European Journal of Medicinal Chemistry 205 (2020) 112687

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Potential treatment methods targeting 2019-nCoV infection

Lu Zheng a, 1, Lina Zhang a, 1, Jiamin Huang a, Kutty Selva Nandakumar a, Shuwen Liu a, b, **,
Kui Cheng a, *
a
Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and
Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
b
State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Southern Medical University, Guangzhou 510515, China

a r t i c l e i n f o a b s t r a c t

Article history: The novel coronavirus, 2019-nCoV, has quickly spread across the world and pose serious threat to public
Received 26 May 2020 health because it can infect people very easily. The major clinical symptoms of 2019-nCoV infection
Received in revised form include fever, dry cough, myalgia, fatigue, and diarrhea. The 2019-nCoV belongs to the betacoronavirus
18 July 2020
family, and gene sequencing results demonstrate that it is a single-stranded RNA virus, closely related to
Accepted 19 July 2020
Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-
Available online 28 July 2020
CoV). It has been observed that the virus invades human body mainly through binding to angiotensin-
converting enzyme 2 (ACE2) receptors similar to SARS-CoV and the main protease (Mpro) acts as a
Keywords:
Coronavirus
critical protease for digesting the polyprotein into functional polypeptides during the replication and
Target protein transcription process of 2019-nCoV. In this review, we summarized the real-time information of 2019-
Chemical drugs nCoV treatment methods and mainly focused on the chemical drugs including lopinavir/ritonavir,
Traditional Chinese medicine (TCM) chloroquine, hydroxychloroquine, arbidol, remdesivir, favipiravir and other potential innovative active
Vaccines molecules. Their potential targets, activity, clinical status and side effects are described. In addition,
Traditional Chinese Medicine (TCM), Convalescent plasma therapy (CPT) and biological reagents avail-
able, as well as the promising vaccine candidates against 2019-nCoV are also discussed.
© 2020 Elsevier Masson SAS. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Chemical drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Lopinavir/ritonavir (LPV/r) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Chloroquine phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.3. Hydroxychloroquine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4. Arbidol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.5. Remdesivir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.6. Favipiravir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.7. Other antiviral agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Traditional Chinese Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Biological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. Convalescent plasma therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2. Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.3. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

* Corresponding author.
** Corresponding author. Guangdong Provincial Key Laboratory of New Drug
Screening and Guangzhou Key Laboratory of Drug Research for Emerging Virus
Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical
University, Guangzhou 510515, China.
E-mail addresses: [email protected] (S. Liu), [email protected] (K. Cheng).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2020.112687
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
2 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687

5. Innovative research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6. Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction depicted in Fig. 1. Moreover, we collated the ongoing clinical trials

