Novel HIV-1 Protease Inhibitors with Morpholine as the P2 Ligand to Enhance Activity against DRV-Resistant Variants

Article abstract of DOI:10.1021/acsmedchemlett.0c00043

Flexible heterocyclic moieties as the P2 ligands of HIV-1 protease inhibitors may be adapted to the minimally distorted active site of mutations easily and enhance activity against DRV-resistant HIV-1 variants. Herein, the design, synthesis, and biological evaluation of a new series of inhibitors containing morpholine derivatives as the P2 ligands were described, among which, carbamate inhibitor 23a and carbamido inhibitor 27a exhibited almost 4- and 2-fold superior activity with enzyme Ki of 0.092 nM and 0.21 nM, as well as antiviral IC50 values of 0.41 nM and 0.95 nM, respectively, compared to DRV. Besides, they exhibited excellent activity with inhibition of 94percent and 91percent, respectively. Furthermore, they also showed appreciable antiviral activity against DRV-resistant HIV-1 variants.

Full text of DOI:10.1021/acsmedchemlett.0c00043

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Letter
Novel HIV‑1 Protease Inhibitors with Morpholine as the P2 Ligand to
Enhance Activity against DRV-Resistant Variants
Mei Zhu,∥ Yue Dou,∥ Ling Ma,∥ Biao Dong, Fan Zhang, Guoning Zhang, Juxian Wang, Jinming Zhou,*
Shan Cen,* and Yucheng Wang*
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ABSTRACT: Flexible heterocyclic moieties as the P2 ligands of HIV-1
protease inhibitors may be adapted to the minimally distorted active site of
mutations easily and enhance activity against DRV-resistant HIV-1 variants.
Herein, the design, synthesis, and biological evaluation of a new series of
inhibitors containing morpholine derivatives as the P2 ligands were described,
among which, carbamate inhibitor 23a and carbamido inhibitor 27a exhibited
almost 4- and 2-fold superior activity with enzyme Ki of 0.092 nM and 0.21
nM, as well as antiviral IC50 values of 0.41 nM and 0.95 nM, respectively,
compared to DRV. Besides, they exhibited excellent activity with inhibition of
94% and 91%, respectively. Furthermore, they also showed appreciable
antiviral activity against DRV-resistant HIV-1 variants.
KEYWORDS: HIV-1 protease inhibitors, morpholine, DRV-resistant HIV-1 variants, inhibition, molecular modeling
IV-1 protease (PR) is critical in the lifecycle of the
Hcausative virus and is an important target for discovering
anti-HIV-1 drugs. Thus far, HIV-1 protease inhibitors (PIs)
approved by the FDA have increased the life expectancy
significantly and reduced the mortality dramatically.1−3
However, the emergence of drug-resistant HIV-1 variants
posed a threat to further treatment. And what’s worse,
Darunavir (DRV, 1, Figure 1), with a high genetic barrier to
many multidrug-resistant (MDR) protease variants, has
emerged highly DRV-resistant HIV-1 variants in treatment-
experienced patients.4,5 Notably, DRV was incapable of
combating the identified mutations.5,6 Although great effort
has been made to explore potent PIs, none of them were found
to inhibit DRV-resistant HIV-1 variants effectively.7−13 Hence,
the discovery of novel PIs with high genetic barrier against
DRV-resistant HIV-1 variants is highly urgent.
Many of the mutations developed resistance to PIs by
altering the amino acid residues, according to the X-ray
analysis of cocrystallization of HIV-1 PR and inhibitors.14−16
The minor changes in mutations not only distorted the active
site backbone conformation but also reduced affinity for
bonding with the inhibitor,17,18 while increasing interactions
between inhibitors and the protease was undoubtedly an
effective means of overcoming drug resistance.19−21 So two
aspects of the design strategy of novel PIs should be taken into
consideration: (1) enhancing flexibility of newly introduced
fragments for better accommodating to a minimally distorted
active site and (2) maximizing interactions with the active site,
especially promoting extensive hydrogen bonding with the
backbone atoms.22,23 On account of the fact that all approved
HIV-1 PIs are competitive inhibitors with different dipeptide
isostere scaffolds as transition state mimetics, except for
Tipranavir (TPV),24 we designed a series of HIV-1 protease
inhibitors with a core scaffold similar to DRV in our previous
study, among which compound 2 with N-2-(2,4-dioxo-3,4-
Received: January 27, 2020
Accepted: March 31, 2020
Published: March 31, 2020
Figure 1. Structure of HIV-1 protease inhibitor Darunavir.
