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Chemistry and Pharmacology of Drug Discovery
2.2. First-Generation HIV Integrase Inhibitors
Even though inhibitors for HIV reverse transcriptase and protease were discovered soon
after the discovery of the virus, it took more than 20 years for the first HIV integrase
inhibitor to appear on the market. One of the challenges was that the HIV integrase
protein has shallow and solvent-exposed binding surface. Early lead structures were
frequently based on catechols, hydrazides, or coumarins, all of which failed to show
antiviral activity in cell culture by a mechanism that could be reliably attributed to
inhibition of virus genome integration. Merck’s raltegravir (3) was approved for
marketing in 2007 as the first HIV integrase inhibitor, a culmination of considerable
effort that was based on clearly defining the biochemical staging of enzyme function. The
second integrase inhibitor Japan Tabacco/Gilead’s elvitegravir (4, Vitekta) was approved
in 2012.
The strand transfer step is the key enzymatic process susceptible to inhibition
rather than assembly of the enzyme on viral substrate or the 3′-cleavage reaction. This
mechanistic insight afforded a more effective screening assay. In 1999, using such an
assay, Merck and a Japanese company Shionogi independently discovered diketoacid
(DKA) derivatives (e.g., 5) as the first specific inhibitors of HIV integrase that
demonstrated antiviral activity in cell culture. These compounds bound to a complex of
HIV and the viral DNA substrate with the DKA moiety. As a phosphate isostere, DKA
binds to the two magnesium divalent ions involved in catalysis, forming a ternary
complex that interferes with the binding to host cell double-stranded DNA. By replacing
the carboxylic acid with a tetrazole bioisostere, Shionogi was able to obtain the first
inhibitor co-crystalized integrase. That was a great contribution to the field even though
the tetrazole analog never became a drug due to stability issues.
7–9
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Chapter 3. Cabotegravir (Vocabria)
Merck Research Laboratories in Rome, Italy, succeeded in finding their
integrase inhibitors by “inter-breeding” two drug discovery programs. In parallel to their
HIV integrase program, Merck Rome also had a hepatitis C virus (HCV) inhibitors
program on-going at the same time. A class of inhibitors of HCV NS5B RNA-dependent
RNA polymerase (RdRp) had the dihydroxypyrimidine pharmacophore with strong
metal-binding capacity even though the compounds per se did not inhibit HIV
integration. Realizing that both HCV NS5B polymerase and HIV integrase rely on
binding to the magnesium ion for their catalytic activity, Merck medicinal chemists used
dihydroxypyrimidine 6 as the bioisostere of the more stable replacement of the DKA
pharmacophore. Simple installation of a hydrophobic benzylamine gave rise to 7 as a
very potent drug in an integrase strand transfer assay. Another major structural core
change was methylation of one of the two nitrogen atom on the pyrimidine ring to
convert the pharmacophore to hydroxypyrimidinone 8. Incremental modifications to
improve physiochemical properties while maintaining cell penetration and limiting
protein binding delivered a drug with an exceptional potency. That became raltegravir
8
(3), which has been marketed as Isentress since 2008.
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Japan Tobacco discovered their HIV integrase inhibitor elvitegravir (4), also
from “inter-breeding” of two drug discovery programs. Their monoketo acid
pharmacophore (10, for instance) was derived from a quinolone antibiotic scaffold
originally designed for bacterial DNA gyrase activity. Even at the very beginning, they
already found that simple 4-quinolone-3-carboxylic acid 10, but not the more
complicated 4-quinolone-3-glyoxlic acid 9, had decent HIV integrase inhibitory activity.
The mono-keto-acid moiety on 10 is a weaker chelator to the magnesium ions, therefore,
10 is probably more selective than diketo-acid 9 because HIV integrase belongs to a large
family of DNA processing enzymes, which contain the same arrangement of three
catalytically essential carboxylates. Unlike raltegravir (3), para-substitutions on the lefthand phenyl ring actually killed the activity to inhibit integrase for this series of
compounds. Luckily, ortho- and meta-substitutions were fruitful, giving rise to decorated
derivative 11, which possessed both integrase inhibitory activity and antiviral activity. It
turned out that the NH on the quinolone core was not essential and alkylation of 11
provided 1-hydroxyethyl analog 12, which gained more than 10-fold of efficacy.
Installation of an isopropyl group offered another 10-fold boost of efficacy. Finally, an
additional 7-methoxyl substitution on the quinolone core led to elvitegravir (4), which
showed significant improvement of inhibition of strand transfer and antiviral activity.
