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Chemistry and Pharmacology of Drug Discovery
The Fast-Track Strategy of UNAIDS targets the end to AIDS by 2030, with the
objective of achieving 95–95–95: 95% of those living with HIV know their status, 95%
are aware of their treatment options, and 95% receive viral load suppression therapy. The
goal is set to decrease annual new adult HIV infections to 200,000 and to eradicate
3
discrimination.
However, the HIV epidemiological data of 2021 suggests that the goals set by
UNAIDS are still far to reach. Globally, an estimated 1.5 million people are infected with
the virus annually, including 160,000 children. And in each year, 650,000 individuals
(ranging from 510,000–860,000) die of AIDS and related complications. As of the end of
2021, approximately 38.4 million people worldwide are living with AIDS, with two-
1
thirds (25.6 million) residing in the African region.
The AIDS epidemic posts severe economic and social issues in Africa. Reports
state that certain areas in South Africa have an alarming prevalence rate of 35% among
all adults aged 15–59. Age and gender stratification reveals that females aged 35–39 have
a prevalence rate as high as 59%.
4
Although worldwide AIDS-related deaths and infections have decreased stately,
epidemiological trend in Eastern Europe and Asia, continue to worsen, increasing by 46%
5
and 49% since 2010
. Approximately daily infections and AIDS-related fatalities in these
regions are 440 and 130, respectively.
Clearly, unmet clinical needs persist in HIV therapy, prevention, and cure.
Ongoing research aims to enhance treatment efficacy by developing new drugs with
novel mechanisms, optimizing current regimens, and discovering methods to eliminate
latent HIV reservoirs. Such advances will offer treatment alternatives for patients who
have developed drug resistance or are intolerant to existing therapies and will reduce
transmission. Potentially a functional cure for HIV can be achieved.
Before 2020, HIV-1 treatment involved six main antiretroviral categories that
target different aspects of the HIV life cycle. Entry inhibitors prevent the binding of HIV
envelope glycoprotein gp120 to co-receptor CCR5 (1) or cell fusion mediated by HIV
gp41 (2). Nucleoside reverse transcriptase inhibitors (NRTIs) (3) and non-nucleoside
reverse transcriptase inhibitors (NNRTIs) (4) inhibit the conversion of viral RNA to
cDNA. Integrase Strand Transfer Inhibitors (INSTIs) (5) and protease inhibitors (PIs) (6)
are antiretroviral drugs that inhibit the integration of proviral DNA into the host genome
and the processing of gag and gag-pol precursors by protease, respectively.
Although antiretroviral therapy (ART), a combination of antiviral medications that
target various molecular targets, can partially relieve HIV-1 progression, it also results in
several deleterious side effects such as psychiatric symptoms, renal function and bone
density changes, elevated cardiovascular risk, and altered body weight.
6
7
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Chapter 5. Fostemsavir (Rukobia)
More important, the emergence of drug-resistant and multidrug-resistant strains
cause ART failure, and lead to a heightened risk of HIV disease progression and
mortality. According to the WHO report, 10% of adults with HIV who received treatment
8
are resistant to NNRTIs.
Additionally, patients who have undergone antiretroviral
therapy in the past are three times more likely to develop resistance to the NNRTI drug
class compared to those who are currently receiving treatment for the first time. The
prevalence of simultaneous resistance to six classes of antiviral medications can be as
high as 5–10% among different groups of antiretroviral-treated patients in North America
and Europe. Consequently, a significant number of these patients will be left without any
9
viable treatment options.
Efficient strategies for confronting HIV drug resistance require a global effort.
These tactics comprise routine viral load monitoring, genotyping for resistance detection,
changing regimens as necessary, and selecting the most potent antiretroviral drug
combination and improved adherence to achieve long-term viral suppressing success.
Correspondently, research priorities include:
(a) Improving the safety and efficacy of antiretroviral therapy;
(b) Enhancing medication adherence by reducing treatment frequency, simplifying
regimens, or utilizing long-acting medications to overcome adherence
challenges;
(c) Developing new medicines with innovative mechanisms of action; and
(d) Preventing HIV transmission, via microbicide or vaccine.
