An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

TLDR: The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome (SARS-CoV-2) has become a global pandemic as discussed by the authors.

Abstract: The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral ther...

Figures

Figure 3. In vivo efficacy of PF-07321332 against SARS-CoV-2 MA10 infection in mice.

Table 1. In vitro and in vivo parameters optimized in identifying oral SARS-CoV-2 Mpro

Figure 4. Preclinical toxicology and healthy adult participant single ascending dose study

Figure 2: PF-07321332 biochemical and antiviral activity. (A) PF-07321332 is a reversible

Figure 1. SARS-CoV-2 Mpro structural biology. (A) Key H-bond interactions of PF-00835231

Full text

1
Title: An Oral SARS-CoV-2 M
pro
Inhibitor Clinical Candidate for the
Treatment of COVID-19
One-Sentence Summary: PF-07321332 is disclosed as a novel, orally active, investigational
small-molecule inhibitor of the SARS-CoV-2 main protease, which is being evaluated in clinical
trials for the treatment of COVID-19.
Authors: Dafydd R. Owen
1
*, Charlotte M. N. Allerton
1
, Annaliesa S. Anderson
2
, Lisa
Aschenbrenner
3
, Melissa Avery
3
, Simon Berritt
3
, Britton Boras
4
, Rhonda D. Cardin
2
, Anthony
Carlo
3
, Karen J. Coffman
3
, Alyssa Dantonio
3
, Li Di
3
, Heather Eng
3
, RoseAnn Ferre
4
, Ketan S.
Gajiwala
4
, Scott A. Gibson
5
, Samantha E. Greasley
4
, Brett L. Hurst
5
, Eugene P. Kadar
3
, Amit S.
Kalgutkar
1
, Jack C. Lee
3
, Jisun Lee
3
, Wei Liu
4
, Stephen W. Mason
2
†, Stephen Noell
3
, Jonathan
J. Novak
3
‡, R. Scott Obach
3
, Kevin Ogilvie
3
, Nandini C. Patel
1
, Martin Pettersson
1
§, Devendra
K. Rai
2
, Matthew R. Reese
3
, Matthew F. Sammons
1
, Jean G. Sathish
2
, Ravi Shankar P. Singh
1
,
Claire M. Steppan
3
, Al E. Stewart
4
, Jamison B. Tuttle
1
, Lawrence Updyke
1
, Patrick R. Verhoest
1
,
Liuqing Wei
3
, Qingyi Yang
1
, Yuao Zhu
2
Affiliations:
1
Pfizer Worldwide Research, Development & Medical; Cambridge, MA 02139, USA.
2
Pfizer Worldwide Research, Development & Medical; Pearl River, NY 10965, USA.
3
Pfizer Worldwide Research, Development & Medical; Groton, CT 06340, USA.
4
Pfizer Worldwide Research, Development & Medical; La Jolla, CA 92121, USA.
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted July 31, 2021. ; https://doi.org/10.1101/2021.07.28.21261232doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

2
5
Institute for Antiviral Research, Department of Animal, Dairy, and Veterinary Sciences,
Utah State University; Logan, UT 84322, USA.
*Corresponding author. Email: Dafydd.owen@pfizer.com
† Present address: Janssen Biopharma; South San Francisco, CA 94080.
‡ Present address: Praxis Precision Medicines; Cambridge, MA 02142.
§ Present address: GRT Therapeutics; Cambridge, MA 02142.
Abstract: The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global
pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare
response to counter the ongoing threat presented by COVID-19. Here, we report the discovery
and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease
inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target
selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-
adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro
antiviral cell potency, in a phase I clinical trial in healthy human participants.
ClinicalTrials.gov Identifier: NCT04756531
All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprintthis version posted July 31, 2021. ; https://doi.org/10.1101/2021.07.28.21261232doi: medRxiv preprint

