Structure–Photoreactivity Relationship Study of Substituted 3-Hydroxyflavones and 3-Hydroxyflavothiones for Improving Carbon Monoxide Photorelease

Carbon monoxide (CO) is notorious for its toxic effects but is also recognized as a gasotransmitter with considerable therapeutic potential. Due to the inherent challenges in its delivery, the utilization of organic CO photoreleasing molecules (photoCORMs) represents an interesting alternative to CO administration characterized by high spatial and temporal precision of release. This paper focused on the design, synthesis, and photophysical and photochemical studies of 20 3-hydroxyflavone (flavonol) and 3-hydroxyflavothione derivatives as photoCORMs. Newly synthesized compounds bearing various electron-donating and electron-withdrawing groups show bathochromically shifted absorption maxima and considerably enhanced CO release yields compared to the parent unsubstituted flavonol, exceeding 0.8 equiv of released CO in derivatives exhibiting excited states with a charge-transfer character. Until now, such outcomes have been limited to flavonol derivatives possessing a π-extended aromatic system. In addition, thione analogs of flavonols, 3-hydroxyflavothiones, show substantial bathochromic shifts of their absorption maxima and enhanced photosensitivity but provide lower yields of CO formation. Our study elucidates in detail the mechanism of CO photorelease from flavonols and flavothiones, utilizing steady-state and time-resolved spectroscopies and photoproduct analyses, with a particular emphasis on unraveling the structure–photoreactivity relationship and understanding competing side processes.


■ INTRODUCTION
Carbon monoxide (CO) is a colorless, tasteless, and odorless gas 1 known for its toxic properties 2 due to competitive binding with the heme moiety of hemoglobin. 3CO is also produced endogenously in mammals due to heme catabolism 4 and acts as a neural messenger 5 and signaling molecule. 6There is growing interest in using CO as a therapeutic agent, for which cardioprotective, 7 cytoprotective, 8 and anti-inflammatory 9 effects have been recognized.Carbon monoxide has also been considered a potential antitumor agent. 10,11O-releasing molecules (CORMs) that liberate CO on demand using, for example, enzymatic 12 or ligand-exchange activations, 13 are developed to deliver CO to an organism in a controlled manner to avoid overexposure.14−17 Photoactivatable CORMs (photoCORMs) offer better spatial and temporal control of CO release.The first generation of photoCORMs was based on metal−carbonyl complexes, 18,19 but several metal-free photoCORMs have already been developed, for example, based on cyclopropenone, 20 BODIPY, 21 or αdiketone derivatives.22 Today, special attention is paid to the development of photoCORMs that absorb in the near-infrared (NIR) range of 650−900 nm, known as the phototherapeutic window.17,23 Significant developments have also been made in the field of prodrugs that release CO under physiological conditions.24−26 3-Hydroxyflavone (flavonol; Figure 1) derivatives belong to the family of flavonoids, common in all plant tissues, that possess antioxidant, anti-inflammatory, antimutagenic, and anticarcinogenic properties. 27PhotoCORMs based on a flavonol scaffold have already been explored for their biocompatibility and easily modifiable structure. Th first report of a π-extended flavonol-based photoCORM (Figure 1a) was introduced by Berreau and co-workers, releasing nearly quantitative amounts of CO upon irradiation by blue light.28 Its thione analog (3-hydroxyflavothione) allowed CO release upon irradiation at longer wavelengths (Figure 1a).29 Recently, we reported on cyanine-flavonol 30 (Figure 1b) and porphyrinflavonol 31 (Figure 1c) hybrid photoCORMs that can liberate CO upon irradiation with red to near-infrared light.
The mechanism of aerobic photodecarbonylation of flavonol derivatives 32−38 involves a singlet excited state that undergoes rapid excited-state intramolecular proton transfer (ESIPT) 39,40 in polar protic solvents followed by intersystem crossing (ISC)   to give a triplet excited state, which reacts with triplet oxygen to release CO, possibly via an endoperoxide intermediate (Scheme 1). 34,35The conjugate base of flavonol liberates CO via an oxygenation reaction with singlet oxygen formed by the triplet sensitization and partially via oxidation of the triplet excited state with 3 O 2 .Both forms also undergo inefficient photorearrangement to release CO in the absence of oxygen (not shown).