Given the current landscape of HIV-1 therapies, we focus on the salvage therapy
of the group comprising 6% of the patient population who have undergone five
unsuccessful rounds of antiretroviral therapy need immediate access to new treatments as
their options are exhausted.
To address this need, we discovered HIV-1 Adhesion Inhibitors (HIV AIs), a
novel class of mechanistic antiviral drugs for HIV-1, inhibit viral infection by interfering
10
with HIV’s cellular adhesion process.
Among them, fostemsavir (6), the prodrug of
temsavir (5) became the first of its kind to receive FDA approval in July 2020, is
TM
currently available under the trade name of Rukobia
TM
Rukobia
can be used in combination with drugs of distinct mechanisms. No
evidence of cross-resistance with these treatments has been found to date.
by ViiV/GSK.
11
2. Pharmacology
The HIV-1 attachment inhibitor (HIV AI) belongs to a distinct mechanistic class of entry
inhibitors for HIV-1. It specifically targets gp120, the membrane surface glycoprotein of
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Chemistry and Pharmacology of Drug Discovery
HIV-1, and prevents gp120’s interaction with CD4+ T cells, thereby halting the HIV-1
11
life cycle during the initial stage of cis-infection mode (Figure
1a).
Figure 1. HIV AI blocks gp120 of HIV-1 binding to CD4 receptor (a) and HIV AI blocks
gp120 of the infected CD4 T cell binding to CD4 receptor of non-infected T cell (b).
The second mode of infection is cell-to-cell fusion. During this process, an
infected CD4
protein on the infected CD4
+
CD4
T-cell form an immunological synapse, which allows their membranes to fuse
together and transfer the viral contents (Figure
1 infection by inhibiting gp120.
3. Structure–Activity Relationship (SAR)
In 1998, Bristol-Myers Squibb revolutionized its drug discovery process by randomly
testing its chemical inventory using high-throughput screening. One of the first projects
adopted a pseudo-type of cell-based assay to identify compounds that could block HIV-1
infection. After reviewing approximately 100,000 compounds within 6 months, several
hits emerged. Upon careful analysis and examination, a single hit, BMS-216 (Figure
1), appeared to have a unique mechanism of action. The compound displayed an EC
+
T-cell transmits the virus to a non-infected CD4+ T-cell. The gp120
+
T-cell membrane and the CD4 receptor on the noninfected
1b). HIV AI also blocks cell-to-cell HIV-
2,
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Chapter 5. Fostemsavir (Rukobia)
value of 153 nM, regardless of the HIV-envelope used, either the CCR5-dependent JRFL
strain or the CXCR4-dependent LAI strain. It was generally non-cytotoxic, with a CC
value of 339 μM in uninfected cells.12
Efforts were made to promptly establish the structure–activity relationship
(SAR) of the indole, piperazine, and benzoyl fractions. Initial results showed that a small
substitution on the 4-position of the indole significantly enhances the antiviral activity.
The lead compound, 4-F indole derivative BMS-705 (2), reduced the EC
10
However, due to its low water solubility and metabolic instability, 2 was deemed
nM.
value to 2.6
50
“impossible” to be formulated for oral dosing.
Theoretically, increasing polarity and reducing electronic potential could
improve aqueous solubility and metabolic stability. Replacement of a carbon atom with a
13
nitrogen atom of aromatic ring should suffice to achieve the goal.
Accordingly, 4-/5-/6-
/7-azaindole analogs of BMS-216-Me were synthesized, resulting in >26-fold
13
improvement in aqueous solubility and >2.3-fold enhancement of metabolic stability.
The further installation of a MeO- group at the 4-position of the 7-azadole led to
11,14
BMS-806 (3).
exposure. This was likely due to 3’s moderate metabolic stability (t
It progressed to Phase I clinical studies but failed to deliver the desired
47 min in HLM)
1/2
and permeability (Caco-2 Pc 51 nm/sec) measured in vitro.
