Pyronaridine Protects against SARS-CoV-2 Infection in Mouse.

There are currently relatively few small-molecule antiviral drugs that are either approved or emergency-approved for use against severe acute respiratory coronavirus 2 (SARS-CoV-2). One of these is remdesivir, which was originally repurposed from its use against Ebola. We evaluated three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg and identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. The in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS-CoV-2-infected mice, reducing lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 μM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

There is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg, and SARS-CoV-2, has now statistically significantly reduced the viral load in mice along with TNF-α, CXCL1, and CCL3 and restored levels of IFN-1β, ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.

At the time of writing, we are in the midst of a major a global health crisis caused by the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that was originally reported in Wuhan, China in late 2019.[1,2] Infection with this virus leads to extensive morbidity, mortality, and a very broad range of clinical symptoms such as cough, loss of smell and taste, respiratory distress, pneumonia, and extrapulmonary events characterized by a sepsis-like disease collectively called 2019 coronavirus disease (COVID-19).[3] In the United States, there are currently three vaccines available, one of which has recently obtained with full approval from the Food and Drug Administration (FDA) to protect against SARS-CoV-2.[4−6] There are however few small-molecule drugs approved for COVID-19,[7] including remdesivir,[8] which originally demonstrated activity in Vero cells,[9,10] human epithelial cells, and in Calu-3 cells[10] infected with SARS-CoV-2 prior to clinical testing. Remdesivir represents a repurposed drug that was originally developed for Hepatitis C virus but was then repurposed for treating Ebola and has since reached clinical trials.[11]

We therefore hypothesized that other drugs that were effective against Ebola might also be prioritized for evaluation in vitro against SARS-CoV-2. Previously, we had used a machine-learning model to identify tilorone, quinacrine, and pyronaridine tetraphosphate[12] for testing against Ebola virus (EBOV), and subsequently, these three inhibited EBOV and Marburg in vitro as well as demonstrating significant efficacy in the mouse-adapted EBOV (ma-EBOV) model.[13−15] All of these molecules were identified as lysosomotropic, a characteristic that suggests that these could be possible entry inhibitors.[16] Pyronaridine tetraphosphate is used as an antimalarial in several countries as part of a combination therapy with artesunate (Pyramax). Pyronaridine alone also demonstrated significant activity in the guinea pig-adapted model of EBOV infection.[17] We and others[18−21] have recently shown that these compounds possess in vitro activity against SARS-CoV-2, and tilorone and pyronaridine are in clinical trials, the latter in combination with artesunate. The Cmax data for pyronaridine in our previous mouse pharmacokinetics studies (i.p. dosing) suggests that plasma levels that are above the average IC50 observed for SARS-CoV-2 inhibition in vitro(13) can be reached with dosing well below the maximum tolerated dose. Pyronaridine also has excellent in vitro ADME properties with a long half-life that makes a single-dose treatment possible.[13,18] We now expand on our earlier in vitro characterization of pyronaridine[18] by assessing the in vivo efficacy in a mouse model of COVID-19. Finally, in an attempt to further explore molecular mechanisms, we tested the activity of pyronaridine in vitro against viral and host targets.

Results and Discussion

In vivo efficacy was assessed in the K18-hACE2 mouse model of COVID-19.[22−24] Pyronaridine (75 mg/kg, i.p.)[13] was administered 1 h prior to infection. Mice that were given pyronaridine received a single treatment. On the third day post-infection, mice were euthanized and lung viral load, cytokine levels, and histopathology were evaluated (Figure A). We chose day 3 post-infection since it is the time point of the highest viral load in the lung of K18-hACE2 mice infected with SARS-CoV-2.[23,25,26] In all groups tested, mice lost weight compared to uninfected animals that received only vehicle formulation (Figure B). Lung viral load was evaluated by RT-qPCR, and the pyronaridine-treated group showed a statistically significant decrease in the lung viral load (Figure C). Moreover, reduced levels of IFN-1β were observed in infected mice, and pyronaridine restored the levels of IFN-1β close to that found in uninfected animals (Figure D). Tukey post-hoc analysis revealed a significant increase in IL-6 levels in infected untreated mice as compared with the uninfected group (uninfected vs infected untreated) (Figure E). Interestingly, we observed no post-hoc difference when comparing pyronaridine-treated infected mice with the uninfected group (uninfected vs infected + pyr) (Figure E). In addition, pyronaridine reduced the high levels of CXCL1 and CCL3 observed in infected animals (Figure F and Figure G, respectively). Pyronaridine did not, however, reduce the high levels of IL-10, CCL2, CCL4, and TNF-α (Figure H–K, respectively) below the elevated levels found in the infected mice. Neither infection nor treatment affected the levels of CXCL2 and CXCL10 (Figure L and Figure M, respectively).

Figure 1

In vivo efficacy of pyronaridine in a mouse model of COVID-19. (A) Experimental timeline: K18-hACE2 mice were infected with SARS-CoV-2 (2 × 104 PFU/40 μL of saline, intranasal) or mock. One group of mice was treated with pyronaridine (75 mg/kg i.p.) 1 h before virus inoculation. (B) Body weight was evaluated daily. (C) At 3 DPI, mice were euthanized and the lung viral load and (D–M) lung cytokine and chemokine levels were determined. *p < 0.05, **p < 0.01, and ***p < 0.001 after one-way ANOVA followed by Tukey post-hoc test. Pyr, pyronaridine.

