A Green Blue LED-Driven Two-Liquid-Phase One-Pot Procedure for the Synthesis of Estrogen-Related Quinol Prodrugs

Quinol derivatives of estrogens are effective pro-drugs in steroid replacement therapy. Here, we report that these compounds can be synthesized in one-pot conditions and high yield by blue LED-driven photo-oxygenation of parent estrogens. The oxidation was performed in buffer and eco-certified 2-methyltetrahydrofuran as the two-liquid-phase reaction solvent, and in the presence of meso-tetraphenyl porphyrin as the photosensitizer. Two steroidal prodrugs 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED) and 10β-Hydroxyestra-1,4-diene-3,17-dione (HEDD) were obtained with high yield and selectivity.


Introduction
Estrogens are steroidal hormones characterized by a variety of biological effects, including anti-cancer activity, prevention of heart diseases, and neuroprotection [1]. In addition, they are applied in Hormone Replacement Therapy (HRT) for the prevention of chronic diseases in post-menopausal women. Unfortunately, current estrogen therapy is limited due to the presence of undesired side effects [2][3][4], such as increased risk of breast cancer [5], thromboembolism, coronary heart disease, and stroke [6,7]. As a result, analogues of estrogens are required to counteract the side effects. Quinol derivatives of estrogens are effective pro-drugs for this HRT. They are converted to corresponding estrogens in the brain, remaining inactive in the rest of the body. This allows the efficient treatment of neurological and psychiatric diseases, without emergence of peripheral side effects [3,8,9]. In this framework, 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), received a particular attention as an alternative to 17β-estradiol. In vitro and in vivo studies showed that DHED has the potential to treat menopausal symptoms [9], ocular neurodegenerations (including glaucoma) [10,11], androgen deprivation-associated hot flushes [12], and Alzheimer's [13] and Parkinson's neurological disorders [14]. Estrogen-related quinols are synthesized by the oxygenation of the phenolic A-ring of the molecule [15][16][17][18][19][20][21], the procedure being limited by the use of stoichiometric oxidants (e.g., oxone [22], hypervalent iodine [23,24], and excess of hydrogen peroxide H 2 O 2 [25]). As an alternative, dye-sensitized photo-oxygenation of 17β-estradiol 1a has been reported to yield mixtures of the corresponding hydro-peroxide 2a and DHED 3a [26] (the structures of compounds 1a, 2a and 3a are reported in Figure 1), the selectivity of the oxidation being dependent from the reaction solvent, substitution pattern [27,28], flow conditions [29], and photosensitizer properties. The synthesis of DHED by multi-step chrysazine-triggered photo-oxygenation in the presence of 1,8-dihydroxyanthraquinone (1, and PPh 3 has been also reported [30]. Recently, we described that singlet oxygen produced from blue-LED irradiation o meso-tetraphenyl porphyrin (meso-TPP) can be trapped by 2-methyltetrahydrofuran (2 MeTHF), favoring the oxidative coupling of phenols by Horseradish Peroxidase (HRP [31]. This procedure avoided the inactivation of HRP by excess H2O2, working under ex perimental conditions simpler than those for in situ reduction of dioxygen [32][33][34][35][36]. Here we describe the application of this procedure in the synthesis of estrogen-related hydrop eroxide and quinol derivatives. The reaction solvent, photosensitizer, and buffer solution have been optimized in order to obtain high conversion of substrate and yield of the de sired product.