Given lipophilicity’s positive impact on permeability, the polarity that is against
lipophilicity was reduced by moving the N atom from the 7- to the 6-position and
simultaneously adding a methoxy group at position 7. This operation hypothetically
should also level the electron potential to retain metabolic stability, compared with 3.
After removing the 2-methyl group from piperazine, BMS-043 (4) was created, which
provided t
Phase II clinical trials and demonstrated the proof-of-concept (POC).
of over 100 min in HLM and Caco-2 Pc 178 nm/sec.13 In 2004, 4 completed
1/2
15,16
However, only
approximately 68% of patients experienced a desired viral load drop, and 1.8 g of 4 was
required co-dosing with a high-fat meal. Further optimization of activity was necessary.
50
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The focus shifted to modifying position 7 of the 6-azaindole. It was discovered
that a sp2 hybridized atom extension at C-7 was critical for potency. Extensive research
was conducted on amides, aromatic rings, C-linked heteroaryl, and N-linked heteroaryl
substituents. Additionally, in a subseries with the same heteroaryl ring, the relative
strength of antiviral activity depended on the arrangement of atoms, which preferred
coplanar between the heteroarene and (aza)indole core. In detail, the optimal compound
in the subseries was the structure that held assembly on both flanking sides of the
heteroaryl, labeled as α and α′ in Figure
arrangement ensured that at the α-position of the C-7 heteroaryl, the heteroatom (O, S, or
N) and the NH of the pyrrole ring of the 6-azaindole could form an internal hydrogen
bond. Meanwhile, the heteroaryl’s α′-position contained a hydrogen that could form an H
or pseudo-H bond with the C-6 azaindole nitrogen, as shown in Figure
of all members of the furan (EC50: 7 0.02 nM, 8 0.11 nM) and pyrimidine (EC50: 9 0.11
nM, 10 0.51 nM, 11 1.28 nM) subseries were compared head-to-head, as illustrated in
3a and b, which clearly supported the assumption. Coplanarity was affected by
Figure
several factors. The strength of an internal H-bond was determined by the substitution on
the C-7 heteroaryl ring. The first, in general, electron-donating ring substitutions (such as
an amino group) increased the electron density of the α-heteroatom, resulting in a
stronger H-bond. Conversely, an electron-withdrawing functionality (such as an alkoxy
group at the alpha-position of the ring nitrogen atom)
the alpha-heteroatom, leading to a weaker H-bond. The greatest impact was expected
Chemistry and Pharmacology of Drug Discovery
Figure 2. Optimization from BMS-216 to fostemsavir
3a, which facilitated coplanarity. This structural
17
decreased the electron density of
3a. The activities
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Chapter 5. Fostemsavir (Rukobia)
from substitutions at the adjacent position to the α-heteroatom. Secondly, the bulkiness of
the substitution at the α- or α′-position of the C-7 heterocycle was also a significant
determinator, as steric repulsion (e.g., 8 and 10) was an obvious negative factor. The third
factor was the location of the in-ring heteroatom. A heteroatom locked in the α-position
experienced the long-pair repulsion from the nitrogen atom of 6-azaindole (e.g., 11),
causing the aromatic ring at C-7 to move out of the plane of the azaindole.
It was observed that the permeability was correlated with the coplanarity
between the heteroarene and azaindole core. For example, the Caco-2 Pc value favored
furan 7 (86 nm/sec) over 8 (46 nm/sec), and 6-pyrimidinyl 9 (378 nm/sec) over 2pyrimidinyl 11 (106 nm/sec) and 5-pyrimidinyl 10 (<15 and 46 nm/sec).
The liver microsome stability in six-membered ring extensions was discovered
to be orthogonal to the coplanarity. For instance, the HLM t
11, and 10 were 15, 32, and over 100 min, respectively. A plausible explanation would be
that the out-of-plane aromatic substitution had stronger electron-withdrawing effect,
which led to lower electronic potential, in turn, better metabolic stability. The
relationship between metabolic stability and coplanarity in the five-membered heteroaryl
subseries was unclear (results not shown).
