Design, Synthesis, and X-ray Studies of Potent HIV-1 Protease Inhibitors with P2-Carboxamide Functionalities

Article abstract of DOI:10.1021/acsmedchemlett.9b00670

The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2′-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.

Full text of DOI:10.1021/acsmedchemlett.9b00670

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Letter
Design, Synthesis, and X‑ray Studies of Potent HIV‑1 Protease
Inhibitors with P2-Carboxamide Functionalities
Arun K. Ghosh,* Alessandro Grillo, Jakka Raghavaiah, Satish Kovela, Megan E. Johnson,
Daniel W. Kneller, Yuan-Fang Wang, Shin-ichiro Hattori, Nobuyo Higashi-Kuwata, Irene T. Weber,
and Hiroaki Mitsuya
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ABSTRACT: The design, synthesis, biological evaluation, and X-ray structural
studies are reported for a series of highly potent HIV-1 protease inhibitors. The
inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic
ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide
transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease
inhibitory and antiviral activity; however, ligand combination is critical for
potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-
derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2′-
ligand displayed very potent antiviral activity and maintained excellent antiviral
activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease
provided molecular insight into the binding properties of the new inhibitor.
KEYWORDS: HIV-1 protease, cyclic ethers, carboxamide, multidrug-resistant, X-ray structure, backbone binding
IV-1 protease inhibitor drugs are important elements of
furan heterocycle as the P2-ligand linked with a urethane
functionality to a (R)-hydroxyethylamine sulfonamide dipep-
tide isosteric scaffold.10 Detailed X-ray structural studies of
darunavir-bound HIV-1 protease revealed critical darunavir-
protease interactions responsible for its high affinity as well as
its potent activity against multidrug-resistant HIV-1 var-
iants.19,20 Structurally, darunavir forms extensive van der
Waals as well as hydrogen-bonding interactions throughout
the protease active site.19,20 In the S2 subsite, the bis-THF
ligand oxygens are within hydrogen bonding distance to
backbone Asp30 and Asp29 amide NHs. The urethane
functionality also plays an important role in directing specific
orientation of the bis-THF ligand oxygens for hydrogen
bonding interactions. Both urethane NH and the carbonyl
groups play an important role. The NH group forms a strong
hydrogen bond with the Gly27 carbonyl of HIV-1 protease.
The bicyclic ring of bis-THF, on the other hand, is engaged in
extensive van der Waals interactions in the active site. The P2
urethane carbonyl functionality is very important. It forms a
water-mediated tetracoordinated hydrogen bonding interaction
with one of the sulfonamide oxygens of darunavir and amide
1,2
Hcurrent combined antiretroviral therapy (cART).
The
use of cART has dramatically reduced both the mortality and
morbidity rates among HIV-infected patients.3,4 However, the
emergence of multidrug-resistant HIV-1 variants has raised
concerns as to the long-term viability of current treatment
options.5,6 In our continuing studies toward X-ray structure-
based design of novel HIV-1 protease inhibitors to combat
multidrug resistance, we recently reported a number of very
potent inhibitors with intriguing structural features.7,8 Our
main inhibitor design strategies are based upon incorporation
of nonpeptide cyclic-ether templates to effectively mimic
peptide bond interactions in the active site.9,10 Also, we strive
to maximize active site ligand-binding site interactions,
particularly to promote a robust network of hydrogen bonding
interactions with the backbone atoms of HIV-1 protease. Over
the years, these design principles have guided our development
of HIV-1 protease inhibitors to combat drug resistance.11,12
The latest FDA-approved protease inhibitor drug, darunavir
(1, Figure 1) has been developed based upon these design
concepts.13,14 Darunavir emerged as a first-line therapy which
̈
is often preferred for cART-naive HIV/AIDS patients due to
its tolerance and a high genetic barrier to the development of
drug-resistant viruses compared to other HIV-1 protease
inhibitors.15,16 This is possibly due to darunavir’s unique dual
mechanism of action as it inhibits the catalytic protease dimer
and also protease monomer dimerization to form the active
enzyme.17,18 Darunavir (1) has been designed with a
stereochemically defined bicylic (3R,3aS,6aR)-bis-tetrahydro-
Special Issue: Medicinal Chemistry: From Targets to
Therapies
Received: December 30, 2019
Accepted: February 27, 2020
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© XXXX American Chemical Society
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inhibitory and antiviral activity. In particular, inhibitor 4h
maintained exceptional antiviral potency against selected
multidrug-resistant HIV-1 variants. Our high resolution X-ray
structural studies of inhibitor 4h-bound HIV-1 protease
provided important molecular insight into the ligand-binding
site interactions.
