European Journal of Medicinal Chemistry 225 (2021) 113787

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Advances in the development of HIV integrase strand transfer

inhibitors
Yue Wang a, Shuang-Xi Gu b, c, *, Qiuqin He a, **, Renhua Fan a
a
Department of Chemistry, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai, 200438, China
b
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology, Wuhan,
430205, China
c
Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, Wuhan Institute of Technology, Wuhan, 430205, China

a r t i c l e i n f o a b s t r a c t

Article history: HIV-1 integrase (IN) is a key enzyme in viral replication that catalyzes the covalent integration of viral
Received 1 May 2021 cDNA into the host genome. Currently, five HIV-1 IN strand transfer inhibitors (INSTIs) are approved for
Received in revised form clinical use. These drugs represent an important addition to the armamentarium for antiretroviral
5 August 2021
therapy. This review briefly illustrates the development history of INSTIs. The characteristics of the
Accepted 5 August 2021
Available online 18 August 2021
currently approved INSTIs, as well as their future perspectives, are critically discussed.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
HIV-1 integrase
Strand transfer inhibitors
Metal chelating
Resistance

1. Introduction
Acquired immunodeficiency syndrome (AIDS) is a chronic, life-
threatening disease caused by the human immunodeficiency vi-
rus (HIV). Despite the availability of more than 40 antiviral drugs
approved for therapeutic use, the HIV epidemic remains one of the
most serious global health threats [1]. The World Health Organi-
zation (WHO) estimated that approximately 38 million people were
living with HIV-1 infection at the end of 2020, with 1.7 million new
HIV infections and 0.7 million deaths related to AIDS [2].
HIV-1 integrase (IN), along with reverse transcriptase (RT) and
protease (PR), is an essential enzyme for retroviral replication [3].
HIV-1 IN has no mammalian counterpart and thus represents an
attractive target for the development of anti-HIV drugs [4,5]. In fact,
although anti-HIV drugs targeting RT and PR have been used for
decades, it was not until 2007 that IN was validated as a viable
target for HIV therapy by discovering the first IN inhibitor, ralte-
gravir (RAL, Fig. 1) [6,7]. Since then, four additional IN inhibitors
have been approved by the Food and Drug Administration (FDA) for
* Corresponding author. Department of Chemistry, Fudan University, 2005
Songhu Road, Yangpu District, Shanghai, 200438, China.
** Corresponding author.
E-mail addresses: shuangxigu@163.com (S.-X. Gu), qqhe@fudan.edu.cn (Q. He).
clinical use (Fig. 1): elvitegravir (EVG) in 2012 [8], dolutegravir
(DTG) in 2013 [9,10], bictegravir (BIC) in 2018 [11] and cabotegravir
(CAB) in 2021 [12]. These FDA-approved drugs act by inhibiting the
IN strand transfer reaction and are referred to as IN strand transfer
inhibitors (INSTIs).
2. HIV-1 IN structure and function
HIV-1 IN is a 32 kDa protein comprised of three structural do-
mains: the N-terminal domain (NTD), the catalytic core domain
(CCD), and the C-terminal domain (CTD) [13e15]. The structure of
each isolated domain has been determined, as well as the bound
structures of CCD with NTD [16] and CTD [17]. Recombinant full-
length IN in solution has also been reported to exist in different
forms ranging from monomer to tetramer to higher-order species
[18e22].
The NTD (residues 1e50) adopts a zinc finger fold containing a
pair of highly conserved histidine and cysteine residues (HHCC
motif) [23e25]. The CTD (residues 213e288) adopts an SH3-like
fold and is implicated in DNA binding [26]. The CCD (residues
51e212) contains the catalytic triad D64, D116, E152 (DDE motif)
within an RNase H-like fold that comprises the active site [27,28].
The DDE motif has been proposed to bind two divalent metals, Mg
(II) or Mn (II), which are essential for catalytic activity. Also, Mg is

