported value for 1¹³⁾ (Fig. 5a). The Ф_NO values of 1–6 were calculated to be over 3.0 × 10⁻³, while those of 7 and 8 were under 3.0 × 10⁻³ (Fig. 5a). As well as the results in Fig. 4, dialkylamino groups suppressed the NO release. To assess the relationship between the NO release efficiency and electron donating/withdrawing capability of each substituent, the Ф_NO values were plotted for Hammett reaction constants of the para-position²²⁾ (Fig. 5b). Although a clear relationship was not observed in the range of −0.5 to 1 for σ_p, Ф_NO values tended to decrease in a range lower than −0.5. These results suggest that strong electron-donating groups on the linker moiety can suppress NO release by competing with the PeT (Fig. 5c). Moreover, substitution of a functional group on the NO releaser, such as an alkoxy or alkyl group, could generate a suitable linker that would not interfere with the liberation of NO.

Fig. 5. (a) Φ_NO of Each Compound; (b) Φ_NO of Each Compound Was Plotted against σ_p of the Substituents Introduced on the Linker Moiety; (c) Plausible Mechanism for the pH-Dependent Changes to the NO Release Efficiency of 8

Based on our finding that an alkoxy group at the linker moiety showed a reduced effect on the NO releasing efficiency of PeT-driven NO releasers, we designed 9 possessing a sulfonate moiety as a model functional group via an alkoxy linker (Fig. 6a, see Supplementary Materials for synthesis of 9). It was reported that a sulfonate group can both improve water solubility and increase bioavailability by disturbing interactions with serum proteins.^23,24) The photochemical properties of 9 (i.e., absorption/fluorescence spectra and Ф_NO) were almost identical to those of 1 (Fig. 6b). These results indicate that an anionic functional group tethered by an alkyl chain does not affect the photochemical properties of PeT-driven NO releasers. In addition, we also investigated whether 9 displayed photoresponsive vasodilation activity using a rat aortic strip (Fig. 6c). NO can activate soluble guanylyl cyclase in endothelial cells to increase production of cyclic guanosine monophosphate followed by relaxation of smooth muscle.¹⁾ For ex vivo experiments of vasodilation, a rat aorta strip was placed in a Magnus tube filled with Krebs buffer. To block endogenous NO generation, an inhibitor of nitric oxide synthase (l-nitroarginine methyl ester; l-NAME) was added prior to noradrenaline (NA)-induced precontraction. Irradiation of the aorta with red light (660 nm) in the absence of 9 did not induce vasodilation. However, light-dependent vasodilation was evident after the introduction of 9 into the tube. Moreover, this light-dependent vasodilation in the presence of 9 was fully reversible. These results indicate that introduction of a functional group via an alkoxy linker on a PeT-driven NO releaser had little effect on either its photochemical properties or its NO release efficiency. Furthermore, the functionalized compound 9 showed potent vasodilation activity.

Fig. 6. (a) Structure of 9 Possessing a Sulfonate Group via an Alkoxy Linker

(b) Absorbance (solid line) and fluorescence (dashed line) spectra of a solution of 9. The λ_max for absorbance, ε_max, λ_max for fluorescence, Ф_F, and Ф_NO of 9 were compared with those of 1 and the results are summarized in the table. (c) An experimental setting of vasodilation test (left) and changes in the tension of rat aorta ex vivo (right) induced by red-light-mediated NO release from 9 (1 μM) in the presence of a nitric oxide synthase inhibitor, N^G-nitro-l-arginine methyl ester (l-NAME, 100 μM). A rat aorta in a glass tube was treated with noradrenaline (NA, 10 μM) to induce precontraction.

In conclusion, we synthesized NORD-1 derivatives with various functional groups attached to the linker moiety and investigated the structure–activity relationship. While dialkylamino groups suppressed NO release efficiency, the other substituents tested in this study had no significant effect on the liberation of NO. It is probable that the strong electron-donating groups induced PeT from the linker moiety to compete with NO release. Compound 9, which possesses a sulfonate group on an alkoxy linker, showed comparable NO release efficiency to that of NORD-1 (1) as well as potent vasodilation activity following exposure to light. These results indicate that a functional group can be bound to the linker moiety via an alkoxy group without significantly altering the activity of the NO releaser. We believe the structure–activity relationship of the linker moiety investigated in this study should act as a valuable guide in further functionalizing PeT-driven NO releasers for biological applications, such as dual-lock type NO releaser triggered by pH and light or novel PeT-type NO releasers having functional groups to target specific organs.