The synthesis of various bis-THF-derived carboxamide P2
ligands started from known optically active alcohol 5.27−29 As
shown in Scheme 1, alcohol 5 was converted to ethyl ester 6 in
a
Scheme 1. Synthesis of Carboxylic Acid P2 Ligands
Figure 1. Structures of HIV-1 protease inhibitors 1−3, 4a, and 4h.
NHs of Ile50 and Ile50′ in the flaps of HIV-1 protease. Similar
water-mediated interactions have been observed with other
HIV-1 protease inhibitors.21,22
Based upon the critical ligand-binding site interactions, we
recently designed a crown-like tetrahydropyranotetrahydrofur-
an with a bridged methylene group as a very effective P2 ligand
as represented in inhibitor 3 (Figure 1) with exceptional
antiviral activity against multidrug-resistant HIV-1 strains.7,23
The crown-THF P2 ligand is linked to an optimized
nonpeptide transition-state mimetic via a urethane function-
ality. The X-ray structural studies of inhibitor 3-bound HIV-1
protease show that compound 3 makes more extensive
molecular interactions compared to darunavir.7,8 The
urethane-functionality, in general, shows good metabolic
stability and drug properties.24 However, one of the major
metabolites of darunavir is the cleavage of the urethane
functionality.24,25 In general, carboxamides are metabolically
more stable than their carbamate counterparts.26 In a
preliminary effort to improve drug properties, we sought to
replace the urethane group. The carbamate group of darunavir
has a direct role in drug−target interactions. In particular, the
carbonyl oxygen forms a water-mediated hydrogen bond with
the backbone Ile50′ amide NH in the S2 subsite.21,22 We have
explored replacement of the urethane with functionalities that
can effectively link the P2 ligand with the transition-state
mimetic amine and adopt favorable conformational and
electronic bias to promote hydrogen bonding and van der
Waals interactions in the active site. Herein we report our
design of a new class of potent HIV-1 protease inhibitors
incorporating stereochemically defined acetamide-based bis-
tetrahydrofuran and hexahydro-methanofuropyran-derived P2-
ligands. A number of inhibitors exhibited very potent enzyme
a
Reaction conditions: (a) DMP, Na2HPO4, CH2Cl2, 23 °C, 3 h; (b)
NaH, (EtO)2P(O)CH2CO2Et, THF, 0 to 23 °C, 4 h (71% over 2-
steps); (c) H2, PtO2 (cat), EtOAc, 23 °C, 2 h, (99%); (d) aq. LiOH,
THF-H2O (1:1) 23 °C, 3 h (96%); (e) pivaloyl chloride, LiCl, Et3N,
(R)-2-benzyl oxazolidinone, THF, 0 to 23 °C, 4 h (72%); (f) pivaloyl
chloride, LiCl, Et3N, 0 °C, then (S)-4-benzyl-2-oxazolidinone, THF, 0
to 23 °C (70%); (g) NaHMDS, THF, −78 °C, 45 min, then MeI,
−78 °C, 4 h (64% for 10; 61% for 12); (h) aq. LiOH, H2O2, 0 to 23
°C, 3 h (87% for 11 and 86% for 13). For ester 15 (71% yield over 3-
steps) and for acid 16 (91%).