Several studies have shown a cytokine and chemokine storm as important during SARS-CoV-2 infection in patients with COVID-19, including PDGF, VEGF,[27] IL-6,[28,29] IL-8, IL-10,[29] TNF-α,[29] IFN-α, and IFN-β.[30−34] Furthermore, an impaired type I interferon response has already been observed in COVID-19[35] followed by increased circulating levels of IL-6 and TNF-α. Many viruses, including SARS-CoV-2, subvert the immune system by inhibiting the production of interferons (IFN), an important family of antiviral mediators,[36−39] which results in an impaired antiviral defense and increased viral replication and infection. Here, we have demonstrated that pyronaridine restored the levels of IFN-1β in infected mice. Moreover, this effect was associated with a reduced viral load after the treatment with pyronaridine. Notably, pyronaridine also reverted the altered levels of CXCL1 and CCL3 observed in infected mice, in addition to avoiding the increase in the levels of IL-6 noticed in individuals infected with SARS-CoV-2. Noteworthy, we did not observe changes in the levels of CXCL10 post SARS-CoV-2 infection, as previously reported.[23] Although discrepant, this difference could be explained by the higher viral inoculum used in the previous study (1 × 105 PFU) in comparison to 2 × 104 PFU used here. Moreover, the difference in the time points after infection could be also a possible explanation. In fact, Oladunni et al.[23] observed increased CXCL10 on 2 dpi decreasing thereafter.

In addition to biochemical changes, histopathological findings are also observed in the lung of patients[40−42] and in other experimental models of COVID-19.[22−24] To determine the severity of lung damage, histological examination of hematoxylin and eosin (H&E)-stained lung tissues was performed. Infected, untreated mice showed severe pathological changes with inflammatory cell infiltrates. In contrast, pyronaridine-treated animals exhibited improved morphology and milder infiltration, appearing comparable to those of uninfected mice (Figure A). Histological observations were confirmed by quantitative morphometric analysis of the H&E-stained slides showing a statistically significant reduction in inflammation (Figure B). Thus, pyronaridine appears to have both antiviral and immunomodulatory effects in this experimental model of COVID-19 as used in the present study.

Figure 2

Lung histopathological analyses of COVID-19 mice treated with pyronaridine. K18-hACE2 mice were infected with SARS-CoV-2 (2 × 104 PFU/40 μL, intranasal) or mock. One group of mice was treated with pyronaridine (75 mg/kg i.p.) 1 h before virus inoculation. At 3 DPI, mice were euthanized, and the lungs were harvested and processed for histopathological analyses. (A) Representative images of lung slices stained with hematoxylin and eosin (H&E). (B) Quantitative morphometric analyses based on the septal area fraction. ***p < 0.001 after one-way ANOVA followed by Tukey post-hoc test. Pyr, pyronaridine. Scale bars: 20× = 125 μm; 40× = 50 μm.

SARS-CoV-2 proteases Mpro and PLpro are essential for viral replication and have been widely studied for the discovery of new direct acting antivirals.[43−45] The FDA approved the emergency use authorization of Pfizer’s Paxlovid, which is a combination of PF-07321332 and the HIV drug ritonavir that slows down the metabolism of PF-07321332 to treat mild-to-moderate COVID-19 in adults and pediatric patients 12 years of age and older (www.fda.gov). PF-07321332 is an Mpro inhibitor. Currently, there are no PLpro inhibitors approved to treat COVID-19.

Pyronaridine was therefore tested against both SARS-CoV-2 recombinant PLpro and Mpro through fluorescence resonance energy transfer (FRET)-based in vitro assays. Pyronaridine inhibited PLpro with an IC50 of 1.86 ± 0.58 μM (Figure A) but did not show any appreciable activity against Mpro at 20 μM (data not shown). As a positive control for Mpro, we used the characterized inhibitor nirmatrelvir (PF-07321332),[46] and as a positive control for PLpro, we used the characterized inhibitor 15c(47) and described IC50 values comparable to published data (Figure S1). Additional analogs of pyronaridine were also synthesized and tested against PLpro. The analogs 12126038, 12126039, and 12126040 (Figure B–D, respectively) showed similar inhibitory activity when compared with pyronaridine (as well as that reported for GRL0617[48]), indicating that the aminophenol moiety together with pyrrole or tertiary amine substitutions at the meta position is tolerated for PLpro inhibition (Table and Figure A). The deletion of these groups in analogs 10326099, 12126035, 12126036, 12126037, and 12126072 caused complete abolishment of the inhibitory activity of the series (Table ). The PLpro active site contains four subsites for peptide recognition, with a strong preference for positively charged amino acids at P3 and P4 subsites.[43−45] There are only a few PLpro inhibitors reported to date, such as daclastavir and sitagliptin with low micromolar IC50 for PLpro[49] and GRL0617,[48] for which the crystal structure and binding mode have been reported. Recently, novel 2-phenylthiophenes with nanomolar inhibitory potency were designed by leveraging the cooperativity of multiple shallow binding sites on the PLpro surface.[50]

Figure 3

Dose–response curves of pyronaridine and active analogs against SARS-CoV-2 PLpro. (A) Pyronaridine, (B) 12126038, (C) 12126039, and (D) 12126040.

Table 1

PLpro IC50 Inhibition Data for Pyronaridine Analogs

Due to the possible effect of pyronaridine on cytokines (Figure ), we have also assessed the effect on host targets as SARS-CoV-2 can cause an imbalance in the immune system that may result in a cytokine storm[51] as well as leading to acute respiratory distress syndrome (ARDS), coagulation disorders, and eventually multiple organ failure.[51,52] Hence, targeting the cytokine storm to address hyperinflammation represents another approach to the treatment of COVID-19 patients.[53−56] In this regard, we have explored the effect of pyronaridine on human kinases, which are responsible for host cell signaling.[57,58] Screening of pyronaridine (tested at 1 μM) against 485 kinases identified only two as having a mean percent inhibition greater than 30%, including CAMK1 (35%) and MELK (31%) (Table S1). Subsequently, the IC50 was determined for CAMK1 (2.4 μM) (Figure ).