3
Main Text:
Human coronavirus infections are common, with at least four examples (229E, NL63, OC43,
HKU1) now considered endemic (1). However, the emergence within the last 20 years of SARS-
CoV-1, Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-2 as novel human
coronaviruses has signaled the significant threat potential of this viral class. The catastrophic
SARS-CoV-2 outbreak of 2019 has resulted in 185 million confirmed cases of COVID-19
causing over 4 million deaths globally as of July 2021. SARS-CoV-2 is a highly infectious,
ribonucleic acid (RNA) beta coronavirus that can cause life-threatening viral pneumonia in the
most serious cases. While effective COVID-19 vaccines have been developed within
unprecedented timelines, a significant number of people are either unable, due to pre-existing
medical conditions, or unwilling to be vaccinated, such that global access challenges remain.
Limited therapeutic options are available to those who are infected. Oral SARS-CoV-2 specific
therapeutics that are applicable for treatment of the broad population upon COVID-19 diagnosis
are urgently needed. Such a treatment approach may prevent more severe disease, hospitalization
and death. Indirectly, it may also reduce further transmission from infected individuals.
Repurposing of approved drugs in the search for small molecule antiviral agents that target
SARS-CoV-2 has thus far been minimally effective (2, 3), however viral RNA-dependent RNA
polymerase inhibitors such as molnupiravir and AT-527 are currently undergoing clinical trials
for the treatment of COVID-19 in patients (4, 5).
The SARS-CoV-2 genome encodes two polyproteins (pp1a and pp1ab) and four structural
proteins (6, 7). These polyproteins are cleaved by the critical SARS-CoV-2 main protease (M
pro
,
also referred to as 3CL protease) at eleven different sites to yield shorter, non-structural proteins
vital to viral replication (8, 9). The coronavirus M
pro
is a three-domain cysteine protease, which
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4
features a Cys145-His41 catalytic dyad located in the cleft between domains I and II. Several
common features are shared among M
pro
substrates, including the presence of a P1 Gln residue.
No known human equivalent cysteine protease exploits a P1 Gln as the cleavage site prompt,
thus offering an intriguing selectivity hypothesis for this viral target over the human proteome
(10-12). In addition, as the SARS-CoV-2 M
pro
and the viral spike protein are distinct protein
entities within the viral proteome, the antiviral efficacy of a small-molecule M
pro
inhibitor is not
expected to be affected by spike protein variants. Viral proteases have also proved to be tractable
targets for small molecule oral therapies in the treatment of human immunodeficiency (HIV) and
hepatitis C (HCV) viruses (13, 14). Moreover, a recent report has also demonstrated oral activity
of an M
pro
inhibitor in a transgenic mouse model of SARS-CoV-2 infection (15). Given the
pivotal role of SARS-CoV-2 M
pro
in viral replication, its potential for mechanistic safety, and
expected lack of spike protein variant resistance challenges, SARS-CoV-2 M
pro
inhibition
represents an attractive small molecule approach for an oral antiviral therapy to treat COVID-19.
As part of the response to the 2002 SARS outbreak, an effort was made to identify inhibitors of
the SARS-CoV-1 M
pro
, which led to the identification of PF-00835231 (1, Table 1) as a potent
inhibitor of recombinant SARS-CoV-1 M
pro
in a fluorescence resonance energy transfer (FRET)-
based cleavage assay (16). PF-00835231 also demonstrated potent inhibition (inhibition constant
(K
i
) = 0.271 nM) of recombinant SARS-CoV-2 M
pro
, as the SARS-CoV-1 and -CoV-2 M
pro
share 100% sequence homology across their respective substrate binding sites (17). Antiviral
activity against SARS-CoV-2 was also observed with PF-00835231 (half-maximal effective
concentration (EC
50
) of 231 nM) in epithelial Vero E6 cells, when co-dosed in the presence of P-
glycoprotein efflux inhibitor CP-100356 (18). The phosphate prodrug form (PF-07304814) of
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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5
PF-00835231 is currently under investigation as an intravenous treatment option for COVID-19
in hospitalized patients (18).
During our oral SARS-CoV-2 M
pro
inhibitor program, two functional groups precedented as
covalent warheads for cysteine proteases were pursued in parallel: nitriles (19, 20) and
benzothiazol-2-yl ketones (21, 22).
These served to replace the hydrogen bond donor (HBD) of
the P1’ α-hydroxymethyl ketone moiety in 1 (Fig. 1A) in order to improve upon the low passive
absorptive permeability (P
app
< 0.207 x 10
-6
cm/s) (23) and poor oral absorption of 1 in animals
(Table 1 and Table S1). Nitrile 2 demonstrated a significant increase in rat oral absorption
(bioavailability (F) = 7.6% and fraction of oral dose absorbed (F
a
x F
g
)
= 38%) (24), while
maintaining reasonable metabolic stability (intrinsic clearance (CL
int
)) towards oxidative
metabolism in human liver microsomes (HLM) (25) relative to 1 (Table 1). However, the in
vitro FRET M
pro
potency (K
i
= 27.7 nM) and SARS-CoV-2 antiviral activity (EC
50
= 1364 nM)
of 2 proved inferior in comparison to 1. Introduction of a 6,6-dimethyl-3-
azabicyclo[3.1.0]hexane as a cyclic leucine mimetic at P2 (Fig. 1B) further reduced HBD count.
When combined with a P1’ benzothiazol-2-yl ketone, the resulting analog 3 (Table 1) displayed
high passive absorptive permeability (P
app
= 10.3 x 10
-6
cm/s) The reduced biochemical SARS-
CoV-2 M
pro
inhibitory potency of 3 (K
i
= 230 nM) relative to other reported
benzothiazole-2-
yl SARS-CoV-2 M
pro
inhibitors (26) containing leucine P2 groups can be rationalized from the
binding mode observed for 3 (Fig. 1C). While the 6,6-dimethyl-3-azabicyclo[3.1.0]hexane
effectively fills the lipophilic S2 pocket formed by Met49, Met169, and His41, productive
hydrogen bonding to Gln189 from a ligand backbone is no longer possible. The inferior SARS-
CoV-2 M
pro
potency and the high CL
int
(337 ml/min/mg) precluded further investments in
compound 3. Similar to 1, the P3 indole of 3 does not protrude into the S3 pocket (Figs. 1A and
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Frequently asked questions

