Antiviral activities of halogenated emodin derivatives against human coronavirus NL63

molecules Article Antiviral Activities of Halogenated Emodin Derivatives against Human Coronavirus NL63 Monika Horvat 1, Martina Avbelj 2, María Beatriz Durán-Alonso 3, Mihailo Banjanac 4, Hrvoje Petkovi´c 2,* and Jernej Iskra 1,*   Citation: Horvat, M.; Avbelj, M.; Durán-Alonso, M.B.; Banjanac, M.; Petkovi´c, H.; Iskra, J. Antiviral Activities of Halogenated Emodin Derivatives against Human Coronavirus NL63. Molecules 2021,26, 6825. https://doi.org/10.3390/ molecules26226825 Academic Editor: Riccardo Petrelli Received: 25 October 2021 Accepted: 8 November 2021 Published: 11 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Faculty of Chemistry and Chemical Technology, University of Ljubljana, Veˇcna pot 113, 1000 Ljubljana, Slovenia; [email protected] 2Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia; [email protected] 3Unit of Excellence, Institute of Biology and Molecular Genetics (IBGM), University of Valladolid-CSIC, 47003 Valladolid, Spain; [email protected] 4Fidelta d.o.o., Prilaz baruna Filipovi´ca 29, 10000 Zagreb, Croatia; [email protected] *Correspondence: [email protected] (H.P.); [email protected] (J.I.) Abstract: The current COVID-19 outbreak has highlighted the need for the development of new vaccines and drugs to combat Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). Recently, various drugs have been proposed as potentially effective against COVID-19, such as remdesivir, infliximab and imatinib. Natural plants have been used as an alternative source of drugs for thousands of years, and some of them are effective for the treatment of various viral diseases. Emodin (1,3,8-trihydroxy-6-methylanthracene-9,10-dione) is a biologically active anthraquinone with antiviral activity that is found in various plants. We studied the selectivity of electrophilic aromatic substitution reactions on an emodin core (halogenation, nitration and sulfonation), which resulted in a library of emodin derivatives. The main aim of this work was to carry out an initial evaluation of the potential to improve the activity of emodin against human coronavirus NL63 (HCoV-NL63) and also to generate a set of initial SAR guidelines. We have prepared emodin derivatives which displayed significant anti-HCoV-NL63 activity. We observed that halogenation of emodin can improve its antiviral activity. The most active compound in this study was the iodinated emodin analogue E_3I, whose anti-HCoV-NL63 activity was comparable to that of remdesivir. Evaluation of the emodin analogues also revealed some unwanted toxicity to Vero cells. Since new synthetic routes are now available that allow modification of the emodin structure, it is reasonable to expect that analogues with significantly improved anti-HCoV-NL63 activity and lowered toxicity may thus be generated. Keywords: emodin; halogenated emodin; human coronavirus NL63; antiviral activities 1. Introduction Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is a family of enveloped positive-sense RNA viruses that cause life-threatening respiratory infections and severe pneumonia in humans [ 1 , 2 ]. Coronavirus (CoV) entry into host cells (pulmonary and parabronchial epithelial cells) is mediated by spike protein, which is responsible for binding to receptors ACE-2 and mediating virus–host membrane fusion [ 3 – 5 ]. The development of effective antiviral drugs with a broad spectrum of activity has been hampered by viral diversity and the ability of SARS-CoV to mutate rapidly, even during an epidemic. It is therefore very important to develop antiviral drugs that effectively and safely inhibit the spread of SARS-CoV, or at least significantly alleviate the symptoms of SARS-CoV infection. In particular, the development of simple, small compounds that can be produced and administered inexpensively would be of great importance. Recently, several potential repurposed drugs against COVID-19 (SARS-CoV-2 virus) have been found, such as remdesivir, infliximab and imatinib. Remdesivir has potent antiviral activity and has already Molecules 2021,26, 6825. https://doi.org/10.3390/molecules26226825 https://www.mdpi.com/journal/molecules Molecules 2021,26, 6825 2 of 16 been approved for urgent use [ 6 , 7 ]. However, to curb the spread of infection, it is important to identify new drug-leads that are more broadly effective against CoV. Nature is not only a source of emerging mutant viruses, but also a reservoir of natural products that play a crucial role in drug development. Emodin (1,3,8-trihydroxy-6- methylanthracene-9,10-dione), a potent natural bioactive anthraquinone, is found in various plants, lichens and molds, such as Cassia obtusifolia and Cassia occidentalis, Rhamnus orbiculatus, Aloe vera, Japanese knotweed, Polygonum multiflorum,Rheum palmatum, Scutellaria baicalensis and Rumex chalepensis [ 8 – 13 ]. Emodin is known for its anti-oxidant, anti-ulcerogenic, antibacterial, anti-fibrotic, anti-inflammatory, anti-cardiovascular, anti-viral and anti-cancer activities [ 14 – 19 ]. It has demonstrated antitumor activity against various cancers such as leukemia, squamous cell carcinoma of human tongue, lung cancer, gallbladder cancer, breast cancer, colon cancer and others [ 9 , 14 , 15 , 20 , 21 ]. Modified emodin compounds have therefore shown relevant pharmacological activity [ 22 – 27 ]. Of particular interest and promise are the results obtained with halogenated derivatives of emodin. In 2014, Huang and co-workers found that halogenated emodin derivatives can exert a potent inhibitory activity on bacterial topoisomerase I and DNA gyrase. The best results were obtained with 2,4-diiodoemodin [ 28 ]. In 2017, the research group led by Sukhatme and Sun reported the structure–activity relationship (SAR) of emodin and emodin derivatives as ATP citrate lyase (ACL) inhibitors. Halogenated emodin analogues (2-iodoemodin, 2-chloroemodin, 4-chloroemodin and 2,4-dibromoemodin) showed significantly increased activity [ 29 ]. Later, Tansakul’s group demonstrated that the hydroxyl and methyl groups were crucial for anti-MRSA (anti-methicillin-resistant Staphylococcus aureus) activity. All compounds containing two halogenated atoms (I, Br or Cl) at positions 2 and 4 were active against MRSA. The best results were obtained in the presence of an iodine atom ( 2,4-diiodoemodin) [30] . 