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A

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b
Scheme 2. Synthesis of Compounds 12 and 16−18
b
Reagents and conditions: (d) 4-nitrophenyl carbonochloridate,
Et2O, 0 °C ∼ rt, overnight, 91%; (e) (i) bromoacetic acid, K2CO3,
anhydrous DMF, argon, rt, overnight; (ii) 4 M HCl, 0 °C, 0.5 h, 85−
88%.
combination with 4-substituted phenylsulfonamides as the
P2′ ligands.
Compounds 8−10 were prepared as reported in the
literature (Scheme 1).26 (2S, 3S)-1, 2-Epoxy-3-(boc-amino)-
4-phenylbutane (3) was reacted with isobutylamine in
acetonitrile at reflux for 6 h to provide the corresponding
amino alcohol (4) in yield of 83%. Treatment of 4 with p-
substituted benzenesulfonyl chlorides in the presence of DIEA
and DMAP furnished sulfonamide derivatives (5−7) in
excellent yields (82%−91%). They were then exposed to
trifluoroacetic acid at 0−25 °C for 3 h to afford the
corresponding amines 8−10 in yields of 78−83%.
2-Morpholinoethan-1-ol(11) was reacted with 4-nitrophenyl
carbonochloridate in diethyl ether to provide activated
carbonate 12 in a yield of 91% in Scheme 2.26 N-Alkylation
of 13−15 with bromoacetic acid proceeded with K2CO3 in
anhydrous DMF, and 4 M HCl (aqueous) was added drop by
drop to give corresponding acids 16−18 with yields of 85−
88%.25
In Scheme 3, alkoxycarbonylation of amines 8−10 with 12
provided compounds 23a−b and 23d in yields of 80−97%.26
Treatment compounds 16−19 with 8−10 generated com-
pounds 24a−27b and 24−27d under the condition of EDCI,
HOBt, and DMAP in yields of 51−80%.25 Compounds 28a−b
and 28d were synthesized by alkylation of 8−10 with 20 under
the catalysis of DIEA and DMAP in 65−69% yields. Reaction
of 8−10 and 21 with K2CO3 provided compounds 29a−b and
29d in yields of 62−73%. The optically active epoxide 22 was
reacted with 8−10 under the catalysis of DIEA to produce
inhibitors 30a−b and 30d in yields of 41−61%. Catalytic
Figure 2. Design and general structure of target compounds.
dihydropyrimidin-1(2H)-yl) acetamide as the P2 ligand in
Figure 2 displayed remarkable enzyme inhibitory (IC50 = 2.53
nM) and appreciable antiviral activity against DRV-resistant
HIV-1 variants with only 2-fold increase in EC50 compared
with that of wild type virus.25 Inspired by the above, we
introduced morpholine derivatives with flexible heterocyclic
moieties instead of pyrimidine bases containing rotation-
restricted double bonds as the P2 ligands. On the one hand,
flexible moieties might be adapted into the distorted protease
active site easily. On the other hand, the oxygen or nitrogen
atoms in morpholine might promote extensive hydrogen
bonding interactions or van der Waals interactions with the
protease in the corresponding S2 subsite. Herein, we reported
the design, synthesis, and biological evaluation of novel HIV-1
PIs with morpholine derivatives as the P2 ligands, in
a
Scheme 1. Synthesis of Amines 8−10
a
Reagents and conditions: (a) i-BuNH2, CH3CN, 80 °C, 6 h, 83%; (b) aryl sulfonyl chloride, DIEA, DMAP(Cat.), THF, 0 °C ∼ rt, 3−5 h, 82−
91%; (c) CH2Cl2−CF3COOH (1:1), 0 °C ∼ rt, 3 h, 78−83%.
B
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a
Scheme 3. Synthesis of Inhibitors 23a−30c
a
Reagents and conditions: (f) DIEA, anhydrous DMF, argon, 0 °C ∼ rt, 10 h, 80−97%; (g) EDCI, HOBt, DMAP, anhydrous DMF, argon, 0 °C ∼
rt, 3 h, 51−80%; (h) DIEA, DMAP, anhydrous EtOH, argon, reflux, 7 h, 65−69%; (i) K2CO3, CH3CN, 60 °C, 1 h, 62−73%; (j) DIEA, anhydrous
EtOH, argon, reflux, 30 h, 41−61%; (k) H2(gas), 50 psi, 5% Pd/C, MeOH-EA (2:1), rt, 4 h, 81−97%.
hydrogenation of 23−30d over 5% Pd/C in the mixture of
methanol and ethyl acetate afforded the aminosulfonamide
inhibitors 23c−30c in 81−97% yields.27
The activity of the inhibitors against HIV-1 wild-type
protease was evaluated as previously reported.28 Morpholine
derivatives as the P2 ligands, 4-substituted phenylsulfonamides
as the P2′ ligands, and types of linkers between the scaffold of
inhibitors and the P2 ligands were all investigated. As can be
seen in Table 1, some of the compounds displayed impressive
enzymatic inhibitory activity with Ki of nanomolar and antiviral
activity with IC50 values of submicromolar to nanomolar in
general.29 In particular, compounds 23a and 27a exhibited
almost 4- and 2-fold superior activity with enzyme Ki of 0.092
nM and 0.21 nM and antiviral IC50 values of 0.41 nM and 0.95
nM compared to DRV, respectively.