Figure 4

Pyronaridine CAMK1 dose response.

Even with vaccines becoming widely available in many countries, COVID-19 continues to exact a very heavy toll on those that are unvaccinated. We are in a race against time before the virus mutates, and vaccines lose their effectiveness. There is therefore an urgent need for new antivirals and in particular small-molecule treatments that are orally delivered and can be used outside of a hospital setting. Finding, developing, and progressing small molecules to the clinic are generally slow and expensive processes;[59] hence, drug repurposing has been attempted by many groups to speed these up (either experimentally or computationally[60]) by identifying already approved or clinical stage candidates used for other applications or quickly follow up the few molecules that are being used already. The traditional prioritization of compounds in vitro before animal models and then humans is still repeated and so far with few successes with many molecules not demonstrating efficacy in vivo.(61,62)

Our understanding of the antiviral mechanism of pyronaridine previously shown to inhibit the Ebola virus in vitro and in vivo(13) via binding to the viral glycoprotein[16] as well as through its potent lysosomotropic activity[63] and now the in vitro activity against SARS-CoV-2[18] is also further expanded. Pyronaridine was previously identified with in vitro activity against SARS-CoV-2 in A549-ACE2 cells that was on a par with remdesivir in this cell line.[18] In the current study, we have demonstrated that pyronaridine also has antiviral activity against SARS-CoV-2 in vivo. A single prophylactic dose of pyronaridine (75 mg/kg i.p.) reduced the viral load in the lung of infected mice 3 days post-infection. In vitro assays suggest that pyronaridine possesses a direct antiviral effect showing activity against PLpro (IC50 = 1.86 μM; Figure A) but did not inhibit SARS-CoV-2 Mpro. Kinase profiling resulted in determination of IC50 for CAMK1 (IC50 = 2.4 μM; Figure ).

A single dose (75 mg/kg i.p.) of pyronaridine has an elimination half-life of 146 h in mice,[13] comparable to the reported half-life of pyronaridine in humans of 195–251 h.[64,65] A study of pyronaridine as a single-oral dose (400 mg) given to a healthy volunteer found a Cmax in plasma of 495.8 ng/mL at a Tmax of 0.5 h,[64,66] which gives a concentration close to 1 μM. In our study, pyronaridine inhibits PLpro at 1.86 μM and CAMK1 at 2.4 μM, which is very close to a Cmax of 1 μM. Pyronaridine preferentially associates with blood cells and is highly plasma protein bound,[66] which suggests that it may not reach the unbound concentration necessary to have an effect on PLpro and CAMK1 at these doses. Activation of CAMK1 has been reported to negatively impact HBV biosynthesis.[67,68] Inhibition of CAMK1 might not however be therapeutically relevant since reports on proteomic and phosphoproteomic profiling of COVID-19[69] and phosphorylation landscape for SARS-CoV-2 infection[70] do not point to an important role of this kinase on COVID-19. Currently, Shin Poong Pharmaceutical Co. Ltd. is recruiting patients for phase III clinical trials for COVID-19 (ClinicalTrials.gov Identifier: NCT05084911) using a combination of pyronaridine and artesunate. In phase II, this combination showed some promising effects in those with severe illness.[71]

In summary, our present study provided additional data on the efficacy of pyronaridine against SARS-CoV-2 infection as well as highlighting reduced lung pathology and inflammation in a mouse model of COVID-19. Furthermore, we have shown that pyronaridine may target PLpro as well as CAMK1. There are few inhibitors of CAMK1 that have been identified to date (such as Barettin[72] or pyridine amides[73]), which have a role in inflammation targeting IL-10.[72] Previous in vitro work has shown that inhibiting CAMK1 in cells reduces IL-10, the master anti-inflammatory interleukin.[74] In the present study, there is no significant difference in the IL-10 levels between the untreated and pyronaridine-treated infected groups so it seems unlikely that CAMK1 inhibition would be involved in the mechanism of action of inhibition of SAR-CoV-2. In conclusion, we propose that pyronaridine could be used alone as a potential therapeutic candidate for COVID-19. Finally, with the emerging virulence of novel SARS-CoV-2 strains, identifying repurposed drugs with novel mechanisms of action and whose antiviral activity translates from in vitro to in vivo is rare[62] and may lead to new treatments as well as their further optimization.

Methods

Chemicals and Reagents

Compound 15c(47) was purchased from Sigma-Aldrich. Nirmatrelvir (PF-07321332)[46] was kindly donated by Prof. Carlos Alberto Montanari (Univeristy of São Paulo, Brazil).

Pyronaridine tetraphosphate [4-[(7-chloro-2-methoxybenzo[b][1,5]naphthyridin-10-yl)amino]-2,6-bis(1-pyrrolidinylmethyl)phenol phosphate (1:4)][12] was purchased from BOC Sciences (Shirley, NY). The purity of this compound was greater than 95%. For pyronaridine analogs, 1H and 13C spectra were measured on Bruker AC-300 (300 MHz, 1H) or Bruker AC-200 (50 MHz, 13C). Chemical shifts were measured in DMSO-d6 or CDCl3 using tetramethylsilane as an internal standard and reported as unit (ppm) values. The following abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; brs, broad singlet; and brm, broad multiplet. The purity of the final compounds was analyzed on an Agilent 1290 Infinity II HPLC system coupled to an Agilent 6460 triple-quadrupole mass spectrometer equipped with an electrospray ionization source. The chromatographic separation was carried out on an Agilent Eclipse Plus C18 RRHD column (2.1 × 50 mm, 1.8 μm) at 40 °C, and the sample injection volume was 0.2 μL. The mobile phase comprising 0.1% formic acid/water (A) and 0.1% formic acid and 85% acetonitrile/water (B) was programmed with gradient elution (0.0–3.0 min, 60% B; 3.0–4.0 min, 60–97% B; 4.0–6.0 min, 97% B; 6.0–6.1 min, 97–60% B) at a flow rate of 0.4 mL/min. The mass spectrometric detection was operated in positive ion mode. Optimal parameters were capillary voltages of 3500 V, a nebulizer pressure of 35 psi, a gas temperature of 350 °C, and a gas flow rate of 12 L/min. All final compounds are >95% pure. Melting points were determined on Electrothermal 9001 (10 °C per min) and are uncorrected. Merck KGaA silica gel 60 F254 plates were used for analytical thin-layer chromatography. Column chromatography was performed on Merck silica gel 60 (70–230 mesh). Yields refer to purified products and are not optimized.