Experimental

General Methods

Proton nuclear magnetic spectra (¹H-NMR) were recorded on a JEOL ECS-400 spectrometer (Tokyo, Japan) in the indicated solvent. Chemical shifts (δ) are reported in parts per million relative to the internal standard, tetramethylsilane. Reagents used in the synthesis were purchased from Tokyo Chemical Industry (Tokyo, Japan), FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Merck (Darmstadt, Germany), Kanto Chemical (Tokyo, Japan), and BLD Pharmatech (Hyderabad, India), unless otherwise noted. When required, solvents were dehydrated using Molecular Sieves 3A 1/16 (FUJIFILM Wako Pure Chemical Corporation). TLC was performed using TLC Silica gel 70 F254 Glass plates (FUJIFILM Wako Pure Chemical Corporation). Medium pressure liquid chromatography (MPLC) was performed using Smart Flash Premium (Yamazen, Osaka, Japan) and Universal Premium (Yamazen, Silica Gel or ODS) as the separation column and injection column, respectively.

General Method for Preparation of 17–23

To a solution of 2-bromobenzaldehyde derivative (10–16, 1.0 equivalent (equiv.)) in anhydrous MeOH (0.50 M) was added trimethyl orthoformate (3.0 equiv.) and H₂SO₄ (0.10 equiv.) under an argon balloon at room temperature. After stirring at 70°C overnight, the mixture was quenched with sat. NaHCO₃. The mixture was extracted with CH₂Cl₂ and the organic layer dried over Na₂SO₄. Filtration and evaporation gave 17–23.

Preparation of 17

Compound 10 (598 mg, 2.0 mmol) was used as a substrate to obtain 17 according to the general method (602 mg, quant.): ¹H-NMR (400 MHz, CDCl₃) δ: 7.86 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 3.36 (s, 6H).

Preparation of 18

Compound 11 (439 mg, 2.0 mmol) was used as a substrate to obtain 18 according to the general method (453 mg, 85%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.61 (d, J = 2.5 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.21–7.12 (m, 1H), 3.31 (s, 6H).

Preparation of 19

Compound 12 (406 mg, 2.0 mmol) was used as a substrate to obtain 19 according to the general method (412 mg, 83%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.50 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 9.4 Hz, 1H), 6.98–6.90 (m, 1H), 3.32 (s, 6H).

Preparation of 20

Compound 13 (398 mg, 2.0 mmol) was used as a substrate to obtain 20 according to the general method (397 mg, 81%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.52–7.36 (m, 2H), 7.03 (d, J = 8.3 Hz, 1H), 3.40 (s, 6H), 2.29 (s, 3H).

Preparation of 21

Compound 14 (430 mg, 2.0 mmol) was used as a substrate to obtain 21 according to the general method (428 mg, 82%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.41 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 3.2 Hz, 1H), 6.77–6.72 (m, 1H), 3.78 (s, 3H), 3.35 (s, 6H).

Preparation of 22

Compound 15 (435 mg, 1.9 mmol) was used as a substrate to obtain 22 according to the general method (512 mg, quant.): ¹H-NMR (400 MHz, CDCl₃) δ: 7.33 (d, J = 9.1 Hz, 1H), 6.96 (s, 1H), 6.55 (d, J = 13.6 Hz, 1H), 3.41 (s, 6H), 2.92 (s, 6H).

Preparation of 23

Compound 16 (1.6 g, 6.1 mmol) was used as a substrate to obtain 23 according to the general method (1.8 g, quant.): ¹H-NMR (400 MHz, CDCl₃) δ: 7.31 (d, J = 8.9 Hz, 1H), 6.87 (dd, J = 7.2, 3.1 Hz, 1H), 6.53–6.48 (m, 1H), 3.44–3.37 (m, 6H), 3.33 (dd, J = 5.5, 1.6 Hz, 4H), 1.14–1.11 (m, 6H).

General Method for Preparation of 25–31

To a solution of 17–23 (1.1 equiv. for 17, 20, 21, 2.0 equiv. for 18, 19, 22, 23) in anhydrous tetrahydrofuran (THF) (0.048 M) was added dropwise sec-BuLi (2.2 equiv.) under an argon balloon at −78°C. The mixture was stirred for 5 min, and then 24 (1.0 equiv.) in THF (0.048 M) was added. The reaction was warmed to room temperature, stirred overnight, and quenched with sat. NaHCO₃. The mixture was extracted with Et₂O and the organic layer dried over Na₂SO₄. After filtration and evaporation, the residue was dissolved in a mixture of MeCN/2 n HCl (1/1, 30 mL). After stirring at room temperature overnight, the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄. Filtration, evaporation, and purification by column chromatography (SiO₂, CH₂Cl₂/MeOH = 90/10 to 50/50) gave 25–31 as a dark blue solid.