In general, the carbamate inhibitors showed the best activity
compared with those carbamido isosteres, acetamide, and
amine derivatives, such as 23a vs 27a, 26a, 29a, and 30a, which
revealed the importance of types of linkers between the
scaffold of inhibitors and the P2 ligands. Inhibitor 23a with a
C
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Table 1. HIV-1 Protease Inhibitory Activity of Inhibitors
a
All assays were conducted in triplicate, and the data shown represent mean values ( 1 standard deviation) derived from the results of three
independent experiments.
Table 2. Inhibition of Inhibitors 23a and 27a on HIV-1 Late
Stage and Early Stage
Inhibition on late stage (%) (10
Inhibition on early stage (%)
a
a
Compd.
μM)
(10 μM)
23a
27a
94
91
7
5
5
12
4
7
DRV
100
a
All assays were conducted in quintuplicate.
high degree of freedom and flexibility may be better bound to
the protease, and the long chain may force the morpholine ring
to extend into the inside of S2 subsite deeply.
However, the activity of inhibitors with aliphatic amine
chains decreased drastically by comparison to those with amide
linkers (carbamate, carbamido, or acetamide) by an order of
magnitude. On the one hand, amide chains could mimic the
peptide characteristics of HIV-1 protease substrate. On the
other hand, the carbonyl group in amide compounds could
form hydrogen bonds or van der Waals interactions with the
backbone of the protease (supported by the molecular
modeling), which further confirmed the significance of
Figure 3. Inhibition of inhibitors 23a and 27a on HIV-1 late stage
compared with early stage.
promoting extensive interactions between P2 ligand and
residues of the protease S2 subsite.22,23
Moreover, the activity of carbamido inhibitors outperformed
significantly that of acetamide inhibitors, such as 27a vs 24a,
25a, and 26a. The length of linker, which obviously influenced
D
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corresponding 4-amino and 4-trifluoromethyl substituents,
except for 28a, 29a, and 30a. The oxygen atom in the 4-
methoxyl group might form a powerful hydrogen bond (O···
H−N) with Asp30′ or Asp29′ in the protease S2′ subsite and
form van der Waals interactions as shown in Figure 5.2130 The
4-amino group could make weak hydrogen bonds involving
directly interactions (N−H···N) with the main chain amides
and water-mediated interactions (NH···H2O···HOOC) with
the side chain oxygen of Asp30′ or Asp29′ in the S2′ active
subsite.31 The 4-trifluoromethyl group may form favorable
halogen interactions within the S2′ subsite, but its weak
electron-withdrawing inductive effect would impair the affinity
slightly.32
Table 3. Antiviral Activity of 23a and 27a against Multidrug
Resistant HIV-1 Variants
a
mean EC50 μM SD
b
Compd.
HIV-1NL4‑3
HIVDRVR’s
fold resistance
23a
27a
DRV
1.20 0.17
2.00 0.48
0.0039 0.0012
9.50 0.89
4.90 0.57
0.24 0.021
7.92
2.45
60.26
a
All assays were conducted in triplicate, and the data shown represent
mean values ( 1 standard deviation) derived from the results of three
b
independent experiments. Fold resistance is defined by EC50(mutant)
/
EC50(WT)
.
To confirm whether the compounds acted on HIV-1 PI,
selected inhibitors 23a and 27a were further evaluated for their
effect on the late stage and early stage of HIV-1 life cycle using
two different cell-based assays as described previously.33,34 The
effect on the late stage but not the early stage would reflect the
effect of inhibitors on HIV-1 protease activity. Notably, they
exhibited excellent activity with inhibition of 94% and 91% on
the late stage (shown in Table 2 and Figure 3), respectively, in
agreement with their potent activity against PI.33 In contrast,
they showed little affection in the infectivity assay on HIV-1
early stage, albeit to a low extent with inhibition of 5% and
12%, respectively.34 Both of the assays on HIV-1 late stage and
early stage provided clear evidence that antiviral activity of
compounds 23a and 27a predominantly derived from their
ability to inhibit HIV-1 protease.
the compatibility between inhibitors and the protease, played
an important role, as shown in the molecular modeling studies
in Figure 5.