The molecules were synthesized, according to Schemes or 2, and the specific methods and analytical results are described in the Supporting Information.

Scheme 1

Synthetic Route for 6-Chloro-2-methoxyacridine Derivatives

Scheme 2

Synthetic Route for Benzo[b]-1,6-naphthyridine Derivatives

Test Article Preparation

Dose formulation for pyronaridine was prepared as previously described[13] under yellow light by mixing the appropriate amount of pyronaridine in melted Kolliphor HS 15 (Solutol) (20% final volume) using a vortex mixer for 30 s. The remaining sterile water (Gibco) was added, and the formulations were mixed using a vortex mixer for 30 s to 5 min until the compound was visually dissolved and then sonicated for 25 min. The final 20% Kolliphor HS 15 dose formulations were observed to be clear, reddish solutions.

Mouse Studies

Ethical Approval

All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of São Paulo and approved by the institutional ethics committee under the protocol number 105/2021.

SARS-CoV-2 Isolate

SARS-CoV-2 was isolated from a COVID-19 positive-tested patient. The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirão Preto Medical School (Ribeirão Preto, Brazil). Cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotic/antimycotic (Penicillin, 10,000 U/mL; Streptomycin, 10,000 μg/mL). The viral inoculum was added to Vero cells in DMEM 2% FBS and incubated at 37 °C with 5% CO2 for 48 h. The cytopathogenic effect (CPE) was observed under a microscope. The cell monolayer was collected, and the supernatant was stored in −70 °C. Virus titration was made by the plaque-forming units (PFU).

K18-hACE2 Mice

To evaluate the effects of pyronaridine in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J).[22−24] K18-hACE2 mice were obtained from the Jackson Laboratory and were bred in the Centro de Criação de Animais Especiais (Ribeirão Preto Medical School/University of São Paulo). This mouse model for SARS-CoV-2-induced disease has been used as it presents clinical signs and biochemical and histopathological changes compatible with the human disease.[23−25,75−78] Mice had access to water and food ad libitum. For the experimental infection, animals were transferred to the BSL3 facility.

SARS-CoV-2 Experimental Infection and Treatments

Female K18-hACE2 mice, 8 weeks old, were infected with 2 × 104 PFU of SARS-CoV-2 (in 40 μL) by the intranasal route. Uninfected mice (N = 5) were inoculated with an equal volume of PBS (phosphate buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4). On the day of infection, 1 h before virus inoculation, animals were treated with pyronaridine (75 mg/kg, i.p.) (n = 6). Five infected animals remained untreated. Body weight was evaluated on the baseline and on all the days post-infection. On day 3 post-infection, animals were humanely euthanized, and lungs were collected. The right lung was collected, harvested, and homogenized in PBS with steel glass beads. The homogenate was added to the TRIzol (Invitrogen, CA, EUA) reagent (1:1), for posterior viral titration via RT-qPCR, or to lysis buffer (1:1), for the ELISA assay, and stored at −70 °C. The left lung was collected in paraformaldehyde (PFA 4%) for posterior histological assessment.

Absolute Viral Copy Quantification

Total RNA from the right lungs was obtained using the Trizol (Invitrogen, CA, EUA) method and quantified using NanoDrop One/Onec (Thermo Fisher Scientific, USA). A total of 800 ng of RNA was used to synthesize cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s protocol. The determination of the absolute number of viral copies was made by a Taqman real-time qPCR assay with the aid of a StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). A standard curve was generated to obtain the exact number of copies in the tested sample using an amplicon containing 944 bp cloned from a plasmid (PTZ57R/T CloneJet Cloning Kit Thermo Fisher Scientific), starting in the nucleotide 14 of the gene N. To quantify the number of copies, a serial dilution of the plasmid in the proportion of 1:10 was performed. Commercial primers and probes for the N1 gene and RNAse P (endogenous control) were used for the quantification (2019-nCov CDC EUA Kit, IDT), following the CDC’s instructions.

ELISA Assay

Lung homogenate was added to RIPA buffer (radioimmunoprecipitation assay buffer: 150 mM NaCl, 1% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris; pH 8.0) in a proportion of 1:1 and then centrifuged at 10,000g at 4 °C for 10 min. The supernatant was collected and stored in −70 °C until use. The sandwich ELISA method was performed to detect the concentration of cytokines and chemokines using kits from R&D Systems (DuoSet), according to the manufacturer’s protocols. The following targets were evaluated: IL-6, IL-10, TNF-α, IFN-1β, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10.

Lung Histopathological Process and Analyses

Five micrometer lung slices were submitted to hematoxylin and eosin staining. A total of 10 photomicrographs in 40× magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to an HDI camera for image capture. The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA). Morphometric analysis was performed in accordance with the protocol established by the American Thoracic Society and European Thoracic Society (ATS/ERS).[79]

All reagents and solvents were purchased from commercial suppliers and used without further purification. 1H and 13C spectra were measured on Bruker AC-300 (300 MHz, 1H) or Bruker AC-200 (50 MHz, 13C). Chemical shifts were measured in DMSO-d6 or CDCl3 using tetramethylsilane as an internal standard and reported as unit (ppm) values. The following abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; brs, broad singlet; and brm, broad multiplet.