Preparation of 25

Compound 17 (96 mg, 0.32 mmol) was used as a substrate to obtain 25 according to the general method (58 mg, 0.11 mmol, 39%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.81 (s, 1H), 7.73 (s, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 9.6 Hz, 1H), 6.66 (d, J = 8.7 Hz, 1H), 3.48 (s, 12H), 0.60 (s, 6H).

Preparation of 26

Compound 18 (170 mg, 0.64 mmol) was used as a substrate to obtain 26 according to the general method (159 mg, 0.33 mmol, 57%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.72 (s, 1H), 8.07 (s, 1H), 7.76–7.76 (m, 1H), 7.46 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.66–6.65 (m, 2H), 6.37 (s, 1H), 3.37 (s, 12H), 0.64 (s, 6H).

Preparation of 27

Compound 19 (160 mg, 0.64 mmol) was used as a substrate to obtain 27 according to the general method (198 mg, 0.43 mmol, 73%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.71 (s, 1H), 8.08–8.02 (m, 1H), 7.77 (d, J = 12.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 6.91–6.86 (m, 3H), 6.65 (d, J = 8.7 Hz, 1H), 3.16 (s, 12H), 0.67 (s, 6H).

Preparation of 28

Compound 20 (78 mg, 0.32 mmol) was used as a substrate to obtain 28 according to the general method (65 mg, 0.14 mmol, 48%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.76 (s, 1H), 7.89 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.23–7.18 (m, 3H), 6.96 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 9.6 Hz, 2H), 3.40 (s, 12H), 2.56 (s, 3H), 0.67–0.62 (m, 6H).

Preparation of 29

Compound 21 (84 mg, 0.32 mmol) was used as a substrate to obtain 29 according to the general method (88 mg, 0.18 mmol, 63%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.71 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 2.7 Hz, 1H), 7.32 (dd, J = 8.2, 2.5 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 9.6 Hz, 2H), 6.65 (dd, J = 9.5, 2.2 Hz, 3H), 3.98 (s, 3H), 3.41 (s, 12H), 0.65 (d, J = 18.1 Hz, 6H).

Preparation of 30

Compound 22 (175 mg, 0.64 mmol) was used as a substrate to obtain 30 according to the general method (37 mg, 0.080 mmol, 13%): ¹H-NMR (400 MHz, CDCl₃) δ: 9.95 (d, J = 0.9 Hz, 1H), 7.41–7.34 (m, 2H), 7.20–7.17 (m, 3H), 7.13–7.00 (m, 2H), 6.84–6.78 (m, 1H), 6.60–6.56 (m, 1H), 3.00 (d, J = 1.6 Hz, 13H), 2.92 (d, J = 1.4 Hz, 6H), 0.60–0.57 (m, 6H).

Preparation of 31

Compound 23 (193 mg, 0.64 mmol) was used as a substrate to obtain 31 according to the general method (121 mg, 0.23 mmol, 36%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.27 (d, J = 9.1 Hz, 1H), 7.07 (d, J = 9.1 Hz, 1H), 6.96 (ddd, J = 9.2, 2.9 Hz, 2H), 6.89–6.84 (m, 1H), 6.72–6.55 (m, 4H), 3.41–3.36 (m, 6H), 2.92 (s, 12H), 1.16 (t, J = 7.0 Hz, 4H), 0.64–0.52 (m, 6H).

General Method for Preparation of 33–39

To a solution of 25–31 in a mixture of anhydrous CH₂Cl₂/AcOH (10/1, 0.1 M) was added 32 (1.1 equiv.). The reaction mixture was stirred for 20 min at room temperature before addition of NaBH(OAc)₃ (3.1 eq). After further stirring for 20 min, the reaction mixture was quenched with 2 M HCl, extracted with CH₂Cl₂, and the organic layer dried over Na₂SO₄. After filtration and evaporation, the residue was dissolved in AcOH (0.05 M) before adding 0.12 M NaNO₂ (aq.) (1.2 equiv.). After stirring for 20 min at 0°C, the mixture was quenched with 1 M HCl and extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄. Filtration, evaporation, and purification by column chromatography (SiO₂, CH₂Cl₂/MeOH = 90/10 to 50/50) gave 33–39 as a dark blue solid.