Photodecarbonylation of π-extended flavonol derivatives (Figure 1b,c) is efficient due to larger molar absorption coefficients (and thus uncaging cross section, Φ CO ε max ) of bathochromically shifted absorption maxima, which is advantageous for potential biological applications.Nevertheless, these compounds are large, and their synthesis is challenging.In this work, we aimed to increase the atom economy of photodecarbonylation utilizing minimal structural modifications of the parent flavonol and 3-hydroxyflavothione. Twenty derivatives substituted by electron-donating and electronwithdrawing groups were studied using steady-state optical, time-resolved, and NMR spectroscopies and high-resolution mass spectrometry (HRMS) to provide insights into the reaction mechanism and structure−photochemical property relationships.We show that elementary structural changes can significantly improve the properties of flavonol photoCORMs.

■ RESULTS AND DISCUSSION
In this work, we synthesized and studied photophysical and photochemical properties of 3-hydroxyflavone (1) derivatives 2−12 and 3-hydroxyflavothione (13) derivatives 14−18, bearing various substituents in positions C6, C7 (ring A), and C4′ (ring B), and 3-methoxyflavone (19) and 3methoxyflavothione (20) (Figure 2) to demonstrate the effects of diverse structural features on their CO photoreleasing abilities.The main aim of this investigation was to understand the mechanism of their phototransformations using steadystate and time-resolved absorption spectroscopies and modify their structures to bathochromically shift their absorption maxima and improve the chemical and quantum yields of CO release by minimizing the formation of undesired sideproducts.Scheme 1. Reported Decarbonylation Mechanism for π-Extended Flavonol 34,35 The Journal of Organic Chemistry Synthesis of 3-Hydroxyflavone Derivatives.3-Hydroxyflavones 1−12 were synthesized via an aldolization reaction from commercially available 2-hydroxyacetophenone (21a−d) and benzaldehyde (22a−c) derivatives under basic conditions according to the reported methods 35,41 to give the corresponding chalcone intermediates (23a−h, Scheme 2).The subsequent in situ Flynn−Algar−Oyamada condensation 42,43 with hydrogen peroxide under basic conditions provided the title 3-hydroxyflavone derivatives (1−5).The ethynyl and nitrile groups were introduced by palladium-catalyzed coupling reactions 44,45 from the corresponding brominated precursors to give compounds 6, 7, and 9−12.The ability of flavonols to coordinate metal ions 46 in these synthetic steps required the use of protecting groups.Methylation of compound 7 with methyl triflate as a methylating agent provided compound 8. Compounds 1−5 and 12 were converted to 3-hydroxyflavothiones 13−18 using Lawesson's reagent. 41,473-Methoxyflavone 19 was synthesized from 1 using methyl iodide and was converted to 3-methoxyflavothione (20) using Lawesson's reagent.
Physicochemical Properties.3-Hydroxyflavones and their thione analogs exist in both acid (1A−18A) and base (1B−18B) forms 35 in polar protic solvents (Scheme 3).A pK a value of 8.7 in pure water for 1A was reported. 48Because of its low solubility in water, the pK a of 3-hydroxyflavothione (13)  was determined in a water/methanol (50:50, v/v) mixture and recalculated to give a value of 10.07 ± 0.12 for pure water using the reported calibration method (Figure S160). 49lavonols with electron-withdrawing groups (EWGs) on rings A and B (e.g., 8 and 9) are partially deprotonated in pure methanol (Figures S108 and S110), while those with an additional electron-donating group (EDG) in position C4′ (7   and 11) are fully protonated (Figures S106 and S114, respectively).