https://doi.org/10.1016/j.ejmech.2021.113787
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
Y. Wang, S.-X. Gu, Q. He et al.
Fig. 1. The FDA approved INSTIs.
considered to be the metal cofactor of HIV-1 IN under physiological
conditions [29].
Over the past decade, intasome structures from several retro-
viruses have been determined by X-ray crystallography [30e33] or
single-particle cryogenic electron microscopy (cryo-EM) [34e38].
The pioneering studies conducted on the prototype foamy virus
(PFV) intasome have provided initial snapshots of the structure and
function of the retroviral intasome. Furthermore, the crystal
structures of PFV intasome-drug complexes have revealed the
mechanistic details of the drug action [30]. However, PFV and HIV-1
INs share only ~15% sequence identity, and many of the sites where
drug-resistance mutations occur in HIV-1 IN are not conserved in
PFV IN Refs. [36,37]. To better understand the interaction of INSTIs
with HIV-1 intasomes, the structures of primate immunodeficiency
virus intasomes including HIV-1 and red-capped mangabey simian
immunodeficiency virus (SIVrcm) in both unbound and INSTI-
bound forms were determined by cryo-EM to near-atomic resolu-
tion [36,37].
The biochemical, genetic, and complementation studies indicate
that HIV-1 IN functions as a multimer [18e20,22]. Minimally, a
tetramer is required for the concerted integration of a pair of vDNA
ends into the target DNA. The HIV-1 IN tetramer stabilizes through
its interaction with two vDNA ends [19,22]. The integration pro-
moted by IN is comprised of two distinct steps. Initially, the enzyme
recognizes the long terminal repeats (LTR) termini of the viral
dsDNA. It selectively cleaves the terminal dinucleotides from the 30 -
ends of vDNA in the cytoplasm of an infected cell (30 -processing
reaction). Subsequently, IN bound to the processed vDNA ends
enters the nucleus, where the joining reaction catalyzed by IN
connects 30 -OH ends of vDNA to the host DNA (strand transfer re-
action, ST) [4,5].
3. HIV-1 INSTIs
3.1. Earlier HIV-1 INSTIs
Since the cofactor Mg2þ plays a vital role in HIV-1 catalytic
functions, the initial investigations on HIV-1 IN inhibitors were
mainly focused on screening the compounds with the structural
elements of a bidentate metal-binding pharmacophore. Although
compounds with various structural features, such as peptides [39],
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European Journal of Medicinal Chemistry 225 (2021) 113787
polyhydroxylated aromatic compounds [40e42], and DNA com-
plexes [43] have been reported to act as IN inhibitors, most of these
compounds only showed inhibitory activity in in vitro assays. The
discovery of the b-diketoacid (DKA) class as HIV-1 IN inhibitors in
the cell-based assay was a major advance in validating IN as a
therapeutically antiretroviral drug target. DKAs, exemplified by L-
731988 [44] (1, Fig. 3), is chemically characterized by a diketo acid
moiety linked to an aromatic ring. The diketo acid moiety (g-ke-
tone, enolizable a-ketone, and carboxylic acid) was believed to
interact with two Mg2þ ions and the end of viral DNA, blocking the
ST reaction (Fig. 2). S-1360 [45] (3, Fig. 3), 5-ClTEP [46] (3, Fig. 3)
and L-870810 [47,48] (4, Fig. 3) also belong to the DKA family as
they can be regarded as diketo acid bioisosteric analogues. All the
bioisosteres have three functional groups that mimic g-ketone,
enolizable a-ketone, and carboxylic acid and possess a coplanar
conformation [49]. S-1360 was the first IN inhibitor to enter clinical
trials, but it failed in the phase II clinical trial owing to its rapid
metabolism [50].
3.2. First-generation INSTIs
RAL, derived from the evolution of DKAs in the HCV polymerase
program by Merck Research Laboratories, was approved for clinical
use as the first HIV-1 IN inhibitor in 2007 [6,7]. RAL is orally
administrated in 400 mg twice daily. It inhibited IN-catalyzed ST
with an IC50 value of 10 nM and blocked the spread of HIV infection
in cell culture with an EC95 value of 33 nM [51]. It retains strong
activity against diverse clinical isolates resistant to other classes of
drugs, including NRTIs, NNRTIs, and PIs. The approval of RAL
Fig. 2. Originally proposed chelating mechanism for DKAs in IN catalytic site.
Y. Wang, S.-X. Gu, Q. He et al.
Fig. 3. The structures of the DKAs and its bioisosteric analogues. The three functional
groups in DKAs are indicated in blue. The three heteroatoms chelating with two Mg2þ
ions are indicated with dashed lines in orange.
provided a second chance to patients who were left with almost no
treatment alternatives after the failure of highly active antiretro-
viral therapy (HAART) at that time. However, clinical resistance to
RAL was observed. HIV-1 mutations that give rise to raltegravir
resistance map to IN involve three primary genetic pathways:
Q148 H/R/K, N155H and less frequently, Y143C [52]. G140S is an
important compensatory mutation for Q148 mutations and the
double mutant G140S/Q148H was observed in clinical trials as one
of the most prevalent profiles of RAL resistance [53]. The cocrystal
structures of full-length IN from PFV intasome with RAL [30]
Fig. 4. Crystallographic RAL (a, from PDB 3OYA) and EVG (b, from PDB 3L2U) in the active site of PFV intasome.
3
European Journal of Medicinal Chemistry 225 (2021) 113787
confirmed that the coplanar three specifically positioned hetero-
atoms coordinate the two Mg2þ (Fig. 4a). The p-F-benzyl group
displaces and substitutes for the 3’ terminal adenosine of the pro-
cessed vDNA, forming a p-stacking interaction with the penulti-
mate cytidine residue. The terminal oxadiazole ring makes a well-
oriented p-stacking interaction with Y212 (corresponding to Y143
in HIV-1 IN). This contact is likely important and unique for RAL,
which could be the reason for the Y143 mutation on the drug po-
tency. In the crystal structures, although PFV IN residues Q217 and
N224 (corresponding to Q148 and N155 in HIV-1 IN, respectively)
do not directly contact RAL, the mutations of these two residues
may induce conformational changes within the catalytic pocket
and result in a decreased binding efficacy.
EVG, the second IN inhibitor, was approved in 2012 by FDA [8]. It
has a reduced dosing frequency (once-daily, 150 mg), but it requires
a pharmacokinetic (PK) booster to increase EVG systemic exposure
[54]. The structural feature of EVG is the 4-quinolone-3-carboxylic
acid scaffold. The coplanar monoketo acid motif in 4-quinolone-3-
carboxylic acid could be an alternative to the diketo acid motif [8].
EVG showed a significant IC50 value of 7.2 nM against HIV-1 IN and
an EC50 value of 0.9 nM in cell-based assay [8]. The binding mode of
EVG in the active site of PFV is similar to that of RAL (Fig. 4b). The
coplanar monoketo acid motif binds two Mg2þ ions. The halobenzyl
moiety displaces 3’ adenine of bound viral DNA and forms a well-
oriented p-stacking interaction with the penultimate cytidine
residue of the processed strand [30]. The reduced susceptibility to
EVG was associated with T66I, E92Q, S147G, Q148 H/K/R and
N155H, suggesting a significant cross-resistance between ralte-
gravir and EVG cross-resistance [55].
3.3. Second-generation INSTIs
An intramolecular tethering design based on carbamoyl pyr-
idone explored by Shionogi company led to the discovery of DTG as
the second-generation HIV-1 IN inhibitor [56]. DTG differs from the
first-generation INSTIs, where its chelating motif is located on a
tricyclic scaffold. In addition, the linker that connects the central
chelating moiety to the halogenated benzyl group is longer than
either in RAL or EVG [57]. It was approved for medical use in 2013
with a once-daily, unboosted dose (50 mg) [58]. Compared to the
first-generation INSTIs, DTG had a distinct resistance profile. It
exhibited in vitro wild-type (WT) or near WT activity against site-
directed mutants with the single primary resistance mutations
observed in vivo at Q148, at N155, and at Y143 [59]. It displayed
remarkably potent in vitro activity against the troublesome Q148K
mutant (IC50 ¼ 0.68 nM). Its long plasma terminal half-life and slow
Y. Wang, S.-X. Gu, Q. He et al.
dissociation rate from the IN complex (dissociative t1/2 ¼ 71 h,
dissociation was 8 times and 26 times slower than RAL and EVG,
respectively) suggested that it should present a high barrier to
resistance development [60]. In 2019, WHO recommended DTG-
containing regimens as the preferred first- and second-line treat-
ment for all patients with HIV [61].
SAR studies on DTG revealed that the modification of oxaza-
bridging combined with introducing an additional fluoro atom on
the 2,4-difluorophenyl ring would significantly improve the anti-
viral potency against G140S/Q148H, leading to the discovery of a
potent and unboosted inhibitor BIC [62]. BIC potently inhibited
G140S/Q148H with an EC50 value of 3.1 nM and displayed a sta-
tistically improved resistance profile compared to RAL, EVG and
DTG against a set panel of patient-derived INSTI-resistant viral
isolates [63]. Additionally, BIC exhibited a substantially longer
dissociation half-life from HIV-1 IN-DNA complexes than DTG [64].
The most recently approved CAB is also structurally similar to
DTG [12]. Although the potency of CAB is lower than DTG and BIC, it
has the unique feature of a long half-life (close to 40 days) [65]. It
can be formulated as a nanoparticle injection so that it can be given
at prolonged intervals [65]. The combination of CAB and rilpivirine
(a NNRTI) was approved by FDA in Jan 2021 as the first long-acting
injection for HIV-1 treatment. Such a combination of two long-
acting injectable agents is called cabenuva. It can be adminis-
trated once a month and replace the currently used daily pills to
treat HIV-1 infection, marking a significant change from the
methods followed in the past [66].
Both BIC and CAB displayed strong inhibitory activity against the
RAL-resistant IN mutants Y143R and N155H, the EVG-resistant IN
mutants T66I and E92Q, and the DTG-resistant IN mutant H51Y and
E138K/R263K with EC50 values of <5 nM. However, only BIC
potently inhibited the well-known RAL-resistant IN double mutant
G140S/Q148H and the DTG-resistant IN mutant G118R, R263K, and
H51Y/R263K with EC50 values of 5 nM [67].
The structural analysis of DTG and BIC-bound in the active site of
SIVrcm has been investigated recently (Fig. 5) [37]. The three cen-
tral electronegative heteroatoms and the halogenated benzyl
moiety make interactions with SIVrcm similar to those found in
RAL-PFV intasome crystals. Importantly, DTG and BIC intimately
contact backbone N117 and G118 from the b4-a2 loop, a crucial
feature of the second-generation INSTIs. Although the second-
generation INIs have been developed to overcome cross-
resistance with the first-generation drugs, several newly identi-
fied resistance mutations such as G118R, R263K, and S153Y have
been selected for the second-generation INIs [68]. Furthermore,
recent studies suggested that the mutations emerging from the
Q148 pathway can confer cross resistance to the second-generation
INIs.
Fig. 5. Binding modes of DTG (a, from PDB 6RWN) and BIC (b, from PDB 6RWM) in the active site of SIVrcm intasome.
4
European Journal of Medicinal Chemistry 225 (2021) 113787
3.4. Other leading INIs
The success of the drugs in targeting INST has sparked signifi-
cant interest in developing the next-generation INSTIs. The main
focus is identifying the new chemical classes based on the metal-
chelating mechanism with an improved genetic barrier to resis-
tance and enhanced patient compliance.
A series of bicyclic inhibitors 5e7 (Fig. 6) were designed using a
hydroxylamide group as a metal-chelating component [69]. A
halobenzylamide functionality was attached through a linker, and
its carboxamide carbonyl group is not part of the key metal-
chelating triad. Compound 5 showed less active in the ST inhibi-
tory potency than the corresponding compounds 6 and 7 (Table 1).
As shown in Table 2, in cellular assays, inhibitors 6 and 7 displayed
EC50 values at nanomolar concentrations against WT virus. More-
over, 6c and 7c retained strong potency against a panel of IN mu-
tants, including Y143R, N155H, G140S/Q148H, G118R, and E138K/
Q148K (Table 3). Further structural modification at the 4、6、7-
positions of compound 7 through incorporating a N-containing
functional group at the 4-position led to 8 (Fig. 6), resulting into
improved potency against vectors harboring the major forms of
drug-resistant IN mutants [70e72]. The most potent 8a exhibited a
superior antiviral activity against a broad range of INSTI-resistant
mutants compared to DTG (only the selected data are shown in
Table 4; for complete data, please check Ref 72). Compared to DTG,
it also exhibited stronger activity against several triple mutants
such as E138K/G140A/Q148K, L74M/G140A/Q148R, and L74 M/
G140C/Q148R. The cryo-EM structures of HIV-1 with bound 8a
revealed the key contacts in the substrate binding region (Fig. 7).
The amino group at the 4-position of 8a forms an intramolecular
hydrogen bond with the halobenzylamide oxygen, stabilizes the
planar conformation, and facilitates metal chelation. Moreover, the
electron and/or inductive effects on the aromatic core enhance the
metal coordination strength. The long chain of the hydroxyl group
points directs the bulk solvent, displaces the loosely bound water,
and provides an additional enthalpic gain. The crystallographic 8a
in the active site of HIV-1 intasome suggests the importance of the
interactions with the b4-a2 loop [36]. The inhibitor that binds
within the envelope defined by both the viral and host DNA sub-
strates assists 8a in retaining broad efficacy.
Systematic optimization of N-methylpyrimidinone carbox-
amides led to the discovery of BMS-707035 (9, Fig. 8) which
incorporated with an embedded morpholine heterocycle [73].
BMS-707035 showed an excellent IC50 value of 3 nM against ST
reaction and an EC50 value of 17 nM in cell-based assays in the
presence of human serum albumin. BMS-707035 displayed low
clearance in the rat, dog and monkey. It also demonstrated good
safety margins in toxicity studies using dogs and rats. However,
Y. Wang, S.-X. Gu, Q. He et al. European Journal of Medicinal Chemistry 225 (2021) 113787

Fig. 6. The design of bicyclic inhibitors 5e8 with a hydroxylamide functionality.

Table 1
Strand transfer inhibitory potency of bicyclic inhibitors 5e7.

No. R IC50 (nM)

0 0
5a 3 -Cl-4 -F 530
5b 30 ,40 -diF 780
5c 20 ,40 -diF 1800
6a 30 -Cl-40 -F 140
6b 30 ,40 -diF 170
6c 20 ,40 -diF 410
7a 30 -Cl-40 -F 270
7b 30 ,40 -diF 400
7c 20 ,40 -diF 550

Table 2
Antiviral potencies of bicyclic inhibitors 6e7 in cellular assays.

No. R EC50 (nM) SIc

a b b b
WT Y143R N155H G140S/Q148H
Fig. 7. Binding mode of compound 8a in the active site of HIV-1 intasome (from PDB
6a 30 -Cl-40 -F 38 34 90 N/Ad >6579 6PUY).
6b 30 ,40 -diF 62 40 2200 N/A >4032
6c 20 ,40 -diF 5.1 4.9 134 438 >49020
7a 30 -Cl-40 -F 35 54 148 489 2914
BMS-707035 was inactive against G140S/Q148H mutant virus with
7b 30 ,40 -diF 20 45 189 507 9600
7c 20 ,40 -diF 6.2 11 31 308 22097 an EC50 value of >8 mM.
a
The mutations associated with the Q148 pathway would cause a
Cells were infected with the lentiviral vector harboring WT IN, and the values
are indicated in EC50.
structural shift of the position of Mg2þ [74]. To enhance the binding
b
Cells were infected with the viral vectors carrying IN mutations, and the values capacity of IN inhibitors, the amide moiety of BMS-707035 was
are indicated in EC50. further replaced with azole heterocycles, generating a set of het-
c
Selectivity index calculated as the ratio of CC50 to EC50. erocycle amide isosteres in the pyrimidinones (10, Fig. 8) [74]. The
d
Not available.
utilizaiton of azole heterocylces would afford a more open geom-
etry and exhibit greater tolerance of the altered active site of Q148
Table 3 mutants. Among the azole heterocycle derivatives, imidazole
Fold change (FC) of 6c and 7c compared with that of RAL. replacement (10a, Table 5) provided the most substantial
improvement in capacity for resistance coverage. It showed potent
No. EC50 (nM)a
activity in cellular assays with EC50 values of 33 nM, 320 nM, and
a
WT Y143Rb N155Hb G140S/Q148Hb G118Rb E138K/Q148Kb
2 nM against Q148R, G140S/Q148H, and N155H mutant virus,
RAL 4 41  38  475  9 375  respectively (Table 5). The binding modes of compounds 9 and 10a
6c 5.1 1 26  86  N/Ab N/A in the active site of the homology model of WT IN and G140S/
7c 6.2 2 5 50  6 32 
Q148H IN, respectively, revealed that the mutation of glutamine
a
Cells were infected with the lentiviral vector harboring WT IN, and the values are 148 to histidine leads to a more open pocket that is no longer well
indicated in EC50. bCells were infected with viral vectors carrying IN mutations, and
occupied by 9. Meanwhile, a more extended geometry of 10 oc-
the indicated values correspond to the fold-change (FC) in EC50 relative to WT. bNot
available. cupies much of the increased space. Combining the wider geometry
of azoles and its effect on Mg2þ binding coupled with better steric
occupation of the mutant active site contributed to improving the
Table 4 resistance coverage of 10.
Comparison of antiviral potencies of 8a relative to DTG. The screening of proprietary collection of HIV-1 proper INIs
No. EC50 (nM)a against WT and G140S/Q148H mutant viruses sponsored by Bristol-
L74 M M50I G140S/Q148H G148H/N155H E138K/Q148K
Myers Squibb Research and Development led to the identification
of bridged tricyclic pyrimidinone series [75]. The [3.2.2]-bridged
DTG 2.2 2.1 3.9 4.0 25
tricyclic analog 11 (Fig. 9) exhibited an EC50 value of 0.7 nM
8a 1.6 1.3 0.9 1.1 16
against WT HIV-1 and 3 nM against the G140S/Q148H mutant virus.
a
Selected EC50 values obtained from cells infected with the indicated IN mutants.

5
Y. Wang, S.-X. Gu, Q. He et al. European Journal of Medicinal Chemistry 225 (2021) 113787

Fig. 8. The replacement of amide moiety of 9 with azole heterocycles. Calculated “bite angel” are indicated with dashed lines in blue.

Table 5 displayed strong IN inhibitory potency (IC50 ¼ 0.7 nM) with

The inhibitory activity of 10a compared with 9.
No. het EC50 (nM)a
WT Q148R G140S/Q148H
9 3 300 >8000
10a 6 33 320
a
Antiviral activity assayed in an HIV infectivity cellular assay.
Fig. 9. The structure of [3.2.2]-bridged tricyclic analog 11.
However, this inhibitor was not considered for further develop-
ment due to unfavorable human dosing projections predicted by
the pharmacokinetic (PK) profiles in the preclinical species.
The combination of structural features from the tricyclic ring
system carbamoylpyridone and naphthyridinone generated the
hydroxyquinoline tetracyclic ring derivatives 12 (Fig. 10) [76]. All
the designed compounds exhibited excellent inhibitory activities
against IN with EC50 values in the range of 0.6e18 nM. Most of them
illustrated a slight loss of potency against the IN signature resis-
tance mutations Q148K and G140S/Q148H. The best compound 12a
reasonable small serum shifts. Furthermore, it showed a minor shift
against the mutations with a 1.4-fold and 2.8-fold loss in potency
against Q148K and G140S/Q148H, respectively. This agent had a
N155H
good t1/2, low i.v. clearance, but poor oral bioavailability in rats and
140 dogs.
2
A novel bicyclic template tetrahydronaphthyridine combining
the structural features from DKAs was designed by Merck & Co., Inc.
A carboxylic amide para to the phenolic oxygen was considered to
be an important structural element since similar substitution in the
1,6-naphthyridine series, exemplified by RAL, greatly improved the
metabolic stability. An intensive exploration of the structure-
activity relationship (SAR) of the tetrahydronaphthyridine system
has led to the discovery of MK-0536 (13, Fig. 11) [77,78]. It exhibited
an excellent antiviral activity in a viral kinetic assay and maintained
good potency against the mutations E92Q, Y143R, Q148R, Q148K,
and N155H, with only a maximum 3-fold change against these
mutants (Table 6). It is believed that the potency against a panel of
mutant virus improves significantly with the substitution of the
steric bulk group at the N-2 position of the tetrahydronaphthyr-
idine system. Additionally, MK-0536 showed a reasonable pre-
clinical PK profile in rat (CLp ¼ 2.3 mL/min/kg, %F ¼ 29) and dog
(CLp ¼ 2.2 mL/min/kg, %F ¼ 44).
To better understand the influence of the structural diversity of
the tetrahydronaphthyridine system, a third ring was introduced
using MK-0536 as a starting point [79]. The resulting fused 5-
membered ring system exhibited an optimal balance between po-
tency and PK. Also, an extensive structural optimization efforts
generated a novel bicycle[3.1.0]hexane ring system (14, Fig. 11) with
a good resistance profile against RAL-resistant mutants. Further
optimization was directed towards improving the serum-shifted
potency by increasing the polar surface area (PSA), which pro-
vided compound 15 containing a chiral secondary alcohol group on

Fig. 10. The design of hydroxyquinoline tetracyclic ring derivatives 12.

6
Y. Wang, S.-X. Gu, Q. He et al. European Journal of Medicinal Chemistry 225 (2021) 113787

Fig. 11. Lead optimization starting from MK-0536.

Table 6 to MK-0636 and the first-generation INSTIs, and resemble the in-
Fold change (FC) of MK-0536 compared with that of RAL. teractions observed with DTG, which may be responsible for the
No. WTa EC50 (nM)b excellent potency of 15 and 16 against a panel of RAL-resistant
E92Q Y143R Q148R Q148K N155H
mutations (Fig. 13).

RAL 9/53 3 13  16  22  8
MK-0536 6/53 1 2 1 1 3 4. Conclusion and perspectives
15 2/67 1.5  1 1.5  1 1.5 
16 3/32 1 1 1 1 1 Since the approval of RAL as the first INSTI in 2007, five drugs
a
Cells were infected with viral vectors carrying IN mutations and the indicated targeting IN ST step have been approved by FDA. Moreover, a
values correspond to the fold change (FC) in EC50 relative to WT as measured in a number of structurally diverse INSTIs which are able to retain the
viral kinetic assay. intrinsic potency against the mutant virus have been identified. In
b
Measured in the presence of 0/50% NHS.
all the cases, there is a halogenated benzyl ring that interacts with
the penultimate cytosine base near the 30 -end of the viral DNA, a
the alkyl chain of the amine nitrogen as well as compound 16 bidentate metal-binding pharmacophore whose function is to
bearing a hydroxymethyl substituent at the optimal position on the chelate the two Mg2þ held in place by the DDE motif, and a linker.
bicycle[3.1.0]hexane ring (Fig. 11). Both compounds exhibited The first-generation INSTIs RAL and EVG suffer from a relatively low
excellent potency against WT virus and extremely low fold-change barrier to resistance development. The second-generation INSTIs
in potency against RAL-resistant mutations (Table 6). Additionally, show improved efficacies against RAL and EVG-resistant strains of
compounds 15 and 16 also displayed favorable PK profiles, which HIV. It is apparent that the second-generation INSTIs have relatively
anticipates a once-daily human dose without the need for a PK longer linkers compared to the first-generation INSTIs and are
booster. However, they suffered from less than dose proportionality better able to adapt to changes in the active-site geometry caused
during oral exposure due to their poor physicochemical properties. by resistance mutations [70]. Clinically significant resistance to the
To address this limitation, a prodrug strategy was undertaken to second-generation INSTIs is rare. The success of the second-
enhance the solubility and permeability, which led to carbonate generation INSTIs and recent structural studies using both HIV-1
acetal/phosphate prodrug 15a and carbonate acetal prodrug 16a and SIVrcm provide implications for the design of next-
with high exposure (Fig. 12). generation INSTIs [36,37].
Both the cocrystal structures of PFV intasome with 15 and 16 The main challenges in designing next-generation INSTIs are
revealed that the bicycle[3.1.0]hexane ring system of 15 and 16 reducing dosing frequency to once daily without a PK booster and
render an additional contact with G187 in the b4-a2 loop compared improving the genetic barrier to resistance, especially for G140S/
Q148H. The INSTIs share a similar mode of action; they chelate two

Fig. 12. A prodrug strategy to advance 15a and 16a.

7
Y. Wang, S.-X. Gu, Q. He et al. European Journal of Medicinal Chemistry 225 (2021) 113787

Fig. 13. Binding modes of compounds 15 (a, from PDB 4ZTF) and 16 (b, from PDB 4ZTJ) in the active site of PFV intasome.

Mg2þ in the IN catalytic site, creating the potential to develop cross- Abbreviations
resistance. To successfully obtain the next-generation INSTIs,
extending the inhibitors toward the IN backbone to fill these spaces HIV-1 human immunodeficiency virus type 1
more fully should be concerned in the design process. The proposed IN integrase
approaches involve: 1) introducing a proper lengthy linker to INSTIs integrase strand transfer inhibitors
connect a bidentate metal-binding pharmacophore and a haloge- AIDS Acquired immunodeficiency syndrome
nated benzyl ring. The nature of this linker can help adapt to WHO The World Health Organization
changes in the active-site geometry caused by resistance muta- RT reverse transcriptase
tions, 2) filling the substrate envelope. The rationale is that the PR protease
inhibitor which interacts more completely with the highly RAL raltegravir
conserved bases will be less prone to inducing resistance, and 3) EVG elvitegravir
making extensive interactions with the b4-a2 loop. DTG dolutegravir
Recently, attempts to identify inhibitors that block the integra- BIC bictegravir
tion process through a distinct mechanism of action have provided CAB cabotegravir
an alternative approach to overcome cross-resistance, leading to FDA Food and Drug Administration
the development of allosteric IN inhibitors (ALLINIs). ALLINIs, NTD N-terminal domain
alternatively referred to as lens epithelium-derived growth factor CCD catalytic core domain
(LEDGF/p75) integrase inhibitors (LEDGINs), noncatalytic site IN CTD C-terminal domain
inhibitors (NCINIs), or IN-LEDGF/p75 allosteric inhibitors (INLAIs), vDNA viral DNA
bind to the dimer interface of IN CCD at the main LEDGF/p75 LTR long terminal repeats
binding site [80e84]. Although originally designed to block IN- HAART highly active antiretroviral therapy
LEDGF/p75 interactions during viral integration, these inhibitors PFV prototype foamy virus
could also induce aberrant multimerization during virion matura- PK pharmacokinetic
tion [80,82,85]. In addition, ALLINIs retarget residual integrants FC fold change
away from transcription units and toward a more repressive ALLINIs allosteric IN inhibitors
chromatin environment, providing a potential “block-and-lock” LEDGF Lens Epithelium Derived Growth Factor
functional cure strategy [86,87]. To date, ALLINIs haven't yet LEDGINs LEDGF/p75 integrase inhibitors
approved for use in humans, but there has been growing interest in NCINIs noncatalytic site integrase inhibitors
the development of ALLINIs due to their immense potential to INLAIs integrase-LEDGF/p75 allosteric inhibitors
overcome HIV-1 drug resistance.
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (21971043, 81861138046, 21877087), the Sci-
ence and Technology Commission of Shanghai Municipality
(17XD1404400, 18XD1400800, 19ZR1403400), Key Laboratory for
Green Chemical Process of Ministry of Education (GCP20200201),
and Hubei Key Laboratory of Novel Reactor and Green Chemical
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