Preparation of 33

Compound 25 (67 mg, 0.13 mmol) was used as a substrate to obtain 33 according to the general method (50 mg, 0.070 mmol, 51%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.69 (d, J = 7.1 Hz, 1H), 7.32–7.30 (m, 4H), 7.16 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 9.6 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.67 (dd, J = 9.4, 2.5 Hz, 2H), 4.85 (s, 2H), 0.97 (s, 9H), 0.64 (s, 6H), 0.16 (s, 6H).

Preparation of 34

Compound 26 (87 mg, 0.18 mmol) was used as a substrate to obtain 34 according to the general method (54 mg, 0.075 mmol, 42%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.40 (d, J = 7.6 Hz, 1H), 7.28 (s, 2H), 7.16 (d, J = 6.6 Hz, 2H), 7.09 (d, J = 7.8 Hz, 1H), 7.04–6.98 (m, 3H), 6.85–6.80 (m, 2H), 6.67 (d, J = 6.9 Hz, 2H), 4.77 (s, 2H), 3.43 (s, 12H), 0.96 (s, 9H), 0.64 (s, 6H), 0.19 (s, 6H).

Preparation of 35

Compound 27 (84 mg, 0.18 mmol) was used as a substrate to obtain 35 according to the general method (85 mg, 0.12 mmol, 67%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.28 (s, 2H), 7.17 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 6.4 Hz, 2H), 7.04–6.98 (m, 2H), 6.82–6.76 (m, 3H), 6.70–6.64 (m, 2H), 4.78 (s, 2H), 3.44 (s, 12H), 0.96 (s, 9H), 0.64 (s, 6H), 0.19 (s, 6H).

Preparation of 36

Compound 28 (60 mg, 0.13 mmol) was used as a substrate to obtain 36 according to the general method (72 mg, 0.10 mmol, 79%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.24–7.19 (m, 3H), 7.16 (d, J = 8.7 Hz, 2H), 7.09–7.06 (m, 2H), 7.00 (d, J = 7.6 Hz, 1H), 6.87–6.82 (m, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.68–6.63 (m, 2H), 4.78 (s, 2H), 3.41 (s, 12H), 2.38 (s, 3H), 0.95 (s, 9H), 0.64 (s, 6H), 0.17 (s, 6H).

Preparation of 37

Compound 29 (62 mg, 0.13 mmol) was used as a substrate to obtain 37 according to the general method (51 mg, 0.070 mmol, 55%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.24-7.21 (m, 2H), 7.17 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 9.6 Hz, 2H), 7.03 (d, J = 8.5 Hz, 1H), 6.92 (dd, J = 8.4, 2.4 Hz, 1H), 6.78 (d, J = 8.7 Hz, 2H), 6.67 (dd, J = 9.6, 2.5 Hz, 2H), 6.59 (d, J = 2.3 Hz, 1H), 4.77 (s, 2H), 3.80 (s, 3H), 3.43 (s, 12H), 0.96 (s, 9H), 0.63 (s, 6H), 0.18 (s, 6H).

Preparation of 38

Compound 30 (37 mg, 0.080 mmol) was used as a substrate to obtain 38 according to the general method (17 mg, 0.020 mmol, 29%): ¹H-NMR (400 MHz, CDCl₃) δ: 7.69 (d, J = 7.1 Hz, 1H), 7.32–7.30 (m, 4H), 7.16 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 9.6 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.67 (dd, J = 9.4, 2.5 Hz, 2H), 4.85 (s, 2H), 0.97 (s, 9H), 0.64 (s, 6H), 0.16 (s, 6H).

General Method for Preparation 2–7

To a solution of 33–38 (1.0 equiv.) in MeCN (33 mM) was added 0.1 M NaF/HF buffer (3.1 equiv.) and the mixture stirred for 3 h at room temperature. The mixture was quenched with 0.2 M HCl and extracted with CHCl₃. The organic layer was dried over Na₂SO₄ and then evaporated. Purification of the residue by column chromatography (SiO₂, CH₂Cl₂/MeOH = 90/10 to 50/50) gave 2–7 as a dark blue solid.