Figure 3. Coplanarity model with C-linked heteroarenes
values of pyrimidines 9,
1/2
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However, despite optimization efforts on the C-linked heteroaryl series, a
desired balance of antiviral activity, metabolic stability, and permeability could not be
achieved, even after surveying hundreds of newly synthesized compounds. Then, the
relationships between (aza)indole and its C-7 aryl/heteroaryl substituent (activity,
metabolic stability, and permeability) were tested in the N-linked heteroaryl series against
coplanarity. This turned out to be a critical design for the success.
Figure
6-azaindole series, which requires a five-membered heteroaryl ring with a nitrogen at the
α-position and a CH at the α′-position to maximize coplanarity. At least two nitrogen
atoms were required in the ring. One nitrogen atom connected the heteroaryl group to the
6-azaindole at the C-7 position. The second nitrogen atom was positioned at the αposition to form an internal hydrogen bond with the azaindole's pyrrole NH. At the α′position, steric or electronic repulsion would be avoided with a non-substituted carbon
atom.
Chemistry and Pharmacology of Drug Discovery
4 illustrates the coplanarity setup of the N-linked heteroaryl 4-methoxy-
Several hypotheses on physicochemical properties have been proposed. The first
hypothesis suggests that the C‒N bond of a N-linked heteroaryl would be shorter than the
C‒C bond of a C-linked heteroaryl, which would bring the heteroaryl closer to the
azaindole ring and reinforce coplanarity. Secondly, the polarity and electronic potential
of 6-azaindole in a C-linked heteroaryl series are further reduced in the corresponding Nlinked heteroaryl series. Additionally, the lipophilicity of the N-linked heteroaryl series
increases contender due to electronic withdrawing effect, in comparison with the Clinked heteroaryl contender. Overall, the N-linked heteroaryl series offers higher
permeability and metabolic stability compared to its C-linked heteroaryl partner. Design
in Figure
1,2,4-triazoles.
4 resulted in the synthesis of C-7 N-linked pyrazoles, as well as 1,2,3- and
Figure 4. Coplanarity model with N-linked heteroarenes
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Chapter 5. Fostemsavir (Rukobia)
After extensive efforts, 1,2,4-triazole derivative BMS-529 (temsavir, 5) was
discovered with an EC
of 0.1 nM (0.10 ± 0.04 nM, n = 177), exhibiting a much broader
50
spectrum of anti-HIV activity than the POC compound 4 did. The preclinical profile was
comparable to that of 4, including all physicochemical properties and safety parameters.
The water solubility of the compound remained low at 0.022 mg/mL at pH = 7.4. This
caused lower-than-dose-proportional increases in exposure during dose-escalation PK
studies. It was concluded that the solubility was due to kinetic factors, which limited
absorption.
The dissolution rate was significantly increased using the phosphonooxymethyl
prodrug strategy. After an individual takes a phosphonooxymethyl prodrug orally, the gut
membrane-bound alkaline phosphatase cleaves the phosphate bond, releasing
formaldehyde and the corresponding parent molecule. When the parent molecule is
highly permeable, it will promptly enter the bloodstream. This was observed in the case
17,18
of BMS-068 (fostemsavir, 6).
6 converted to 5 in the intestinal tract, and 5 was
rapidly absorbed. Only a negligible quantity of 6 was detected in the early stages after
oral administration (Figure
5).
Figure 5. Fostemsavir converted to temsavir in the presence of alkaline phosphatase
AUC p.o. 24 h
(μM*h)
Temsavir (5)
BMS-043 (4)
2.6 (low)
2.4 (low)
7.5 (low)
4.3 (low)
3.2 (low)
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Chemistry and Pharmacology of Drug Discovery
4. Pharmacokinetics and Drug Metabolism
4.1. Pharmacokinetics of Temsavir
In rat PK studies, temsavir (5) was compared to the benchmark BMS-043 (4). Table 1
lists some of the results. 5 had a 7-fold better AUC after p.o. dosing at 5 mg/kg. Its
intrinsic clearance was 1/10 of 4’s, while its volume of distribution was 1/3 of 4’s, which
together displayed a 1.5-fold longer half-life. Oral bioavailabilities were similar, with 5 at
82% and 4 at 90%.