Among the acetamide isosteres, chiral methyl substitution in
the 2-position led to a decrease in the enzymatic inhibitory
activity and antiviral activity (24a−c and 25b−c, Ki of 93.86−
14.25 nM, IC50 value of 419.0−63.6 nM), compared with
inhibitors without substitute groups (26a−c, Ki of 9.90−7.44
nM, IC50 value of 44.2−33.2 nM), with the exception of 25a
that exhibited slightly lower Ki of 5.71 nM and IC50 value of
25.5 nM. The reason may be that bulkier hindrance could
impose restriction on conjunction inhibitors with the cavity of
the S2 subsite.
Moreover, among the amine derivatives, compounds with
linkers of nonsubstitution aliphatic chains exhibited slightly
better activity than the corresponding compounds with
carbonyl or hydroxyl substituted aliphatic chains, such as
29a−c vs 28a and 30a−c, with the exception of 28b and 28c,
which may be due to the large degree of freedom and the
reduction of steric hindrance.
Furthermore, inhibitors 23a and 27a were tested for antiviral
activity against DRV-sensitive or resistant pseudotyped HIV-1
in a single-round infection assay.6 Four amino acid
substitutions (V32I, L33F, I54M, and I84V) in HIV-1 PR
were introduced into pNL4-3-E−R− (pHIV-1NL4‑3), resulting in
R
S
The functional P2′ ligands also carried significant impact on
the potency of inhibitors. For instance, compounds with 4-
methoxyl substituent exhibited higher potency than the
DRV-resistant HIV-1 proviral DNA pHIV-1DRV in the assay.
The inhibitory potency of 23a and 27a against DRV-resistant
mutations differed slightly from potency against PI-resistant
Figure 4. Antiviral activity of 23a and 27a against multidrug resistant HIV-1 variants. (A−C) Dose−response of compounds DRV, 23a, and 27a
against wild type HIV-1 and DRV-resistant mutant. (D) Fold resistance is defined by EC50(mutant)/EC50(WT)
.
E
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Figure 5. Molecular modeling for inhibitors 27a and 26a. (A) Docked pose of 27a. (B) Ligplot interaction of 27a. (C) Docked pose of 26a. (D)
Ligplot interaction of 26a. Ligand exposures were represented as purple spheres, and hydrogen bonding was depicted as blue arrows.
HIV-1variant HIV-1NL4‑3 with only a 2- or 8-fold increase in
EC50, compared with DRV which exhibited a 60-fold increase
in EC50 against DRV-resistant mutations than that against wild
type virus (Table 3 and Figure 4). The results indicated that
incorporating morpholine derivatives as the P2 ligands was in
favor of enhancing activity against DRV-resistant mutants.
For the purpose of predicting the binding mode of inhibitors
with HIV-1 PR and revealing the discrepancy in antiviral
potency, molecular modeling of 27a and 26a was done in the
Molecular Operating Environment (MOE) (version
2009.06).35 The protease structure (PDB-ID: 4mc9) was
obtained from the protein data bank.36 Remarkably, 27a fitted
into the HIV-1 PR binding site perfectly and showed a binding
score of −13.46 kJ/mol. As can be seen in Figure 5, favorable
van der Waals interactions or hydrophobic interactions formed
between morpholine and the outer atoms of PR. So
introduction of a flexible heterocyclic moiety as the P2 ligand
could adapt into the active cavity well and form van der Waals
interactions with the residues of PR. Besides, several hydrogen-
bonding interactions between the scaffold of the inhibitor and
residues Gly48, Gly48′, Ile50, and Ile50′ of PR were observed.
In addition, the phenyl and methoxyl groups in the P2′ ligand
could produce van der Waals interactions with the S2′ subsite
of protease.
Inhibitor 26a formed hydrogen bonding interactions
between the hydroxyl and the residues Gly27 and Gly27′, as
well as van der Waals interactions or hydrophobic interactions
of the P2 ligand with the enzyme. However, inhibitor 26a was
not fitted into the binding site very well because of the long
chain in the P2 ligand. Furthermore, compared with 27a,
inhibitor 26a exhibited a lower negative docking score of
−13.17 kJ/mol, which implied weaker bindings with the
enzyme.37 In brief, the molecular modeling studies confirmed
that the antiviral activity of carbamido inhibitors was
significantly superior to that of acetamide inhibitors (27a vs
26a).
