key: cord-0735774-d8p6ntsr authors: Zhang, Xintong; Shi, Yulong; Guo, Zhihao; Zhao, Xiaoqiang; Wu, Jiajing; Cao, Shouchun; Liu, Yonghua; Li, Yuhua; Huang, Weijin; Wang, Youchun; Liu, Qiang; Li, Yinghong; Song, Danqing title: Clofazimine derivatives as potent broad-spectrum antiviral agents with dual-target mechanism date: 2022-03-05 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2022.114209 sha: 48069ee1ed679b894bb9d3f7f1ece82f2b2b3c35 doc_id: 735774 cord_uid: d8p6ntsr Thirty-two clofazimine derivatives, of which twenty-two were new, were synthesized and evaluated for their antiviral effects against both rabies virus and pseudo-typed SARS-CoV-2, taking clofazimine (1) as the lead. Among them, compound 15f bearing 4-methoxy-2-pyridyl at the N5-position showed superior or comparable antiviral activities to lead 1, with the EC(50) values of 1.45 μM and 14.6 μM and the SI values of 223 and 6.1, respectively. Compound 15f inhibited rabies and SARS-CoV-2 by targeting G or S protein to block membrane fusion, as well as binding to L protein or nsp13 to inhibit intracellular biosynthesis respectively, and thus synergistically exerted a broad-spectrum antiviral effect. The results provided useful scientific data for the development of clofazimine derivatives into a new class of broad-spectrum antiviral candidates. Rabies virus, a negative-stranded RNA virus, causes fatal brain damage and other systematic symptoms, which leads to a mortality rate of nearly 100% [1] [2] [3] . To date, pre-or post-exposure prophylaxis, along with rabies immunoglobulin (RIG) is the only available way to protect the infected crowd [3] . However, multiple administrations of rabies vaccine and the public's lack of awareness hampere their in-time protection against the rabies virus [4] [5] [6] , and an average of 60,000 people die from rabies each year globally t [2] . The most favored Milwaukee protocol (M therapy) and its modified regimens obtained disappointing clinical outcomes [7, 8] . Up to now, no drug has been approved for rabies treatment albeit with multiple efforts on drug discovery. Therefore， it is of great significance to find effective drugs for the treatment of rabies virus infection. Recently, we found that riminophenazine alkaloid clofazimine (1, Figure 1 ), an anti-leprosy and anti-tuberculous drug for clinical use, exhibited a good anti-rabies efficacy by affecting the g-glycoprotein (G protein)-mediated membrane fusion process [9] . At the meantime, Yuen's group discovered that it also effectively inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) both in vitro and in vivo, by interfering with spike glycoprotein (S protein)-mediated cell fusion and viral helicase activity [10] . These results indicated that 1 had a broad-spectrum antiviral activity, and deserved further investigation. However, highly lipophilic compound 1 upon therapeutic administration accumulates in lipid-rich tissues, and produces the most documented the red skin-coloring side effect [11] , which might be alleviated by the reduction on lipophilicity [12, 13] . Therefore, in the present study, taking 1 as the lead compound, several series of clofazimine analogues with reduced ClogP values were synthesized and evaluated for J o u r n a l P r e -p r o o f anti-rabies as well as anti-pseudo-typed SARS-CoV-2 (pSARS-CoV-2) activities. The anti-rabies structure-activity relationship (SAR) analysis, safety profile and the dualtarget antiviral mechanism exploration of the key compound were presented. A total of 32 clofazimine derivatives, of which 22 were new, were prepared as depicted in Schemes 1 and 2 respectively. The synthesis of compounds 6a, 6b, 7a-c, 7l-o and 15b were reported previously [13] [14] [15] [16] [17] [18] [19] . As shown in Scheme 1, nucleophilic substitution of starting material 2a or 2b with equimolar 1,5-difluoro-2,4-dinitrobenzene (DFDNB) achieved 3a or 3b, which underwent a subsequent nucleophilic substitution reaction with various of