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8
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Chemistry and Pharmacology of Drug Discovery
Figure 3. The structure of coronavirus’s 3-CL protease, drawn from PDB 6UL7
Unlike the conventional Ser(Cys)–His–Asp(Glu) triad found in other
pro
chymotrypsin-like enzymes, 3-CL
of SARS-CoV-2 has a catalytic dyad formed by
His41 and Cys145 that catalyzes the hydrolysis of the peptide bond at highly specific
sites of a polypeptide chain through a common nucleophilic-type reaction. It was
suggested that a water molecule might complete the catalytic triad by mediating crucial
interactions between His41 and other important conserved residues, such as His164 and
Asp187.
8
A nomenclature carton is shown below to better orient us with regard to the
binding pockets of protease and substituents of endogenous ligands or protease inhibitors
(Figure
the first binding pocket on the left of the active site as S
4). In essence, a protease normally has an active catalytic site. It was defined that
and the second on the left as S
1
and so on. The first binding pocket on the right of the catalytic metal is defined as S1′ and
the second one on the right as S
chain, the fragment occupying the S
peptide fragment that is occupying the S
known as the Schechter–Berger nomenclature,
′. For the endogenous ligand in the form as a peptide
2
pocket is defined as P1 region. Meanwhile, the
1
′ pocket is known as the P1′ region. This is
1
9
which will be used throughout this
chapter to illustrate drugs’ binding and structure–activity relationship (SAR), etc.
2
Figure 4. Schechter–Berger nomenclature for protease and its substrate-binding subsites
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Chapter 1. Nirmatrelvir (Paxlovid)
2.3. The Mechanism of Action of Nirmatrelvir
Nirmatrelvir (1) is a reversible covalent inhibitor of coronavirus’s 3CL
pro
prolonged enzyme inhibition. The recovery of >50% 3CL
1 indicates that inhibition of SARS-CoV-2 3CL
pro
is reversible.
activity after incubation with
pro
, eliciting
Figure 5. Reversible covalent bond between nirmatrelvir (1) and 3CL
pro
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The substrates for 3CL
including the omnipresence of a glutamine (Glu) residue at P
Chemistry and Pharmacology of Drug Discovery
pro
, pp1a and pp1b, share several common features
. No known human cysteine
1
protease cleaves after Glu, thus offering potential selectivity for this viral target over the
human proteome. Once nirmatrelvir (1) properly binds to the 3CL
pro
, cysteine-145 of the
enzyme attacks the nitrile of the inhibitor in a fashion similar to the Pinner reaction to
form a reversible S–C covalent bond on thioimidate 10 (Figure
Once 3CL
pro
is inhibited, the protease is then unable to cleave the polyproteins.
5).
10
As a consequence, the cell fails to produce various shorter, nonstructural proteins vital to
viral replication.
3. Structure–Activity Relationship (SAR)
The 3-CL
pro
sequence between SARS-CoV-1 and SARS-CoV-2 is highly conserved. In
fact, they are 100% identical in the catalytic domain that carries out polyprotein cleavage.
Consequently, some previously discovered compounds developed over 15 years ago to
treat SARS-CoV-1, such as PF-00835231 (3), showed high in vitro potency against
2,10
SARS-CoV-2 as well.
PF-00835231 (3) was a good starting point for lead optimization. But at first, let
us examine the origin of the three key fragments on nirmatrelvir (1), namely, the
pyrrolidone substituent at P
nitrile warhead at P
′.
1
, the rigid bicyclic dimethylcyclopropylproline at P2 and the
1
3.1. The Pyrrolidone Substituent at P1
The pyrrolidone substituent in the P1 pocket has been ubiquitous in many 3-CL
inhibitors. Why?
Wang and coworkers provided an excellent pedagogical explanation.
pro
Coronavirus’s 3-CL
substrate. In theory, glutamine would be an ideal choice of the P
invariably cleaves the Glu residue at the P1 position of the
substituent of the
1
protease inhibitors. But glutamine’s side chain amide can react with the warhead to form
the cyclized product as a piperidinone thus not a good choice after all.