The substituents considerably affect the absorption and other physical properties of hydroxyflavones and hydroxyflavothiones (Table 1).−52 With moderately (ethynyl, 6A, and 7A) or more strongly (cyano, 10A−12A) electron-withdrawing 53 groups on C6 or C7, ESICT is manifested even more (Figures S104, S106, S112, S114, and S116).The absorption bands of 3hydroxyflavothiones are bathochromically shifted 54 compared to those of 3-hydroxyflavones; the absorption bands of base forms 1B−18B are shifted by 30−80 nm for 3-hydroxyflavones and 30−70 nm for their thione analogs compared to those of the corresponding acid forms (Figures S96−S130).For example, 12B exhibits absorption at λ max = 510 nm, reaching 600 nm at the tail of the absorption band due to ESICT involving both EWGs and EDGs on rings A and B, respectively, as well as resonance effects (Figure S117). 53uch an absorption shift has only been possible for flavonol derivatives with a π-extended conjugated system. 30As a moderate EWG, the bromine atom 53 in position C6 (4, 5, 16,  17) does not affect the absorption spectra significantly (Figures S101, S102, S124, and S126).
Kasha and co-workers rationalized the spectroscopy of 3hydroxyflavone (1A) by intramolecular ESIPT to form a singlet-excited phototautomer (Scheme 1), 39,40 which is known to take place in less than 125 fs 60 and possesses a lifetime of <10 ps (in ethanol). 61It explains why the emission spectra of 3-hydroxyflavone such as 1A show an emission band from the locally excited state at shorter wavelengths in nonpolar solvents or a lower temperature 52 and a new, bathochromically shifted band of the phototautomer in protic solvents such as methanol (Figure S96).On the other hand, emission spectra of 4′-dimethylamino derivatives in methanol have the bands bathochromically shifted (Table 1 and Figure S99) due to ESICT. 62Polar solvents stabilize a zwitterionic excited state, the energy of which may decrease to such an extent that the proton transfer (ESIPT) is no longer favorable. 51The compounds with electron-withdrawing cyano groups 9A−12A were weakly fluorescent.The most bathochromically shifted emission maximum at 675 nm, corresponding to a very large Stokes shift of 224 nm (7358 cm −1 ), was observed for a dicyano derivative 12A.To investigate the emissive states further, we measured the emission spectra of 1A, 3A, 7A, 9A, and 12A in solvents of different polarities.ESIPT was dominant in flavonols without an EDG in position C4′ (1A and 9A; Stokes shifts of ∼9568 cm −1 ), and the emission maxima were not affected by the solvent polarity (Figures S131 and S134).On the other hand, the derivatives exhibiting ESICT (3A, 7A, and 12A) showed a significant emission band of a charge-transfer character, which was bathochromically shifted with increased polarity of the solvent (Figures S132, S133, and S135).As reported before, 51 these derivatives showed emission from the normal state in methanol.3-Hydroxyflavothione derivatives (13A−18A) are either not emissive or show a very low emission 63 (Figure S128) due to more efficient intersystem crossing related to the heavy atom effect of the sulfur atom. 64The observed Stokes shifts were always below 100 nm in polar methanol; thus, the The Journal of Organic Chemistry emission was ascribed to the normal form emission, as also demonstrated by Chou and co-workers for 3A. 65The base forms of 3-hydroxyflavones showed one emission band (Scheme 1; ESIPT is not possible) with moderate to large Stokes shifts ranging from 4073 cm −1 for 1B to 5302 cm −1 for 12B).