The purity of the final compounds was analyzed on an Agilent 1290 Infinity II HPLC system coupled to an Agilent 6460 triple-quadrupole mass spectrometer equipped with an electrospray ionization source. The chromatographic separation was carried out on an Agilent Eclipse Plus C18 RRHD column (2.1 × 50 mm, 1.8 μm) at 40 °C, and the sample injection volume was 0.2 μL. The mobile phase comprising 0.1% formic acid/water (A) and 0.1% formic acid and 85% acetonitrile/water (B) was programmed with gradient elution (0.0–3.0 min, 60% B; 3.0–4.0 min, 60–97% B; 4.0–6.0 min, 97% B; 6.0–6.1 min, 97–60% B) at a flow rate of 0.4 mL/min. The mass spectrometric detection was operated in positive ion mode. Optimal parameters were capillary voltages of 3500 V, a nebulizer pressure of 35 psi, a gas temperature of 350 °C, and a gas flow rate of 12 L/min. All final compounds are >95% pure. Melting points were determined on Electrothermal 9001 (10 °C per min) and are uncorrected. Merck KGaA silica gel 60 F254 plates were used for analytical thin-layer chromatography. Column chromatography was performed on Merck silica gel 60 (70–230 mesh). Yields refer to purified products and are not optimized.

The molecules were synthesized, according to Schemes or 2, and the specific methods and analytical results are described in the Supporting Information.

Molecule Synthesis

4-[(6-Chloro-2-methoxyacridin-9-yl)amino]phenol 2 (12126037)

Solid 2-aminophenol (0.39 g, 3.6 mmol) was added to a suspension of starting 6,9-dichloro-2-methoxyacridine 1 (0.5 g, 1.8 mmol) in 10 mL of dimethylformamide. The reaction mass was stirred at reflux for 1 h and cooled, and the precipitate was filtered off and washed with DMF, EtOH, and diethyl ether. The yield was 72%. Mp 290 °C with decomposition, (DMF). Mass (EI), m/z (Irelat. (%)): 350.7981 [M]+ (92). C20H15ClN2O2. 1H NMR (DMSO-d6; δ, ppm): 9.98 (1H, s, NH), 8.16 (3H, m, 3CH), 7.73 (2H, m, 2CH), 7.47 (1H, d, J = 4.5, CH), 7.23 (2H, d, J = 6.5, 2CH), 6.92 (2H, d, J = 6.5, 2CH), 3.81 (3H, s, OCH3). 13C NMR (DMSO-d6; δ, ppm): 156.0, 152.9, 147.8, 147.6, 145.1, 144.7, 133.7, 131.3, 131.1, 128.1, 122.8, 121.8, 121.5, 121.4, 120.9, 120.3, 120.1, 117.9, 101.0, 55.7.

4-[(6-Chloro-2-methoxyacridin-9-yl)amino]-2,6-bis[(dimethylamino)methyl]phenol 3 (12126038)

A mixture of 40% aqueous solution of dimethylamine (1.1 mL, 9.0 mmol), 1 mL of acetic acid, and 37% aqueous solution of formaldehyde (0.39 mL, 5.4 mmol) was added at once to a suspension of 4-[(6-chloro-2-methoxyacridin-9-yl)amino]phenol 2 (0.9 mmol) in 10 mL of dimethylformamide. The suspension was heated to boiling and boiled for 4 h. The reaction mixture was cooled, and an aqueous solution of NaHCO3 (17 mmol) was added, transferred to a separatory funnel, and extracted with ethyl acetate (2 × 30 mL). The combined organic layer was washed with water (2 × 100 mL) and dried over sodium sulfate. The solvent was removed in vacuo, and residue oil was treated with a mixture of 20 mL of hexane and 1 mL of diethyl ether. The oil was ground and kept for 10 h at 40 °C. The solid was filtered off, washed with hexane, and purified by column chromatography (methanol). The yield of the aim product was 25%. Mp 148–152 oC. Mass (EI), m/z (Irelat.(%)): 464.9869 [M]+ (88). C26H29ClN4O2. 1H NMR (DMSO-d6; δ, ppm): 9.98 (1H, s, NH), 8.17 (3H, m, 3CH), 7.84 (2H, m, 2CH), 7.45 (1H, d, J = 4.5, CH), 7.06 (2H, s, 2CH), 3.81 (3H, s, OCH3), 3.61 (2H, s, 2CH2), 2.78 (12H, br s, 2 N(CH3)2. 13C NMR (DMSO-d6; δ, ppm): 157.2, 156.0, 154.5, 147.8, 145.1, 144.6, 133.7, 131.3, 131.1, 128.0, 128.1, 125.5, 122.8, 121.4, 118.6, 114.9, 112.2, 101.0, 55.8, 55.7, 44.9.

4-[(6-Chloro-2-methoxyacridin-9-yl)amino]-2-(pyrrolidin-1-ylmethyl)phenol 4 and 4-[(6-Chloro-2-methoxyacridin-9-yl)amino]-2,6-bis(pyrrolidin-1-ylmethyl)phenol 5

A mixture of pyrrolidine (0.52 mL, 6.0 mmol), 0.7 mL of acetic acid, and 37% aqueous formaldehyde solution (0.28 mL, 3.6 mmol) was added to a suspension of 4-[(6-chloro-2-methoxyacridin-9-yl)amino]phenol 2 (0.6 mmol) in 10 mL of dimethylformamide. The solution was stirred at reflux for 1 h. The reaction mixture was cooled, and an aqueous solution of NaHCO3 (11 mmol) was added and extracted with ethyl acetate (2 × 30 mL). The combined organic layer was washed with water (2 × 100 mL) and dried with sodium sulfate, and the solvent was evaporated in vacuo. The resulting oil was dissolved in chloroform and applied to a chromatography column (chloroform/methanol 9:1), and product 4 was isolated. Pure methanol was used as an eluent for separation product 5.