11
In contrast, the cyclic pyrrolidone provides rigidity over the higher flexibility of
the glutamine side chain, likely providing a reduced loss of conformational entropy upon
binding to the target.
11
pro
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Chapter 1. Nirmatrelvir (Paxlovid)
Indeed, Pfizer employed the constrained lactam at the P1 site as the isostere of
the highly conserved P
on PF-00835231 (3) is well positioned in the S
glutamine present in all SARS CoV substrates. The pyrrolidone
1
pocket, making a favorable hydrogen
1
bond to His163. The NH of the lactam is within the hydrogen bond distance to Glu166
and the backbone oxygen of Phe140 in the 3CL
pro.2,10
3.2. Dimethylcyclopropylproline at P2
In order to have good oral bioavailability, a drug has to penetrate into the cell through the
cell membrane at first. That requires balanced physiochemical properties of the drug that
allow good permeability.
PF-00835231 (3) has five hydrogen bond donors, which make the drug too polar
to have good passive absorptive permeability for the cell membrane. The obvious tactic
would be removal of one or more hydrogen bond donors—the trick is to maintain the
peptidomimetics’ antiviral activity at the same time. Pfizer eliminated one hydrogen bond
donor by switching the warhead from α-hydroxymethyl ketone to benzothiazol-2-yl
ketone. Concurrently, they used a fused cyclopropyl bicycle to replace PF-00835231
(3)’s leucine portion at the middle of the molecule, the P
region, to remove another
2
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Chemistry and Pharmacology of Drug Discovery
hydrogen bond donor. The resultant benzothiazol-2-yl ketone 11, now only has three
hydrogen bond donors.
Regrettably, even though benzothiazol-2-yl ketone 11 saw a 49-fold increase in
permeability over PF-00835231 (3). But its binding was significantly lower than the
prototype because 11 lost an important hydrogen bond interaction with glutamine-189
that was present on 3. The lost potency was made up later on the left side P
in the form
3
of trifluoroacetamide (vide infra).
The rigid bicyclic ring was actually an old trick. In the past, dimethylcyclopropylproline (the 6,6-dimethyl-3-azabicyclo[3.1.0]-hexane) substituent was employed in
two marketed HCV NS3/4A serine protease inhibitors, e.g., boceprevir (8) and
narlaprevir (9). Last year, Wang
inhibited SARS-CoV-2’s 3CL
inhibitor GC-376 (12), which is an investigational veterinary drug developed for feline
infectious peritonitis caused by a coronavirus. It was designed to target the viral 3CL
protease and had potent antiviral activity against multiple viruses including MERS, feline
infectious peritonitis coronavirus, and norovirus.
12
and others13 discovered that boceprevir (8) also
pro
as did an older broad-spectrum viral 3CL protease
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Chapter 1. Nirmatrelvir (Paxlovid)
3.3. The Nitrile Warhead at P1′
There are numerous reports of reversible cysteine protease inhibitors. Their warheads
include aldehydes, thio- or oxymethyl ketones [e.g., PF-00835231 (3)], cyclic ketones,
amidomethyl ketones, nitriles, or various 1,2-dicarbonyl motifs. The electrophilic carbon
of these chemotypes reacts reversibly with the sulfur atom of an active-site cysteine,
forming a covalently bound tetrahedral complex.
Rather than keeping the benzothiazol-2-yl ketone as the warhead on 11, Pfizer
chose nitrile instead as the warhead at the end for nirmatrelvir (1). Nitrile has several
advantages. It is easier to make. It is smaller thus might have better physiochemical
properties. Indeed, the nitrile was more soluble than the benzothiazol-2-yl ketone in this
series of compounds.
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Historically, nitrile warhead has been present in several successful drugs on the
market. DPP-4 inhibitors alone boast four nitrile-containing drugs including vildagliptin
(7), saxagliptin (Onglyza), alogliptin (Nesina), and anagliptin (Suiny). Take vildagliptin
(7) as an example. A reversible Pinner reaction between its nitrile warhead and the
serine-630 in the S
thioimidate 13 with the aid of tyrosine-547 by protonation of the nitrile.