Photochemistry of Flavonol Derivatives.Near-UV irradiation of 1A in aerated methanol was reported to give CO and 2-(benzoyloxy)benzoic acid as a side product in relatively low yield (below 0.2 equiv; Scheme 1). 36We found that substituents in compounds 2A−12A affected this photochemistry of flavonol significantly (Table 1).4′-DMA substitution in 3A increased CO yields to 0.5 equiv, but when combined with the cyano EWG at C6 and/or C7 positions Scheme 2. Synthesis of 3-Hydroxyflavone and 3-Hydroxyflavothione Derivatives Scheme 3. Acid−Base Forms of 3-Hydroxyflavone 1 and 3-Hydroxyflavothione 13 The Journal of Organic Chemistry (10A−12A), for example, the yield reached almost unity (up to 0.99 equiv for 11A).Such high yields have only been reported in π-extended flavonols. 28,34The CO-photorelease enhancement was also significant in 6-(6A; 0.58 equiv) and especially 7-ethynyl (7A; 0.83 equiv) derivatives bearing the 4′-DMA group.On the other hand, when the electrondonating 4′-DMA group was replaced by trimethylammonium as an EWG in 8A, the CO yield was negligible (no ESICT).7-CN and 6-Br substitution alone (9A and 4A, respectively) had no effect (∼0.2 equiv) but in connection with the 4′-DMA group in 5A and 11A, CO yields increased significantly.The high CO yield (∼0.8 equiv) in 5A is attributed to the combination of ESICT and heavy atom effect, promoting the triplet excited state, which has a pivotal role in the photodecarbonylation mechanism. 36Interestingly, irradiating the corresponding base forms in methanol provided very similar CO yields.Quantum yields of CO release on the order of 0.001 for acid forms and 0.01 for base forms were not affected significantly by the substituents (Table 1).The corresponding uncaging cross-section values (Φ CO ε max ) for the selected flavonols are listed in the same table.3-Methoxyflavone (19) has a slightly hypsochromically shifted absorption maximum compared to 1A due to the electron-donating methyl group and produces only trace amounts of CO upon irradiation (Table 1).The protection of the 3-OH group (from which CO originates; Scheme 1) by the methyl group suppresses CO release. 32o rationalize a significant increase in the CO yields in donor−acceptor-substituted flavonols, we irradiated representative derivatives 7A and 12A to compare their photochemistry with that of the parent compound 1A and its 4′-DMA derivative 3A.Compounds 7A and 12A were chosen as the  The Journal of Organic Chemistry most efficient photoCORMs from all studied derivatives; compound 12A has the most bathochromically shifted absorption spectrum.
Oxygen in both ground 33 and excited 66 states is a critical element in the photochemistry of flavonols and the production of CO and 3,4-flavandione, involving a triplet excited state as a productive common intermediate. 34,35,67Nanosecond transient spectroscopy of 1A in degassed methanol showed a long-lived species possessing a lifetime of 12.5 ± 0.6 μs (Figure 3a).It is efficiently quenched by oxygen in an aerated solution (Figure S148), and we assigned it to a triplet state in agreement with previous observations. 33,35The transients formed from 7A and 12A exhibited significantly shorter lifetimes in degassed methanol (0.233 ± 0.007 and 0.071 ± 0.036 μs, Figure 3b and Figure S155, respectively) and bathochromically shifted signals.The lifetime of the former species in less polar degassed acetonitrile was found to be ∼200-fold longer (48 ± 2 μs, Figure 3c), while that of 1A remains the same in nonpolar 33 and polar protic solvents (Figure 3a).Because these transients were also quenched by triplet oxygen (Figures S151 and S154), we attributed them to a triplet state, possibly of a charge-/proton-transfer character. 50Despite their shortened lifetimes, partially due to their interaction with oxygen, oxygen is also indispensable for the oxidative formation of CO.
The shorter triplet lifetimes in the case of 7A and 12A correlate with the lower quantum yields of CO production and in particular with the formation of 2-methoxyflavo-3,4-dione (30) and 3-aryl-3-methoxy-1,2-indandione (32) as photoproducts (Scheme 4).The latter process involves a nucleophilic bimolecular attack of methanol, which, in principle, should be significantly suppressed when directly connected to the productive excited state.We found that the photodecomposition of these compounds in degassed methanol is very slow (Figure S136), and because it no longer competes with the oxidative production of CO, CO yield approaches 0.9 equiv (Table 1).
Self-sensitized photo-oxygenation is an important channel of CO production from flavonol base forms, while acid forms were reported to be unreactive. 35We determined the role of singlet oxygen in CO release from 7 and 12. Photochemical CO yields (Table 2) from derivatives 1, 3, 7, and 12 in their acid and base forms in the presence of singlet oxygen traps (NaN 3 or furfuryl alcohol 68,69 ) were found to be essentially the same or similar to those obtained in their absence.On the other hand, the affinities of 7 and 12 in their acid and base forms toward singlet oxygen (Table 2) are similar to or even higher than those reported for flavonol derivatives with a πextended aromatic system. 34We concluded that the amount of singlet oxygen produced by the compounds is insufficient The Journal of Organic Chemistry (quantum yield of singlet oxygen production (Φ Δ ) is ∼1 × 10 −2 for both forms) to compete with the reaction of triplet state with triplet oxygen (Scheme 1).
Photochemistry of Flavothione Derivatives.Irradiation of the acid form of unsubstituted flavothione 13A in aerated methanol at 420 nm (c ≈ 1.0 × 10 −4 M; Φ dec = 0.0232 ± 0.008) resulted in the formation of a mixture of photoproducts possessing absorption below 400 nm, 2hydroxy-3,4-flavandione (33) and 2-(benzoyloxy)benzoic dithioperoxyanhydride (34) and carbon monoxide (Figures 4 and Figures S144 and S145; Table 1).In addition, the product of the thione → carbonyl conversion, 3-hydroxyflavone (1A), was found in trace amounts.(It is important to note that the photodegradation of all 3-hydroxyflavothione derivatives led to many other unidentified minor compounds (LC-HRMS, Figure S144), formed probably via secondary (photo)oxygenation processes.)Because 33 and 34 are the products of oxidation, the effect of O 2 on the reaction was investigated.The photodegradation efficiency of 13A was much lower in a pump-freeze-degassed methanolic solution, suggesting that oxygen is again essential for the transformations (Figure S137).
Flavothione derivatives 13A−17A produced 0.1−0.4equiv of carbon monoxide, which is up to 3 times lower than for analogous 3-hydroxyflavones (Table 1).An electron-donating group in position 4′ had only a small effect on the CO production yields.The bromine substituent in position 6 in 6bromo-3-hydroxyflavothione (16A; 0.43 equiv) increased CO yield compared to that of 13A, while a very low CO yield (0.13 equiv) was observed for 6-bromo-4′-DMA derivative 17A.The photodegradation efficiency of 13A was concomitant with that of CO production; therefore, CO was not formed in a secondary process.6,7-Dicyano substitution, which enhanced the CO yield in the case of 12A, resulted in poor yields for 18A    The Journal of Organic Chemistry (0.04 equiv).(Note: the decomposition quantum yields of thiones were larger than those of the corresponding flavonols; Table 1.) Nanosecond transient absorption (ns TA) spectroscopy of 13A showed a long-lived species (λ max ≈ 500 nm; Figure S158) with a lifetime of ∼110 ns in an aerated solution, which extends 40-fold to 5.2 μs in a degassed sample (Figure S159).Elisei and co-workers assigned this signal to a triplet excited state, 70 which is not fully quenched by oxygen due to its relatively short lifetime.Still, the singlet oxygen production by 3-hydroxyflavothione is very high (Φ Δ = 0.56 70 ).Thus, the potential dual role of oxygen as a triplet quencher and oxidizing agent, facilitated in the later steps of the mechanism, was further explored.The photodegradation of 13A in the presence of furfuryl alcohol and dimethyl furan used as a singlet oxygen trap did not change the CO yield or the degradation kinetics.Interestingly, all protonated flavothiones (13A−17A), bearing less electronegative sulfur, were found to be inert to singlet oxygen generated by the thermal decomposition of naphthalene 1,4-endoperoxide in the dark (Figure S161).
The photochemistry of 13B−17B was more complicated to study because of their poor stability in the dark (Figure S163) and higher sensitivity to ambient light.For 13B, photodegradation products were not fully identified but were different from those found in the case of 13A (Figure S146).Unlike the acid forms, conjugate bases 13B−17B react with singlet oxygen (generated from naphthalene 1,4-endoperoxide) to give the same photoproducts as those observed during direct irradiation (Figure S147).The presence of α-terpinene (1000 equiv, c ≈ 6.0 × 10 −2 mol L −1 ) as a singlet oxygen trap during the photodecomposition of 13B did not interfere with the photoreaction or with the nature of the photoproducts; thus, 1 O 2 did not play any significant role in photodegradation.The Journal of Organic Chemistry 3-Hydroxyflavothione (13A) photodegradation occurs through the triplet excited state (Scheme 5), with a quantum yield of the triplet-state formation close to the unity. 70This triplet excited state reacts with triplet oxygen to form an adduct or sensitize it to singlet oxygen (Φ Δ = 0.56 in ethanol 70 ).Our experiments with singlet oxygen traps confirmed the absence of any significant involvement of singlet oxygen in the primary photochemical steps.This is unusual behavior for thiones, which are often reported to react with electrophilic 1 O 2 via its addition to the thione bond to form sulfine or ketone. 71,72ased on our photoproduct analyses and kinetic and quenching experiments described above, we propose that the triplet excited state of 13A in methanol reacts via three pathways.Two of them (paths A and B, Scheme 5) share a common intermediate 35 formed by the reaction of the triplet excited state of 13A with triplet oxygen.