a three step sequence. Oxidation of alcohol 5 was carried out
with Dess−Martin periodinane (DMP)30 in CH2Cl2 in the
presence of Na2HPO4 at 23 °C for 3 h to provide ketone. The
resulting ketone was subjected to Horner−Wadsworth−
Emmons reaction31 with sodium hydride and triethyl
phosphonoacetate in THF at 0 to 23 °C for 4 h to afford a
mixture of α,β-unsaturated ester in 71% yield over two steps.
The resulting unsaturated ester was hydrogenated with a
catalytic amount of PtO2 under a hydrogen-filled balloon at 23
°C for 2 h to provide near quantitative yield of the saturated
ester 6 (91:9 mixture, 71% yield over 3-steps). The mixture
B
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could not be separated by column chromatography.
Saponification of the diastereomeric mixture of ester 6 with
aqueous LiOH in THF at 0 to 23 °C for 3 h furnished
carboxylic acid ligand 7 in 96% yield. Carboxylic acid 7 was
utilized to incorporate methyl group to make van der Waals
interactions in the active site of HIV-1 protease.
Scheme 2. Synthesis of Carboxamide-Derived Inhibitor 4a−
a
d
Thus, carboxylic acid 7 was converted to carboxamide 8 as
follows.32 Commercially available (R)-4-benzyl-2-oxazolidi-
none was mixed with acid 7 and LiCl in THF. The resulting
lithio derivative was reacted with pivaloyl chloride and Et3N at
0 to 23 °C for 4 h to provide oxazolidinone derivative 8 in 72%
yield. Oxazolidinone derivative 9 with (S)-4-benzyl-2-oxazoli-
dinone was prepared in a similar manner. For stereoselective
incorporation of methyl group, oxazolidinone derivative 8 was
treated with NaHMDS in THF at −78 °C for 45 min to
provide the corresponding sodium enolate. Reaction of the
enolate with methyl iodide at −78 °C for 4 h furnished
methylated product 10 in 64% yield.33 The corresponding
diastereomeric oxazolidinone 12 afforded alkylation product 9
in 61% yield.34 The removal of the oxazolidinone group was
carried out by exposure to lithium hydroperoxide at 0 to 23 °C
for 3 h to provide methylated ligand carboxylic acids 11 and 13
in 87% and 86% yields, respectively. The synthesis of
carboxylic acid ligand containing crown-THF scaffold was
carried out from the known optically active alcohol 14.7,8 This
was converted to ethyl ester derivative in a three step process
involving (1) DMP oxidation of alcohol 14 to the
corresponding ketone; (2) Horner−Emmons reaction of the
resulting ketone with triethyl phosphonoacetate to provide the
corresponding α,β-unsaturated ester; and (3) hydrogenation of
α,β-unsaturated ester using 10% Pd/C as the catalyst to
provide 15 in 71% yield over 3-steps. Saponification of ethyl
ester 15 using aqueous LiOH in THF furnished optically active
ligand acid 16 in 91% yield.
a
Reaction conditions: (a) acid 7, HATU, DIPEA, DMF, 23 °C, 24 h,
(77% for 4a and 79% for 4b); (b) acid 11 or acid 13, HATU, DIPEA,
DMF, 23 °C, 24 h (65% for 4c and 69% for 4d).
The synthesis of various inhibitors containing bis-tetrahy-
drofuranyl acetamide as the P2 ligand is shown in Scheme 2.