After the FDA approval in 2012, Japan Tobacco and Gilead co-marketed it with a trade
Vitekta.9
name
Chemistry and Pharmacology of Drug Discovery
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Chapter 3. Cabotegravir (Vocabria)
2.3. Second-Generation HIV Integrase Inhibitors
The first-generation INSTIs suffer from cross-resistance between each other, i.e.,
raltegravir (3) and elvitegravir (4). Both of them are also associated with some adverse
side effects.
Second-generation integrase inhibitors doletegravir (14) and bictegravir (15) are
superior to the first-generation integrase inhibitors. They include good tolerability, oncedaily dosing with no need for a pharmaco-enhancer and relatively little cross-resistance
that plagued the first-generation integrase inhibitors.
Shionogi and GSK scientists embarked on their quest of the second-generation
integrase inhibitors starting with monocyclic carbamoyl pyridone 16. Their intentional
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Chemistry and Pharmacology of Drug Discovery
use of a triad of all oxygen-derived lone pairs to serve as the chelating donor atoms
toward the two divalent metals.
Although monocyclic 16’s enzymatic and antiviral activities are superior to
raltegravir (3) and elvitegravir (4), its antiviral efficacy against resistant mutants
(particularly Q148K) decreased. Nevertheless, bicyclic piperazinone 17 fixed the
problem.
If two rings are good, three rings must be better. Indeed, although racemic
tricyclic hemiaminal 18 did not elevate the antiviral potency but positively modified the
rat PK profile. Between the enantiomers, 18S had a 43-fold loss of potency with added
human serum albumin while the R isomer had a very modest 4-fold loss. An additional Smethyl substituent gave rise to dolutegravir (14) that was approved in 2013 with trade
name Trivicay.
10–13
Five years later in 2018, Gilead′s me-too drug bictegravir (15), which was a very
close cousin of dolutegravir (14), was approved. Gilead sold it as a combination drug
with
emtricitabine and tenofovir alafenamide with trade name Biktarvy, now a constant
feature of commercials on TV every day and everywhere.
Cabotegravir (1) is a new long-acting parenteral and a highly effective integrase
inhibitor with a half-life of 54 days, allowing parenteral administration every other
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month. It features low water solubility, high activity, long half-life, and slow metabolic
clearance.
Figure 4. A typical integrase strand transfer inhibitor binding to the catalytic triad of HIV
Chapter 3. Cabotegravir (Vocabria)
integrase
A pattern has emerged in the field of integrase inhibitors. As shown in Figure
a successful competitive (orthosteric) HIV INSTi almost always contains a chelation
warhead to bind to the two metal ions. The warhead is then connected to a hydrophobic
region connected by a flexible linker. The second-generation INSTIs have an additional
region for optimization of pharmacokinetics and drug-like properties required for oral
bioavailability.
13
4,
2.4. Integrase–LEDGF/p75 Allosteric Inhibitors
The five INSTIs on the market are orthosteric inhibitors. They are catalytic site inhibitors
that specifically target the strand transfer step necessary for viral DNA insertion into the
host chromatin. Due to ubiquitous drug resistance, there is always a concern using the
drugs with the same MoA. Therefore, allosteric HIV-1 integrase inhibitors (ALLINIs)
have garnered special interest because of their novel MoA.
In the life cycle of an HIV, after reverse transcription, the PIC binds to the host
(cellular cofactor) lens epithelium-derived growth factor (LEDGF)/p75 protein in the
nucleus. As shown in Figure 5, LEDGF is characterized by a conserved N-terminal
PWWP domain. Its signature Pro–Trp–Trp–Pro motif is a chromatin reader and is able to
bind simultaneously and synergistically to DNA and methyl lysines present on histone
tails.
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Chemistry and Pharmacology of Drug Discovery
Figure 5. Allosteric HIV-1 integrase inhibitors targeting LEDGF/p75
The region following the PWWP is presumed to be a nuclear localization signal
(NLS) motif, two AT-hooks (a minor-groove DNA-binding motif consisting of a Pro–
Arg–Gly–Arg–Pro core that preferentially binds AT-rich sequences). LEDGF is
connected by a supercoiled DNA recognition domain, which is followed by an integrasebinding domain (IBD), a compact right-handed bundle of five α-helices. LEDGF/p75 is
the most studied cellular cofactor that is essential for tethering the integrase PIC to host
chromatin and also for the recruitment of other cellular factors to the PIC, thereby
14
facilitating effective integration.