In summary, our attention has been given to the design of
HIV-1 PIs with flexible heterocyclic moieties as the P2 ligands,
with the aim of adapting into the minimally distorted protease
active site easily to some extent and enhancing genetic barrier
against DRV-resistant HIV-1 variants. Herein, we designed a
series of novel inhibitors with morpholine derivatives as the P2
F
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ligands. Particularly, carbamate inhibitor 23a and carbamido
inhibitor 27a exhibited almost 4- and 2-fold superior activity
with enzyme Ki of 0.092 nM and 0.21 nM, and antiviral IC50
values of 0.41 nM and 0.95 nM compared to DRV,
respectively. Furthermore, their inhibition of infectivity on
HIV-1 late stage was comparable to DRV, as well as
appreciable antiviral activity against DRV-resistant HIV-1
variants. The SAR studies and molecular modeling studies
revealed the importance of the types and length of linkers
between the scaffold of inhibitors and the P2 ligands.
The state-of-the-art inhibitors were not obtained in this
study; even so, inspiration for further optimization of HIV-1
PIs could be gained according to the molecular modeling
studies. Although there existed van der Waals interactions or
hydrophobic interactions between the P2 ligand and the S2
subsite of PR, the nitrogen atom of morpholine in the P2
ligand was wrapped inward and lost the ability of forming
hydrogen bonds with residues of the protease. Exposure of the
nitrogen atom in morpholine may be of high importance for
enhancing antiviral activity and thus becomes the focus of our
next study.
Biao Dong − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China
Fan Zhang − Department of Pharmacy, Jinzhou Medical
University, Jinzhou 121001, China
Guoning Zhang − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China
Juxian Wang − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00043
Author Contributions
∥M.Z., Y.D., and L.M. made equal contributions to this work.
M.Z. designed and performed the experiments; Y.D. and G.Z.
designed and performed the experiments; L.M. and B.D.
performed the inhibitory activity assay; J.W. and F.Z. proposed
the work; J.Z. performed molecular modeling and analyzed the
data; Y.W. and S.C. designed the research. All authors
discussed, edited, and approved the final version.
ASSOCIATED CONTENT
Funding
■
sı
This work was supported by National Natural Science
Foundation of China (81703366), National Mega-Project for
Infectious Disease (2018ZX10301408), CAMS Innovation
Fund for Medical Sciences (CAMS-I2M-1-012), and National
key research and development program (2018YFE0107600).
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00043.
Procedures for synthesis and spectroscopic character-
ization including 1H NMR and 13C NMR for all
compounds and HR MS of the target compounds.
Descriptions of assay for HIV-1 protease inhibition,
infectivity assay on HIV-1 late stage and early stage, as
well as construction of DRV-resistant pNL4-3-E-R-
cloning. (PDF)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Zonggen Peng from Institute of Medicinal
Biotechnology, Chinese Academy of Medical Science and
Peking Union Medical College for his creative suggestions and
kind help. Baixiang Du from Jiangsu Normal University is
acknowledged for assistance for mass spectrometry.
AUTHOR INFORMATION
■
Corresponding Authors
Jinming Zhou − Key Laboratory of the Ministry of Education
for Advanced Catalysis Materials, Department of Chemistry,
Zhejiang Normal University, Jinhua 321004, China;
Phone: +86-0579-82291295; Email: zhoujinming@
zjnu.edu.cn
Shan Cen − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China; Phone: +86- 010-63037279;
Email: shancen@imb.pumc.edu.cn
Yucheng Wang − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China; orcid.org/0000-0002-5203-7628;
Phone: +86- 010-63165263; Email: wangyucheng@
imb.pumc.edu.cn
ABBREVIATIONS
■
PIs protease inhibitors; HAART highly active antiretroviral
therapy; PR protease; DRV Darunavir; VSVg vesicular
stomatitis virus G protein; TLC thin layer chromatography;
DMF N,N-dimethylformamide; DMAP 4-dimethylaminopyr-
idine; EDCI N-(3-(dimethylamino)propyl)-N′-ethylcarbodii-
mide hydrochloride; HOBt 1-hydroxybenzotriazole; rt room
temperature; FRET fluorescence resonance energy transfer;
MOE Molecular Operating Environment
REFERENCES
■
(1) Ghosh, A. K.; Osswald, H. L.; Prato, G. Recent progress in the
development of HIV-1 protease inhibitors for the treatment of HIV/
AIDS. J. Med. Chem. 2016, 59, 5172−5208.
(2) Lohse, N.; Hansen, A. B.; Gerstoft, J.; Obel, N. Improved
survival in HIV-infected persons: consequences and perspectives. J.
Antimicrob. Chemother. 2007, 60, 461−463.
(3) Montaner, J. S. G.; Lima, V. D.; Barrios, R.; Yip, B.; Wood, E.;
Kerr, T.; Shannon, K.; Harrigan, P. R.; Hogg, R. S.; Daly, P.; Kendall,
P. Association of highly active antiretroviral therapy coverage,
population viral load, and yearly new HIV diagnoses in British
Columbia, Canada: a population-based study. Lancet 2010, 376, 532−
539.