aimed to treat COVID-19 patients from Clinicaltrials.gov in Table 2.
Since December 2019, the novel coronavirus (2019-nCoV, SARS-
CoV-2, COVID-19) causes an epidemic of acute respiratory syn- 2.1. Lopinavir/ritonavir (LPV/r)
drome and has rapidly become a worldwide threat to public health.
As of 15 July 2020, at least 13,691,570 cases and 586,820 deaths Lopinavir/ritonavir (LPV/r), a protease inhibitor manufactured
were identified around the world. The major clinical symptoms of by AbbVie Corporation, is used for treating HIV-1 infection. The
2019-nCoV infection include fever, dry cough, short of breath, chest chemical structure of Lopinavir is (2S)eN-[(2S,4S,5S)-5-[[2-(2,6-
pain, fatigue, and diarrhea [1]. Although few patients were identi- dimethylphenoxy)acetyl]amino]-4-hydroxy-1,6-diphenyl-hexan-
fied without any abnormal radiological findings [2]. Many severe 2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide (Table 1).
patients have also developed dyspnea and lymphopenia. However, It is the active component of this drug combination, which blocks
until now no specific medicine is identified that could efficiently the division of Gag-Pol polyproteins resulting in the production of
control or eliminate the virus. On 22 Feb 2020, researchers have immature virus particles incapable of infecting the patients further.
successfully isolated the 2019-nCoV and sequenced its gene. The However, LPV has no effect on cells having integrated viral DNA,
results showed that 2019-nCoV belongs to betacoronavirus with a and only prevents subsequent infections of other susceptible cells.
single-stranded RNA [3], which is closely related to severe acute Pharmacokinetic studies have demonstrated that lopinavir is
respiratory syndrome CoV (SARS-CoV) and Middle East respiratory mainly metabolized by CYP3A4 and produces low systemic con-
syndrome Cov (MERS-CoV) [4]. Therefore the clinical experiences centrations when used alone. However, the metabolism of lopinavir
gained against SARS and MERS provide possible ways to treat 2019- could be inhibited by ritonavir. Hence, a combination of these two
nCoV infection effectively. SARS-CoV-2 encodes at least 27 proteins, drugs could prolong the systemic exposure to lopinavir and keep
including 15 non-structural proteins, 4 major structural proteins, the lopinavir concentration in the circulation for a longer period of
and 8 auxiliary proteins [5]. The 4 major structural proteins are the time. The recommended dose for adults is 400/100 mg twice daily.
spike (S), membrane (M), envelope (E) and the nucleocapsid (N) The most common adverse effects in adults associated with LPV/r
proteins, with the S protein mainly affecting the fusion and cell medication are diarrhea and other gastrointestinal disturbances,
entry process of the virus, the M protein mainly defines the shape asthenia, headache and skin rashes [8]. Based on the Diagnosis and
of the viral envelope, and both the E and N proteins are actively Treatment Guidelines of the 2019-nCoV in China (the seventh
participating in the viral assembly and budding events [6]. edition), LPV/r is included for treating the coronavirus pneumonia,
Angiotensin-converting enzyme 2 (ACE2) is the receptor of SARS- however its efficacy and safety remains to be established clearly.
CoV-2, and the S protein primed by the cellular serine protease Antiviral effects of lopinavir at EC50 value of 26.1 mM but not rito-
TMPRSS2 might facilitate the entry process of the virus into the navir against SARS-CoV-2 in vitro was reported earlier (Table 1) [9].
host cells. Therefore, the serine protease TMPRSS2 inhibitor Later in a clinical trial, 47 patients with COVID-19 infection,
camostat mesylate (Table 1) might prevent the entry of SARS-CoV-2 admitted to Rui’an People’s Hospital, were tested along with a
into cells [7]. With the imminent threat of the virus growing control group with LPV/r. This combination treatment with LPV/r
exponentially, it is high priority to develop specific vaccines and had a more clearly therapeutic effect in lowering the body tem-
drugs to combat COVID-19 infection. Fortunately, experimental perature and restoring homeostasis with no obvious toxic and side
vaccines are promising, while, the clinical trials of small chemical effects [10]. However, a paper published in the New England Journal
molecules showed certain beneficial effects. Currently, many of Medicine pointed out that LPV/r treatment did not significantly
medications and drugs are being used in clinics worldwide to test accelerate the clinical improvement and reduced mortality in pa-
their effectiveness including lopinavir/ritonavir, chloroquine, tients with serious COVID-19 [11]. Recently, a clinical trial
hydroxychloroquine, arbidol, remdesivir, favipiravir and Traditional (NCT04321174) is recruiting participants to further evaluate the
Chinese Medicine (TCM). Besides, scientists are exploring several efficacy of LPV/r for treating Covid-19 patients (Table 2).
specific molecules and combinations of different drugs to treat the
patients having SARS-CoV-2 infection. In this review, we collected
and sorted out the relevant information available currently to 2.2. Chloroquine phosphate
combat SARS-CoV-2 and we mainly focused on the chemical drugs
(Table 1). At the same time, we have also discussed the biological Chloroquine phosphate is a water-soluble compound with the
methods including convalescent plasma therapy (CPT) and the chemical structure of 7-chloro-4-((40 -diethylamino-1-methylbutyl)
promising vaccines under development against 2019-nCoV. amino)quinolinediphosphate (Table 1), which is widely used for
treating malarial infection and autoimmune diseases, like lupus
(both discoid lupus erythematosus and systemic lupus erythema-
2. Chemical drugs tosus) and arthritis. It is one of the first-line disease-modifying
anti-rheumatic drugs used for treating rheumatoid arthritis (RA)
Many chemical drugs have shown promising antiviral activities and it acts by inhibiting antigen presentation capacity of dendritic
against SARS-CoV-2. Here we summarized their chemical struc- cells, cytokine production in macrophages, as well as calcium and
tures, effects, targets and severe adverse effects in Table 1. In Toll-like receptor (TLR) signaling in B, T and other immune cells
addition, their possible mode of action against 2019-nCoV is [12]. Later, studies also discovered the broad-spectrum virus-
 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687 3

Table 1
Chemical drug in clinical against COVID-19.

Compounds Chemical structures Drug Target against COVID-19 EC50 Severe adverse effects

Lopinavir/ritonavir (LPV/r) 3CLprp 26.63 mM No obvious severe adverse reports

CYP3A4 >100 mM

Chloroquine (CQ) inhibit viral membrane fusion process 1.13 mM Retinopathy, cardiomyopathy, and
neuromyopathy

Hydroxychloroquine inhibit viral membrane fusion process 0.72 mM Retinopathy, cardiomyopathy, and
(HCQ) neuromyopathy

Arbidol (Umifenovir) Spike glycoprotein 4.11 mM Hepatic insufficiency

Remdesivir (GS-5734) RdRp 0.77 mM No obvious severe adverse reports

Favipiravir (T-705) RdRp 61.88 mM Teratogenicity and embryotoxicity

Baricitinib JAK e A risk of reactivation of latent infections

Ribavirin RdRp >100 mM Hemolytic anemia and teratogenic

Galidesivir (BCX4430) RdRp >100 mM No obvious severe adverse reports

Darunavir 3CLprp e Hepatic insufficiency

(continued on next page)