4-[(6-Chloro-2-methoxyacridin-9-yl)amino]-2-(pyrrolidin-1-ylmethyl)phenol 4 (12126040)

The yield is 5%. Mp 178–182 oC. Mass (EI), m/z (Irelat.(%)): 433.9298 [M]+ (76). C25H24ClN3O2. 1H NMR (DMSO-d6; δ, ppm): 10.21 (1H, s, NH), 8.17 (3H, m, 3CH), 7.81 (2H, m, 2CH), 7.45 (1H, d, J = 4.5, CH), 7.06 (2H, s, 2CH), 3.83 (3H, s, OCH3), 3.60 (2H, s, 2CH2), 2.5–2.7 (8H, br m, 2 N(CH2)2), 1.6–1.8 (8H, br m, 2(CH2)2). 13C NMR (DMSO-d6; δ, ppm): 157.7, 156.2, 154.5, 147.8, 145.1, 144.3, 133.7, 131.3, 131.1, 125.5, 122.8, 121.4, 118.7, 114.9, 111.5, 101.1, 55.7, 54.1, 52.3, 23.4.

4-[(6-Chloro-2-methoxyacridin-9-yl)amino]-2,6-bis(pyrrolidin-1-ylmethyl)phenol 5 (12126039)

The yield is 10%. Mp 145–150 oC. Mass (EI), m/z (Irelat.(%)): 517.0615 [M]+ (79). C30H33ClN4O2. 1H NMR (DMSO-d6; δ, ppm): 10.04 (1H, s, NH), 8.16 (3H, m, 3CH), 7.79 (2H, m, 2CH), 7.47 (1H, d, J = 4.5, CH), 7.06 (1H, d, J = 4.5, CH), 6.93 (1H, s, CH), 3.83 (3H, s, OCH3), 3.60 (2H, s, 2CH2), 2.5–2.7 (8H, br m, 2 N(CH2)2), 1.6–1.8 (8H, br m, 2(CH2)2). 13C NMR (DMSO-d6; δ, ppm): 156.0, 152.4, 149.3, 147.7, 145.1, 139.2, 131.2, 131.1, 128.7, 124.1, 122.8, 121.2, 120.8, 116.4, 116.1, 112.4, 101.6, 55.8, 51.9, 23.42.

6-Chloro-2-methoxyacridin-9-ol 6 (12126072)

The mixture of pyrrolidine (1.0 mL, 13.0 mmol), 1.4 mL of acetic acid, and 37% aqueous formaldehyde solution (0.56 mL, 7.8 mmol) was added to a suspension of 4-[(6-chloro-2-methoxyacridin-9-yl)amino]phenol 2 (1.3 mmol) in 10 mL of ethanol. The suspension was boiled for 36 h. The reaction mixture was cooled, an aqueous solution of NaHCO3 (2.6 mmol) was added, and the precipitate of the aim compound was filtered off. The yield is 38%. Mp 300 oC (DMF). Mass (EI), m/z (Irelat.(%)): 259.0615 [M]+ (79). C14H10ClNO2. 1H NMR (DMSO-d6; δ, ppm): 8.56 (1H, d, J = 9.0, CH), 7.72 (H, s, CH), 7.62 (1H, d, J = 4.5, CH), 7.44 (1H, d, J = 4.5, CH), 7.27 (1H, s, CH), 7.07 (1H, d, J = 9.0, CH), 3.86 (3H, s, OCH3). 13C NMR (DMSO-d6; δ, ppm): 157.8, 148.2, 147.9, 140.6, 138.0, 136.7, 128.7, 126.9, 125.5, 124.6, 123.3, 102.9, 55.3.

6-Chloro-2-methoxy-9-morpholin-4-ylacridine 7 (12126036)

Morpholine (0.23 mL, 2.7 mmol) was added to a suspension of starting 6,9-dichloro-2-methoxyacridine 1 (0.25 g, 0.9 mmol) in 7 mL of dimethylformamide. The reaction mixture was stirred at reflux for 30 min and cooled, and the formed precipitate was filtered off and washed with dimethylformamide and acetone. The yield is 70%. Mp 208–212 oC (DMF). Mass (EI), m/z (Irelat.(%)): 328.7926 [M]+ (74). C18H17ClN2O2. 1H NMR (DMSO-d6; δ, ppm): 8.56 (1H, d, J = 9.0, CH), 7.72 (H, s, CH), 7.62 (1H, d, J = 4.5, CH), 7.44 (1H, d, J = 4.5, CH), 7.27 (1H, s, CH), 7.07 (1H, d, J = 9.0, CH), 3.86 (3H, s, OCH3). 13C NMR (DMSO-d6; δ, ppm): 155.7, 149.8, 142.6, 134.5, 131.7, 130.9, 130.6, 127.6, 122.4, 119.7, 119.1, 114.8, 100.5, 66.8, 55.7, 50.8.

(2E)-2-Cyano-3-(dimethylamino)but-2-enamide 2

Dimethyl acetal dimethylacetamide (1 mL, 7.2 mmol) was added to a suspension of cyanoacetamide 1 (0.5 g, 6 mmol) in 15 mL of absolute alcohol. The suspension was boiled for 3 h and cooled, and the obtained precipitate was filtered off and washed with alcohol and diethyl ether. Aim product 2 was obtained with a yield of 82%. Mp 188–192 oC (isopropanol).