amines, such as anilines, benzylamines or 5-amino-2-methoxypyridine, to give key intermediates As described in Scheme 2, the nucleophilic substitution between 8a or 8b at 90-100 ℃ in DMF achieved 9a or 9b in yields of 55-61% instead of the conventional fusing reaction operation [13] , followed by a reduction via Pd-C hydrogenation to yield key intermediate 10a or 10b in good yields. Next, following a similar five-step procedure as shown in Scheme 1, including nucleophilic substitution with DFDNB, nitro reduction, nucleophilic substitution with varied anilines, nitro reduction via Pd-C hydrogenation and ambient oxidation, compounds 14a-g were gained in yields of 46-80%. Finally, products 15a-j were acquired via condensation of compounds 14a-g with amines in the presence of acetic acid in yields of 48-80%. The products were isolated for the flash column chromatography with petroleum ether/CH2Cl2 or CH2Cl2/ethyl J o u r n a l P r e -p r o o f acetate as eluent. All target compounds were measured for their in vitro anti-rabies activities in BSR cells using rapid fluorescent focus inhibition test (RFFIT) assay constructed in our lab, taking 1 as the control [9] . The combination of the EC50 and selectivity index (SI, CC50/EC50 ratio) values of each compound was taken as the evaluation reference for antiviral potency. The structures and anti-rabies activities of all the target compounds were listed in Table 1 . Table 1 Anti-rabies activity and cytotoxicity of all the target compounds J o u r n a l P r e -p r o o f anti-rabies activity displayed declined activity. It was speculated that isopropyl group on the imine N atom might be beneficial for activity. Next, the 3-isopropyl imine was retained, and the p-chloroaniline moiety attached on C-2 position was replaced by different anilines, 6-methoxy-3-pyridylamine or phenylethylamine, to generate compounds 7c, 7d and 7g-k. Most of them gave decreased activity to varying degrees, except that compounds 7i with ptrifluoromethoxyaniline and 7k with p-chlorobenzylamine displayed comparable activities to the lead 1, with the IC50 values of 1.77 μM and 0.76 μM and SI values of 182 and 123, respectively. Then, the 3-isopropyl imine was kept, modification on the N-5 substitutent was carried out, and p-chlorophenyl moiety was altered into phenyl, pfluorophenyl or 6-methoxy-3-pyridylamine, and the corresponding analogues 7l, 7m, 7o-q and 15a-g were generated and evaluated. Among them, compounds 7l, 7p and 7q bearing a p-chloroaniline, benzylamine and p-fluorobenzylamine on their C-2 position gave elevated antiviral activity with the IC50 values of 1.40, 1.40 and 1.90 μM respectively, as well as obviously increased cytotoxicity. Compounds 15f and 15g displayed the highest antiviral activity, with IC50 value of 1.45 μM and 1.41 μM, and SI value of 223 and 229, respectively. Meanwhile, the replacement of 3-isopropyl imine with imine, cyclopropyl imine, propyl imine or 4-methoxycyclohexyl imine led to decreased activity as in all cases of compounds 6b, 6d, 6e, 6l, 7e, 7f, 7n and 15h-j, and 3-isopropyl imine was thus considered as a beneficial moiety for the anti-rabies activity. Meanwhile, the anti-SARS-CoV-2 activities of target clofazimine derivatives were evaluated on an S protein-based pSARS-CoV-2 model in Huh 7 cells constructed in our lab [20, 21] , taking 1 as the positive control. As indicated in Table 2 , the anti-pSARS-CoV-2 activity tendency of most compounds was similar to the anti-rabies J o u r n a l P r e -p r o o f tendency as disclosed above, and compounds 7m, 15f and 15g gave elevated activities compared to 1. Especially, compounds 15f and 15g gave the EC50 values of 14.6 μM and < 10 μM, and SI values of 6.1 and > 8.9, respectively, suggesting that 15f and 15g might own a broad-spectrum antiviral activity against rabies viruses and pSARS-Cov-2, worthy of the further investigation. Table 2 Anti-pSARS-CoV-2 activity and cytotoxicity of all the target compounds To evaluate the