4 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687

Table 1 (continued )

Compounds Chemical structures Drug Target against COVID-19 EC50 Severe adverse effects

Oseltamivir unknown >100 mM Severe rash and hemorrhagic colitis

Camostat mesylate TMPRSS2 e No obvious severe adverse reports

11a 3CLprp 0.53 mM Unknown

11b 3CLprp 0.72 mM Unknown

13b 3CLprp 4e5 mM Unknown

N3 3CLprp 16.77 mM Unknown

resistance activity of chloroquine (CQ). Chloroquine is mainly chloroquine, while still retaining the antiviral activity. The immu-
absorbed from the gastrointestinal tract, and it might block the nomodulatory mechanisms are similar to chloroquine by elevating
virus infection by increasing the endosomal pH required for virus/ the pH through accumulation in lysosomes. Thus, HCQ interferes in
cell fusion, as well as by interfering with the glycosylation of the process of antigen presentation (Fig. 1) and possibly attenuating
cellular receptors of SARS-CoV [13]. An article in Cell Research re- the inflammatory response by significantly decreasing the pro-
ported that CQ could prevent 2019-nCoV infection at the cellular duction of pro-inflammatory factors in COVID-19 patients. Similar
level (EC50 ¼ 1.13 mM, CC50 > 100 mM, SI > 88.50) (Table 1). to CQ, HCQ has beneficial effects in immunodeficient conditions
Immunofluorescence studies on virus infection after chloroquine and also reduces development of metabolic and cardiovascular
treatment also proved the anti-2019-nCoV activity of CQ (Fig. 2) complications apart from having anti-tumor and anti-thrombotic
[14]. Subsequently, the clinical studies confirmed its effectiveness effects [12]. HCQ with a EC50 of 0.72 mM was reported to have
against 2019-nCoV at a dose of 500 mg/day with no obvious serious anti-viral activity against COVID-19 in vitro, which suggested the
adverse reactions [15]. Although patients showed few adverse ef- potential use of HCQ in clinics [16]. Results from an open label non-
fects in the clinics at the standard dose of chloroquine, the main randomized clinical trial of limited size in France claimed HCQ
severe side effects include retinopathy, cardiomyopathy and neu- (200 mg, three times per day in 10 days) used in COVID-19 patients
romyopathy, while used for longer periods, and the acute toxicity of might be efficient in clearing nasopharyngeal carriage of SARS-
chloroquine occurred when high dose was rapidly administered by CoV-2 within three to six days. A combination of HCQ and azi-
parenteral routes [12]. Therefore clinicians should closely observe thromycin also showed synergistic effect providing an alternate
adverse reactions with chloroquine treatment, especially with treatment strategy for SARS-CoV-2 infection [17]. However, at
elderly patients. Recently, many clinical trials with chloroquine present there is still no clear evidence to support the effectiveness
against 2019-nCoV were initiated in many health care centers of HCQ against SARS-CoV-2 infection [18], which remains to be
(Table 2). confirmed by clinical trials (Table 2). Although HCQ is a less toxic
molecule, poisoning occurs with its prolonged use and over-dosage,
particularly irreversible retinopathy possibly leading to loss of
2.3. Hydroxychloroquine vision. Therefore, clinical monitoring and early recognition of toxic
symptoms are essential for an effective management strategy. Cli-
Hydroxychloroquine (HCQ) is an aminoquinoline compound nicians should monitor the dose taken by the patients and pay close
modified by chloroquine, with an N-hydroxyethyl side chain attention to the side effects including retinal toxicity, neuro-
instead of the N-diethyl group of chloroquine. An increase in hy- myopathy, and cardiac disease during clinical trials.
droxyl moiety makes it highly water soluble and less toxic than
 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687 5

Fig. 1. The mechanism of clinical drugs against COVID-19. SARS-CoV-2 infects the host cells by binding to ACE2 receptor. S protein of SARS-CoV-2 primed by the cellular serine
protease TMPRSS2 play an essential role in facilitating this entry process. Camostat mesylate inhibit TMPRSS2 to hamper virus invasion. Arbidol, chloroquine, and hydroxy-
chloroquine mainly inhibit the membrane fusion processes, which prohibit the virial entry. 3CLpro digests the polyprotein of SARS-CoV-2 into functional polypeptides. Both
Lopinavir and Darunavir hinder the viral replication processes by inhibiting 3CLpro. Ribavirin, Remdesivir and Favipiravir block the viral RNA replication by inhibiting the RdRp
enzyme. Traditional Chinese Medicines dampen the immune response by decreasing the production of cytokines and regulating the immune related pathways.

2.4. Arbidol potential against herpes viruses (HHV-8), ebola virus (EBOV), are-
naviruses (Tacaribe virus), poliovirus and HBV [22]. Therefore, ARB
Arbidol (ARB), also known as umifenovir, is recommended as a is considered as a potential compound to combat COVID-19. Later
specific anti-influenza drug to control influenza A and influenza B ARB (EC50 ¼ 4.11 mM, CC50 ¼ 31.79 mM, SI ¼ 7.73) [23] was shown to
viral infections. It has been approved in Russia and China for human inhibit COVID-19 infection (Fig. 3a) more efficiently and it interferes
use, with no major adverse effects reported to date. It prevents with the release of SARS-CoV-2 from intracellular vesicles (Fig. 3b).
contact between the virus and host cells and penetration of virus Subsequently, a retrospective study approved by the Third People’s
particles into the cell by inhibiting the fusion of the virus lipid shell Hospital of Changzhou, China demonstrated the efficacy of ARB
to the cell membrane (Fig. 1) [19]. ARB is an indole analogue and, monotherapy, which might be superior to LPV/r against COVID-19
the amine in position 4 and the hydroxyl moiety in position 5 are [24]. However, the existing data with clinical trials is insufficient
important for the antiviral action of ARB. Insertion of a methyl to precisely prove the effectiveness of ARB against COVID-19
group between the indole ring and 5-hydroxyl group has consid- because of the limited sample size, and the unknown antiviral
erably increased its antiviral potency, whereas introduction of mechanisms. Another study confirmed that ARB combined with
particular azote-based heterocyclic groups at position 4 improved LPV/r might benefit the patients by delaying the progression of lung
anti-HBV activity. Replacement of the S-phenyl group at position 2 lesions and lowering the possibility of respiratory and gastroin-
by a phenyl-sulfonyl group decreased its cytotoxicity, while testinal transmissions [25]. Hence, a combination therapy could be
increasing the anti-HBV activity of the compound [20]. Interest- the most effective way for managing COVID-19, which needs to be
ingly, arbidol has substantial antiviral activities against Zika virus validated in the near future. At present, phase IV clinical trials are
(ZIKV), West Nile virus (WNV) and Tick-borne encephalitis virus being carried out to test the efficacy of ARB against COVID-19
(TBEV) with EC50 values ranging from 10.57 ± 0.74 to including NCT04350684 and NCT04260594 (Table 2).
19.16 ± 0.29 mM in HBCA and Vero cells [21]. It also has antiviral
6 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687

Table 2
Clinical trials identified at Clinicaltrials.gov related to drug repositioning for COVID-19 treatment.

Compound N test Clinical condition (sample size of 2019-nCoV Sponsors Phase

participants)

Lopinavir/ritonavir NCT04321174 1220 participants Darrell Tan III

Lopinavir/Ritonavir, Ribavirin and IFN-beta Combination NCT04276688 127 participants University of Hong Kong, Hospital II
Authority
Chloroquine Phosphate (CQ) NCT04328493 250 participants Oxford University Clinical Research II
Unit, Vietnam
NCT04333628 210 participants (Mild) HaEmek Medical Center, Israel II
hydroxychloroquine (HCQ) NCT04330144 2486 participants Gangnam Severance Hospital III
Azithromycin- Hydroxychloroquine Combination or NCT04336332 160 participants Rutgers, The State University of New II
Hydroxychloroquine Jersey
Remdesivir (GS-5734) NCT04323761 Expanded Access Gilead Sciences e
NCT04292899 6000 participants (Severe) Gilead Sciences III
Favipiravir (T-705) NCT04336904 100 participants Giuliano Rizzardini III
NCT04346628 120 participants (Mild) Stanford University II
Baricitinib NCT04320277 200 participants Hospital of Prato II and
III
NCT04340232 80 participants University of Colorado, Denver II and
III
Galidesivir (BCX4430) NCT03891420 66 participants BioCryst Pharmaceuticals I
Darunavir and Cobicistat NCT04252274 30 participants Shanghai Public Health Clinical Center III
Hydroxychloroquine, Oseltamivir, or Azithromyci NCT04338698 500 participants Shehnoor Azhar III
CamostatMesylate NCT04321096 580 participants University of Aarhus I
Arbidol (Umifenovir) NCT04350684 40 participants Shahid Beheshti University of Medical Ⅳ
Sciences
NCT04260594 380 participants Jieming QU Ⅳ

2.5. Remdesivir 2.6. Favipiravir

DNA or RNA polymerase enzymes are essential components in Favipiravir (T-705), 6-Fluoro-3-hydroxypyrazine-2-carboxamide,
the process of viral replication. Nucleoside analogues are used to is a novel RNA-dependent RNA polymerase (RdRp) Inhibitor from
control the viral infections in clinics, which target the DNA or RNA Toyama Chemical Co., Ltd, and used to treat influenza viruses in Japan
polymerase. Remdesivir (GS-5734), a prodrug developed by Gilead [28]. Favipiravir-RTP (favipiravir ribofuranosyl-50 -triphosphate) is the
Science, is well recognized as a potential antiviral drug against a active form of T-705 converted by host enzymes, which acts as a
wide array of infections with RNA viruses in cell cultures, mice and nucleotide analogue selectively inhibiting the RdRp. At the same time,
non-human primate (NHP) models. The predicted mechanism of T-705 can also get incorporated into the nascent viral RNA resulting in
action is by the incorporation of an active triphosphate into the termination of the viral replication processes (Fig. 1) [29]. Since the
viral RNA resulting in premature termination of RNA synthesis that catalytic domain of RdRp is conserved among various types of RNA
decreased the level of viral RNA (Fig. 1) [26]. A study reported the viruses, T-705 has a broad spectrum of anti-viral activities. It has
efficacy of Remdesvir (GS-5734), which is highly active against inhibited the replication of ZIKV in Vero cells (EC50 ¼ 3.5e3.8 mg/mL)
coronaviruses (EC50 ¼ 0.074 ± 0.023 mM in MERS-CoV-infected and and Zaire Ebola virus in Vero E6 cells (EC50 ¼ 10.5 mg/mL) [29]. It is also
EC50 ¼ 0.069 ± 0.036 mM in SARS-CoV-infected HAE cultures) at used as a potential promising candidate for the treatment of COVID-
early times post infection. Interestingly, drug resistance was not 19. Favipiravir (EC50 ¼ 61.88 mM, CC50 > 400 mM, SI > 6.46) has
observed, which precludes development of resistant superviruses effectively reduced the COVID-19 infection in Vero E6 cells (Table 1)
[27]. Therefore GS-5734 is a promising molecule to defeat COVID- [14]. Another study compared the effects of FPV and LPV/r for treating
19. Subsequently, a study from Wuhan Institute of Virology re- COVID-19 patients. FPV had better treatment effects on COVID-19
ported the EC90 value of GS-5734 against 2019-nCoV in Vero E6 compared to LPV/RTV having faster viral clearance rate and a higher
cells as 1.76 mM [14], suggesting the feasibility of achieving its level of improvement from lung complications as evidenced by im-
working concentration in NHP models. Preliminary data showed aging techniques [30]. However, the production and application of T-
that remdesivir has also inhibited 2019-nCoV infection efficiently in 705 are severely restricted in Japan because of the prevailing risks of
a human cell line (human liver cancer Huh-7 cells). The study also teratogenicity and embryotoxicity. Currently, a Phase III clinical trial of
proved the anti-SARS-CoV-2 activity of GS-5734 using immuno- T-705 (NCT04336904) is already underway (Table 2) [31].
fluorescence microscopy (Fig. 2) [14]. However, a report published
in New England Journal of Medicine described the effect of GS-5734
was still inadequate to assess in the first COVID-19 patient recov- 2.7. Other antiviral agents
ered after remdesivir injection in the United States, because the
viral load of the patient was decreased before remdesivir was used. Ribavirin (Table 1) is known as an effective antiviral agent to
Hence, the patient recovery was attributable to the drug or the role combat hepatitis C virus (HCV) but it is highly cytotoxic [32]. Even
of self-defense mechanisms and supportive treatments was not then, it was included in the recommended combination therapy
made clear [26]. Currently, clinical trials using GS-5734 as a ther- with interferon or LPV/r in the Diagnosis and Treatment Guidelines
apeutic against 2019-nCoV infection are in progress (NCT04323761 of the 2019-nCoV in China (the seventh edition). However, there
and NCT04292899), and the effectiveness and adverse reactions of was insufficient evidence available for its clinical efficacy after
this drug are worthy of our attention (Table 2). administration to COVID-19 patients. A janus kinase inhibitor,
baricitinib (Table 1), which binds to the cyclin G-associated kinase,
is a regulator of endocytosis. Researchers used AI to search for the
 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687 7

treatment of pestilence for thousands of years in China, and also it

is a vastly unexploited treasure in modern medicine. In recent
years, development and application of TCM-derived herbs and
formulations for evidence-based therapy is increasing exponen-
tially [36]. TCM had notable therapeutic effects against SARS
epidemic in 2003 [37]. Now, traditional Chinese medicine prepa-
rations like Lianhuaqingwen (LH) Capsule and Qingfeipaidu
Decoction (QPD) have shown promising therapeutic effects against
2019-nCoV infection in clinics. According to the theory of TCM,
these preparations work mainly by regulating the immune related
pathways involving cytokine functions to alleviate excessive im-
mune response and related-complications (Fig. 1) [38]. LH inhibited
the replication of 2019-nCoV with an IC50 value of 411.2 mg/mL by
CPE assay, and the image of viral particles in ultra-thin sections of
infected cells under electron microscopy supported these obser-
vations (Fig. 4) [39]. Another study tested the antiviral activity of LH
against 2019-nCoV by CPE and plaque reduction assay using Vero
E6 cells, and the results showed that LH could inhibit the replica-
tion of 2019-nCoV and also reduce the production of pro-
inflammatory cytokines (TNF-a, IL-6, CCL-2/MCP-1 and CXCL-10/
IP-10) [39]. Subsequently, a retrospective clinical analysis on the
treatment of SARS-CoV-2 with LH at the Ninth Hospital of Wuhan
and CR & WISCO General Hospital was conducted andthe results
showed that LH has significantly relieved the cough and fever
symptoms associated with the infection [40]. Similarly, QPD was
also widely used to treat SARS-CoV-2 in China. According to Na-
tional Administration of Traditional Chinese Medicine, the disease
symptoms of patients were markedly stabilized with the disap-
pearance of cough and fever after QPD treatment. Recently, Re-
searchers are focused to investigate and analyse the potential
protective mechanisms of QPD against SARS-CoV-2, the possible
side effects and new formulations. In the future, an integrated
approach combining Chinese and Western drugs will be tested to
cure 2019-nCoV infected patients.

4. Biological methods

4.1. Convalescent plasma therapy

Fig. 2. Immunofluorescence images of virus infection in Vero cells with and Convalescent plasma therapy (CPT) is a passive immune therapy
without treatment with remdesivir and chloroquine. Virus infection and drug used to fight the novel coronavirus by injecting the plasma of the
treatment were performed in vitro using Vero cells. At 48 h p.i., the infected cells were recovered person containing anti-virus antibodies [41]. It has been
fixed, and probed with rabbit sera against the nucleocapsid protein (NP) of a bat SARS-
successfully used for controlling SARS infection, but the limited
related CoV2 as the primary antibody. Alexa 488-labeled goat anti-rabbit IgG (1:500;
Abcam) was used as the secondary antibody. The nuclei were stained with Hoechst quantity of the plasma available impedes wider clinical applica-
dye. Bars, 100 mm. (Reproduced with permission from ref.14). tions. CPT was used to treat critically ill patients in China [42], and
experts are still evaluating its efficacy and safety in clinical trials.
Recently, it was observed that after CPT therapy SARS-CoV-2 RNA
approved drugs that could help in the treatment of COVID-19 has disappeared completely along with a clear rise in neutralizing
infection and found baricitinib as a potential drug to treat 2019- antibody titers in nearly all the patients, which could reduce mor-
nCoV acute respiratory disease [33]. Recently, the clinical trials of tality rate in severe COVID-19 patients [43].
baricitinib against COVID-19 (NCT04320277 and NCT04340232) are
ongoing and the results are eagerly awaited (Table 2). Lanjuan Li 4.2. Interferons
team has shown Darunavir (Table 1), a HIV-1 protease inhibitor
[34], has the activity against 2019-nCoV. Darunavir could possibly Interferons (IFNs) are a broad class of cytokines divided into
be administered along with either ritonavir or cobicistat, but the type I, type II, and type III secreted by the host when subjected to
effectiveness and safety profile are yet to be explored. Currently, the stress or infections, and also during the development of auto-
phase III clinical trial of Darunavir together with Cobicistat against inflammatory and autoimmune diseases [44]. Interferons are
COVID-19 is under way (Table 2). Oseltamivir (Table 1) is a drug crucial for antiviral responses, antigen presentation, development
used for preventing and treating influenza virus infection in chil- of autoimmunity and inflammation by activating the Jak-STAT
dren. It was tried for the treatment of SARS-CoV-2 patients [35], but signaling pathway [45]. All IFNs share the ability to promote anti-
its efficacy remains uncertain (Fig. 3a). viral activities initiated by their interactions with cognate receptors
[46]. It’s well known that type I IFNs could effectively inhibit virus
3. Traditional Chinese Medicine replication, and also show critical effects on the development and
activation of immune cell subsets. Given both the antiviral and
Traditional Chinese Medicine (TCM) has played a vital role in the immunomodulatory effects, type I IFNs are used either alone or in
8 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687

Fig. 3. The antiviral activities of Arbidol and Oseltamivir against 2019-nCoV in vitro. (A) Antiviral activities of Arbidol and Oseltamivir in Vero E6 cells were determined by qRT-
PCR analysis of virus yield at 48 h p.i. (B) Effect of Arbidol on intracellular trafficking of SARS-CoV-2. The colocalization of virions with early endosomes (EEs) or late endosomes (LEs)
was analyzed by immunofluorescence. Representative confocal microscopic images of virions (red) and LAMP1þ ELs (green) in each group were shown. The nuclei (blue) were
stained with Hoechst 33258 dye. White arrows: virions co-localized with ELs; bars: 10 mm. (Reproduced and arranged with permission from ref.23). (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Virions in the ultrathin sections of infected Vero E6 cells under electron microscope. (A, F) Uninfected and (B, G) mock-treated SARS-CoV-2 virus infected cells. (C, D, H,
I) Infected cells after Lianhuaqingwen (LH) or (E, J) Remdesivir treatment. White arrows indicate the spindle shape of viral particles within the infected cells after LH treatment.
(Reproduced with permission from ref.39).

combination with other medicines to treat a variety of chronic and However, prolonged clinical use of IFNs is limited because of their
acute viral infections in clinics [47]. During the SARS-CoV and EBOV adverse side effects including neutropenia, thrombocytopenia,
disease outbreak, Type I IFNs were used as auxiliary drugs together hyper-and hypothyroidism, pancreatitis, type I diabetes mellitus,
with other antiviral medicines to alleviate patients’ complications. and irreversible pulmonary hypertension [47]. Recently, IFN-a (5
 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687 9

million U each time for adults twice per day) was recommended to transfer (FRET) assay to screen a library of ~10,000 compounds, and
use along with Lopinavir/ritonavir or Ribavirin to combat 2019- found 7 clinical drug candidates including Ebselen (IC50 ¼ 0.67 mM),
nCoV infection [48]. Disulfiram, Carmofur, Shikonin, Tideglusib, PX-12 and TDZD-8 [53].
Thus this study provided a reliable strategy for rapid drug discovery
4.3. Vaccines against COVID-19 in the near future. Based on the structure of the
substrate-binding pocket of SARS-CoV Mpro, Hualiang Jiang and
Vaccines are effective weapons to fight against the viruses like Hong Liu team have designed and synthesized novel inhibitors
smallpox, measles, polio and other infectious diseases that were targeting the SARS-CoV-2 Mpro and obtained compounds 11a
once raged around the world. The isolation of new coronavirus (Table 1) (EC50 ¼ 0.53 ± 0.01 mM, CC50 > 100 mM, SI > 189) and 11b
makes vaccine development a possibility, but success is limited (Table 1) (EC50 ¼ 0.72 ± 0.09 mM, CC50 > 100 mM, SI > 139). Both
because of the strenuous development processes involved in con- these compounds showed excellent inhibitory potency against
structing and validating a vaccine [49]. At present, researchers have SARS-CoV-2 Mpro enzyme [53]. The pharmacokinetic properties of
developed five technical routes for the development of new COVID- 11a and 11b demonstrate their good bioavailability with no obvious
19 virus vaccines, including inactivated vaccines, recombinant toxicity. Their data indicated that these 2 compounds are good
genetically engineered vaccines, adenovirus vector vaccines, candidates for further clinical studies (Fig. 5B and C). Rolf Hilgenfeld
nucleic acid vaccines, and vaccines made from attenuated influenza and his team reported the x-ray structures of the un-liganded
virus vaccine vectors [50]. It may require more than a year to start SARS-CoV-2 Mpro and its complex with an a-ketoamide inhibitor
phase I clinical trials for most of the candidate vaccines. Up to May [54]. Compound 11r is a broad-spectrum inhibitor of main pro-
12, 2020, 10 candidates of COVID-19 vaccines are undergoing clin- teases, which could inhibit betacoronaviruses, alphacoronaviruses
ical trials, including Adenovirus Type 5 Vector (CanSino Biologics as well as the 3CLpro of enteroviruses. They have optimized 11r and
Inc), ChAdOx1 nCoV-19 (Moderna), INO-4800 (Inovio Pharmaceu- synthesized the compounds 13a, 13b (Figs. 5D) and 14b. All of them
ticals), mRNA-1273 (University of Oxford), BNT162 (BioNTech), were tolerated well in mice. More interestingly, compounds 13a
bacTRL-Spike (Symvivo Corporation), Pathogen-specific aAPC and 13b tend to concentrate more in the lungs, which implied the
(Shenzhen Geno-Immune Medical Institute), LV-SMENP-DC possibility of direct administration of these compounds to the
(Shenzhen Geno-Immune Medical Institute) and other 2 vaccines lungs. This study provided a solid framework for the development
developed by Sinovac Biotech, and Wuhan Institute of Biological of pyridone-containing inhibitors against the virus. Shuai Xia and
Products. Notably, on May 22, 2020, an article published in The his team have successfully solved the X-ray crystal structure of six-
Lancet Journal reported the first-in-human trial of a recombinant helical bundle (6-HB) core of the HR1 and HR2 domains of the
adenovirus type-5 (Ad5) vector COVID-19 vaccine (NCT04313127) SARS-CoV-2 S protein S2 subunit, and generated a series of lip-
[51]. This study mainly assessed the safety, tolerability, and opeptides derived from EK1 (a pan-coronavirus fusion inhibitor). It
immunogenicity of the vaccine in which 108 healthy participants was found that EK1C4 is the most potent fusion inhibitor available
were participated. The participants were divided into 3 groups on against SARS-CoV-2 S protein-mediated membrane fusion and
the basis of dosage [low dose (n ¼ 36), middle dose (n ¼ 36), or high pseudovirus infection with IC50 of 1.3 and 15.8 nM, respectively
dose (n ¼ 36)]. This vaccine was well tolerated and the most [55]. This suggests the possibility of using EK1C4 for prevention and
common adverse reactions were mild or moderate in severity treatment of 2019-nCoV infection.
including fever, fatigue, headache and muscle pain. In addition,
humoral responses against COVID-19 were peaked at day 28 post- 6. Conclusion and perspectives
vaccination and rapid specific T-cell responses were noted from day
14 post-vaccination. This data implied the potential of Ad5 vectored At present the 2019-nCoV infection is rapidly spreading
COVID-19 vaccine in preventing COVID-19 infection. However, this worldwide and as of 15 July 2020, the death toll has reached
study was limited by the sample size, short follow-up period and 586,820. Hence, there is an urgent requirement to identify more
the absence of a randomized control groups. Another ongoing effective antiviral agents to combat the disease and validate their
phase II trial in China (NCT04341389) will provide more informa- clinical effects. Although the development of new drugs has shown
tion on the safety and immunogenicity of the Ad5 vector COVID-19 some progress, currently, no specific treatment against this new
vaccine and the results are expected soon. virus is available. Certainly, vaccines are the most effective tools to
fight the viruses, and many scientists all over the world are actively
5. Innovative research involved in developing specific vaccines. The phase I clinical trial
data of Ad5 vectored COVID-19 vaccine is promising and there is
During this 2019-nCoV outbreak, scientists all over the world hope to defeat the virus eventually. Considering the possible in-
are focused on actively exploring different aspects of virus struc- validity of vaccines because of the rapid gene mutations in COVID-
ture, mode of transmission, pathology and clinical complications in 19, the chemical drugs and active molecules offer exciting oppor-
the infected patients and, made some outstanding contributions. tunities. China is one of the first-line countries in developing anti-
The main protease (Mpro), also termed 3CLpro, acts as a critical epidemic strategies and, it has quickly and efficiently controlled the
protease for digesting the polyprotein into functional polypeptides domestic epidemic situation. Living conditions and production
involved in 2019-nCoV replication and transcription processes. It is work have gradually begun to get normalized to pre-COVID19 sit-
also an ideal antiviral target because of no homologues present in uation. For treating this virus infection, many antiviral drugs were
the humans. Professor Rao and his team have identified N3 as a used in China, and this experience gained about beneficial strate-
potent irreversible inhibitor of COVID-19 virus Mpro. They have gies and methods is being shared to other countries to contain the
determined the crystal structure of COVID-19 virus Mpro in com- COVID-19 infection. Enormous data generated is also facilitating
plex with N3 to 2.1 Å resolution (Fig. 5A) [52]. This structure pro- further scientific studies in several laboratories. According to the
vides a model for discovering lead inhibitors to target COVID-19 Diagnosis and Treatment Guidelines of 2019-nCoV in China (the
virus Mpro through computer-aided drug designs as well as virtual seventh edition), Lopinavir/ritonavir (combined with Ribavirin),
and high-throughput screening methods, which is fundamental for chloroquine and arbidol are recommended as antiviral drugs for
identifying potential active molecules against COVID-19. Subse- mild COVID-19 patients. In addition, IFN-a can be used as an
quently, these authors developed a fluorescence resonance energy adjuvant therapy by inhalation. For severe patients, convalescent
10 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687

Fig. 5. Structure and binding pocket of different inhibitors to COVID-19 main protease, Mpro. (A) The crystal structure of COVID-19 virus Mpro-N3 complex (PDB: 6LU7) and the
chemical structure of N3. (B) The crystal structure of COVID-19 virus Mpro-11a complex (PDB: 6LZE) and the chemical structure of 11a. (C) The crystal structure of COVID-19 virus
Mpro-11b complex (PDB: 6MOK) and the chemical structure of 11b. (D) The crystal structure of COVID-19 virus Mpro-13b complex (PDB: 6Y2F) and the chemical structure of 13b.

plasma therapy and blood purification treatment are advisable. As to the traditional therapy with chemical drugs, monotherapy
Tocilizumab is suitable to use for severely 2019-nCoV infected pa- may be inadequate to control the virus, hence combination thera-
tients having critical pulmonary lesions, especially with the in- pies should be considered seriously for combating COVID-19 more
crease of IL-6R expression. Chinese Traditional medicines like efficiently. Different drug combinations and dosages as well as
Lianhuaqingwen capsules, Shufengjiedu capsules, Jinhuaqinggan potential toxic effects are worth exploring further. TCM also shows
granules and Qingfeipaidu Decoction are also suggested to control huge potential for alleviating and moderating the symptoms of
the progression of disease. Although some chemical drugs tested patients infected with 2019-nCoV. However, the composition of
in vitro have shown anti-coronavirus effects, their potential remain TCM is complex and they also act on unknown targets. Hence,
to be investigated in vivo. It is worth noting that more scientific development of precise TCM prescriptions and analysis of accom-
research and reasonable design of clinical trials are essential to panied potential toxicities will be the major focus in future studies.
assess the effectiveness of drugs. Some existing clinical reports are Furthermore, the crystal structure of COVID-19 virus Mpro provides
lacking in their credibility because of the limitation in sample size. a new and efficient strategy to develop potential antiviral drugs
Researchers should design scientific and logical experiments with that are based on computer-aided drug designs, virtual and high-
reasonable sample size for clinical trials to evaluate the accuracy of throughput screening in the near future, which could greatly
the results in the future. speed up the drug development processes.
 L. Zheng et al. / European Journal of Medicinal Chemistry 205 (2020) 112687 11

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