Chemistry and Pharmacology of Drug Discovery
pocket of the DPP-4 enzyme forms a time-dependent covalent adduct
1
14
3.4. Lead Optimization
Switching the α-hydroxymethyl ketone warhead on PF-00835231 (3) to nitrile 14
resulted in a boost of rat oral bioavailability with a reasonable intrinsic clearance and
stability in human liver microsomes (HLMs).
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Chapter 1. Nirmatrelvir (Paxlovid)
But nitrile 14 suffered lower affinity toward 3CL
pro
and inferior antiviral
efficacy in comparison to 3. As mentioned before, benzothiazol-2-yl ketone 11 saw 49fold increase in permeability over 3. But the former was significantly less potent than the
latter because benzothiazol-2-yl ketone 11 could no longer form a productive hydrogen
bond with glutamine-189 even though its rigid bicycle at P
lipophilic S
pocket. Compound 11 also suffered from high clearance in HLMs (CL
2
337 μL/min/mg), making it not suitable for further investment.
could effectively occupy the
2
10
int
=
In order to regain the interaction with glutamine-189, Branched, acyclic P
groups were introduced in place of indole. The (S)-val-sulfonamide in compound 15
extends underneath Gln-189, productively engaging P
pocket residues and achieves
3
improved hydrogen-bonding interactions with the Glu-166 backbone. Sulfonamide 15
pro
regained back much affinity to 3CL
and VeroE6 cell antiviral activity. As an added
benefit, it was also much more stable in HLMs in comparison to its precursor 11. Further
optimization of the P
-capping group led to trifluoroacetamide 16, which saw
3
significantly improved antiviral efficacy although the biochemical assay showed similar
potency to sulfonamide 15.
3
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Chemistry and Pharmacology of Drug Discovery
More excitingly, trifluoroacetamide 16 had increased metabolic stability in
HLMs and greatly improved oral pharmacokinetics in both rats and monkeys. The
trifluoroacetamide stood out in its ability to permeate the gut barrier in assays in
comparison to other bioisosteres. Even through there was some uncertainty with regard to
its stability, trifluoroacetamide here proved to be robust. Finally, introduction of the P
nitrile and replacing the (S)-valine group with the (S)-tert-leucine group at the P
substituent to this scaffold led to the identification of the clinical candidate nirmatrelvir
10
(1).
The rest, like they say, is history.
′
1
3
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Chapter 1. Nirmatrelvir (Paxlovid)
4. Pharmacokinetics and Drug Metabolism
4.1. Pharmacokinetics of Nirmatrelvir
Although nirmatrelvir (1) had moderate oral bioavailability in rats (10 mpk, F = 50%). It
exhibited poor oral bioavailability in monkeys (10 mpk, F = 8.5%) due to first-pass
metabolism along the gastrointestinal tract by cytochrome P450 (CYP) enzymes. This is
consistent with a rapid (t
= 20.5 min, CL
1/2
metabolic turnover of nirmatrelvir (1) in monkey intestinal microsomes. Nirmatrelvir (1)
exhibited moderate plasma clearance (CL
lives (t
) of 5 h and <1 h, respectively, after intravenous dosing.10
1/2
4.2. Metabolism of Nirmatrelvir
Nirmatrelvir (1) demonstrated moderate clearance, which was significantly inhibited
(≥82%) by the selective CYP3A4/5 inhibitor ketoconazole. Moreover, the oxidative
metabolic profile of nirmatrelvir (1) in HLMs, which includes modifications on the P
6,6-dimethyl-3-azabicyclo[3.1.0]hexane (e.g., 17), the tert-butyl group at the P3 position
as represented by 18 and the P
incubations of nirmatrelvir (1) with recombinant human CYP3A4.
pyrrolidinone ring (19, as an example), was reproduced in
1
= 33.8 mL/min/mg), NADPH-dependent
int
) in rats and monkeys, with elimination half-
p
10
2
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