safety profiles of 15g and 15f, an acute toxicity test was performed in Kunming mice. Both compounds were given orally in a single-dosing experiment at 0, 200, 400, 600 and 800 mg•kg -1 , respectively. The mice were closely monitored for 7 days, and all the surviving mice had glossy hair, fleshy body and good appetite. Therefore, the median lethal dose (LD50) value for both 15g and 15f was over 800 mg•kg -1 , indicating their good safety profiles in vivo. J o u r n a l P r e -p r o o f In order to figure out the precise step of rabies proliferation that is blocked by the key compounds, we first evaluated their anti-rabies activity using a time-of-addition assay in a single infectious cycle in BSR cells. As illustrated in Figure suggested that they might have dual-target or multi-target mechanism against rabies virus, and the inhibitory behavior of compound 15f was highly similar to that of the lead 1. Our earlier molecular docking analysis indicated that compound 1 might target J o u r n a l P r e -p r o o f rabies G protein to interfere with viral membrane fusion [9] , therefore, the recombinant rabies G protein was prepared, and surface plasmon resonance (SPR) assay was conducted to further confirm the direct interaction between rabies G protein and compound 15f. As depicted in Fig. 3A and 3B, it displayed the concentration-dependent binding with immobilized G protein with Kd value of 0.28 μM, while 1 gave the Kd value of 0.73 μM. Then, molecular docking analysis between rabies G protein (PDB ID: 6LGW) and 15f was performed by Discovery Studio 4.5. As disclosed in Fig. 3C and 3D, van der waals force and hydrophobic interactions contributed to the strong interaction between 15f and G protein, and gave the Libdock scores of 106.1. These results demonstrated that compound 15f blocked rabies viral membrane fusion by binding to G protein. S1A and S1B, the multiple hydrogen bonds with Leu1835, Ile1843 and Gln1844 between 15f and L protein might contribute to the higher affinity of 15f over 1, suggesting that compound 15f might directly bind to rabies L protein, and thus inhibit the viral biosynthesis. Our results indicated that compound 15f might exert the antirabies efficiency by simultaneously targeting G protein and L protein in the membrane fusion and intracellular biosynthesis stages respectively, consistent with the above timeof-addition results. Similarly, the direct interaction between recombinant SARS-CoV-2 S protein and 15f was conducted by SPR assay, taking 1 as the control. As demonstrated in Fig. 4A and 4B, compound 15f could directly bind to immobilized SARS-CoV-2 S protein in a J o u r n a l P r e -p r o o f concentration-dependent manner, and gave the Kd value of 2.52 μM, while 1 gave the that of 3.82 μM. The molecular docking analysis between compound 15f and SARS-CoV-2 S protein (PDB ID: 6VSB) was performed ( Fig. 4C and 4D) , taking 1 as the control (supplementary Fig. S1C and S1D). Compound 15f gave the Libdock scores of 124.1, while lead 1 gave the score of 118.6. As disclosed in Fig. 4C and 4D , besides the hydrogen bond between their N2 atoms with Phe377 and Val483 respectively, they fit in a T-shaped cavity of SARS-CoV-2 S protein, which was accomplished by the planar tricyclic scaffold and the pyridine or benzene moiety respectively. And the residues Thr385, Lys458, Arg547 and Ser383 might mainly contribute to strong val der waals force between 15f and the target S protein. Since lead 1 inhibited the unwinding activity of nsp13 in the SARS-CoV-2 viral biosynthesis [10] , the direct interaction between nsp13 (PDB ID: 7NN0) and compound 15f was predicted by docking analysis (Fig. 4E and 4F) , taking 1 as the control (supplementary Fig. S1E and S1F). Compounds 15f and 1 gave reasonable Libdock scores of 110.0 and 103.0, respectively. Apparently, they fit well in the same cavity, and formed a common hydrogen bond between the N10 atom and Trp506, but compound 15f also formed a second hydrogen bond