The known7,8 4-methoxybenzenesulfonamide 17 and 4-amino-
benzenesulfonamide 18 isosteres were reacted with carboxylic
acid 7 in the presence of HATU, DIPEA in DMF at 23 °C for
24 h to provide carboxamide inhibitors 4a and 4b in 77% and
79% yield, respectively. For the synthesis of inhibitors 4c and
4d, the known7 amine 19 was coupled with acids 11 and 13
using HATU and DIPEA as described above to provide
inhibitors 4c and 4d in 65% and 69% yield, respectively. The
synthesis of various inhibitors containing crown-THF-derived
acetamide as the P2 ligand is shown in Scheme 3. Amines 17,
18, 19, and 20 were reacted with optically active carboxylic
acid 16 in the presence of HATU, DIPEA in DMF at 23 °C as
described above affording carboxamide inhibitors 4e, 4f, 4g,
and 4h in very good yields.
Scheme 3. Synthesis of Carboxamide-Derived Inhibitors
4e−g
a
We have evaluated various carboxamide derivatives in Table
1 in both enzyme inhibitory and antiviral assays. HIV-1
protease inhibitory Ki was determined using the procedure
described by Toth and Marshall.35 Antiviral IC50 values were
determined using MT-2 human T-lymphoid cells exposed to
36,37
HIV-1LAI
.
Interestingly, both bis-tetrahydrofuranylaceta-
mide derivatives 4a and 4b displayed significantly reduced
HIV-1 protease inhibitory activity (Ki values of 0.28 nM and
17.4 nM, respectively) compared to darunavir (1) or its
methoxy derivative 2 (entries 1 and 2) which showed Ki of 14
pM. Both carboxamide derivatives 4a and 4b did not show any
appreciable antiviral IC50 value, while darunavir displayed
antiviral IC50 value of 3.6 nM in the same assay. We then
a
Reaction conditions: (a) acid 16, HATU, DIPEA, DMF, 23 °C, 24 h
(73% for 4e, 77% for 4f, 70% for 4g, and 68% for 4h).
evaluated the effect of methyl group on the carboxamide
ligand. Compound 4c with a (R)-methyl substituent on the P2
C
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The resulting inhibitor 4e with a 4-methoxy sulfonamide as the
P2′-ligand exhibited HIV-1 protease inhibitory Ki of 32 pM
and antiviral IC50 value of 2.9 nM, which are comparable to the
corresponding carbamate-derived inhibitors. Inhibitor 4f with
4-aminosulfonamide as the P2′-ligand also showed very potent
enzyme Ki as well as antiviral activity similar to darunavir and
its methoxy derivatives (1 and 2). Combination of crown-THF
containing acetamide derivative as the P2 ligand and
cyclopropylamino benzothiazole as the P2′-ligand resulted in
inhibitor 4g which displayed with very potent Ki value as well
as antiviral IC50 value (entry 7). We then examined the
combined effect of the difluorophenylmethyl group as the P1-
ligand in inhibitor, crown-THF-derived acetamide as the P2
ligand, and a cyclopropylaminobenzothiazole P2′-ligand. The
resulting inhibitor 4h displayed very potent activity with a Ki
value of 30 pM and antiviral IC50 value of 79 pM (entry 8).
Based upon the antiviral data in preliminary studies, we then
selected inhibitors 4e and 4h for further evaluation against a
panel of highly multidrug-resistant HIV-1 variants that had
been selected in vitro with widely used FDA-approved HIV-1
protease inhibitors, LPV, ATV, and DRV. Each of these HIV-1
variants were selected in vitro by propagating HIV-1NL4−3 or
HIV8MIX, a mixture of eight multidrug resistant clinical isolates,
as starting viral populations in the presence of increasing
concentrations of each drug (up to 5 μM) in MT-4 cells as
reported by us previously.36,38 The results are shown in Table
2. As can be seen, two current clinically used protease inhibitor
drugs, LPV and ATV, lost significant activity against three
highly PI-resistant HIV-1 variants examined. DRV displayed
relatively better results; however, it too failed to effectively
block replication of highly DRV-resistant HIV-1 variants.