Authors
Mei Zhu − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China
Yue Dou − Department of Pharmacy, Jinzhou Medical
University, Jinzhou 121001, China
Ling Ma − Institute of Medicinal Biotechnology, Chinese
Academy of Medical Science and Peking Union Medical College,
Beijing 100050, China
G
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ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(4) Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H. Darunavir, a
conceptually new HIV-1 protease inhibitor for the treatment of drug-
resistant HIV. Bioorg. Med. Chem. 2007, 15, 7576−7580.
(5) Ghosh, A. K.; Rao, K. V.; Nyalapatla, P. R.; Osswald, H. L.;
Martyr, C. D.; Aoki, M.; Hayashi, H.; Agniswamy, J.; Wang, Y. F.;
Bulut, H.; Das, D.; Weber, I. T.; Mitsuya, H. Design and development
of highly potent HIV-1 protease inhibitors with a crown-like
oxotricyclic core as the P2-ligand to combat multidrug-resistant
HIV variants. J. Med. Chem. 2017, 60, 4267−4278.
(6) Aoki, M.; Das, D.; Hayashi, H.; Aoki-Ogata, H.; Takamatsu, Y.;
Ghosh, A. K.; Mitsuya, H. Mechanism of Darunavir (DRV)’s high
genetic barrier to HIV-1 resistance: a key V32I substitution in
protease rarely occurs, but once it occurs, it predisposes HIV-1 to
develop DRV resistance. mBio 2018, 9, No. e02425-17.
(7) Ghosh, A. K.; P, R. N.; Kovela, S.; Rao, K. V.; Brindisi, M.;
Osswald, H. L.; Amano, M.; Aoki, M.; Agniswamy, J.; Wang, Y. F.;
Weber, I. T.; Mitsuya, H. Design and synthesis of highly potent HIV-1
protease inhibitors containing tricyclic fused ring systems as novel P2
ligands: structure-activity studies, biological and X-ray structural
analysis. J. Med. Chem. 2018, 61, 4561−4577.
(18) Kovalevsky, A. Y.; Tie, Y.; Liu, F.; Boross, P. I.; Wang, Y. F.;
Leshchenko, S.; Ghosh, A. K.; Harrison, R. W.; Weber, I. T.
Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1
protease with highly drug resistant mutations D30N, I50V, and
L90M. J. Med. Chem. 2006, 49, 1379−1387.
(19) Ghosh, A. K.; Ramu Sridhar, P.; Kumaragurubaran, N.; Koh, Y.;
Weber, I. T.; Mitsuya, H. Bis-tetrahydrofuran: a privileged ligand for
Darunavir and a new generation of HIV protease inhibitors that
combat drug resistance. ChemMedChem 2006, 1, 939−950.
(20) Ghosh, A. K.; Leshchenko-Yashchuk, S.; Anderson, D. D.;
Baldridge, A.; Noetzel, M.; Miller, H. B.; Tie, Y.; Wang, Y.-F.; Koh, Y.;
Weber, I. T.; Mitsuya, H. Design of HIV-1 protease inhibitors with
pyrrolidinones and oxazolidinones as novel P1′-ligands to enhance
backbone-binding interactions with protease: synthesis, biological
evaluation, and protein-ligand X-ray studies. J. Med. Chem. 2009, 52,
3902−3914.
(21) Agniswamy, J.; Shen, C. H.; Wang, Y. F.; Ghosh, A. K.; Rao, K.
V.; Xu, C. X.; Sayer, J. M.; Louis, J. M.; Weber, I. T. Extreme
multidrug resistant HIV-1 protease with 20 mutations is resistant to
novel protease inhibitors with P1′-pyrrolidinone or P2-tris-tetrahy-
drofuran. J. Med. Chem. 2013, 56, 4017−4027.
(22) Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Design
of HIV protease inhibitors targeting protein backbone: an effective
strategy for combating drug resistance. Acc. Chem. Res. 2008, 41, 78−
86.
(8) Ghosh, A. K.; Chapsal, B. D.; Baldridge, A.; Steffey, M. P.;
Walters, D. E.; Koh, Y.; Amano, M.; Mitsuya, H. Design and synthesis
of potent HIV-1 protease inhibitors incorporating hexahydrofuropyr-
anol-derived high affinity P(2) ligands: structure-activity studies and
biological evaluation. J. Med. Chem. 2011, 54, 622−634.
(23) Ghosh, A. K.; Anderson, D. D.; Weber, I. T.; Mitsuya, H.