between fluorine atom and Arg272, which might contribute to its higher affinity. Besides, both of them formed π-π stacking effect with His464 and Phe472, indicating that 15f might directly target nsp13 to inhibit SARS-CoV-2 biosynthesis. As shown in Fig To To which was suspended or dissolved in ethanol (20 mL) and then was stirred under air overnight. The precipitation was filtered to give 6a-h, which was purified by flash chromatography using a gradient of CH2Cl2/CH3OH as the eluent. The title compound was prepared following a similar procedure in 4.1.2, taking 2aminodiphenylamine (2b) as the starting material. To a suspension of 6a-m (0.24 mmol) in dioxane (3 mL Yield Intermediate 9b (12.9 mmol) and 10% Pd/C (0.2 equiv) were suspended in ethanol (90 mL), and the mixture was shaken under hydrogen atmosphere for 6 h. The mixture was then filtered to achieve a solution of 10b, which was taken as starting material instead of 2a, following similar procedures in 4. J o u r n a l P r e -p r o o f by subtracting responses from the reference flow cell and from the blank cycles. All data were analyzed by the kinetic model in the Biacore™ T200 Evaluation Software 2.0 (GE Healthcare, US), and Kd was applied to evaluate the binding affinity. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the reaction of 10b with corresponding intermediates 12c-g. Then, the mixture of 12c-g (1 g) and 10% Pd/C (0.2 equiv.) in methanol (30 mL) and THF (30 mL) was shaken overnight under hydrogen atmosphere. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The residue suspended or dissolved in ethanol/CH2Cl2, and then stood still under air overnight. The precipitate was filtered, and then washed with ethanol to give compounds 14c-g respectively °C; 1 H NMR (500 MHz, CDCl3) δ 11.03 (1H, s, NH), 8.29 (1H, s, Ar-H), 8.17 (1H, d, J =8.2 Hz 2-p-Methoxyanilino-3-isopropylimino-5-(6-methoxy-3-pyridinyl)-3,5-dihydro phenazine (15e) Yield: 51% Ar-H), 7.66 (1H, d, J = 7.7 Hz, Ar-H) 27 (2H, m, Ar-H), 7.16 (1H, t, J = 7.3 Hz, Ar-H) Ar-H), 6.94-6.89 (2H, m, Ar-H), 6.66 (1H, s, 1-CH), 6.48 (1H, d 2-m-Fluoroanilino-3-isopropylimino-5-(6-methoxy-3-pyridinyl)-3,5-dihydro phenazine (15f) Yield: 57% Ar-H), 7.70 (1H, dd, J =7.7, 1.6 Hz Ar-H), 6.92 (1H, s, 1-CH), 6.80-6.74 (1H, m, Ar-H) 2-p-Fluoroanilino-3-isopropylimino-5-(6-methoxy-3-pyridinyl)-3,5-dihydro phenazine (15g) Yield: 55% Ar-H), 7.67 (1H, dd, J =7.8, 1.4 Hz Ar-H), 5.33 (1H, s, 4-CH), 4.09 (3H, s, OMe), 3.56-3.47 (1H, m, CHMe2), 1.10 (6H, dd, J =10.3, 6.3 Hz, CHMe2); 13 C NMR (126 MHz, CDCl3) 2-p-Fluoroanilino-3-cyclopropylimino-5-(6-methoxy-3-pyridinyl)-3,5-dihydro phenazine (15h) Yield: 57% Ar-H), 7.65 (1H, dd, J =7.7, 1.5 Hz Ar-H), 6.65 (1H, s, 1-CH), 6.47 (1H, d, J =8.0 Hz, Ar-H) 2-p-Fluoroanilino-3-propylimino-5-(6-methoxy-3-pyridinyl)-3,5-dihydro phenazine (15i) Yield: 49% Ar-H), 7.69 (1H, d, J =6.7 Hz, Ar-H), 7.57 (1H, dd, J =8 (2H, m, Ar-H), 7.22-7.12 (2H, m, Ar-H), 7.11-7.04 (3H, m, Ar-H), 6.72 (1H, s, 1-CH), 6.51 (1H, d, J =7.0 Hz -methoxycyclohexylimino)-5-(6-methoxy-3-pyridinyl)-3,5 -dihydrophenazine (15j) Yield: 54%; orange solid MHz, CDCl3) δ 8.16 (1H, d, J =2.4 Hz, Ar-H), 7.70-7.65 (1H, m, Ar-H), 7.56 (1H, dd, J =8.7, 2.6 Hz, Ar-H), 7.33-7.28 (2H, m, Ar-H), 7.20-7.11 (2H, m, Ar-H) 72 (1H, s, 1-H), 6.53 (1H, d, J =7.5 Hz, Ar-H) 2H, m, CH2), 1.76-1.66 (2H, m, CH2 2456, found: 524.2456. lines, plasmids, and viruses BSR and 293T cells were cultured in 5% CO2 at 37 °C in high glucose Dulbecco's UT) supplemented with 10% FBS Cells were passaged every 2 days. Virulent rabies virus CVS strain was adapted by BSR (variant strain of BHK) cells, and stored at -70 °C. The pSARS-Cov-2 virus was constructed as described previously Anti-rabies activity evaluation Following a similar RFFIT method as described previously FFU/well) in duplicate at 37°C for 1 h, then mixed with BSR cells (1 × 10 6 /ml) and subsequently incubated for 24 h. Then, after being fixed with pre-chilled 