Q1. What are the contributions in this paper?

Here, the authors report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. ( which was not certified by peer review ) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Q2. How long did the plate remain incubated?

For agonist studies 15 µl of HBSS/HEPES containing compound was added to the cells and the plate was incubated at room temperature for 90 min. 

Q3. How many vehicle periods were applied to establish a stable baseline?

Three vehicle periods each lasting 5 minutes were applied to establish a stable baseline, each well followed by the addition of increasing concentrations of test compound, with each exposure lasting 5 minutes. 

Q4. What was the potential of PF-07321332 to induce chromosome damage?

The potential of PF-07321332 to induce chromosome damage was determined by the increased frequency of micronucleated reticulocytes in peripheral blood samples from vehicle- and PF07321332-treated rats from the 2-week GLP toxicity study. 

Q5. how many mmol of t18 was added to a solution of aque?

Methyl N-(triethylammoniosulfonyl)carbamate, inner salt (Burgess reagent; 69.3 g, 276 mmol) was added to a solution of T18 (61 g, 111 mmol) in dichloromethane (550 ml). 

Q6. What was the terminal rate constant of the log-linear concentration-time curve?

∞The terminal rate constant (kel) was calculated by linear regression of the terminal phase of the log-linear concentration-time curve and the terminal elimination t1/2 was calculated as:𝑡1 2= 0.693𝑘𝑒 

Q7. What dose was used in the PF-07321332/RTV co-administration paradigm?

In the PF-07321332/RTV co-administration dosing paradigm, each subject (active and placebo) received one tablet (100 mg) of RTV at -12 h, 0 h and 12 h. 

Q8. What was the dose of PF-07321332 given in the co-administration paradigm?

In the PF-07321332/RTV co-administration dosing paradigm, each subject (active and placebo)received one tablet (100 mg) of RTV at -12 h, 0 h and 12 h.