4-chloroemodin was found to significantly inhibit the growth of gram-positive bacteria, especially that of common drug-resistant MRSA and VRE (vancomycin-resistant enterococci) isolates, through a dual antibacterial mechanism that interacts with the bacterial cell membrane and DNA [31]. In addition, emerging evidence suggests that emodin displays broad spectrum antiviral activities against herpes simplex viruses (HSV-1 and HSV-2) [ 32 , 33 ], hepatitis B virus (HBV) [ 34 , 35 ], Japanese encephalitis virus (JEV) [ 13 ], Human cytomegalovirus [ 36 ], Influenza A [ 37 ], Zika virus [ 38 ], Coxsackie B virus [ 39 , 40 ], Poliovirus [ 41 ], Cypridine herpesvirus 3 (CyHV-3) [ 42 ] and in a number of viral diseases. Through its antiviral activity, emodin can also prevent or reduce SARS-CoV infection [ 43 – 48 ]. Hsiang and co-workers reported that emodin can block the interaction of SARS-CoV spike protein with ACE-2 and infectivity of spike protein-pseudotyped retrovirus on Vero E6 cells [ 49 ]. In 2011, Schwarz and co-workers showed that emodin can inhibit the 3a ion channel of coronavirus as well as the release of SARS-CoV from infected cells [ 19 ]. Singha Roy and Das performed a blind molecular docking analysis of natural anthraquinones against SARS-CoV-2 main protease Mpro. The results suggest that natural emodins may prove to be effective inhibitors of COVID-19 by binding to the catalytic dyad, HIS41 and CYS145, through non-covalent forces near the active site [50]. The aim of the current study was to synthesize a series of emodin analogues and investigate their activities against human coronavirus NL63 (HCoV-NL63) [ 51 ]. For this work, we focused on the HCoV-NL63 virus, which causes mild to moderate upper respiratory tract infections in children, severe lower respiratory tract infection, croup and bronchiolitis [ 52 ]. HCoV-NL63 and SARS-CoV-2 both belong to the group of common human coronaviruses and they both use the ACE2 receptor to infect cells. HCoV-NL63 is thus a low pathogenic common coronavirus that may be used to study SARS under BSL2 conditions. This is the reason why HCoV-NL63 has been suggested as a suitable surrogate virus for studying SARS-CoV-2 [53]. Herein, we report the strategies followed for the selective introduction of NO 2 , SO 3 H and halogen atoms into the anthraquinone ring of emodin. Taking into account the potency of halogenated emodins, our work focused on the selective decoration of emodin, Molecules 2021,26, 6825 3 of 16 containing different halogen atoms and substitution patterns (Figure 1) and the evaluation of their antiviral activity against HCoV-NL63. Our results demonstrate that the presence of different functional groups in the emodin scaffold has a significant impact on their anti-HCoV-NL63 activity. Molecules 2021, 26, x FOR PEER REVIEW 3 of 17 Herein, we report the strategies followed for the selective introduction of NO2, SO3H and halogen atoms into the anthraquinone ring of emodin. Taking into account the potency of halogenated emodins, our work focused on the selective decoration of emodin, containing different halogen atoms and substitution patterns (Figure 1) and the evaluation of their antiviral activity against HCoV-NL63. Our results demonstrate that the presence of different functional groups in the emodin scaffold has a significant impact on their anti- HCoV-NL63 activity. Figure 1. Derivatization of emodin. 2. Results and Discussion 2.1. Synthesis of Emodin Derivatives The natural product emodin E_H provides an entry point for the introduction of various functional groups on aromatic rings by electrophilic aromatic substitution. Our aim was to create a library of emodin derivatives by modifying the aromatic ring through halogenation, nitration, amination and sulfonation. First, we investigated the selective halogenation of emodin E_H by classical reagents (N-chlorosuccinimide—NCS, N-bromosuccinimide—NBS, N-iodoosuccinimide—NIS) and by an alternative method—oxidative halogenation with hydrogen peroxide as oxidant. 2-Iodoemodin E_I was prepared according to the published methods [28] using I2 and NaHCO3 in 73% yield (Table 1, entry 1) and in the presence of NIS reagent in 84% yield (Table 1, entry 6). Oxidative iodination with I2 and 30% hydrogen peroxide in 2- MeTHF also allowed the selective synthesis of the same product with a better yield of 93% (Table 1, entry 16). For the oxidative iodination, 2 equivalents of iodine and 2.5 equivalents of hydrogen peroxide were used. While conducting the experimental work, it was observed that the reaction does not proceed in the absence of the oxidant H2O2 (entry 11) and that a higher amount of iodine is required for the quantitative conversion to E_I (entries 12–15). We also found that the amount of hydrogen peroxide has a minimal effect on the increase in conversion. A larger number of iodine substituents could not be introduced into the aromatic core of emodin using oxidative iodination. To introduce additional iodine atoms, I2/NaHCO3 or NIS had to be used. Nevertheless, the diiodinated emodin E_2I could not be selectively prepared, regardless of the amount of iodine or NIS used. 2,4,7- Triiodoemodin E_3I was selectively formed by applying the classical method (I2, Na- HCO3) [28] in 81% yield or using NIS reagent in 79% yield as a brown-orange powder (Table 1, entries 3 and 10). We also attempted to synthesize the fully iodinated emodin E_4I, but this compound could not be prepared regardless of the reaction conditions. We tried increasing the temperature and adding the activator H2SO4 to the reagent NIS. In all cases, the triiodinated product appeared selectively. The positions of iodine on the aromatic rings in compounds E_I and E_3I were confirmed by 2D NMR spectroscopy (Figures S6–S13, Supplementary Information). Figure 1. Derivatization of emodin. 