In path A, the intermediate is converted to endoperoxide 36 that decarbonylates and forms unstable 2-(benzoyloxy)benzothioic S-acid, which is air-oxidized to 2-(benzoyloxy)benzoic dithioperoxyanhydride (33).This reaction is common for thioacids 73 and analogous to the reported decarbonylation mechanism of 3-hydroxyflavone and their derivatives. 28,35,37ath B starts with 35, which forms exoperoxide 37, analogous to that suggested for 3-hydroxyflavone photodegradation. 33This exoperoxide undergoes autoxidation 74 cleavage, turning the peroxide into the hydroxy group.The resulting intermediate, 2-hydroxy-2-phenyl-4-thioxochroman-3-one, undergoes another oxidation step on the sulfur atom with triplet oxygen via a 1,2,3-thiadioxetane intermediate, 75 analogous to the reactions between thiones and 1 O 2 . 71,72This mechanism finally leads to the formation of 2-hydroxy-3,4flavandione (34), identified as a final product.2-Hydroxy-3,4flavandione derivatives have already been reported in the literature as the products obtained from 3-hydroxyflavone derivatives by chemical oxidation 76 and photolysis. 67Photodegradation of 13A in methanol in the presence of 5% labeled water D 2 ( 18 O) did not show any sign of integrating the 18 O atom into the structure of 34.
Path C consists of the direct attack of triplet oxygen to the thione group of 13A in the triplet excited state to give a 1,2,3thiadioxetane intermediate 38 (shared by path B) and the subsequent loss of sulfur monoxide to yield to 3-hydroxyflavone (1A).This is a minor pathway, giving only traces of flavone.We initially assumed that 34 is produced by the degradation of 1A.However, irradiation of a mixture of 13A and 1A (1:1) showed that the reaction of 13A is not affected by the presence of 1A; thus, we dismissed this assumption.
Photodegradation of 3-methoxyflavothione (20) produced only trace amounts of CO and gave 19, probably via path C. Inertness of 20 toward the singlet oxygen attack confirmed that desulfuration in paths A and B involves the reaction of the triplet excited state with triplet oxygen.Sulfur monoxide (SO) is a probable side product of thione oxidation (paths B and C) formed upon photo-oxidation of thiones. 71,72,77Unfortunately, its short lifetime and high reactivity make its detection difficult. 78CONCLUSIONS Twenty 3-hydroxyflavone and 3-hydroxyflavothione derivatives were synthesized and subjected to a detailed investigation of their photophysical and photochemical properties.The strategically placed electron-donating and electron-withdrawing groups were used to fine-tune the charge-transfer character of the flavonol excited state, which showcases bathochromically shifted absorption bands up to 600 nm, high CO liberation yields (up to 0.99 equiv), and uncaging cross-section values.Such results, previously observed only for π-extended derivatives, were achieved for the simple (parent) 3hydroxyflavone skeleton, which is easily functionalized and shows a significantly improved atom economy of CO release.Although 3-hydroxyflavothione substituents improved CO yields, its modification never reached the qualities of 3hydroxyflavone as a photoCORM platform.With the considerably improved photoCORM properties and detailed understanding of the CO release mechanism, we demonstrate here that derivatives of 3-hydroxyflavone are a promising and potentially useful alternative to the more complex, π-extended flavonol derivatives.

Table 1 .
Physicochemical and Photochemical Properties of the Studied Flavone Derivatives a