Results and Discussion
17α-Ethinylestradiol 1b was first studied as a model substrate. Compound 1b (0.2 mmoL) was dissolved in 2-MeTHF (32 mL) in the presence of meso-TPP (1.0 mol% with respect to substrate), followed by the addition of HRP (407 U) in sodium phosphate buffer (PBS; 16 mL 0.1 M, pH 6.0). The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) and air atmosphere for 24 h at 28 °C. The photore actor consisted of an internal jar (4.5 cm diameter) inserted in a supplementary externa jar (7.5 cm diameter), and blue-LED strips were wrapped around the external jar and cov ered by aluminum foil ( Figure S1). Under these experimental conditions, the hydro-per oxide 2b was isolated as the only recovered product in low yield, besides the unreacted substrate (Scheme 1; Table 1, entry 1). No trace amounts of dimeric products, possibly derived from oxidative radical homo-coupling processes, were detected in the reaction mixture. The structure of hydro-peroxide 2b was confirmed by spectroscopic and spec trometric analyses (including 2D NMR analysis; SI-Section 8), and by comparison with data previously reported [37]. Scheme 1. Blue LED−driven two−liquid−phase photo-oxygenation of estrogens 1a-d to hydro-per oxides 2a-d.
When the reaction was carried out in the absence of HRP, the hydro-peroxide 2b was again obtained as the only recovered product in acceptable yield, suggesting that the en zyme was not involved in the oxidation of the substrate (Table 1, entry 2). In addition Recently, we described that singlet oxygen produced from blue-LED irradiation of meso-tetraphenyl porphyrin (meso-TPP) can be trapped by 2-methyltetrahydrofuran (2-MeTHF), favoring the oxidative coupling of phenols by Horseradish Peroxidase (HRP) [31]. This procedure avoided the inactivation of HRP by excess H 2 O 2 , working under experimental conditions simpler than those for in situ reduction of dioxygen [32][33][34][35][36]. Here, we describe the application of this procedure in the synthesis of estrogen-related hydroperoxide and quinol derivatives. The reaction solvent, photosensitizer, and buffer solution have been optimized in order to obtain high conversion of substrate and yield of the desired product.

Results and Discussion
17α-Ethinylestradiol 1b was first studied as a model substrate. Compound 1b (0.2 mmoL) was dissolved in 2-MeTHF (32 mL) in the presence of meso-TPP (1.0 mol% with respect to substrate), followed by the addition of HRP (407 U) in sodium phosphate buffer (PBS; 16 mL 0.1 M, pH 6.0). The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) and air atmosphere for 24 h at 28 • C. The photoreactor consisted of an internal jar (4.5 cm diameter) inserted in a supplementary external jar (7.5 cm diameter), and blue-LED strips were wrapped around the external jar and covered by aluminum foil ( Figure S1). Under these experimental conditions, the hydro-peroxide 2b was isolated as the only recovered product in low yield, besides the unreacted substrate (Scheme 1; Table 1, entry 1). No trace amounts of dimeric products, possibly derived from oxidative radical homo-coupling processes, were detected in the reaction mixture. The structure of hydro-peroxide 2b was confirmed by spectroscopic and spectrometric analyses (including 2D NMR analysis; SI-Section 8), and by comparison with data previously reported [37].
Recently, we described that singlet oxygen produced from blue-LED irradiatio meso-tetraphenyl porphyrin (meso-TPP) can be trapped by 2-methyltetrahydrofura MeTHF), favoring the oxidative coupling of phenols by Horseradish Peroxidase (H [31]. This procedure avoided the inactivation of HRP by excess H2O2, working unde perimental conditions simpler than those for in situ reduction of dioxygen [32][33][34][35][36]. H we describe the application of this procedure in the synthesis of estrogen-related hyd eroxide and quinol derivatives. The reaction solvent, photosensitizer, and buffer solu have been optimized in order to obtain high conversion of substrate and yield of the sired product.