2. Results and Discussion 2.1. Synthesis of Emodin Derivatives The natural product emodin E_H provides an entry point for the introduction of various functional groups on aromatic rings by electrophilic aromatic substitution. Our aim was to create a library of emodin derivatives by modifying the aromatic ring through halogenation, nitration, amination and sulfonation. First, we investigated the selective halogenation of emodin E_H by classical reagents (N-chlorosuccinimide—NCS, N-bromosuccinimide—NBS, N-iodoosuccinimide—NIS) and by an alternative method—oxidative halogenation with hydrogen peroxide as oxidant. 2-Iodoemodin E_I was prepared according to the published methods [ 28 ] using I 2 and NaHCO 3 in 73% yield (Table 1, entry 1) and in the presence of NIS reagent in 84% yield (Table 1, entry 6). Oxidative iodination with I 2 and 30% hydrogen peroxide in 2-MeTHF also allowed the selective synthesis of the same product with a better yield of 93% (Table 1 , entry 16). For the oxidative iodination, 2 equivalents of iodine and 2.5 equivalents of hydrogen peroxide were used. While conducting the experimental work, it was observed that the reaction does not proceed in the absence of the oxidant H 2 O 2 (entry 11) and that a higher amount of iodine is required for the quantitative conversion to E_I (entries 12–15) . We also found that the amount of hydrogen peroxide has a minimal effect on the increase in conversion. A larger number of iodine substituents could not be introduced into the aromatic core of emodin using oxidative iodination. To introduce additional iodine atoms, I 2 /NaHCO 3 or NIS had to be used. Nevertheless, the diiodinated emodin E_2I could not be selectively prepared, regardless of the amount of iodine or NIS used. 2,4,7-Triiodoemodin E_3I was selectively formed by applying the classical method (I 2 , NaHCO 3 ) [ 28 ] in 81% yield or using NIS reagent in 79% yield as a brown-orange powder (Table 1, entries 3 and 10). We also attempted to synthesize the fully iodinated emodin E_4I , but this compound could not be prepared regardless of the reaction conditions. We tried increasing the temperature and adding the activator H 2 SO 4 to the reagent NIS. In all cases, the triiodinated product appeared selectively. The positions of iodine on the aromatic rings in compounds E_I and E_3I were confirmed by 2D NMR spectroscopy (Figures S6–S13, Supplementary Material). Next, bromination was carried out using NBS and by oxidative halogenation using H 2 O 2 /HBr. Neither of these methods could be effective for selective synthesis of monobromo-derivative E_Br , as shown by the results in Table 2. Although a reaction with 1 equivalent of NBS was carried out in an ice bath, both E_Br and E_2Br were formed simultaneously (Table 2, entries 1 and 2). The same results were observed for oxidative bromination with HBr and H 2 O 2 . Regardless of the amounts of HBr and hydrogen peroxide used, a mixture of the two products was always formed. Using 2D NMR spectroscopy, we found that in the case of E_Br , bromine binds to site 2 in emodin, either when the NBS reagent or HBr/H2O2is used. Molecules 2021,26, 6825 4 of 16 Table 1. Iodination of emodin E_H with I2/NaHCO3, NIS or I2/H2O2. Molecules 2021, 26, x FOR PEER REVIEW 4 of 17 Table 1. Iodination of emodin E_H with I2/NaHCO3, NIS or I2/H2O2. Entry Reagent (equiv.) Solvent Conditions Relative Distribution a E_H E_I E_2I E_3I 1 I2 (2), NaHCO3 THF/H2O rt., 1 h - 100 (73%) - - 2 I2 (4), NaHCO3 THF/H2O rt., 1 h - 100 - - 3 I2 (10) , NaHCO3 THF/H2O rt., 24 h - - - 100 (81%) 4 I2 (10), NaHCO3 THF/H2O 60 °C, 24 h - - - 100 5 NIS (1) THF rt., 24 h 3 97 - - 6 NIS (1.3) THF rt., 3 h - 100 (84%) - - 7 NIS (1.3) THF rt., 24 h - 93 7 - 8 NIS (2) THF rt., 24 h - 86 14 - 9 NIS (4) THF rt., 24 h - - 56 44 10 NIS (4) THF 60 °C, 24 h - - - 100 (79%) 11 I2 (1) 2-MeTHF rt., 24 h 100 - - - 12 I2 (0.5), H2O2 (8) 2-MeTHF rt., 24 h 56 44 - - 13 I2 (1), H2O2 (8) 2-MeTHF rt., 24 h 21 79 - - 14 I2 (1.5) , H2O2 (2.5) 2-MeTHF rt., 24 h 16 84 - - 15 I2 (1.5), H2O2 (4) 2-MeTHF rt., 24 h 13 87 - - 16 I2 (2), H2O2 (2.5) 2-MeTHF rt., 24 h - 100 (93%) - - 17 I2 (8), H2O2 (8) 2-MeTHF rt., 24 h - 100 - - Reaction conditions: Emodin (0.1 mmol), reagent (I2 (0.2–1.0 mmol), NIS (N-iodosuccinimide) (0.1–0.4 mmol), H2O2 (30%, 0.25–0.8 mmol)), solvent (1 mL), a Conversion to product was determined by 1H NMR. Next, bromination was carried out using NBS and by oxidative halogenation using H2O2/HBr. Neither of these methods could be effective for selective synthesis of monobromo-derivative E_Br, as shown by the results in Table 2. Although a reaction with 1 equivalent of NBS was carried out in an ice bath, both E_Br and E_2Br were formed simultaneously (Table 2, entries 1 and 2). The same results were observed for oxidative bromination with HBr and H2O2. Regardless of the amounts of HBr and hydrogen peroxide used, a mixture of the two products was always formed. Using 2D NMR spectroscopy, we found that in the case of E_Br, bromine binds to site 2 in emodin, either when the NBS reagent or HBr/H2O2 is used. E_2Br was selectively prepared according to a published procedure [29] with a slight modification consisting of treating emodin E_H with NBS in THF at 0 °C (88% yield). Due to the high reactivity of emodin, the bromination required a relatively short reaction time (30 min) and a low temperature (0 °C). The same product was obtained by the oxidative halogenation method (HBr, H2O2) in 2,2,2 trifluoroethanol (TFE) in 91% yield (Table 2, entry 14). Unfortunately, the oxidative method, although more environmentally friendly, did not allow the introduction of more bromine atoms. Despite the higher amount of HBr and the use