Inhibitor 3 containing a carbamate-linked crown-THF P2-
ligand, an aminobenzotriazole P2′-ligand, and a difluorophe-
Table 1. HIV-1 Protease Inhibitory and Antiviral Activity of
4a−h
nylmethyl as the P1-ligand showed variants tested except for
R
highly DRV-resistant HIVDRV
variant selected in the
P51
presence of DRV over 51 viral passages. Inhibitor 4e
containing carboxamide-linked P2-crown-THF ligand and 4-
methoxysulfonamide as the P2′-ligand showed comparable
antiviral activity to DRV. Inhibitor 4h containing a
carboxamide-linked crown-THF P2-ligand, an aminobenzo-
triazole P2′-ligand and a difluorophenylmethyl as the P1-ligand
also maintained superior activity against all HIV-1 variants
comparable to one of our most potent carbamate-derived
a
Ki (HIV-1 protease) values represent at least 5 data points. Standard
error in all cases was less than 7%. Darunavir exhibited Ki = 16 pM.
inhibitor 3. Carboxamide derived inhibitor 4h exerted
b
R
IC50 (HIV-1LAI) values are means of at least three experiments.
exceptionally potent antiviral activity against HIV-1LPV
,
5 μM
R
R
Standard error in all cases was less than 5%. Darunavir exhibited
antiviral IC50 = 3.2 nM, saquinavir IC50 = 21 nM; inhibitor 3, Ki = 14
pM, IC50 = 17 pM.
HIV-1ATV 5 μM, and HIV-1DRV
with IC50 values ranging
P20
from 1.1 to 5.4 pM. Furthermore, against highly DRV-resistant
R
P30
R
P51,
variants, HIV-1DRV
and HIVDRV
inhibitor 4h main-
tained antiviral IC50 values of 39 pM and 14 nM, respectively.
Our X-ray crystallographic studies of inhibitor 4h-bound HIV-
1 protease provided molecular insight into the carboxamide-
derived inhibitor’s potency.
ligand and aminobenzothiazole as the P2′-ligand showed
reduction of Ki value compared to unsubstituted compound
4a. Incorporation of methyl group did not improve antiviral
activity of (R)-methyl derivative 4a. Inhibitor 4d with a (S)-
methyl substituent on the P2 ligand however, showed
improvement in both Ki and antiviral activity (entry 4)
compared to its stereoisomeric derivative 4c (entry 4). We
have shown that the crown-THF ligand maintained similar
backbone hydrogen bonding interactions as bis-THF ligand. In
addition, crown-THF forms enhanced van der Waals
interactions in the active site. We therefore, investigated
inhibitors incorporating crown-THF-derived carboxamide
derivatives as the P2 ligand. In inhibitor 4e, we incorporated
crown-THF containing acetamide derivative as the P2 ligand.