80% acetone, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-rabies N monoclonal antibody Anti-pSARS-CoV-2 activity evaluation Following a similar method as described previously Cytotoxicity testing Serial dilutions of test compounds, starting from 200 µM were incubated with BSR cells or 293T cells in 96-well plates, then 50 μL of medium was added instead of virus After being incubated at 37 °C for 24 h, the cell viability was recorded by an Ensight microplate luminometer (PerkinElmer, Singapore), and the the CC50 values of test compounds were calculate by GraphPad Prism The experiment was performed in accordance with the guidelines established by the Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Science for the care and use of laboratory animals. The mice were fed with regular rodent chow and housed in an air-conditioned room. The mice were randomly divided into different groups with 6 mice each. Each compound was given orally in a single-dosing experiment at 0, 200, 400, 600 or 800 mg•kg -1 (ddH2O as control), respectively. The mice were closely monitored for 7 days The "time-of-addition" experiment was performed as described previously Specifically, compounds 15f and 15g were applied as the test compound, and 1 was used as positive control. Data were analyzed based on three replicates Molecular modeling analysis The protein crystal structures were obtained from the RCSB Protein Data Bank AA residues) with a GST-His tag was cloned into pET28a vector and grown in E. coli BL21 (DE3) cells. The bacteria were grown at US) according to the manufacturer's instructions. SARS-CoV-2 S recombinant protein was purchased from Sino biological Inc The cell biology of rabies virus: using stealth to reach the brain Overexpression of interleukin-33 in recombinant rabies virus enhances innate and humoral immune responses through activation of dendritic cell-germinal center reactions WHO/Department of control of neglected tropical diseases, WHO expert consultation on rabies A social justice perspective on access to human rabies vaccines Seroconversion after three doses of intramuscular rabies vaccine as a post-exposure treatment Rabies post-exposure prophylaxis initiation and adherence among patients in Vietnam Update on rabies diagnosis and treatment Overview, prevention, and treatment of rabies Clofazimine: a promising inhibitor of rabies virus Clofazimine broadly inhibits coronaviruses including SARS-CoV-2 The physicochemical basis of clofazimine-induced skin pigmentation Optimization of the clofazimine structure leads to a highly water-soluble C3-aminopyridinyl riminophenazine endowed with improved anti-Wnt and anticancer activity in vitro and in vivo Identification of less lipophilic riminophenazine derivatives for the treatment of drugresistant tuberculosis Novel hydrophilic riminophenazines as potent antiprotozoal agents Novel therapeutic agents Use of a riminophenazine for treating MDR resistance Riminophenazines with 2-(heteroaryl)amino substituents and their antimicrobial activity Investigation of the structural properties of dihydrophenazines which contribute to their pro-oxidative interactions with human phagocytes Application of imino phenazines as rabies virus inhibitors Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 Discovery and evolution of 12N-substituted aloperine derivatives as anti-SARS-CoV-2 agents through targeting late entry stage Rabies infection: an overview of lyssavirus-host protein interactions 15f exerts a broad-spectrum antiviral effect against rabies virus and pSARS-CoV-2. 4. 15f inhibits rabies virus by simultaneously targeting G and L proteins 15f inhibits SARS-CoV-2 by simultaneously targeting S protein and nsp13 This work was supported by the National Natural Science Foundation of China (81872912) and CAMS Innovation Fund for Medical Sciences (2021-12M-1-048). Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.xxxx.xxxxxx. J o u r n a l P r e -p r o o f The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.