of the activating solvent TFE, the reaction stopped at the dibrominated product. The use of an NBS reagent allowed a greater number of bromine atoms to be introduced, but we encountered problems with the selectivity in the preparation of E_3Br. Regardless of the temperature at which the reaction was carried out and the amount of reagent used, E_2Br and/or E_4Br were also formed (Table 2, entries 4–6). Selectively, we prepared the orange-colored product 2,4,6,8-tetrabromo-1,3,5-trihydroxy-7-methylan- thracene-9,10-dione E_4Br (Table 2, entry 7). Entry Reagent (equiv.) Solvent Conditions Relative Distribution a E_H E_I E_2I E_3I 1 I2(2), NaHCO3THF/H2O rt., 1 h - 100 (73%) - - 2 I2(4), NaHCO3THF/H2O rt., 1 h - 100 - - 3 I2(10), NaHCO3THF/H2O rt., 24 h - - - 100 (81%) 4 I2(10), NaHCO3THF/H2O 60 ◦C, 24 h - - - 100 5 NIS (1) THF rt., 24 h 3 97 - - 6 NIS (1.3) THF rt., 3 h - 100 (84%) - - 7 NIS (1.3) THF rt., 24 h - 93 7 - 8 NIS (2) THF rt., 24 h - 86 14 - 9 NIS (4) THF rt., 24 h - - 56 44 10 NIS (4) THF 60 ◦C, 24 h - - - 100 (79%) 11 I2(1) 2-MeTHF rt., 24 h 100 - - - 12 I2(0.5), H2O2(8) 2-MeTHF rt., 24 h 56 44 - - 13 I2(1), H2O2(8) 2-MeTHF rt., 24 h 21 79 - - 14 I2(1.5), H2O2(2.5) 2-MeTHF rt., 24 h 16 84 - - 15 I2(1.5), H2O2(4) 2-MeTHF rt., 24 h 13 87 - - 16 I2(2), H2O2(2.5) 2-MeTHF rt., 24 h - 100 (93%) - - 17 I2(8), H2O2(8) 2-MeTHF rt., 24 h - 100 - - Reaction conditions: Emodin (0.1 mmol), reagent (I 2 (0.2–1.0 mmol), NIS (N-iodosuccinimide) (0.1–0.4 mmol), H 2 O 2 (30%, 0.25–0.8 mmol)), solvent (1 mL), aConversion to product was determined by 1H NMR. Table 2. Bromination of emodin E_H with NBS and HBr/H2O2. Molecules 2021, 26, x FOR PEER REVIEW 5 of 17 Table 2. Bromination of emodin E_H with NBS and HBr/H2O2. Entry Reagent (Equiv.) Solvent Conditions Relative Distribution a E_H E_Br E_2Br E_3Br E_4Br 1 NBS (1) THF 0 °C, 10 min 34 40 26 - - 2 NBS (1.5) THF 0 °C, 15 min 17 43 40 - - 3 NBS (2.2) THF 0 °C, 30 min - - 100 (88%) - - 4 NBS (3) THF 0 °C, 24 h - - 63 37 - 5 NBS (3) THF rt., 24 h - - 27 73 - 6 NBS (4) THF rt., 24 h - - - 60 40 7 NBS (5) THF rt., 24 h - - - - 100 (83%) 8 HBr (1), H2O2 (2.5) TFE 0 °C, 2 h 29 71 - - - 9 HBr (1.3) , H2O2 (5) TFE 0 °C, 2 h 67 31 2 - - 10 HBr (2.5), H2O2 (5) TFE 0 °C, 2 h 8 81 11 - - 11 HBr (1), H2O2 (5) TFE rt., 24 h 9 79 12 - - 12 HBr (2), H2O2 (5) TFE rt., 24 h - 28 72 - - 13 HBr (2.4), H2O2 (5) TFE rt., 24 h - 14 86 - - 14 HBr (4), H2O2 (5) TFE rt., 24 h - - 100 (91%) - - Reaction conditions: Emodin (0.1 mmol), reagent (NBS (N-bromosuccinimide) (0.1–0.5 mmol), HBr (48%, 0.1–0.4 mmol), H2O2 (30%, 0.25–0.5 mmol)), solvent (1 mL). a Conversion to product was determined by 1H NMR. Chlorination of emodin gave similar results to bromination. The monosubstituted product could not be prepared selectively with the NCS reagent or oxidatively with HCl/H2O2, since the dichloro derivative E_2Cl was also formed. It was observed that the regioselectivity of chlorination to E_Cl depends on the method used—HCl/H2O2 or NCS—as determined by conducting 1D and 2D NMR spectroscopy on the crude reaction mixture consisting of E_H, E Cl or/and E_2Cl. The results showed that when NCS was used, the Cl was bound at position 2 (E_Cl-2), whereas when HCl/H2O2 was used, it was bound at position 4 (E_Cl-4) (Figures S1–S3). Good yields of the disubstituted emodin 2,4- dichloroemodin E_2Cl were obtained selectively with NCS [30] using H2SO4 as catalyst in a 3-h process at room temperature (Table 3, entry 3). When emodin was treated with 6 equivalents of NCS in the presence of H2SO4 at a reflux temperature, no formation of three- or four-chloroemodin occurred. Only two chlorine atoms could be introduced into emodin. Oxidative chlorination was proved to be an unsuitable procedure for the selective synthesis of chlorine products. Despite the combination of different ratios of HCl and H2O2, emodin was always converted into a mixture of different products that could not be separated (Table 3, entries 5–9). Entry Reagent (Equiv.) Solvent Conditions Relative Distribution a E_H E_Br E_2Br E_3Br E_4Br 1 NBS (1) THF 0 ◦C, 10 min 34 40 26 - - 2 NBS (1.5) THF 0 ◦C, 15 min 17 43 40 - - 3 NBS (2.2) THF 0 ◦C, 30 min - - 100 (88%) - - 4 NBS (3) THF 0 ◦C, 24 h - - 63 37 - 5 NBS (3) THF rt., 24 h - - 27 73 - 6 NBS (4) THF rt., 24 h - - - 60 40 7 NBS (5) THF rt., 24 h - - - - 100 (83%) 8 HBr (1), H2O2(2.5) TFE 0 ◦C, 2 h 29 71 - - - 9 HBr (1.3), H2O2(5) TFE 0 ◦C, 2 h 67 31 2 - - 10 HBr (2.5), H2O2(5) TFE 0 ◦C, 2 h 8 81 11 - - 11 HBr (1), H2O2(5) TFE rt., 24 h 9 79 12 - - 12 HBr (2), H2O2(5) TFE rt., 24 h - 28 72 - - 13 HBr (2.4), H2O2(5) TFE rt., 24 h - 14 86 - - 14 HBr (4), H2O2(5) TFE rt., 24 h - - 100 (91%) - - Reaction conditions: Emodin (0.1 mmol), reagent (NBS (N-bromosuccinimide) (0.1–0.5 mmol), HBr (48%, 0.1–0.4 mmol), H 2 O 2 (30%, 0.25–0.5 mmol)), solvent (1 mL). aConversion to product was determined by 1H NMR. Molecules 2021,26, 6825 5 of 16 E_2Br was selectively prepared according to a published procedure [ 29 ] with a slight modification consisting of treating emodin E_H with NBS in THF at 0 ◦ C (88% yield). Due to the high reactivity of emodin, the bromination required a relatively short reaction time (30 min) and a low temperature (0 ◦ C). The same product was obtained by the oxidative halogenation method (HBr, H 2 O 2 ) in 2,2,2 trifluoroethanol (TFE) in 91% yield (Table 2, entry 14). Unfortunately, the oxidative method, although more environmentally friendly, did not allow the introduction of more bromine atoms. Despite the higher amount of HBr and the use of the activating solvent TFE, the reaction stopped at the dibrominated product. The use of an NBS reagent allowed a greater number of bromine atoms to be introduced, but we encountered problems with the selectivity in the preparation of E_3Br . Regardless of the temperature at which the reaction was carried out and the amount of reagent used, E_2Br and/or E_4Br were also formed (Table 2, entries 4–6). Selectively, we prepared