Preparation of 2

Compound 33 (50 mg, 0.070 mmol) was used as a substrate to obtain 1 according to the general method (35 mg, 0.050 mmol, 78%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.28 (s, 1H), 7.71 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.17–7.12 (m, 3H), 6.93 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 9.6 Hz, 2H), 6.40 (dd, J = 9.6, 2.3 Hz, 2H), 4.94 (s, 2H), 3.42 (s, 12H), 0.69 (s, 3H), 0.52 (s, 3H). HRMS (ESI⁺) calcd, 603.23976; found, 603.23934.

Preparation of 3

Compound 34 (54 mg, 0.075 mmol) was used as a substrate to obtain 3 according to the general method (29 mg, 0.050 mmol, 64%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.30 (s, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.34 (dd, J = 8.2, 2.1 Hz, 1H), 7.08 (dd, J = 9.4, 2.5 Hz, 2H), 6.97–6.92 (m, 3H), 6.67 (d, J = 8.5 Hz, 2H), 6.61 (d, J = 9.6 Hz, 2H), 6.40 (dd, J = 9.6, 2.7 Hz, 2H), 4.86 (s, 2H), 3.41 (s, 12H), 0.67 (s, 3H), 0.52 (s, 3H). HRMS (ESI⁺) calcd, 569,21341; found, 569.21301.

Preparation of 4

Compound 35 (85 mg, 0.12 mmol) was used as a substrate to obtain 4 according to the general method (48 mg, 0.050 mmol, 68%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.35 (s, 1H), 7.21–7.17 (m, 1H), 7.10 (d, J = 3.0 Hz, 2H), 7.08–7.06 (m, 1H), 7.00–6.94 (m, 3H), 6.66 (d, J = 8.7 Hz, 2H), 6.62 (d, J = 9.6 Hz, 2H), 6.40 (dd, J = 9.6, 2.7 Hz, 2H), 4.87 (s, 2H), 3.42 (s, 12H), 0.67 (s, 3H), 0.51 (s, 3H). HRMS (ESI⁺) calcd, 553.24296; found, 553.24296.

Preparation of 5

Compound 36 (72 mg, 0.10 mmol) was used as a substrate to obtain 5 according to the general method (54 mg, 0.14 mmol, 92%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.26 (s, 1H), 7.19–7.14 (m, 2H), 7.05 (dd, J = 7.6, 2.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 7.8 Hz, 1H), 6.71–6.67 (m, 4H), 6.42–6.38 (m, 2H), 4.86 (s, 2H), 3.39 (s, 12H), 2.41 (s, 3H), 0.65 (s, 3H), 0.51 (s, 3H). HRMS (ESI⁺) calcd, 549.26803; found, 549.26715 (M⁺).

Preparation of 6

Compound 37 (51 mg, 0.070 mmol) was used as a substrate to obtain 6 according to the general method (41 mg, 0.14 mmol, 97%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.25 (s, 1H), 7.07 (s, 2H), 6.94–6.91 (m, 3H), 6.88 (d, J = 3.0 Hz, 2H), 6.73–6.67 (m, 4H), 6.41 (d, J = 9.6 Hz, 2H), 4.86 (s, 2H), 3.85 (s, 3H), 3.40 (s, 12H), 0.66 (s, 3H), 0.52 (s, 3H). HRMS (ESI⁺) calcd, 565.26294; found, 565.26235.

Preparation of 7

Compound 38 (17 mg, 0.020 mmol) was used as a substrate to obtain 7 according to the general method (3 mg, 0.0050 mmol, 24%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.26 (s, 1H), 7.12–7.05 (m, 3H), 6.92 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 9.4 Hz, 2H), 6.78 (d, J = 8.5 Hz, 1H), 6.72–6.67 (m, 2H), 6.64 (d, J = 7.6 Hz, 2H), 6.45 (d, J = 9.6 Hz, 2H), 4.85 (s, 2H), 3.39 (s, 12H), 3.01 (s, 6H), 0.66 (s, 3H), 0.51 (s, 3H). HRMS (ESI⁺) calcd, 578.29458; found, 578.29369.