Most of the effective integrase-LEDGF/p75 inhibitors contain a hydrophobic
moiety and a carboxylic acid functionality in common, which mimic the hot spot residues
Ile365 and Asp366 within the interface of LEDGF/p75 IBD. The H-bonding interactions
between the carboxylic acid and the backbone amide protons of residues E170 and H171
of integrase was critical to antiviral potency and that there was no tolerated isosteric
replacement for the acid. ALLINIs elicit antiviral activity by binding to the highly
conserved allosteric pocket on the integrase catalytic core that also serves as the binding
pocket for LEDGF. By targeting the protein–protein interactions (PPIs) between HIV
integrase and LEDGF/p75, ALLINIs induce aberrant integrase multimerization, leading
to the production of replication-deficient viral particles.
Around 2014, Boehringer Ingelheim was the first to bring an ALLINI, BI
15
224436 (19), to clinical trial, which was terminated during phase I.
GSK described
GSK1264 (20) that disrupts the interaction between HIV-1 integrase and the cellular
factor LEDGF)/p75. Interestingly, GSK1264 (20) was only modestly potent to inhibit the
early infection steps and had little effect on integration targeting, which is guided by the
16
integrase–LEDGF/p75 interaction.
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BMS carried out a drug discovery program on integrase–LEDGF/p75 allosteric
inhibitors. The carried out their optimization with a specific emphasis on the inhibition of
the 124/125 polymorphs such that the designed compounds showed excellent potency in
vitro against majority of the 124/125 variants. BMS-986180 (21, GSK3739936) emerged
as a promising preclinical lead with a good PK profile. Regrettably, findings in rat
toxicology studies precluded further development of 21 in humans.
Chapter 3. Cabotegravir (Vocabria)
17
Though the PPI inhibitors are less potent than the active site-directed enzyme
inhibitors, allosteric inhibitors can provide new MoA to address the drug-resistance issue.
3. Structure–Activity Relationship (SAR)
In the early 2000s, Shionogi and GSK carried out extensive structure–activity
relationship (SAR) investigations of carbamoyl pyridones as HIV-1 integrase inhibitors.
Dolutegravir (14) was the fruit of their labor. Even though dolutegravir (14) was a very
good drug, efforts were made to explore to replace its six-membered 1,3-oxazinane ring
with a five-membered oxazolidine ring as represented by
cabotegravir (1).12
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Chemistry and Pharmacology of Drug Discovery
The SAR on the effects of tricyclic carbamoyl pyridones around structure 22 is
summarized in Table 1. Since 3,5-difluorobenzyl motif was invariably superior to the 3-
monofluorobenzyl counterpart, only the difluorobenzyl derivatives are compiled here for
clarity.
Table 1. Effects of tricyclic carbamoyl pyridones
Compound Structure
1
23 5.4 39 35
24 0.9 63 ND
25 8.5 35 63
26 330 3100 >15
pHIV
IC
50
(nM)
pHIV
PAIC
(nM) Q148K (FC)
50
0.3 30 3.9
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Chapter 3. Cabotegravir (Vocabria)
27 10 37 12
28 3.1 18 ND
29 430 370 >10
30 72 620 44
31 13 380 13
32 0.6 4.5 19
The potency (IC50) is measured using a pseudo-typed virus assay (pHIV).
Protein-adjusted potency (PAIC
) is also listed to reflect serum protein-binding shift.
50
Lastly, fold change (FC) is measured for the most important mutation, the Q148K
mutant.
As shown in Table
1, cabotegravir (1) with the S-configuration is significantly
more potent than its enantiomer 23 with the R-configuration. Similarly, the S-ethyl analog
24 is more potent than the corresponding the R-ethyl analog 25 as well. Once the Rsubstitution becomes larger, the potency falls as demonstrated by cyclohexylmethyl
derivative 26. Alcohol 27 also lost substantial potency (20×) compared to the methyl
analogue 1. However, thioether 28 retained low nM potency with less loss of potency
compared to 1, it made up for this loss with an improved protein adjusted value of
. The sulfone 29 showed very poor activity with or without added proteins in the
PAIC
50
pHIV assay system. The rigid 3-phenyl derivative 30 was 143× less potent than the
corresponding methyl analogue 1. With an extra methylene group, the benzyl derivative
31 was somewhat more potent than 30. Very surprisingly, even though S-derivatives were
more potent than the R-derivatives when the substituents were simple methyl, ethyl, and
propyl, the R-benzyl derivative 31 was significantly more potent than the corresponding
S-benzyl derivative 33.
The Q148K mutant data for the five-membered series consistently showed a
sharp decline in potency against the mutant as the substituent present in the fivemembered saturated ring increased in size beyond a methyl group. This was different
from the six-membered series, which might explain why six-membered dolutegravir (14)
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