Results and Discussion
17α-Ethinylestradiol 1b was first studied as a model substrate. Compound 1b mmoL) was dissolved in 2-MeTHF (32 mL) in the presence of meso-TPP (1.0 mol% respect to substrate), followed by the addition of HRP (407 U) in sodium phosphate b (PBS; 16 mL 0.1 M, pH 6.0). The solution was gently stirred (200 rpm) under blueirradiation (blue-LED stripes, 470 nm) and air atmosphere for 24 h at 28 °C. The pho actor consisted of an internal jar (4.5 cm diameter) inserted in a supplementary exte jar (7.5 cm diameter), and blue-LED strips were wrapped around the external jar and ered by aluminum foil ( Figure S1). Under these experimental conditions, the hydro oxide 2b was isolated as the only recovered product in low yield, besides the unrea substrate (Scheme 1; Table 1, entry 1). No trace amounts of dimeric products, pos derived from oxidative radical homo-coupling processes, were detected in the reac mixture. The structure of hydro-peroxide 2b was confirmed by spectroscopic and s trometric analyses (including 2D NMR analysis; SI-Section 8), and by comparison data previously reported [37]. When the reaction was carried out in the absence of HRP, the hydro-peroxide 2b again obtained as the only recovered product in acceptable yield, suggesting that th zyme was not involved in the oxidation of the substrate (Table 1, entry 2). In addi Scheme 1. Blue LED−driven two−liquid−phase photo-oxygenation of estrogens 1a-d to hydroperoxides 2a-d.
When the reaction was carried out in the absence of HRP, the hydro-peroxide 2b was again obtained as the only recovered product in acceptable yield, suggesting that the enzyme was not involved in the oxidation of the substrate (Table 1, entry 2). In addition, compound 1b was unreactive when the reaction was carried out in the absence of buffer  (Table 1, entry 3), under dark conditions (Table 1, entry 4), and without meso-TPP (Table 1, entry 5) highlighting the key role played by blue-photons, pH, and photosensitizer in the transformation.
The activity of meso-TPP was compared with that of other useful photosensitizers, such as tris(2-phenyl-pyridine) iridium [Ir(ppy) 3 ] and Rose Bengal (structure and UV-vis adsorption spectra of the photosensitizers are in Figures S7-S9). As reported in Table 1, meso-TPP showed the highest activity in the photo-oxygenation of compound 1b (entry 2 versus entries 6 and 7). The possible formation of 2-MeTHF hydro-peroxide from 2-MeTHF during blue LED irradiation, previously observed by us [31], was evaluated by the pyrogallol assay at different reaction times (1, 2, 4, 6, and 24 h) and in the presence-or alternatively in the absence-of compound 1b. As reported in Figure S2, compound 1b lowered the concentration of 2-MeTHF hydro-peroxide, suggesting higher reactivity of compound 1b with singlet oxygen with respect to the organic solvent. To optimize the photo-oxygenation procedure we analyzed the effect played by the concentration of substrate, the amount of the buffer (and relative pH), and the nature of the reaction solvent, on the process. Correspondingly to the other experimental parameters, hydro-peroxide 2b was obtained in higher yield starting from 60 mM of substrate (Table 2, entry 1). This result was in accordance with the effect played by the concentration of the substrate on the intensity of the blue LED-photons in the bulk of the solution [38]. The high yield of hydro-peroxide 2b was retained in the presence of a low amount of buffer (160 µL, 5% v/v with respect to 2-MeTHF) ( Table 2, entry 2) at pH 6, while it decreased at pH 8 (5% NaHCO 3 ss; Table 2, entry 3), and at pH 2 (AcOH 0.5%; Table 2, entry 4). Finally, we studied the effect of a panel of reaction solvents, characterized by a different stabilization effect for singlet oxygen, including CH 2 Cl 2 , EtOAc, and HFIP [39]. The highest yield of hydro-peroxide 2b was obtained in CH 2 Cl 2 (>98%; Table 2, entry 6) confirming the high stabilizing effect previously reported (Table 2, entry 6 versus entries 2, 5 and 7) [40]. The general order of reactivity was as follows: CH 2 Cl 2 > 2-MeTHF > HFIP > EtOAc.
Next, we studied the photo-oxygenation of 17β-estradiol 1a, estrone 1c, and estriol 1d under optimal experimental conditions (that is: 60 mM of substrate, CH 2 Cl 2 , PBS, and meso-TPP). Unfortunately, estradiol 1a and estriol 1d showed very low conversion of substrate due to the limited solubility of CH 2 Cl 2 , while hydro-peroxide 2c was obtained in quantitative conversion of the substrate and yield of the product ( Table 3, entry 1).  The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5).  The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd Molecules 2022, 27, x FOR PEER REVIEW 4 of 13 The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd Molecules 2022, 27, x FOR PEER REVIEW 4 of 13 The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd  The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd Molecules 2022, 27, x FOR PEER REVIEW 4 of 13 The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd  The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd Molecules 2022, 27, x FOR PEER REVIEW 4 of 13 The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd  The reaction was successively repeated in the second most reactive organic solvent previously observed in the oxidation of compound 1b, 2-MeTHF, also taking advantage of its sustainability [35,36]. Under these experimental conditions, hydro-peroxides 2a and 2c-d were obtained, ranging from acceptable to high yields ( Table 3, entry 2 and entries 4-5). Table 3. Substrate scope of novel blue LED-driven two-liquid-phase photo-oxygenation a .

