Discovery of Novel Cysteine Protease Inhibitors for the Treatment of Coronavirus (COVID-19)

Abstract

The application describes compounds, such as compounds of general Formula, with warheads and their use in treating medical diseases or disorders, such as viral infections. Pharmaceutical compositions and synthetic methods of various compounds with warheads are included. The compounds are inhibitors of proteases, such as the 3C, CL- or 3CL-like protease.

1. COMPOUND CLASS (GENERAL FORMULA)

Title: Inhibitors of cysteine proteases and methods of use there of

Patent Publication Number: US11312704B2

URL: https://patents.google.com/patent/US11312704B2/en?oq=US11312704B2

Patent Grant Date: April 26, 2022

Patent Priority Number: US202063012039P

Priority Date: April 17, 2020

Inventors: Arnold, L. D.; Jennings, A.; Keung, W.

Assignee Company: Pardes Biosciences Inc, San Diego, California, USA

Disease Area: Coronavirus and other viruses

Biological Target: SARS-CoV-2 main protease and HCoV 229E

Number of Claims: 7

Number of Compounds Claims: 781

2. SUMMARY

The Coronaviridae family of viruses are enveloped, single-stranded, and positive-sense RNA viruses. There are 141 species that are categorized into four general groups according to their phylogenetic relationships: α-, β-, γ-, and δ-coronavirus. Coronaviruses (CoVs) are zoonotic viruses. These viruses infect a variety of animals from whales to birds, bats, cats, and humans resulting in mild to moderate respiratory tract infections [1-12]. Severe acute respiratory syndrome coronavirus (SARS-CoV) is a human coronavirus that was affected in the first pandemic of the 21^st century, resulting in over 8,000 people with a 10% mortality rate. Middle East respiratory syndrome coronavirus (MERS-CoV) was infected in November 2012 and has since infected over 1,600 people in 26 countries with a 36% mortality rate. Recently, COVID-19 (SARS CoV2) coronavirus became a global pandemic and it was first identified in China in 2019. As of 6 April 2023, there have been 762,201,169 confirmed cases of COVID-19, and 6,893,190 deaths globally according to the World Health Organization. It is an urgent and unmet need to discover a new broad-spectrum anti-coronaviral agent to treat the current infections and emerging coronaviruses in the future.

Most coronaviruses have >800 kDa replicase polyprotein, two or three cysteine proteases, the papain-like proteases (PLPpro, or PLP1 and PLP2), and the 3C-like protease (3CLpro, nsp5, or Mpro). These proteases make the CoV replicase polyprotein by cleaving it into 16 non-structural proteins. The CoV 3CLpro makes 11 cleavage sites within the replicase polyprotein and is essential for CoV survival. The overall active site architecture and substrate recognition pockets are structurally conserved across CoV 3CLpros, making it a valid target for the development of broad-spectrum anti-CoV therapeutics.

The sulfur of the cysteine in the protease behaves as a nucleophile and an electrophilic nitrile moiety consists of this series of compounds in the C-terminus. When the compound binds into the 3CLpro active site, the Cys145 thiol group attacks on the nitrile group, forming a covalent bond formation thus inhibiting the viral protease activities as shown in Fig. (1).

Fig. (1).

Modification of Cysteine of the protease with nitrile group of the compound, forming a covalent bond.

3. DEFINITIONS

From the general formula, wherein:

R_1a is selected from hydrogen and C₁-C₈alkyl;

R_1b is selected from hydrogen and C₁-C₈alkyl;

R_1a and R₂ are joined together to form, together with the carbon to which they are attached, a 4-10 membered monocyclic or bicyclic heterocycle having a ring nitrogen, wherein the heterocycle may be optionally substituted on a free carbon by one, two, or three substituents;

R₃ is selected from 4-10 membered heterocycles;

R_3b is selected from hydrogen or C₁-C₈alkyl.

4. SYNTHESIS

The synthesis of compound 101 starts from compound 1 as shown in Scheme 1. Deprotection of the Boc group in the presence of HCl in ethyl acetate provides compound 2 in 74% yield. Compound 2 is coupled with Boc-L-Leu-OH using EDC, and DMAP in DMF for 14 h to give compound 3 in 75% yield. The Boc group is removed in the presence of TFA in DCM to afford compound 4 in 84% yield. Compound 4 is coupled with 4-methoxy-1H-indole-2-carboxylic acid using EDC, and DMAP in DMF to give compound 5 in 48% yield. The Methyl ester of compound 5 is converted to the corresponding amide using ammonia in methanol to give compound 6 in 76% yield. The conversion of amide to the nitrile using Burgess reagent provides the final compound 101 in 25% yield.

Scheme 1.

Synthesis of compound 101.

5. KEY STRUCTURES

This series of compounds has three moieties as shown in Fig. (2). Indole is attached to a leucine via an amide bond followed by 2-oxopyrrolidine-3-yl nitrile with another amide bond.

Fig. (2).

Chemical structure of compound 101.

6. KEY COMPOUNDS

The chemical structures of the most potent compounds are shown in Fig. (3).

Fig. (3).

Selected potent compounds against COVID-19 and human coronavirus.

7. BIOLOGICAL ASSAY

7.1. Evaluation of Antiviral Activity of Compounds against COVID-19 (nCoV-2019, SARS-CoV2) Mpro in the Enzymatic Assay

Compounds were assayed using standard methods to determine compound activity and IC₅₀ values. This patent provided a detailed experimental procedure.

Evaluation of antiviral activity of compounds against human Coronavirus (HCov) 229E and OC43 in the Cytopathic Effect (CPE) assays, for detailed experimental procedures, please read the patent.

7.2. Biological Data

Key compounds’ inhibition data are summarized in Table 1.

Table 1.

Inhibition data for key compounds against COVID-19 and human coronavirus.

Compound Number	229E CPE EC50 (μM)	HCoV 229E, Mpro IC50 (µM)	SARS-CoV-2, (COVID-19) Mpro IC50 (µM)
101	D	D	D
145	Not tested	D	D
205	D	D	D
305	Not tested	D	D
361	D	D	D
377	D	D	D
491	D	D	D
515	Not tested	D	D
551	C	D	D
625	Not tested	D	D
737	D	D	D

A > 30 μM; B > 10 μM and ≤ 30 μM; C ≥ 2 μM and ≤ 10 μM; D < 2 μM.

CONCLUSION

This application provides several potent small molecules inhibiting COVID-19 and human coronavirus. Currently, Paxlovid and Molnupiravir are used to treat coronavirus infection. Both drugs have some adverse reactions and some patients get rebound effects. There are no complete cure medicines to tackle any pandemic. There is an urgent need for a potent drug with fewer adverse reactions. These small molecules have high drug-like properties with a good inhibition profile. We anticipate these compounds could be the potential benefit for the treatment of COVID-19, human coronavirus, and other viruses. The key compounds have a selectivity index (CC₅₀/EC₅₀) >50 times.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The author declares no conflict of interest, financial or otherwise.