We determined high resolution X-ray crystal structure of
inhibitor 4h-bound wild-type HIV-1 protease. The X-ray
structure was determined at the near-atomic resolution of 1.18
Å to establish accurate protein−inhibitor interactions.39 The
complex crystallized in orthorhombic space group P21212 with
one protease homodimer per asymmetric unit. The inhibitor
binds in the active site in two alternate conformations related
by 180° rotation with relative occupancy of 0.55/0.45. The two
alternate conformations are very similar so only the major one
will be described here. Inhibitor 4h contains a crown-THF-
derived carboxamide P2 ligand. It is similar to inhibitor 3
D
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Table 2. Antiviral Activity of Compounds 4e and 4g against a Panel of Multidrug-Resistant HIV-1 Variants
ab
,
IC50 (nM) SD
Virus species
LPV
2
>1,000
340 44
>1,000
>1,000
>1,000
ATV
5.0
309 25
>1,000
>1,000
>1,000
>1,000
DRV
Compound 3
Compound 4e
Compound 4h
HIV-1NL4-3
27
1
4.6 0.5
50 13
11 1.2
316 41
424 100
>1,000
0.0042 0.00006
0.00018 0.00006
0.0025 0.0003
0.00055 0.00007
0.0047 0.0004
5.0 0.4
1.0 0.04
88 22
9.0 1.4
299 23
425 108
>1,000
0.0045 0.0004
0.0011 0.0004
0.0054 0.0006
0.0040 0.0001
0.039 0.012
14 1.2
R
HIV-1LPV
5 μM
R
HIV-1ATV
5 μM
R
HIV-1DRV
HIV-1DRV
P20
R
P30
R
HIV-1DRV
P51
a
The amino acid substitutions identified in the protease-encoding region compared to the wild-type HIV-1NL4−3 were L23I, E34Q, K43I, M46I,
I50L, G51A, L63P, A71V, V82A, and T91A in HIV-1ATV‑5 μM; L10F, V32I, M46I, I47A, A71V, and I84V in HIV-1LPV‑5 μM; L10I, I15V, K20R, L24I,
R
V32I, M36I, M46L, L63P, V82A, and L89M in HIV-1DRV P20; L10I, I15V, K20R, L24I, V32I, M36I, M46L, L63P, K70Q, V82A, I84V, and L89M
b
R
R
in HIV-1DRV P30; L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82I, I84V, and L89M in HIV-1DRV P51. The IC50 (50%
inhibitory concentration) values were determined by using MT-4 cells as target cells. MT-4 cells (105/mL) were exposed to 100 TCID50s of each
HIV-1, and the inhibition of p24 Gag protein production by each drug was used as an end point. All assays were conducted in duplicate, and the
data shown represent mean values ( S.D.) derived from the results of two independent experiments. LPV, lopinavir; ATV, atazanavir; DRV,
darunavir.
Figure 2. (A) Inhibitor 4h-bound X-ray structure of HIV-1 protease. The major orientation of the inhibitor is shown. The inhibitor carbon atoms
are shown in green, the water molecule is a red sphere, and the hydrogen bonds are indicated by dotted lines. (B) The van der Waals surface and
interactions of the fluorinated P1 ligand are shown (PDB ID: 6VCE).
Figure 3. Side by side comparison of the new carboxamide P2-ligand containing Crown-THF moiety of inhibitor 4h (A, green carbon chain) with
the urethane Crown-THF moiety of inhibitor 3 (B, cyan carbon chain) inside the S2 subpocket. Both ligands form extensive van der Waals
interactions (Val32 and Ile47) in the S2 subsite. Also, they are located close to the periphery of the protease active site and form three strong
hydrogen bonds in a similar fashion (black dotted lines).
except that the P2 urethane linker has been replaced with a
carboxamide linker. The overall dimer structure is similar to
inhibitor 3-HIV-1 protease complex8 with a low RMSD of 0.13
Å for 198 equiv Cα atoms. The largest deviation of 0.6 Å
between the two structures occurs at residue Gly49′ of the flap
region. As highlighted in Figure 2, all major hydrogen bonds
E
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observed between inhibitor 3 and the backbone atoms of
protease are retained in the structure of 4h-bound HIV-1
protease.
Synthesis of inhibitors, full NMR spectroscopic data for
all final compounds, X-ray structural data for inhibitor
4h-bound HIV-1 protease, molecular formula strings
and some data, and Virus and cell biology protocols
(PDF)
The two fluorine atoms in the P1 group of inhibitor 4h
preserve the important halogen bond interactions with the
protease seen for the structure of 3-bound HIV-1 protease.
One fluorine atom forms a halogen bond with the amide NH
of Ile50. The second fluorine atom forms multipolar
interactions with one conformation of the guanidium group
of Arg8′, which is involved in a critical intersubunit ion pair
with Asp29. Both alternate conformations of the guanidium
group of Arg8′ preserve the ion pair with Asp29. Compared to
the urethane linker of inhibitor 3, the C atom in the
carboxamide linker of inhibitor 4h shifts toward Cδ1 of Ile84
by almost 0.9 Å and forms a new van der Waals interaction.