the orange-colored product 2,4,6,8-tetrabromo-1,3,5-trihydroxy-7-methylanthracene-9,10-dione E_4Br (Table 2, entry 7). Chlorination of emodin gave similar results to bromination. The monosubstituted product could not be prepared selectively with the NCS reagent or oxidatively with HCl/H 2 O 2 , since the dichloro derivative E_2Cl was also formed. It was observed that the regioselectivity of chlorination to E_Cl depends on the method used—HCl/H 2 O 2 or NCS—as determined by conducting 1D and 2D NMR spectroscopy on the crude reaction mixture consisting of E_H , E Cl or/and E_2Cl . The results showed that when NCS was used, the Cl was bound at position 2 ( E_Cl-2 ), whereas when HCl/H 2 O 2 was used, it was bound at position 4 ( E_Cl-4 ) (Figures S1–S3). Good yields of the disubstituted emodin 2,4-dichloroemodin E_2Cl were obtained selectively with NCS [ 30 ] using H 2 SO 4 as catalyst in a 3-h process at room temperature (Table 3, entry 3). When emodin was treated with 6 equivalents of NCS in the presence of H 2 SO 4 at a reflux temperature, no formation of three- or four-chloroemodin occurred. Only two chlorine atoms could be introduced into emodin. Oxidative chlorination was proved to be an unsuitable procedure for the selective synthesis of chlorine products. Despite the combination of different ratios of HCl and H 2 O 2 , emodin was always converted into a mixture of different products that could not be separated (Table 3, entries 5–9). Table 3. Chlorination of emodin E_H with NCS and HCl/H2O2. Molecules 2021, 26, x FOR PEER REVIEW 6 of 17 Table 3. Chlorination of emodin E_H with NCS and HCl/H2O2. Entry Reagent (Equiv.) Solvent Conditions Relative Distribution a E_H E_Cl E_2Cl E_3Cl 1 NCS (1) THF rt., 24 h 100 - - - 2 NCS (2) THF rt., 24 h 49 42 9 - 3 NCS b (2.5) THF rt., 3 h - - 100 (81%) - 4 NCS b (6) THF 60 °C, 24 h - - 100 - 5 HCl (1), H2O2 (2) TFE rt., 24 h 56 36 8 - 6 HCl (2), H2O2 (5) TFE rt., 24 h 10 68 22 - 7 HCl (3), H2O2 (5) TFE rt., 24 h - 44 39 17 8 HCl (4), H2O2 (5) TFE rt., 24 h - 41 37 22 9 HCl (5), H2O2 (10) TFE rt., 24 h - 17 50 33 Reaction conditions: Emodin (0.1 mmol), reagent (NCS (N-chlorosuccinimide) (0.1–0.6 mmol), HCl (37%, 0.1–0.5 mmol), H2O2 (30%, 0.2–1.0 mmol)), solvent (1 mL), a Conversion to product was determined by 1H NMR, b H2SO4. Next, we investigated the selective nitration of E_H. We attempted to selectively prepare nitroemodin with one, two or three NO2 functional groups, but none of the conditions tested led to successful results. The results showed that despite the use of small amounts of nitric and sulfuric acids, a mixture of various nitrated products E_X formed that could not be separated (Table 4, entries 1–7). It was concluded that the nitration reaction is not selective towards a particular product despite the absence of H2SO4. We prepared the tetranitroemodin E_4NO2 using 6.2 equiv. HNO3 and 10 equiv. H2SO4 in 91% yield (Table 4, entry 8), reducing excess reagents by an order of magnitude according to a published method [24] (Table 4, entry 9). Table 4. Nitration of emodin with HNO3 and H2SO4. Entry Reagent (Equiv.) a Conditions Conv. b E_X E_4NO2 1 HNO3 (1), H2SO4 (4), MeCN 0 °C, 1 h 2% - 2 HNO3 (2.5) , H2SO4 (10) 0 °C, 1 h 78% 22% 3 HNO3 (2.5), H2SO4 (20) 0 °C, 1 h 79% 21% 4 HNO3 (3), H2SO4 (10) 0 °C, 1 h 59% 41% 5 HNO3 (3), H2SO4 (20) 0 °C, 1 h 27% 73% 6 HNO3 (4.4), H2SO4 (20) 0 °C, 1 h 10% 90% 7 HNO3 (6.2), H2SO4 (5) 0 °C, 0.25 h 13% 87% 8 HNO3 (6.2) , H2SO4 (10) 0 °C, 0.5 h - 100 (91%) 9 HNO3 (63), H2SO4 (80) 0 °C, 1 h; rt, 4 h - 100 % [24] a Emodin (0.1 mmol), reagent (HNO3 (0.1–6.3 mmol), H2SO4 (0.4–8.0 mmol)), MeCN/no solvent, b Conversion to product was determined by 1H NMR relative to E_H. E_X is a mixture of mono-, di- and trinitroemodin. In addition, we also prepared amino-substituted emodin E_NH2 by the method described in an earlier publication (Figure 2) [24]. Entry Reagent (Equiv.) Solvent Conditions Relative Distribution a E_H E_Cl E_2Cl E_3Cl 1 NCS (1) THF rt., 24 h 100 - - - 2 NCS (2) THF rt., 24 h 49 42 9 - 3NCS b(2.5) THF rt., 3 h - - 100 (81%) - 4NCS b(6) THF 60 ◦C, 24 h - - 100 - 5 HCl (1), H2O2(2) TFE rt., 24 h 56 36 8 - 6 HCl (2), H2O2(5) TFE rt., 24 h 10 68 22 - 7 HCl (3), H2O2(5) TFE rt., 24 h - 44 39 17 8 HCl (4), H2O2(5) TFE rt., 24 h - 41 37 22 9 HCl (5), H2O2(10) TFE rt., 24 h - 17 50 33 Reaction conditions: Emodin (0.1 mmol), reagent (NCS (N-chlorosuccinimide) (0.1–0.6 mmol), HCl (37%, 0.1–0.5 mmol), H 2 O 2 (30%, 0.2–1.0 mmol)), solvent (1 mL), aConversion to product was determined by 1H NMR, bH2SO4. Next, we investigated the selective nitration of E_H . We attempted to selectively prepare nitroemodin with one, two or three NO 2 functional groups, but none of the conditions tested led to successful results. The results showed that despite the use of small Molecules 2021,26, 6825 6 of 16 amounts of nitric and sulfuric acids, a mixture of various nitrated products E_X formed that could not be separated (Table 4, entries 1–7). It was concluded that the nitration reaction is not selective towards a particular product despite the absence of H 2 SO 4 . We prepared the tetranitroemodin E_4NO2 using 6.2 equiv. HNO 3 and 10 equiv. H 2 SO 4 in 91% yield (Table 4 , entry 8), reducing excess reagents by an order of magnitude according to a published method [24] (Table 4, entry 9). Table 4. Nitration of emodin with HNO3and H2SO4. Entry Reagent (Equiv.) aConditions Conv. b E_X E_4NO2 1 HNO3(1), H2SO4(4), MeCN 0 ◦C, 1 h 2% - 2 HNO3(2.5), H2SO4(10) 0 ◦C, 1 h 78% 22% 3 HNO3(2.5), H2SO4(20) 0 ◦C, 1 h 79% 21% 4 HNO3(3), H2SO4(10) 0 ◦C, 1 h 59% 41% 5 HNO3(3), H2SO4(20) 0 ◦C, 1 h 27% 73% 6 HNO3(4.4), H2SO4(20) 0 ◦C, 1 h 10% 90% 7 HNO3(6.2), H2SO4(5) 0 ◦C, 0.25 h 13% 87% 8 HNO3(6.2), H2SO4(10) 0 ◦C, 0.5 h - 100 (91%) 9 HNO3(63), H2SO4(80) 0 ◦C, 1 h; rt, 4 h - 100 % [24] a Emodin (0.1 mmol), reagent (HNO 3 (0.1–6.3 mmol), H 2 SO 4 (0.4–8.0 mmol)), MeCN/no solvent, b Conversion to product was determined by 1H NMR relative to