(2E)-3-Anilino-2-cyanobut-2-enamide 3

Aniline (0.45 mL, 5 mmol) was added to a suspension of (2E)-2-cyano-3-(dimethylamino)but-2-enamide 2 (0.3 g, 2 mmol) in acetic acid (4 mL). The suspension was boiled for 3 h and cooled, and acetic acid was removed under vacuum. The residue was treated with water, and the formed precipitate was filtered off and washed with water, isopropyl alcohol, and diethyl ether. Aim product 3 was obtained with a yield of 87%. Mp 179–182 oC (isopropanol).

A Mixture of 6-[(E)-2-(Dimethylamino)vinyl]-4-oxo-1-phenyl-1,4-dihydropyrimidine-5-carbonitrile 4 and (2Z,4E)-3-Anilino-2-cyano-5-(dimethylamino)-N-[(1E)-(dimethylamino)methylene]penta-2,4-dienamide 5

Dimethylformamide diethyl acetal (1.3 mL, 7.5 mmol) was added to a suspension of (2E)-3-anilino-2-cyanobut-2-enamide 3 (0.6 g, 3 mmol) in 5 mL of absolute ethanol. The dark red solution was boiled for 6 h. Part of the solvent was removed under vacuum until a thick suspension was obtained. The precipitate was filtered off and washed with absolute alcohol and dry diethyl ether. A mixture of compounds 4 and 5 was obtained.

4-Anilino-5-formyl-2-oxo-1,2-dihydropyridine-3-carbonitrile 6

A mixture of 6-[(E)-2-(dimethylamino)vinyl]-4-oxo-1-phenyl-1,4-dihydropyrimidine-5-carbonitrile 4 and (2Z,4E)-3-anilino-2-cyano-5-(dimethylamino)-N-[(1E)-(dimethylamino)methylene]penta-2,4-dienamide 5 (5.6 g) was dissolved in 56 mL of 90% acetic acid. A precipitate formed after 30 min, and the reaction mixture was stirred at room temperature for 20 h. The precipitate was filtered off and washed with water, ethyl alcohol, and acetone. The yield is 2.3 g of aim product 6. Mp 225–230 °C (with decomposition).

3-Chloro-2,3-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 7

The 4-anilino-5-formyl-2-oxo-1,2-dihydropyridine-3-carbonitrile 6 (2.3 g) was refluxed in phosphorus oxychloride (22 mL) for 1 h. The reaction mixture was poured onto ice and stirred for 30 min. The formed precipitate was filtered off and washed with water, ethyl alcohol, and diethyl ether. Aim product 7 was obtained in 80% yield. Mp 300–304 oC (DMF).

3-Chloro-10-oxo-5,10-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 8

To a suspension of 3-chloro-2,3-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 7 (0.44 g) in 15 mL of acetone was added m-chloroperbenzoic acid 55% (1.45 g) in small portions and refluxed for 9 h. The reaction mixture was cooled, and the obtained precipitate was filtered off and washed with acetone, toluene, and acetone. The aim product 8 was obtained with a yield of 50%. Mp 305 °C.

3-Morpholin-4-ylbenzo[b]-1,6-naphthyridine-4-carbonitrile 9 (12126035)

Morpholine (0.16 mL, 1.8 mmol) was added to a suspension of 3-chloro-2,3-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 7 (0.15 g, 0.6 mmol) in 4 mL of dry dimethylformamide. The solution was boiled for 1 h, cooled, poured into 40 mL of water, and extracted two times with 30 mL of ethyl acetate. The organic fractions were additionally washed with water two times. Ethyl acetate was removed under vacuum, ethyl alcohol was added to the dry residue, and the precipitate was filtered off and washed with diethyl ether. The aim product 9 was obtained with a yield of 78%. Mp 222–225 oC (EtOH:DMF 2:1). Mass (EI), m/z (Irelat.(%)): 290.3194 [M]+ (79). C17H14N4O. 1H NMR (DMSO-d6; δ, ppm): 9.46 (1H, s, CH), 9.13 (1H, s, CH), 8.16 (1H, d, J = 6.5, CH), 7.91 (1H, s, CH), 7.44 (1H, d, J = 6.5, CH), 4.16 (4H, m, O(CH2)2), 3.78 (4H, m, N(CH2)2). 13C NMR (DMSO-d6; δ, ppm): 144.9, 143.5, 137.8, 136.4, 136.0, 133.1, 129.5, 128.4, 126.8, 126.1, 114.5, 101.4, 66.7, 53.8.

3-Morpholin-4-yl-10-oxo-5,10-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 10 (10326099)

Morpholine (0.14 mL, 1.6 mmol) was added to a solution of 3-chloro-10-oxo-5,10-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 8 (0.2 g, 0.8 mmol) in 5 mL of dry dimethylformamide. The formed suspension was stirred at reflux for 1 h and cooled, and the precipitate was filtered off and washed with dimethylformamide and acetone. Mp 290 oC (DMF). The yield is 38%. Mass (EI), m/z (Irelat.(%)): 306.3188 [M]+ (68). C17H14N4O2. 1H NMR (DMSO-d6; δ, ppm): 8.02 (1H, s, CH), 7.91 (1H, d, J = 6.5, CH), 7.59 (1H, s, CH), 7.09 (1H, d, J = 6.5, CH), 4.08 (4H, m, O(CH2)2), 3.72 (4H, m, N(CH2)2). 13C NMR (DMSO-d6; δ, ppm): 182.5, 146.4, 146.1, 139.7, 139.1, 138.7, 137.0, 124.9, 122.7, 118.3, 115.4, 108.8, 94.9, 66.8, 53.7.

PLpro Cloning, Expression, Purification, and Assays

The viral cDNA template (GenBank MT126808.1) was kindly provided by Dr. Edison Durigon. Amplification of the nucleotide sequence coding for the PLpro domain (residues 1564–1879 of SARS-CoV-2) was performed by polymerase chain reaction (PCR) using forward (5′- ATTCCATGGGCGAAGTGAGGACTATTAAGGTGTTTAC-3′) and reverse (5′- ATTGCTCGAGTGGTTTTATGGTTGTTGTGTAACT-3′) primers, with restriction sites for NcoI and XhoI. The PCR product was digested with NcoI and XhoI and cloned into pET28a (Novagen) in frame with a C-terminal his-tag coding sequence. Escherichia coli BL21 transformed with plasmids was grown in LB to an optical density (OD600) of 0.6 at 37 °C and 200 rpm. Protein expression was induced by adding 0.5 mM IPTG and 1 mM zinc chloride and grown overnight at 18°. The cell pellet was resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM imidazole, and 1 mM DTT, pH 8.5), disrupted by sonication, and centrifuged at 12,500 rpm for 40 min at 4 °C. Protein was isolated from the lysate using 5 mL of Ni-NTA resin (Qiagen) pre-equilibrated with lysis buffer and then washed with 15 column volumes (CV) with the same buffer. The his-tagged protein was eluted 4 CV of elution buffer (lysis buffer supplemented with 250 mM imidazole) and further purified by size exclusion chromatography in a Superdex 200 10/30 (GE Healthcare) equilibrated with 20 mM Tris pH 7.4, 100 mM NaCl, and 2 mM DTT.

The PLpro inhibition assay was performed using the FRET-based fluorescent peptide substrate Abz-TLKGG↓APIKEDDPS-EDDnp, kindly provided by Dr. Maria Aparecida Juliano (Federal University of São Paulo, Brazil). The assay was standardized with an enzyme concentration of 70 nM and 27 μM fluorescent substrate in PLpro assay buffer (50 mM HEPES pH 7.5, 0.01% Triton X-100, and 5 mM DTT), at 37 °C for 30 min. Activity was measured in the plate reader system Spectramax Gemini EM (Molecular Devices), with λex = 320 nm and λem = 420 nm, in the presence of different inhibitors and 1% DMSO.

Mpro Cloning, Expression, Purification, and Assays

The Mpro cloning is described elsewhere.[80] For expression, BL21 cells transformed with plasmid were grown in ZYM-5052 to an OD600 of 0.6–0.8 at 37 °C and 200 rpm. Protein expression was induced lowering the temperature to 18 °C, and cells were then grown for 16 h and harvested by centrifugation. The cell pellet was resuspended in lysis buffer (20 mM Tris pH 7.8, 150 mM NaCl, and 1 mM DTT), disrupted by sonication, and centrifuged at 12,000g for 40 min at 4 °C. Uncleaved protein with 6× HisTag was isolated from the lysate using Ni-NTA resin (Qiagen). The flow-through (FT) was then used to purify cleaved protein with ammonium sulfate precipitation. Ammonium sulfate was added into the FT to a final concentration of 1 M, incubated on ice for 10 min, and centrifuged at 12,000g for 30 min at 4 °C. The precipitated protein was resuspended in gel filtration buffer (20 mM Tris pH 7.8, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT) and purified by size-exclusion chromatography in a HiLoad 26/100 Superdex 200 column (GE Healthcare) pre-equilibrated with gel filtration buffer. Afterward, the protein was buffer-exchanged to 20 mM Tris pH 8.0 and 1.0 mM DTT, loaded into a Mono-Q 5/50 GL column (GE Healthcare) in the same buffer, and eluted with a linear gradient of a buffer containing 20 mM Tris pH 8.0, 1.0 M NaCl, and 1.0 mM DTT. Proteins were concentrated at 1.0 mg/mL, flash-frozen, and stored at −80 °C for inhibition assays.

The Mpro inhibition assay was performed using the FRET-based fluorescent peptide substrate DABCYL-KTSAVLQ↓SGFRKM-E(EDANS)-NH2 (purchased from Genscript). The assay was standardized with an enzyme concentration of 140 nM and 30 μM fluorescent substrate in Mpro assay buffer containing 20 mM Tris pH 7.3, 1 mM EDTA, and 1 mM DTT, at 37 °C for 30 min. Activity was detected in the spectrofluorometer system Spectramax Gemini EM (Molecular Devices), with λex = 360 nm and λem = 460 nm, in the presence of different inhibitors and 1% DMSO.

Kinase Profiling of Pyronaridine

Kinase profiling was performed for pyronaridine (1 μM) in duplicate by Thermo Fisher Scientific (Life Technologies Corporation, Chicago, IL 60693) using Z’Lyte,[81] Adapta,[82] and LanthaScreen[83] assays for 485 purified kinases.

CAMK1 Inhibition

Pyronaridine inhibition of CAMK1 was performed by Selected Services (Thermo Fisher Scientific) using the Adapta universal kinase assay, which is a homogeneous, fluorescent-based immunoassay for the detection of ADP. The 2× CAMK1 (CaMK1) and ZIPtide mixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 4 mM CaCl2, 800 U/mL calmodulin, and 0.02% NaN3. The final 10 μL kinase reaction consists of 0.25–1.2 ng of CAMK1 (CaMK1) and 200 μM ZIPtide in 32.5 mM HEPES pH 7.5, 0.005% BRIJ-35, 5 mM MgCl2, 500 μM EGTA, 2 mM CaCl2, 400 U/mL calmodulin, and 0.01% NaN3. After 1 h of kinase reaction incubation, 5 μL of detection mix was added.