The positions of nearby atoms from the carbonyl group in the
linker to the whole crown-like P2 group also shift and
consequently alter the van der Waals interactions of inhibitor
with Gly49 and Ile50. The semirigid crown-like P2 group
retains the shape and relative positions of P2 group of inhibitor
3. However, inhibitor 4h may show slight improvement in
interactions with protease. We have compared hydrogen
bonding and van der Waals interactions of the crown-THF
urethane ligand of inhibitor 3 with crown-THF-derived
acetamide ligand of inhibitor 4h in the X-ray structures. As
shown in Figure 3, the hydrogen bonding distances of acetal
oxygens with Asp29 and Asp30 backbone amide NHs are
nearly identical. All van der Waals interactions with Ile47 and
Val32 are also very similar, although inhibitor 4h forms a
stronger van der Waals contact with Val32. Furthermore,
aminobenzothiazole P2′ ligands form similar hydrogen
bonding with Asp30′ backbone amide NH and van der
Waals contacts in the S2′ subsite. These extensive active site
interactions are responsible for the inhibitor’s high affinity for
HIV-1 protease as well as its potent antiviral activity against
multidrug-resistant HIV-1 strains.
Accession Codes
Inhibitor 4h-bound HIV-1 protease X-ray structure is 6VCE.
Authors will release the atomic coordinates and experimental
data upon article publication.
AUTHOR INFORMATION
Corresponding Author
■
Arun K. Ghosh − Department of Chemistry and Department of
Medicinal Chemistry, Purdue University, West Lafayette,
Indiana 47907, United States; orcid.org/0000-0003-2472-
1841; Email: akghosh@purdue.edu
Authors
Alessandro Grillo − Department of Chemistry and Department
of Medicinal Chemistry, Purdue University, West Lafayette,
Indiana 47907, United States
Jakka Raghavaiah − Department of Chemistry, Purdue
University, West Lafayette, Indiana 47907, United States
Satish Kovela − Department of Chemistry, Purdue University,
West Lafayette, Indiana 47907, United States
Megan E. Johnson − Department of Chemistry, Purdue
University, West Lafayette, Indiana 47907, United States
Daniel W. Kneller − Department of Biology, Georgia State
University, Atlanta, Georgia 30303, United States
Yuan-Fang Wang − Department of Biology, Georgia State
University, Atlanta, Georgia 30303, United States
Shin-ichiro Hattori − Department of Refractory Viral Infections,
National Center for Global Health and Medicine Research
Institute, Tokyo 162-8655, Japan
Nobuyo Higashi-Kuwata − Department of Refractory Viral
Infections, National Center for Global Health and Medicine
Research Institute, Tokyo 162-8655, Japan
In conclusion, we investigated the replacement of urethane
functionality of darunavir with designed carboxamide deriva-
tives that can maintain key backbone binding interactions in
the HIV-1 protease active site. Compounds with bis-
tetrahydrofuran-derived carboxamide resulted in substantial
reduction of potency compared to the corresponding
carbamate derivatives. However, incorporation of crown-
THF-derived carboxamide derivatives provided very potent
inhibitors. In particular, compound 4h is remarkably potent (Ki
= 30 pM; IC50 = 79 pM), several orders of magnitude more
potent than the latest approved PI drug, darunavir. Inhibitor
4h also maintained very potent antiviral activity against a wide
spectrum of multidrug-resistant HIV-1 variants. The observed
antiviral activity of compound 4h is superior to darunavir. An
inhibitor 4h-bound HIV-1 protease X-ray structure revealed
extensive interactions of the inhibitor in active site.
Particularly, the compound maintained a network of hydrogen
bonding interactions with the backbone amide NHs in the
active site. Further optimization of inhibitor’s properties is in
progress.
Irene T. Weber − Department of Biology, Georgia State
University, Atlanta, Georgia 30303, United States;
orcid.org/0000-0003-4876-7393
Hiroaki Mitsuya − Department of Refractory Viral Infections,
National Center for Global Health and Medicine Research
Institute, Tokyo 162-8655, Japan; Experimental Retrovirology
Section, HIV and AIDS Malignancy Branch, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland
20892, United States; Department of Clinical Sciences,
Kumamoto University Hospital, Kumamoto 860-8556, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00670
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (Grant AI150466, AKG and Grant AI150461, ITW)
and a Georgia State University Molecular Basis of Disease
fellowship (DWK). X-ray data were collected at the Southeast
Regional Collaborative Access Team (SER-CAT) beamline
22ID at the Advanced Photon Source, Argonne National
Laboratory. Use of the Advanced Photon Source was
supported by the US Department of Energy, Basic Energy
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00670.
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Letter
(11) 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.
(12) 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.
Sciences, Office of Science, under Contract No. W-31-109-
Eng-38. This work was also supported by the Intramural
Research Program of the Center for Cancer Research, National
Cancer Institute, National Institutes of Health (HM), and in
part by grants for the promotion of AIDS research from the
Ministry of Health; grants from Welfare and Labor of Japan
(HM); grants for the Research Program on HIV/AIDS from
the Japan Agency for Medical Research and Development
(AMED) under grant numbers JP15fk0410001 and
JP16fk0410101 (HM); a grant from the National Center for
Global Health and Medicine (NCGM) Research Institute
(HM); and a Grant-in-Aid for Scientific Research (Priority
Areas) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (Monbu Kagakusho)(HM).
The authors would like to thank the Purdue University Center
for Cancer Research, which supports the shared NMR and
mass spectrometry facilities. The authors also thank Manabu
Aoki for discussion.
(13) 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.
́
(14) de Bethune, M. P.; Sekar, V.; Spinosa-Guzman, S.; Vanstockem,
M.; De Meyer, S.; Wigerinck, P.; Lefebvre, E. Darunavir (Prezista,
TMC114): From bench to clinic, improving treatment options for
HIV-infected patients. In Antiviral Drugs: From Basic Discovery
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(15) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.;
Devasamundram, T.; Kincaid, J. F.; Boross, P.; Wang, Y. F.; Tie, Y.;
Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber, I. T.; Ghosh, A. K.;
Mitsuya, H. Novel bis-tetrahydrofuranylurethane-containing non-
peptidic protease inhibitor (PI) UIC-94017 (TMC114) with potent
activity against multi-PI-resistant human immunodeficiency virus in
vitro. Antimicrob. Agents Chemother. 2003, 47, 3123−3129.
ABBREVIATIONS
■
ATV, atazanavir; bis-THF, bis-tetrahydrofuran; cART, com-
bined antiretroviral therapies; DIPEA, N,N-diisopropyletyl-
amine; DRV, darunavir; HATU, hexafluorophosphate azaben-
zotriazole tetramethyl uronium; HMDS, hexamethyldisilazane;
LPV, lopinavir
(16) De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.;
́
Pauwels, R.; Wigerinck, P.; de Bethune, M. P. TMC114, a novel
human immunodeficiency virus type 1 protease inhibitor active
against protease inhibitor-resistant viruses, including a broad range of
clinical isolates. Antimicrob. Agents Chemother. 2005, 49, 2314−2321.
(17) Koh, Y.; Matsumi, S.; Das, D.; Amano, M.; Davis, D. A.; Li, J.;
Leschenko, S.; Baldridge, A.; Shioda, T.; Yarchoan, R.; Ghosh, A. K.;
Mitsuya, H. Potent inhibition of HIV-1 replication by novel non-
peptidyl small molecule inhibitors of protease dimerization. J. Biol.
Chem. 2007, 282, 28709−28720.
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