E_H.E_X is a mixture of mono-, di- and trinitroemodin. In addition, we also prepared amino-substituted emodin E_NH2 by the method described in an earlier publication (Figure 2) [24]. Molecules 2021, 26, x FOR PEER REVIEW 7 of 17 Figure 2. Synthesis of amino emodin E_NH2. Sulfonation was carried out with oleum at room temperature. After 24 h, the reaction mixture was analyzed by NMR; three different products were observed. According to the NMR spectra obtained from the crude reaction mixture, trisulfonated, disulfonated and monosulfonated emodin were formed. However, the composition of the mixture changed with time and reverse sulfonation occurred. Hence, following the work-up procedure, the crude reaction mixture was stirred in MeCN/hexane and after 3 h the product was completely converted to a stable monosubstituted E_SO3H, which was isolated as a brown solid in 76% yield (Figure 3). Figure 3. Synthesis of E_SO3H. 1,3,8-Trimethoxy-6-methyl-9,10-anthraquinone E_OCH3 was obtained in good yield (81%) by applying a previously described reaction procedure (Me2SO4 and base K2CO3) [30]. Brominated trimethoxyemodin E_Br_OCH3 was synthesized from tetrabrominated emodin E_4Br by the same method. After purification, the product E_4Br_OCH3 was obtained in 87% yield. 2.2. Antiviral Activity of Emodin Analogues The primary objective of this study was to determine the potential of a library of 11 emodin analogues (Figure 4) to combat the cytopathic effects of HCoV-NL63. The nontransformed primate kidney Vero cell line was used for this work. An additional series of studies was performed to evaluate the effects of each compound on the viability of Vero cells. Figure 2. Synthesis of amino emodin E_NH2. Sulfonation was carried out with oleum at room temperature. After 24 h, the reaction mixture was analyzed by NMR; three different products were observed. According to the NMR spectra obtained from the crude reaction mixture, trisulfonated, disulfonated and monosulfonated emodin were formed. However, the composition of the mixture changed with time and reverse sulfonation occurred. Hence, following the work-up procedure, the crude reaction mixture was stirred in MeCN/hexane and after 3 h the product was completely converted to a stable monosubstituted E_SO3H , which was isolated as a brown solid in 76% yield (Figure 3). Molecules 2021, 26, x FOR PEER REVIEW 7 of 17 Figure 2. Synthesis of amino emodin E_NH2. Sulfonation was carried out with oleum at room temperature. After 24 h, the reaction mixture was analyzed by NMR; three different products were observed. According to the NMR spectra obtained from the crude reaction mixture, trisulfonated, disulfonated and monosulfonated emodin were formed. However, the composition of the mixture changed with time and reverse sulfonation occurred. Hence, following the work-up procedure, the crude reaction mixture was stirred in MeCN/hexane and after 3 h the product was completely converted to a stable monosubstituted E_SO3H, which was isolated as a brown solid in 76% yield (Figure 3). Figure 3. Synthesis of E_SO3H. 1,3,8-Trimethoxy-6-methyl-9,10-anthraquinone E_OCH3 was obtained in good yield (81%) by applying a previously described reaction procedure (Me2SO4 and base K2CO3) [30]. Brominated trimethoxyemodin E_Br_OCH3 was synthesized from tetrabrominated emodin E_4Br by the same method. After purification, the product E_4Br_OCH3 was obtained in 87% yield. 2.2. Antiviral Activity of Emodin Analogues The primary objective of this study was to determine the potential of a library of 11 emodin analogues (Figure 4) to combat the cytopathic effects of HCoV-NL63. The nontransformed primate kidney Vero cell line was used for this work. An additional series of studies was performed to evaluate the effects of each compound on the viability of Vero cells. Figure 3. Synthesis of E_SO3H. 1,3,8-Trimethoxy-6-methyl-9,10-anthraquinone E_OCH3 was obtained in good yield (81%) by applying a previously described reaction procedure (Me 2 SO 4 and base K 2 CO 3 ) [ 30 ]. Brominated trimethoxyemodin E_Br_OCH3 was synthesized from tetrabrominated emodin Molecules 2021,26, 6825 7 of 16 E_4Br by the same method. After purification, the product E_4Br_OCH3 was obtained in 87% yield. 2.2. Antiviral Activity of Emodin Analogues The primary objective of this study was to determine the potential of a library of 11 emodin analogues (Figure 4) to combat the cytopathic effects of HCoV-NL63. The nontransformed primate kidney Vero cell line was used for this work. An additional series of studies was performed to evaluate the effects of each compound on the viability of Vero cells. Molecules 2021, 26, x FOR PEER REVIEW 8 of 17 Figure 4. Emodin and emodin derivatives prepared in the course of this study. 2.2.1. Evaluation of the Viability of Vero Cells in the Presence of Emodin and Emodin Analogues Published reports indicate that emodin displays a broad spectrum of activities, including cytotoxicity [14]. Therefore, in addition to evaluating the activity of emodin and the emodin analogues against HCoV-NL63, it was important to test the potential cytotoxicity of these compounds. Viability assays were performed using Vero cells; IC50 curves for each compound and other additional data are shown in Figure 5 and in Table S1 (raw data in Table S4). Figure 5. IC50 curves reflecting the effect of the tested compounds on Vero cell viability. Interestingly, only E_I showed higher toxicity to Vero cells compared to emodin (E_H). E_4Br, E_Cl and E_OMe were less toxic than emodin, while virtually no toxicity was observed with E_SO3H, E_NH2 and E_Br_OMe. However, it is important to consider that although all emodin analogues were soluble in DMSO at 50 mM concentrations, some analogues exhibited a tendency to precipitate when added to the cell culture medium; compounds containing methoxy groups were the most difficult to dissolve in DMSO and were therefore probably the least soluble in aqueous solutions. It is therefore possible that these compounds gave lower cytotoxicity indexes due to their poor solubility in the cell culture medium. Figure 4. Emodin and emodin derivatives prepared in the course of this study. 2.2.1. Evaluation of the Viability of Vero Cells in the Presence of Emodin and Emodin Analogues Published reports indicate that emodin displays a broad spectrum of activities, including cytotoxicity [ 14 ]. Therefore, in addition to evaluating the activity of emodin and the emodin analogues against HCoV-NL63, it was important to test the potential cytotoxicity of these compounds. Viability assays were performed using Vero cells; IC50 curves for each compound and other additional data are shown in Figure 5and in Table S1 (raw data in Table S4). Molecules 2021, 26, x FOR PEER REVIEW 8 of 17 Figure 4. Emodin and emodin derivatives prepared in the course of this study. 2.2.1. Evaluation of the Viability of Vero Cells in the Presence of Emodin and Emodin Analogues Published reports indicate that emodin displays a broad spectrum of activities, including cytotoxicity [14]. Therefore, in addition to evaluating the activity of emodin and the emodin analogues against HCoV-NL63, it was important to test the potential cytotoxicity of these compounds. Viability assays were performed using Vero cells; IC50 curves for each compound and other additional data are shown in Figure 5 and in Table S1 (raw data in Table S4). Figure 5. IC50 curves reflecting the effect of the tested compounds on Vero cell viability. Interestingly, only E_I showed higher toxicity to Vero cells compared to emodin (E_H). E_4Br, E_Cl and E_OMe were less toxic than emodin, while virtually no toxicity was observed with E_SO3H, E_NH2 and E_Br_OMe. However, it is important to consider that although all emodin analogues were soluble in DMSO at 50 mM concentrations, some analogues exhibited a tendency to precipitate when added to the cell culture medium; compounds containing methoxy groups were the most difficult to dissolve in DMSO and were therefore probably the least soluble in aqueous solutions. It is therefore possible that these compounds gave lower cytotoxicity indexes due to their poor solubility in the cell culture medium. Figure 5. IC50 curves reflecting the effect of the tested compounds on Vero cell viability. Molecules 2021,26, 6825 8 of 16 Interestingly, only E_I showed higher toxicity to Vero cells compared to emodin ( E_H ). E_4Br , E_Cl and E_OMe were less toxic than emodin, while virtually no toxicity was observed with E_SO3H , E_NH2 and E_Br_OMe . However, it is important to consider that although all emodin analogues were soluble in DMSO at 50 mM concentrations, some analogues exhibited a tendency to precipitate when added to the cell culture medium; compounds containing methoxy groups were the most difficult to dissolve in DMSO and were therefore probably the least soluble in aqueous solutions. It is therefore possible that these compounds gave lower cytotoxicity indexes due to their poor solubility in the cell culture medium. 2.2.2. Anti-Viral Activity of Emodin and Emodin Derivatives In another series of studies, we evaluated the potential of emodin and emodin derivatives to protect Vero cells against the cytopathic effects induced by HCoV-NL63. Dead cell protease activity was used as a measure of impaired cell viability, as described in the Methods section; chloroquine and remdesivir were used as controls. The results obtained are presented in Figure 6and Table 5. Additional data can be found in Supplementary Materials (Table S2; raw data in Table S5). Three compounds, E_OMe , E_SO3H and E_Br_OMe , did not exhibit significant anti-viral activity; as described above, this could be due to their lower solubility in aqueous solutions. Emodin and the emodin analogues E_4NO2 and E_I impaired Vero cell viability and had anti-viral effects at very similar concentrations. Five other compounds, E_3I , E_4Br , E_2Br , E_2Cl and E_NH2 , showed anti-viral activity at concentrations lower than those at which they impaired Vero cell viability; among these, E_NH2 exhibited the least toxicity to Vero cells. On the other hand, E_3I was the compound that demonstrated the strongest anti-HCoV-NL63 activity, along with the largest difference (about 10-fold) between the IC 50 value for anti-HCoV-NL63 activity and that for cytotoxicity, thus providing the largest therapeutic window. Nevertheless, the IC 50 value for cytotoxicity of E_3I was still considerably high under the applied experimental conditions, namely identical to that of E_H (Table S1). Molecules 2021, 26, x FOR PEER REVIEW 9 of 17 2.2.2. Anti-Viral Activity of Emodin and Emodin Derivatives In another series of studies, we evaluated the potential of emodin and emodin derivatives to protect Vero cells against the cytopathic effects induced by HCoV-NL63. Dead cell protease activity was used as a measure of impaired cell viability, as described in the Methods section; chloroquine and remdesivir were used as controls. The results obtained are presented in Figure 6 and Table 5. Additional data can be found in Supplementary Information (Table S2; raw data in Table S5). Three compounds, E_OMe, E_SO3H and E_Br_OMe, did not exhibit significant anti-viral activity; as described above, this could be due to their lower solubility in aqueous solutions. Emodin and the emodin analogues E_4NO2 and E_I impaired Vero cell viability and had anti-viral effects at very similar concentrations. Five other compounds, E_3I, E_4Br, E_2Br, E_2Cl and E_NH2, showed antiviral activity at concentrations lower than those at which they impaired Vero cell viability; among these, E_NH2 exhibited the least toxicity to Vero cells. On the other hand, E_3I was the compound that demonstrated the strongest anti-HCoV-NL63 activity, along with the largest difference (about 10-fold) between the IC50 value for anti-HCoV-NL63 activity and that for cytotoxicity, thus providing the largest therapeutic window. Nevertheless, the IC50 value for cytotoxicity of E_3I was still considerably high under the applied experimental conditions, namely identical to that of E_H (Table S1). Figure 6. IC50 curves representing the anti-HCoV-NL63 effects of emodin and emodin derivatives. Table 5. IC50 (µM) values corresponding to the anti HCoV-NL63 effects (AV) and the effects on Vero cell viability (expressed as IC50 values, CV) of each of the tested compounds. IC50 (µM) Exp. CT E_H 2.5 4.9 E_4NO2 6.1 6.1 E_I 1.3 3.6 E_3I 0.5 4.9 E_4Br 1.7 7.2 E_2Br 1.0 5.4 E_2Cl 1.1 7.5 E_OMe >50 8.7 E_SO3H 22.0 >50 E_NH2 6.3 41.8 E_Br_OMe >50 >50 Remdesivir 0.61 Chloroquine 19.2 As mentioned earlier, chloroquine and remdesivir were used as controls, since these drugs are being considered as potentially effective against COVID-19. Interestingly, the anti-HCoV-NL63 activity of some of the emodin analogues was much higher than that of chloroquine. On the other hand, the emodin analogues E_3I and E_2Br displayed anti- Figure 6. IC50 curves representing the anti-HCoV-NL63 effects of emodin and emodin derivatives. Table 5. IC 50 ( µ M) values corresponding to the anti HCoV-NL63 effects (AV) and the effects on Vero cell viability (expressed as IC50 values, CV) of each of the tested compounds. IC50 (µM) Exp. CT E_H 2.5 4.9 E_4NO26.1 6.1 E_I 1.3 3.6 E_3I 0.5 4.9 E_4Br 1.7 7.2 E_2Br 1.0 5.4 E_2Cl 1.1 7.5 E_OMe >50 8.7 E_SO3H22.0 >50 E_NH26.3 41.8 E_Br_OMe >50 >50 Remdesivir 0.61 Chloroquine 19.2 Molecules 2021,26, 6825 9 of 16 As mentioned earlier, chloroquine and remdesivir were used as controls, since these drugs are being considered as potentially effective against COVID-19. Interestingly, the anti-HCoV-NL63 activity of some of the emodin analogues was much higher than that of chloroquine. On the other hand, the emodin analogues E_3I and E_2Br displayed anti- HCoV-NL63 activity comparable to that of remdesivir; all the data obtained on the activities of chloroquine and remdesivir are presented in Supplementary Materials (Table S3 and Figure S5; raw data in Tables S6 and S7). 3. Materials and Methods Emodin was purchased from Fluorochem Ltd. (Glossop, UK). All other reagents and solvents were of reagent-grade quality and were obtained from commercial suppliers Honeywell (Seelze, Germany) and Sigma-Aldrich (Taufkirchen, Germany). TLC was performed on Merck-60-F 254 plates (Merck, Darmstadt, Germany) using mixtures of EtOAc:EtOH (10:1), CH 2 Cl 2 :EtOH (100:1) or EtOAc:MeOH (20:1). Crude emodin preparations were purified by column chromatography on silica gel (63–200 µ m, 70–230 mesh ASTM; Honeywell, Seelze, Germany). The isolated compounds were characterized by 1 H, 13 C NMR spectra, HRMS and IR analysis. 1 H and 13 C NMR spectra were recorded on Bruker Avance III 500 instruments (Bruker, Billerica, MA, USA). IR spectra were recorded on Bruker Alpha II FTIR Instrument (Bruker, Billerica, MA, USA). HR-MS were recorded on LC MS system Agilent 6224 Accurate Mass TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA). 3.1. Compound Synthesis and Structure Confirmation 1,3,8-trihydroxy-2-iodo-6-methylanthracene-9,10-dione (E_I) [ 28 ]. Iodine (254 mg, 1.0 mmol) and 30% H 2 O 2 (255 µ L, 2.5 mmol) were added to a stirred solution of emodin (135 mg, 0.5 mmol) in 2-MeTHF (5 mL). The reaction mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC (CH 2 Cl 2 :EtOH = 100:1). After the reaction was complete, the reaction mixture was washed with NaHSO 3 and extracted with dichloromethane (3 × 30 mL). The organic layer was washed with water ( 1×30 mL ), dried over anhydrous Na 2 SO 4 and evaporated under a vacuum. The crude reaction product was washed with hexane (5 mL) and acetonitrile (5 mL) to remove soluble impurities. The product was dried in vacuum to provide the product (184 mg, 93%) as an orange solid. 1 H NMR (500 MHz, DMSO-d 6 , 25 ◦ C): δ = 13.07 (s, 1H, OH), 12.26 (s, 1H, OH), 11.83 (s, 1H, OH), 7.47 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.16 (s, 1H, ArH), 2.41 (s, 3H, CH 3 ) ppm. 13 C NMR ( 126 MHz , DMSO-d 6 , 25 ◦ C): δ = 189.4, 181.2, 165.2, 163.5, 161.4, 148.6, 134.2, 132.7, 124.3, 120.6, 113.1, 108.5, 106.8, 83.2, 21.6 ppm. IR: 3339, 1665, 1615, 1473, 1379, 1263, 1168, 949 cm−1 . HRMS (ESI − ): m/z calcd for C 15 H 9 IO 5 394.9422 [M-H] − , found: 394.9428 [M-H]−. 1,3,8-trihydroxy-2,4,7-triiodo-6-methylanthracene-9,10-dione (E_3I) [ 28 ]. Iodine ( 1.3 g , 5.0 mmol) was added at a temperature of 0 ◦ C to a stirred solution of emodin (135 mg, 0.5 mmol ) in THF (13 mL) and water (13 mL). NaHCO 3 (3.5 g, 42.0 mmol) was then added in a stepwise manner. The reaction mixture was stirred for 24 h at room temperature. The reaction was monitored by TLC (CH 2 Cl 2 :EtOH = 100:1). After completion of the reaction, the mixture was extracted with dichloromethane (3 × 30 mL). The organic layer was washed with water (1 × 30 mL), dried over anhydrous Na 2 SO 4 and evaporated under vacuum. The crude reaction product was washed with hexane (5 mL) and acetonitrile ( 3×5 mL ) to remove soluble impurities. The product was dried in vacuum to provide the product (262 mg, 81%) as an orange–brown solid. 1 H NMR (500 MHz, DMSO-d 6 , 25 ◦ C): δ= 13.61 (s, 1H, OH), 12.60 (s, 1H, OH), 7.47 (s, 1H, ArH), 2.48 (s, 3H, CH 3 ) ppm. 13 C NMR (126 MHz, DMSO-d 6 , 25 ◦ C): δ = 187.2, 180.2, 165.1, 163.8, 159.5, 151.8, 133.3, 132.0, 120.4, 111.8, 110.0, 101.3, 84.1, 82.5, 29.3 ppm. IR: 3359, 1614, 1377, 1236, 1111, 1043 cm −1 . HRMS (ESI−): m/z calcd for C15H7I3O5646.7355 [M-H]−, found: 646.7380 [M-H]−. 2,4-dibromo-1,3,8-trihydroxy-6-methylanthracene-9,10-dione (E_2Br) [ 28 ]. HBr (48%, 2.0 mmol) and 30% H 2 O 2 (255 µ L, 2.5 mmol) were added to a stirred solution of emodin (135 mg, 0.5 mmol) in TFE (5 mL). The reaction mixture was stirred at room temperature for Molecules 2021,26, 6825 16 of 16 42. Wang, Z.; Zheng, N.; Liang, J.; Wang, Q.; Zu, X.; Wang, H.; Yuan, H.; Zhang, R.; Guo, S.; Liu, Y.; et al. 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