Entry
Cpd 1c 80 Table 3. Cont. The mechanism of dye-mediated photo-oxygenation of phenols has been reviewed and discussed; it includes the transfer of the excited state from the photosensitizer to the substrate (Type I mechanism), or alternatively the inter-crossing system between the photosensitizer and dioxygen, with formation of singlet oxygen ( 1 O2) (Type II mechanism) [38,[41][42][43]. Under our experimental conditions, the Type I mechanism was most probably not operating, as suggested by the loss of reactivity of the substrate in the absence of meso-TPP (Table 1, entry 5), associated with the low absorption coefficient of estrogens in the interaction with blue-LED photons ( Figures S3-S6) [44][45][46][47]. Additional experiments were performed to investigate the possible involvement of the Type II mechanism [43,48]. Hydroxy and superoxide radicals were not produced during the reaction as evaluated by the "coumarin" assay [48][49][50][51] (Figure S10) and the TEMPO assay ( Figure S11) [52], respectively, while the NaN3 assay ( Figure S12) confirmed the involvement of 1 O2 [53][54][55]. In addition, the reaction was not effective under an argon atmosphere in the presence of degassed solvents (SI-Section 6). Although the possibility of the Type I mechanism cannot be completely ruled out, these data support the formation of 1 O2 as the primary oxidant in the photo-oxygenation of estrogens 1a-d. This result is in accordance with the reported ability of meso-TPP to produce 1 O2 in aerated systems [42,43].

Entry
The tentative reaction pathway for the photo-oxygenation of compounds 1a-d is reported in Scheme 2, it includes: (i) blue-LED photo-activation of meso-TPP to form the singlet excited state ( 1 meso-TPP*); (ii) intersystem crossing (ISC) to form the triplet excited state ( 3 meso-TPP*); (iii) energy transfer, and formation of singlet oxygen ( 1 O2); (iv) selective insertion of 1 O2 on substrate to yield an unstable adduct A (not isolated in our case); and (v) rearrangement of adduct A to yield the corresponding hydro-peroxide. The mechanism of dye-mediated photo-oxygenation of phenols has been reviewed and discussed; it includes the transfer of the excited state from the photosensitizer to the substrate (Type I mechanism), or alternatively the inter-crossing system between the photosensitizer and dioxygen, with formation of singlet oxygen ( 1 O2) (Type II mechanism) [38,[41][42][43]. Under our experimental conditions, the Type I mechanism was most probably not operating, as suggested by the loss of reactivity of the substrate in the absence of meso-TPP (Table 1, entry 5), associated with the low absorption coefficient of estrogens in the interaction with blue-LED photons ( Figures S3-S6) [44][45][46][47]. Additional experiments were performed to investigate the possible involvement of the Type II mechanism [43,48]. Hydroxy and superoxide radicals were not produced during the reaction as evaluated by the "coumarin" assay [48][49][50][51] (Figure S10) and the TEMPO assay ( Figure S11) [52], respectively, while the NaN3 assay ( Figure S12) confirmed the involvement of 1 O2 [53][54][55]. In addition, the reaction was not effective under an argon atmosphere in the presence of degassed solvents (SI-Section 6). Although the possibility of the Type I mechanism cannot be completely ruled out, these data support the formation of 1 O2 as the primary oxidant in the photo-oxygenation of estrogens 1a-d. This result is in accordance with the reported ability of meso-TPP to produce 1 O2 in aerated systems [42,43].
The tentative reaction pathway for the photo-oxygenation of compounds 1a-d is reported in Scheme 2, it includes: (i) blue-LED photo-activation of meso-TPP to form the singlet excited state ( 1 meso-TPP*); (ii) intersystem crossing (ISC) to form the triplet excited state ( 3 meso-TPP*); (iii) energy transfer, and formation of singlet oxygen ( 1 O2); (iv) selective insertion of 1 O2 on substrate to yield an unstable adduct A (not isolated in our case); and (v) rearrangement of adduct A to yield the corresponding hydro-peroxide. The mechanism of dye-mediated photo-oxygenation of phenols has been reviewed and discussed; it includes the transfer of the excited state from the photosensitizer to the substrate (Type I mechanism), or alternatively the inter-crossing system between the photosensitizer and dioxygen, with formation of singlet oxygen ( 1 O 2 ) (Type II mechanism) [38,[41][42][43]. Under our experimental conditions, the Type I mechanism was most probably not operating, as suggested by the loss of reactivity of the substrate in the absence of meso-TPP (Table 1, entry 5), associated with the low absorption coefficient of estrogens in the interaction with blue-LED photons ( Figures S3-S6) [44][45][46][47]. Additional experiments were performed to investigate the possible involvement of the Type II mechanism [43,48]. Hydroxy and superoxide radicals were not produced during the reaction as evaluated by the "coumarin" assay [48][49][50][51] (Figure S10) and the TEMPO assay ( Figure S11) [52], respectively, while the NaN 3 assay ( Figure S12) confirmed the involvement of 1 O 2 [53][54][55]. In addition, the reaction was not effective under an argon atmosphere in the presence of degassed solvents (SI-Section 6). Although the possibility of the Type I mechanism cannot be completely ruled out, these data support the formation of 1 O 2 as the primary oxidant in the photo-oxygenation of estrogens 1a-d. This result is in accordance with the reported ability of meso-TPP to produce 1 O 2 in aerated systems [42,43].
The tentative reaction pathway for the photo-oxygenation of compounds 1a-d is reported in Scheme 2, it includes: (i) blue-LED photo-activation of meso-TPP to form the singlet excited state ( 1 meso-TPP*); (ii) intersystem crossing (ISC) to form the triplet excited state ( 3 meso-TPP*); (iii) energy transfer, and formation of singlet oxygen ( 1 O 2 ); (iv) selective insertion of 1 O 2 on substrate to yield an unstable adduct A (not isolated in our case); and (v) rearrangement of adduct A to yield the corresponding hydro-peroxide.
The reduction of hydro-peroxide 2b was performed with different redox agents, Na 2 S 2 O 3 , KI, and PPh 3 . In this latter case, the progress of the reduction was monitored by the analysis of the C-10 signal (81.020 ppm) in the 13 C NMR spectrum of the substrate, due to the high structural similarity between compound 2b, and the corresponding quinol derivative 3b (experimental procedures are in SI-Section 7). Among the reagents studied, PPh 3 afforded quinol 3b in quantitative yield and conversion of substrate. PPh 3 was then used for the design of a novel one-pot synthesis of quinol 3b by contemporary oxidation of 17α-ethinylestradiol 1b, and in situ reduction of hydro-peroxide 2b (Scheme 3).
Compound 1b (60 mM) and meso-TPP (1.0 mol% with respect to substrate) were dissolved in 2-MeTHF, followed by addition of PBS (0.1M, pH 6; 5% with respect to organic solvent). The solution was gently stirred under blue-LED irradiation at 28 • C, and PPh 3 was added to the reaction mixture at indicated reaction times. The presence of PPh 3 at the starting point of the reaction totally inhibited the formation of quinol 3b, with hydroperoxide 2b being the only recovered product (Table 4, entry 1), probably due to the fast oxidation of PPh 3 to triphenyl-phosphinoxide (TPPO). A better result was obtained when PPh 3 was added to the reaction mixture after 2 h. In this latter case, quinol 3b was obtained with 50% yield and 70% conversion of substrate (Table 4, entry 2). The addition of PPh 3 after 3 h further increased the yield of quinol 3b, and conversion of substrate (Table 4, entry  3). Longer addition times (e.g., 24 h) did not further increase the yield of quinol 3b ( Scheme 2. Tentative reaction pathway for the photo-oxygenation of estrogen 1 by blue-LED irradiation in the presence of meso-TPP and bi-phasic system. "*" represents the excited structure of meso-TPP. The reduction of hydro-peroxide 2b was performed with different redox agents, Na2S2O3, KI, and PPh3. In this latter case, the progress of the reduction was monitored by the analysis of the C-10 signal (81.020 ppm) in the 13 C NMR spectrum of the substrate, due to the high structural similarity between compound 2b, and the corresponding quinol derivative 3b (experimental procedures are in SI-Section 7). Among the reagents studied, PPh3 afforded quinol 3b in quantitative yield and conversion of substrate. PPh3 was then used for the design of a novel one-pot synthesis of quinol 3b by contemporary oxidation of 17α-ethinylestradiol 1b, and in situ reduction of hydro-peroxide 2b (Scheme 3). Compound 1b (60 mM) and meso-TPP (1.0 mol% with respect to substrate) were dissolved in 2-MeTHF, followed by addition of PBS (0.1M, pH 6; 5% with respect to organic solvent). The solution was gently stirred under blue-LED irradiation at 28 °C, and PPh3 was added to the reaction mixture at indicated reaction times. The presence of PPh3 at the Scheme 2. Tentative reaction pathway for the photo-oxygenation of estrogen 1 by blue-LED irradiation in the presence of meso-TPP and bi-phasic system. "*" represents the excited structure of meso-TPP.
Molecules 2022, 27, x FOR PEER REVIEW 6 of 13 Scheme 2. Tentative reaction pathway for the photo-oxygenation of estrogen 1 by blue-LED irradiation in the presence of meso-TPP and bi-phasic system. "*" represents the excited structure of meso-TPP.
The reduction of hydro-peroxide 2b was performed with different redox agents, Na2S2O3, KI, and PPh3. In this latter case, the progress of the reduction was monitored by the analysis of the C-10 signal (81.020 ppm) in the 13 C NMR spectrum of the substrate, due to the high structural similarity between compound 2b, and the corresponding quinol derivative 3b (experimental procedures are in SI-Section 7). Among the reagents studied, PPh3 afforded quinol 3b in quantitative yield and conversion of substrate. PPh3 was then used for the design of a novel one-pot synthesis of quinol 3b by contemporary oxidation of 17α-ethinylestradiol 1b, and in situ reduction of hydro-peroxide 2b (Scheme 3). Compound 1b (60 mM) and meso-TPP (1.0 mol% with respect to substrate) were dissolved in 2-MeTHF, followed by addition of PBS (0.1M, pH 6; 5% with respect to organic solvent). The solution was gently stirred under blue-LED irradiation at 28 °C, and PPh3 was added to the reaction mixture at indicated reaction times. The presence of PPh3 at the Scheme 3. One−pot synthesis of quinols 3a-d. Table 4. One-pot synthesis of quinol 3b at different times of addition of PPh 3 a . The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh 3 . As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries [3][4]. Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .
The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh3. As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries 3 -4). Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .

Entry
Cpd 90 88 a The reaction was performed solubilizing 1b (0.2 mmol) and meso-TPP (1.0 mol%) in 2-MeTHF (3.2 mL), followed by the addition of PBS (0.1 M, pH 6) and of PPh3 (0.3 mmol) at different reaction times. The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) at 28 ± 1 °C; b Conversion of substrate and yield of quinol 3b were calculated on the basis of mmol of starting substrate. c Yield of hydro-peroxide 2b.
The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh3. As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries 3 -4). Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .

Entry
Cpd 24 48 90 88 a The reaction was performed solubilizing 1b (0.2 mmol) and meso-TPP (1.0 mol%) in 2-MeTHF (3.2 mL), followed by the addition of PBS (0.1 M, pH 6) and of PPh3 (0.3 mmol) at different reaction times. The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) at 28 ± 1 °C; b Conversion of substrate and yield of quinol 3b were calculated on the basis of mmol of starting substrate. c Yield of hydro-peroxide 2b.
The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh3. As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries 3 -4). Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .

Entry
Cpd 24 48 90 88 a The reaction was performed solubilizing 1b (0.2 mmol) and meso-TPP (1.0 mol%) in 2-MeTHF (3.2 mL), followed by the addition of PBS (0.1 M, pH 6) and of PPh3 (0.3 mmol) at different reaction times. The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) at 28 ± 1 °C; b Conversion of substrate and yield of quinol 3b were calculated on the basis of mmol of starting substrate. c Yield of hydro-peroxide 2b.
The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh3. As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries 3 -4). Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .

Entry
Cpd 24 48 90 88 a The reaction was performed solubilizing 1b (0.2 mmol) and meso-TPP (1.0 mol%) in 2-MeTHF (3.2 mL), followed by the addition of PBS (0.1 M, pH 6) and of PPh3 (0.3 mmol) at different reaction times. The solution was gently stirred (200 rpm) under blue-LED irradiation (blue-LED stripes, 470 nm) at 28 ± 1 °C; b Conversion of substrate and yield of quinol 3b were calculated on the basis of mmol of starting substrate. c Yield of hydro-peroxide 2b.
The one-pot procedure was then applied to estrogens 1a and 1c-d assuming 3 h as the optimal reaction time for the addition of PPh3. As reported in Table 5, quinols 3a and 3c-d were obtained ranging from acceptable to high yield (Table 5, entry 1 and entries 3 -4). Quinols 3a (DHED) and 3c (HEDD) are well recognized pro-drugs in Hormone Replacement Therapy [9][10][11][12][13][14]. Table 5. Substrate scope of novel blue LED-driven two-liquid-phase in One-Pot condition a .

Entry
Cpd

General Considerations
Commercially available reagents were used without further purification. Chromatographic separations were performed on Merck silica gel 60 (230-400 mesh). Rf values are referred to TLC carried out on 0.25 mm silica gel plates (F254) using the eluent indicated for column chromatography. All products were dried in high vacuum (10-3 mbar) before characterization. 1 H NMR, 13

General Considerations
Commercially available reagents were used without further purification. Chromatographic separations were performed on Merck silica gel 60 (230-400 mesh). Rf values are referred to TLC carried out on 0.25 mm silica gel plates (F254) using the eluent indicated for column chromatography. All products were dried in high vacuum (10-3 mbar) before characterization. 1 H NMR, 13

General Considerations
Commercially available reagents were used without further purification. Chromatographic separations were performed on Merck silica gel 60 (230-400 mesh). Rf values are referred to TLC carried out on 0.25 mm silica gel plates (F254) using the eluent indicated for column chromatography. All products were dried in high vacuum (10-3 mbar) before characterization. 1 H NMR, 13 C NMR, and 2D NMR were recorded on a Bruker Advance DRX400 (400 MHz/100 MHz) spectrometer. Chemical shifts are in parts per million (δ scale) and internally referenced the CD 3 OD signal at δ 3.31 and 49.00 ± 0.01 ppm, respectively. Coupling constants (J) are reported in Hz. UV-visible (UV-vis) spectra were recorded using Cary 60 UV-Vis spectrophotometer, Agilent, Santa Clara, USA. Blue-LED apparatus

General Procedure for the Synthesis of Hydro-Peroxides 2a-d
The selected estrogen (0.2 mmol) and meso-TPP (1 mol%) were dissolved in 2-Me-THF (3.2 mL), followed by the addition of PBS (0.16 mL; 0.1 M, pH 6), and the mixture was gently stirred (200 rpm) under blue-LED irradiation and air atmosphere at 28 ± 1 • C for 24 h. The reaction mixture was washed with brine (3 × 2 mL), dried over sodium sulphate, and evaporated under vacuum. The crude mixture was purified by column chromatography.

General Procedure for the Synthesis of Estrogen-Related Quinols 3a-d
The selected estrogen (0.2 mmol) and meso-TPP (1.0 mol%) were dissolved in 2-Me-THF (3.2 mL), followed by the addition of PBS (0.16 mL; 0.1 M, pH 6), and the mixture was gently stirred (200 rpm) under blue-LED irradiation and air atmosphere at 28 ± 1 • C for 3 hrs. Then PPh 3 (0.3 mmol) was added, and the reaction was left under magnetic stirring for 21 h. After washing with brine (3 × 2 mL), the reaction mixture was dried over sodium sulphate, and evaporated under vacuum. The crude mixture was purified by column chromatography.

Conclusions
In conclusion, we developed a novel one-pot approach for the synthesis of estrogenrelated quinols by using blue LED-driven photo-oxygenation in a two-liquid-phase system. The reaction proceeded under mild and sustainable conditions, including with a catalytic amount of meso-TPP, eco-certified 2-MeTHF, and buffer as solvents, and PPh 3 as the reducing agent. The reaction pathway involved blue-LED photo-activation of meso-TPP, and the generation of singlet oxygen ( 1 O 2 ) (Type II mechanism), followed by oxidation of estrogen to the corresponding hydro-peroxide, and in situ reduction of hydro-peroxide to the desired quinol. Under these experimental conditions, quinols were synthesized ranging from acceptable to very high yield, including two well recognized pro-drugs in Hormone Replacement Therapy, DHED and HEDD. The irrelevance of the Type I mechanism was suggested by the un-reactivity of the system, in the absence of the photosensitizer associated with the low adsorption capacity of estrogens towards blue-LED photons. The presence of 2-MeTHF hydro-peroxide, OH, and superoxide radicals in the reaction pathway was investigated, and ruled-out by means of different specific assays. The additional scopes and applications of this photocatalytic process will be further investigated in our laboratory.