Table 1. PK profile of BMS-043 and temsavir in rat
IV 1 mg/kg
PO 5 mg/kg
BMS-043 (4) 15 ± 6.3 13 ± 4.0 1.1 ± 0.22 2.4 ± 0.33 90
BMS-529 (5) 111 ± 25 1.3 ± 0.19 0.36 ± 0.098 4.3 ± 1.1 82
The next evaluations were conducted on higher species, including dogs,
monkeys, and chimpanzees (Table
dogs (29 vs 18 μg*h/mL), but not in monkeys and chimpanzees. The clearance rates of
both compounds were similar in dogs, with higher rates for 5 in monkeys and
chimpanzees. However, 5's exceptional in vitro metabolic stability in microsomes
produced better oral bioavailability in dogs and monkeys. 5 was advanced to clinical
trials, given its well-balanced anti-HIV-1 activity spectrum, metabolic stability,
permeability, and in vivo exposure.
Table 2. PK profiles of temsavir and BMS-043 in higher species
Oral F (%)
Dog
Monkey 64 60
Chimpanzee 16 (susp) 25 (susp)
Oral AUC at 5 mg/kg (μg*h/mL)
Dog
Monkey 6.8 12
Chimpanzee 2.1 7.1
Total CL (mL/min/kg)
Dog
Monkey
Chimpanzee 6.6 (inter)
In humans, the clearance of 5 was identified 51% via renal path.19
CL i.v.
(mL/min/kg)
2). 5 showed an advantage over 4 in term of AUC in
89 57
29 18
Vss i.v.
(L/kg)
t
i.v. (h) F (%)
1/2
Rat
0 50 100 150 200
0
750
1500
2250
3000
Dosed with BMS-663068
Dosed with BMS-626529
BMS626529 Equivalent Dosage (mg/kg)
Mean BMS-626529 AUC ( M h)
Dog
0 50 100 150 200
0
800
1600
2400
Dosed with BMS-663068 (QD)
Dosed with BMS-626529 (QD)
Dosed with BMS-663068 (BID)
Dosed with BMS-626529 (BID)
BMS626529 Equivalent Total Dosage (mg/kg)
Mean BMS-626529 AUC ( M h)
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Chapter 5. Fostemsavir (Rukobia)
4.2. Pharmacokinetics of Fostemsavir (6)
Figure 6. Plasma exposure profiles of temsavir (BMS-626529) as temsavir and
fostemsavir (BMS-663068) at escalating doses in the rat (a) and dog (b)
At pH 7.4, temsavir (5, BMS-626529)’s aqueous solubility in a crystalline form was
0.022 mg/mL, which classified it in the BSC II category due to its low aqueous solubility
and high Caco-2 permeability. When an un-optimized suspension was compared with
aqueous solution, its relative oral bioavailability was 52%, indicating its absorption was
limited by dissolution or solubility. In the additional dose escalation studies conducted on
rats and dogs (red and green lines in Figure
6), the exposure increased at a lower rate
than the escalated dose, which confirmed the concern of absorption being limited by
dissolution or solubility.
Fostemsavir (6, BMS-663068) was a phosphonooxymethyl prodrug designed to
enhance the dissolution rate of 5. 6 increased 5’s aqueous solubility from 22 μg/mL at pH
7.4 to over 11 mg/mL at pH 1.5–8.2 at room temperature. 6 was stable in both its solid
state and acidic or neutral solutions for more than 24 h at 37 °C. As previously described,
alkaline phosphatases located at the brush border membranes of the intestinal lumen
hydrolyzed 6 into 5 which was rapidly absorbed into the bloodstream. In vitro studies
showed that 6 is converted to 5 by human placental alkaline phosphatase and hepatocytes,
which suggested a similar process occurring in vivo. Good to excellent oral
bioavailability (80–122%) of 5 was observed after administering low doses of 6 solutions
to rats, dogs, and monkeys. Minimal 6 exposure was detected in the blood.
Figure
At lower dosages (e.g., ≤25 mg/kg), both compounds had similar AUCs because they
6a’s blue line compares the AUC exposure of 5 dosed with 5 or 6 in rats.
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