Preparation of 8

To a solution of 31 (121 mg, 0.23 mmol) in anhydrous CH₂Cl₂ (2.6 mL) was added 32 (33 mg, 0.25 mmol) and AcOH (0.26 mL). The mixture was stirred for 20 min at room temperature before addition of NaBH(OAc)₃ (85 mg, 0.71 mmol). After stirring for a further 20 min, the reaction mixture was quenched with 2 M HCl and extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄. After filtration and evaporation, the residue was dissolved in AcOH (2.6 mL). To the solution was added 0.12 M NaNO₂ (aq.) (1.3 mL) at 0°C. After stirring for 20 min, the mixture was quenched with 1 M HCl and extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄. After filtration and evaporation, the residue (crude 39) was dissolved in a mixture of MeCN (4.0 mL) and 0.1 M NaF/HF buffer (4.0 mL). After stirring for 3 h at room temperature, the reaction mixture was quenched with 0.2 M HCl and extracted with CHCl₃. The organic layer was dried over Na₂SO₄. Filtration and evaporation followed by purification of the residue by column chromatography (SiO₂, CH₂Cl₂/MeOH = 90/10 to 50/50) gave 8 as a dark blue solid (70.9 mg, 0.11 mmol, 47%): ¹H-NMR (400 MHz, CDCl₃) δ: 10.46–9.94 (1H), 7.05 (d, J = 3.2 Hz, 2H), 6.92 (dd, J = 9.1, 5.9 Hz, 4H), 6.74 (d, J = 9.1 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H), 6.58 (t, J = 3.2 Hz, 2H), 6.49 (dd, J = 9.7, 2.9 Hz, 2H), 4.82 (s, 2H), 3.42–3.35 (m, 16H), 1.25–1.17 (m, 6H), 0.64 (s, 3H), 0.51 (s, 3H). HRMS (ESI⁺) calcd, 606.32588; found, 606.32474.

Measurements of Absorption and Fluorescence Spectra

Absorption spectra of a solution of each compound (10 μM) in HEPES buffer (100 mM, pH 7.3, 0.1% DMSO) were recorded on an Agilent 8453 spectroscopy system (Santa Clara, CA, U.S.A.). Fluorescence spectra of a solution of each compound (10 μM) in HEPES buffer (100 mM, pH 7.3, 0.1% DMSO) or phosphate buffer (100 mM, indicated pH, 0.1% DMSO) were recorded on a fluorescence spectrometer (RF5300-PC; Shimadzu, Kyoto, Japan). The Φ_F values were calculated with reference to the Φ_F of NORD-1 (1) as reported previously.¹⁸⁾

Determination of NO Concentration Using an NO Electrode

A solution (total volume 10 mL) of each compound (10 μM) in HEPES buffer (100 mM, pH 7.3, DMSO 0.1%) was irradiated at 37°C using a LED (CL-1501; Asahi Spectra, Tokyo, Japan) equipped with a 660 nm LED head-unit (CL-H1-660-9-1). The light intensity at 660 nm was 144 mW cm⁻². NO release was measured with an NO electrode, ISO-NOP (World Precision Instruments, Sarasota, FL, U.S.A.), and recorded on a LabChart7 (ADInstruments, Dunedin, New Zealand).

Calculation of NO Release Quantum Yield (Φ_NO)

A solution of each compound (10 μM) in 100 mM HEPES buffer (pH 7.3, total volume: 3 mL) containing 0.1% DMSO was placed in a quartz cuvette and irradiated at 655 nm for 1 min with the Xe lamp of a fluorescence spectrometer, RF5300-PC (Shimadzu). The amount of NO released was measured with an ISO-NOP (World Precision Instruments) and recorded on a LabChart7 (ADInstruments). The Φ_NO values were calculated with reference to the Φ_NO of NORD-1 (1) as reported previously.¹³⁾

Photoinduced Vasodilation with 9

All animal experiments were performed following the Guiding Principles for the Care and Use of Laboratory Animals of the Science and International Affairs Bureau of the Japanese Ministry of Education, Culture, Sports, Science and Technology. The study design was reviewed and approved by the Animal Experimentation Ethics Committee of Nagoya City University. A rat aortic strip was placed in a glass tube filled with Krebs buffer (5 mL) at 37°C. The strip was pretreated with N^G-nitro-l-arginine methyl ester hydrochloride (l-NAME, 100 μM) and noradrenaline (NA, 10 μM). After equilibration, the strip was irradiated for 3 min using a 660 nm LED (40 mW cm⁻²) and 9 (1 μM) was then added to the glass tube. Upon incubation for 3 min, the system was irradiated for a further 3 min.

Acknowledgments

This work was supported by JST-CREST (Grant No.: JPMJCR1902 to M.O.), the JSPS KAKENHI (Grant No.: 23K08766 to Y.H., Grant No.: 23K27303 to H.N.), and by Takeda Science Foundation (Grant to N.I.). We thank the Instrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University, for ESI-MS Executive Mass Spectrometer assistance and their insight and expertise that greatly contributed to this study.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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