Enhancing protein backbone binding–a fruitful concept for combating
drug-resistant HIV. Angew. Chem., Int. Ed. 2012, 51, 1778−1802.
(24) Ali, A.; Bandaranayake, R. M.; Cai, Y.; King, N. M.; Kolli, M.;
Mittal, S.; Murzycki, J. F.; Nalam, M. N.; Nalivaika, E. A.; Ozen, A.;
Prabu-Jeyabalan, M. M.; Thayer, K.; Schiffer, C. A. Molecular Basis
for Drug Resistance in HIV-1 Protease. Viruses 2010, 2, 2509−2535.
(25) Zhu, M.; Ma, L.; Zhou, H.; Dong, B.; Wang, Y.; Wang, Z.;
Zhou, J.; Zhang, G.; Wang, J.; Liang, C.; Cen, S.; Wang, Y.
Preliminary SAR and biological evaluation of potent HIV-1 protease
inhibitors with pyrimidine bases as novel P2 ligands to enhance
activity against DRV-resistant HIV-1 variants. Eur. J. Med. Chem.
2020, 185, 111866.
(26) Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li,
J.; Kovalevsky, A. Y.; Walters, D. E.; Wedekind, J. E.; Grum-Tokars,
V.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.; Weber, I. T.; Mitsuya,
H. Structure-based design of novel HIV-1 protease inhibitors to
combat drug resistance. J. Med. Chem. 2006, 49, 5252−5261.
(27) Olmo, E. D.; Diaz-Gonzalez, R.; Escarcena, R.; Carvalho, L.;
Bustos, L. A.; Navarro, M.; Feliciano, A. S. Diamine and aminoalcohol
derivatives active against Trypanosoma brucei. Bioorg. Med. Chem.
Lett. 2012, 22, 440−443.
(28) Matayoshi, E.; Wang, G.; Krafft, G.; Erickson, J. Novel
fluorogenic substrates for assaying retroviral proteases by resonance
energy transfer. Science 1990, 247, 954−958.
(29) Cer, R. Z.; Mudunuri, U.; Stephens, R.; Lebeda, F. J. IC50-to-Ki:
a web-based tool for converting IC50 to Ki values for inhibitors of
enzyme activity and ligand binding. Nucleic Acids Res. 2009, 37,
W441−W445.
(30) Meier, C.; Jessen, H. J.; Schulz, T.; Weinschenk, L.;
Pertenbreiter, F.; Balzarini, J. Rational development of nucleoside
diphosphate prodrugs: DiPPro-compounds. Curr. Med. Chem. 2015,
22, 3933−3950.
(31) Perez, M. A.; Fernandes, P. A.; Ramos, M. J. Drug design: new
inhibitors for HIV-1 protease based on Nelfinavir as lead. J. Mol.
Graphics Modell. 2007, 26, 634−642.
(9) Ghosh, A. K.; Parham, G. L.; Martyr, C. D.; Nyalapatla, P. R.;
Osswald, H. L.; Agniswamy, J.; Wang, Y. F.; Amano, M.; Weber, I. T.;
Mitsuya, H. Highly potent HIV-1 protease inhibitors with novel
tricyclic P2 ligands: design, synthesis, and protein-ligand X-ray
studies. J. Med. Chem. 2013, 56, 6792−6802.
(10) Ghosh, A. K.; Osswald, H. L.; Glauninger, K.; Agniswamy, J.;
Wang, Y. F.; Hayashi, H.; Aoki, M.; Weber, I. T.; Mitsuya, H. Probing
lipophilic adamantyl group as the P1-ligand for HIV-1 protease
inhibitors: design, synthesis, protein X-ray structural studies, and
biological evaluation. J. Med. Chem. 2016, 59, 6826−6837.
(11) Hohlfeld, K.; Wegner, J. K.; Kesteleyn, B.; Linclau, B.; Unge, J.
Disubstituted bis-THF moieties as new P2 ligands in nonpeptidal
HIV-1 protease inhibitors (II). J. Med. Chem. 2015, 58, 4029−4038.
(12) Rusere, L. N.; Lockbaum, G. J.; Lee, S. K.; Henes, M.;
Kosovrasti, K.; Spielvogel, E.; Nalivaika, E. A.; Swanstrom, R.; Yilmaz,
N. K.; Schiffer, C. A.; Ali, A. HIV-1 protease inhibitors incorporating
stereochemically defined P2′ ligands to optimize hydrogen bonding in
the substrate envelope. J. Med. Chem. 2019, 62, 8062−8079.
(13) Ghosh, A. K.; Williams, J. N.; Ho, R. Y.; Simpson, H. M.;
Hattori, S. I.; Hayashi, H.; Agniswamy, J.; Wang, Y. F.; Weber, I. T.;
Mitsuya, H. Design and synthesis of potent HIV-1 protease inhibitors
containing bicyclic oxazolidinone scaffold as the P2 ligands: structure-
activity studies and biological and X-ray structural studies. J. Med.
Chem. 2018, 61, 9722−9737.
(14) Wlodawer, A.; Miller, M.; Jaskolski, M.; Sathyanarayana, B.;
Baldwin, E.; Weber, I.; Selk, L.; Clawson, L.; Schneider, J.; Kent, S.
Conserved folding in retroviral proteases: crystal structure of a
synthetic HIV-1 protease. Science 1989, 245, 616−621.
(15) Lapatto, R.; Blundell, T.; Hemmings, A.; Overington, J.;
Wilderspin, A.; Wood, S.; Merson, J. R.; Whittle, P. J.; Danley, D. E.;
Geoghegan, K. F.; Hawrylik, S. J.; Lee, S. E.; Scheld, K. G.; Hobart, P.
M. X-ray analysis of HIV-1 proteinase at 2.7 Å resolution confirms
structural homology among retroviral enzymes. Nature 1989, 342,
299−302.
(16) Wang, Y. F.; Tie, Y.; Boross, P. I.; Tozser, J.; Ghosh, A. K.;
Harrison, R. W.; Weber, I. T. Potent new antiviral compound shows
similar inhibition and structural interactions with drug resistant
mutants and wild type HIV-1 protease. J. Med. Chem. 2007, 50,
4509−4515.
(17) Tie, Y.; Kovalevsky, A. Y.; Boross, P.; Wang, Y. F.; Ghosh, A.
K.; Tozser, J.; Harrison, R. W.; Weber, I. T. Atomic resolution crystal
structures of HIV-1 protease and mutants V82A and I84V with
saquinavir. Proteins: Struct., Funct., Genet. 2007, 67, 232−242.
(32) Aoki, M.; Hayashi, H.; Rao, K. V.; Das, D.; Higashi-Kuwata, N.;
Bulut, H.; Aoki-Ogata, H.; Takamatsu, Y.; Yedidi, R. S.; Davis, D. A.;
Hattori, S. I.; Nishida, N.; Hasegawa, K.; Takamune, N.; Nyalapatla,
P. R.; Osswald, H. L.; Jono, H.; Saito, H.; Yarchoan, R.; Misumi, S.;
Ghosh, A. K.; Mitsuya, H. A novel central nervous system-penetrating
protease inhibitor overcomes human immunodeficiency virus 1
resistance with unprecedented aM to pM potency. eLife 2017, 6,
No. e28020.
H
https://dx.doi.org/10.1021/acsmedchemlett.0c00043
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(33) Garcia, J. M.; Gao, A.; He, P. L.; Choi, J.; Tang, W.; Bruzzone,
R.; Schwartz, O.; Naya, H.; Nan, F. J.; Li, J.; Altmeyer, R.; Zuo, J. P.
High-throughput screening using pseudotyped lentiviral particles: a
strategy for the identification of HIV-1 inhibitors in a cell-based assay.
Antiviral Res. 2009, 81, 239−247.
(34) Mitsuya, H.; Broder, S. Strategies for antiviral therapy in AIDS.
Nature 1987, 325, 773−778.
(35) Mohammed, A. F.; Abdel-Moty, S. G.; Hussein, M. A.; Abdel-
Alim, A. A. Design, synthesis and molecular docking of some new
1,2,4-triazolobenzimidazol-3-yl acetohydrazide derivatives with anti-
inflammatory-analgesic activities. Arch. Pharmacal Res. 2013, 36,
1465−1479.
(36) Ganguly, A. K.; Alluri, S. S.; Wang, C.-H.; Antropow, A.; White,
A.; Caroccia, D.; Biswas, D.; Kang, E.; Zhang, L. K.; Carroll, S. S.;
Burlein, C.; Fay, J.; Orth, P.; Strickland, C. Structural optimization of
cyclic sulfonamide based novel HIV-1 protease inhibitors to
picomolar affinities guided by X-ray crystallographic analysis.
Tetrahedron 2014, 70, 2894−2904.
(37) Zhu, M.; Ma, L.; Dong, B.; Zhang, G.; Wang, J.; Zhou, J.; Cen,
S.; Wang, Y. Synthesis and evaluation of potent human
immunodeficiency virus 1 protease inhibitors with epimeric
isopropanol as novel P1′ ligands. Future Med. Chem. 2020,
DOI: 10.4155/fmc-2019-0331.
I
https://dx.doi.org/10.1021/acsmedchemlett.0c00043
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX