Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands

Article abstract of DOI:10.1016/j.bmcl.2017.09.003

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (K_i = 13.2 nM, IC₅₀ = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (K_i = 62 pM and 14 pM, respectively) and antiviral activity (IC₅₀ = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

Full text of DOI:10.1016/j.bmcl.2017.09.003

Accepted Manuscript
Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic
HIV-1 protease inhibitors involving the P1′-P2′ ligands
Arun K. Ghosh, W. Sean Fyvie, Margherita Brindisi, Melinda Steffey, Johnson
Agniswamy, Yuan-Fang Wang, Manabu Aoki, Masayuki Amano, Irene T.
Weber, Hiroaki Mitsuya
PII:
S0960-894X(17)30892-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.003
BMCL 25267
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
28 July 2017
3 September 2017
Please cite this article as: Ghosh, A.K., Sean Fyvie, W., Brindisi, M., Steffey, M., Agniswamy, J., Wang, Y-F.,
Aoki, M., Amano, M., Weber, I.T., Mitsuya, H., Design, synthesis, X-ray studies, and biological evaluation of novel
macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.003
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of novel macrocyclic HIV-1 protease inhibitors involving
P1′-P2′-ligands
∗
Arun K. Ghosh,a,b W. Sean Fyvie,a Margherita Brindisi,a Melinda Steffey,a Johnson Agniswamy,c Yuan-Fang
Wang,c Manabu Aoki,d Masayuki Amano,d Irene T. Weber,c and Hiroaki Mitsuya d,e,f
aDepartment of Chemistry, Purdue University, West Lafayette, IN 47907; bDepartment of Medicinal Chemistry, Purdue University, West Lafayette, IN
c
47907 (USA); Departments of Biology and Chemistry, Georgia State University, Atlanta, GA 30303 (USA); dDepartments of Hematology and Infectious
e
Diseases, Kumamoto University of Medicine, Kumamoto 860-8556 (Japan); Experimental Retrovirology Section, HIV and AIDS Malignancy Branch
f
National Cancer Institute, Bethesda, MD 20892 (USA); Center for Clinical Sciences, National Center for Global Health and Medicine, Shinjuku, Tokyo
162-8655 (Japan).

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design, synthesis, X-ray studies, and biological evaluation of novel
macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands
Arun K. Ghosh,a,b ∗ W. Sean Fyvie,a Margherita Brindisi,a Melinda Steffey,a Johnson Agniswamy,c Yuan-
Fang Wang,c Manabu Aoki,d Masayuki Amano,d Irene T. Weber,c and Hiroaki Mitsuya d,e,f
aDepartment of Chemistry, Purdue University, West Lafayette, IN 47907; bDepartment of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907
(USA); cDepartments of Biology and Chemistry, Georgia State University, Atlanta, GA 30303 (USA); dDepartments of Hematology and Infectious Diseases,
e
Kumamoto University of Medicine, Kumamoto 860-8556 (Japan); Experimental Retrovirology Section, HIV and AIDS Malignancy Branch National Cancer
Institute, Bethesda, MD 20892 (USA); fCenter for Clinical Sciences, National Center for Global Health and Medicine, Shinjuku, Tokyo 162-8655 (Japan).
ARTICLE INFO
ABSTRACT
Article history:
Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse
Received XX Month 2017
Revised XX Month 2017
Accepted XX Month 2017
Available online XX Month 2017
flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived
macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50
=
22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic
inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62
pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively).
Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.
Keywords:
HIV protease
2009 Elsevier Ltd. All rights reserved
.
Drug resistance
P1′- P2′ ligands
Macrocyclic inhibitors
Structure-based design
———
∗ Corresponding author. Tel.: +1-765-494-5323; fax: +1-765-496-1612; e-mail: akghosh@purdue.edu (A. K. Ghosh).

The introduction of combined active antiretroviral treatment
(cART) in late nineteen-nineties marked the beginning of a
breakthrough treatment for patients with HIV infection and
AIDS.1,2 cART treatment regimens with protease inhibitors and
reverse transcriptase inhibitors dramatically improved HIV-
related disease progression and mortality.3,4 The cART is not a
cure, however, it has significantly improved quality of life and
transformed the HIV/AIDS pandemic to a manageable chronic
ailment with normal life expectancy.5,6 The impact of cART is
remarkable, however, current cART suffers from a number of
major drawbacks. The most concerning is the rapid emergence of
drug-resistant HIV-1 variants making cART ineffective for some
HIV/AIDS patient groups.7-,9 Current patients who achieve initial
viral suppression may ultimately experience treatment failure.10,11
Furthermore, it has been suggested that these drug-resistant
variants can be transmitted to new individuals. The ability to
provide long-term cART benefits remains a complex issue. HIV
protease inhibitors (PIs) are critical components of cART
regimens particularly for salvage treatments. Therefore, design
and development of new, more potent, safer therapeutics with
high genetic barrier against HIVs acquisition of drug resistance
are very important.
Our laboratories have been involved in the design and
synthesis of nonpeptide PIs that are active against HIV-1 variants
resistant to the currently approved PIs.12-14 One of the PIs is
darunavir (DRV, 1), an FDA approved first-line therapeutic agent
for the treatment of HIV/AIDS patients.15,16 DRV contains a
structure-based designed privileged template, (3R,3aS,6aR-bis-
tetrahydrofuranyl urethane (bis-THF) as the P2 ligand imbedded
in a hydroxyl ethylamine sulfonamide isostere (1, Figure 1).14,17
DRV showed a high genetic barrier, to acquire drug-resistance
associated mutations.18-20 One of our key design strategies is to
promote the extensive network of hydrogen bonding interactions
with the active site backbone atoms of HIV-1 protease.13,17
Based upon these strategies, we developed a range of PIs with
broad-spectrum activity against multidrug-resistant HIV-1
variants.2,17
In an alternative approach to develop PIs with broad-spectrum
activity, we have designed a number of macrocyclic PIs with
exceptional antiviral activity and drug-resistance profiles.21-24
Among them, we have reported macrocyclic inhibitors typified
by 13-membered unsaturated derivatives 3 and 4, modifying P1′-
P2′-ligands of darunavir-like PIs (Figure 1). Both geometrical
isomers displayed excellent inhibitory potency as well as
antiviral activity. The corresponding saturated derivatives are
significantly less potent. The rationale underlying the design of
these inhibitors is based on crystallographic data and modeling
studies indicating decreased van der Waals interactions and
inhibitor side-chain repacking across representative mutant
strains in the vicinity of the S1’ subsite area.25,26 In this context,
the introduction of specific heterocyclic scaffolds or heteroatoms
on the P1′-P2′ tether of the macrocycle could allow effective
inhibitor adaptation to a range of side chain mutations. With the
aim of developing novel broad-spectrum HIV-1 PIs we explored
the combination of a flexible macrocyclic P1′-P2′ tether with the
pyrrolidinone ring as the potential source for additional backbone
interactions. Furthermore, we sought to investigate the outcome
of a small set of flexible macrocycles incorporating suitably
functionalized nitrogen and oxygen heteroatoms to interact with
the backbone residues. Herein, we report design, synthesis and
biological results of a series of potent macrocyclic HIV-1
protease inhibitors.
Based upon our previous studies, our current design plan was
to further explore 13- and 14-membered macrocycles.21 Early
approaches of the work focused on the incorporation of 2-
pyrrolidinone heterocycles with (S)- and (R)-configuration to
promote hydrogen bonding interaction with Gly27 backbone
amide NH.27 Furthermore, we planned to incorporate N-methyl
sulfonamide and alkyl ether functionalities to promote
interactions with backbone atoms in the active site. The synthesis
of the pyrrolidinone-containing macrocyclic inhibitors are
described
in
Scheme
1.
Commercially
available
allylpyrrolidinone 6a was converted to the corresponding tosylate
derivative by treatment with tosyl chloride and triethylamine in
the presence of a catalytic amount of DMAP in CH2Cl2 at 23 °C.
The resulting tosylate was reacted with NaH and di-tert-butyl
iminodicarboxylate in DMF at 0 °C. The resulting mixture was
then heated at 65 °C for 12 h to provide Boc-derivative 7a in
excellent yield. Exposure of 7a to trifluoroacetic acid (TFA) at 23
°C provided the amine 8a in 82% yield. Reaction of amine 8a
with commercially available optically active oxirane 9 in
isopropanol at 60 °C for 24 h provided Boc-aminoalcohol
derivative 10a in good yield. Amine 10a was reacted with the
known21 sulfonyl chloride 11a in CH2Cl2 in the presence of
aqueous NaHCO3 solution at 23 °C to provide diene derivative
12a. Exposure of diene 12a to ring closing metathesis (RCM)
using Grubb’s second-generation catalyst (Grubbs II) (5 mol%)
in CH2Cl2 at 23 °C for 14 h afforded macrocyclic derivative 13a
as a E/Z mixture nearly 60:40 by HPLC analysis. Removal of the
Boc group by treatment with TFA provided the corresponding
amine. The reaction of the resulting amine with activated
carbonate derivative 14 afforded a mixture of unsaturated
derivatives. These E/Z isomers were separated by HPLC using a
reverse phase C18 column to provide the pure E- and Z-isomers
5a and 5b, respectively. Catalytic hydrogenation of E/Z mixture
in the presence of 10% Pd-C in ethyl acetate under a hydrogen-
filled balloon afforded the saturated inhibitor 5c in good yield.
The corresponding saturated derivative 5d containing (S)-
pyrrolidinone was synthesized following the same sequence of
reactions using (S)-allylpyrrolidinone 6b as the starting material.
Figure 1. Structure of inhibitors 1-4 and general structures of macrocyclic
inhibitors 5a-o.

macrocyclic inhibitors 5e-h as follows. The acyclic dienes 22-25
were exposed to RCM using Grubbs’ 2nd generation catalyst16 to
give the corresponding unsaturated macrocyclic derivatives. The
Boc-group was deprotected using TFA and the resulting amines
were reacted with the mixed carbonate of activated bis-THF
derivative 14 providing the corresponding unsaturated
macrocyclic derivatives. The resulting unsaturated compounds
were hydrogenated over 10% Pd-C as catalyst to yield inhibitors
5e-h.
Scheme 1. Reagents and conditions: (a) p-TsCl, DMAP, Et3N, CH2Cl2, 16 h,
(R) 63%, (S) 74%; (b) NHBoc2, NaH, dry DMF, 0 °C to 65 °C, 12 h, (R)
90%, (S) 93%; (c) TFA, CH2Cl2, 23 °C, 13 h, (R) 82%, (S) 75%; (d) 9,
iPrOH, 60 °C, 24 h, (R) 56%, (S) 57%; (e) 11a, aqueous NaHCO3, CH2Cl2, 23
° C, 14 h, (R) 55%, (S) 80%; (f) Grubbs II, dry CH2Cl2, 23 °C, 12 h, (R) 64%
(E/Z mixture 57:43 by HPLC), (S) 53%,(E/Z mixture); (g) TFA, CH2Cl2, 23
°C, 8 h; (h) 14, N,N-DIPEA, MeCN, 23 °C, 5 days, (R) 64%, (S) 92%, (E/Z
mixture); (i) H2, 10 % Pd/C, EtOAc, 23 °C, 12 h, (R) 73%, (S) 89%.
Scheme 2. Reagents and conditions: (a) MsCl, aq. NaHCO3, CH2Cl2, 23 °C,
16 h, 86%; (d) LiOH.H2O, aq. THF, 23 °C, 18 h, 95%; (c) DPPA, Et3N,
toluene, 23 °C to reflux, 5 h then aq. NaOH, 98 °C 12 h, 67%; (d) 9, i-PrOH,
56 °C, 14 h, 56-63%; (e) 11a or 11b, aq. NaHCO3, CH2Cl2, 23 °C, 14 h, 90-
98%; (f) Grubbs II, dry CH2Cl2, 40 °C, 3-6 h, 52-92% (E/Z mixture); (g)
TFA, CH2Cl2, 23 °C, 48 h; (h) 14, DIPEA, MeCN, 23 °C, 3-7 days, 23-60%;
(i) H2, 10 % Pd/C, EtOAc, 23 °C, 12 h, 75-90%.
The synthesis of heteroatom-containing macrocyclic
inhibitors is described in Scheme 2. Commercially available
methyl allylaminopropionate 16 was reacted with MsCl in
CH2Cl2 in the presence of aqueous NaHCO3 at 23 °C to provide
the corresponding mesylate derivative. Saponification of the
methyl ester with LiOH in aqueous THF at 23 °C furnished
carboxylic acid 17. Curtius rearrangement of acid 17 with
diphenylphosphoryl azide (DPPA) in the presence of
triethylamine in toluene afforded amine 18 in good yield. For
incorporation of ether functionality within the macrocycle, we
utilized commercially available allyloxyethylamine 19. Both
amines 18 and 19 were reacted with commercially available
optically active oxirane 9 in isopropanol at 56 °C for 14 h to
provide Boc-aminoalcohol derivatives 20 and 21, respectively.
Amine 20 was then reacted with known21 sulfonyl chlorides 11a
and 11b in CH2Cl2 in the presence of aqueous NaHCO3 solution
at 23 °C to afford diene sulfonamide derivatives 22 and 23,
respectively. Similarly, reaction of amine 21 with sulfonyl
chlorides 11a and 11b furnished diene sulfonamide derivatives
24 and 25 in excellent yields. Dienes 22-25 were converted
Based upon X-ray structure of our previous macrocyclic
inhibitor-bound HIV-1 protease, we sought to investigate
macrocycles with isomeric benzyl ether oxygen which would be
within proximity to form hydrogen bonds with backbone atoms
in the S2’-site. These benzyl ether derivatives may exhibit better
stability than the phenyl ether derivatives. The synthesis of these
benzyl ether-derived macrocyclic inhibitors is described in
Scheme 3. Preparation of sulfonyl chloride 27 was carried out
from commercially available 3-allyloxymethylanisole 26 as
described by Blotny and co-workers28 by treatment with
chlorosulfuric acid at 0 °C followed by reaction of the resulting
sulfonic acid with cyanuric chloride in dry acetone in the
presence of triethylamine to provide sulfonyl chloride 27 in 37%
yield. For the synthesis of benzyl ether derivative with 4-amino
substitution, the corresponding sulfonyl chloride derivative 29
was prepared from commercially available 2-chloro-5-
nitrobenzyl alcohol 28. Reaction of alcohol with NaH in the

presence of TBSCl in dry THF provided the TBS ether. This was
converted to the corresponding thiophenol derivative by reaction
with sodium disulfide, freshly prepared from sodium sulfide and
elemental sulfur in ethanolic solution in the presence of NaOH to
provide a mixture of the corresponding thiol along with its
oxidized disulfide derivative.29 Oxidation of this mixture by a
combination of N-chlorosuccinimide and dilute hydrochloric acid
in MeCN afforded the corresponding sulfonyl chloride 29 in
moderate yield.30 Commercially available 5-hexenylamine and
6-heptenylamine 30 and 31 were reacted with chiral epoxide 9
derivative 27 afforded the corresponding diene sulfonamide
derivatives 34 and 35. For the synthesis of diene 36, sulfonamide
intermediate derived from sulfonyl chloride 29 was subjected to
n-Bu4N+F- in THF. The resulting alcohol was subjected to O-
allylation with allyl-tert-butylcarbonate in the presence of
catalytic Pd(PPh3)4 to provide 36. For the synthesis of the
unsaturated inhibitors with p-OMe sulfonamides 5i-l, diene
derivatives 34 and 35 were exposed to RCM to provide the
corresponding unsaturated macrocycles with E/Z mixtures
(approximately 1:2 E/Z ratio). Deprotection of Boc-group with
TFA followed by reaction of the resulting amines with activated
bis-THF derivative 14 furnished unsaturated inhibitors as a
mixture of E/Z isomers. The mixture of isomers were separated
by HPLC using a C18 column to furnish macrocyclic inhibitors
5i-5l (34-60% yield). Diene sulfonamide with a p-NO2 group
was converted to macrocyclic inhibitors 37 by similar sequence
of reactions. Catalytic hydrogenation of unsaturated macrocycles
over 10% Pd-C provided saturated inhibitors 5m and 5n. For the
synthesis of the terminal p-NH2-derived inhibitor 5o, unsaturated
macrocycle 37 was exposed to SnCl2-mediated reduction in
EtOH to afford the corresponding aniline derivative in 82% yield.
Hydrogenation of the resulting olefin mixture over 10% Pd-C
furnished saturated macrocyclic inhibitor 5o.
HIV-1 protease inhibitory potency of all synthesized
inhibitors was evaluated using the assay protocol reported by
Toth and Marshall.31 These results are shown in Tables 1 and 2.
A selected number of compounds was further evaluated in
antiviral assay following a previously published assay protocol
32
using MT-2 cells exposed to HIV-1LAI
.
We first investigated a
set of macrocyclic inhibitors containing both E- and Z-olefins
along with R-pyrrolidinone on the macrocyclic tether to form
backbone hydrogen bonding with Gly27 in the S1’-subsite. We
specifically investigated (R)-pyrrolidinone in inhibitors 5a to 5c
(entries 1-3, Table 1) as this stereochemistry showed enhanced
potency over the (S)-isomer in acyclic inhibitors. As can be seen,
inhibitor 5b with an E-isomer is significantly potent in enzyme
inhibitory assay. However, inhibitor 5a with Z-isomer showed
better antiviral activity. Saturated inhibitor 5c displayed good
enzyme activity however its antiviral activity was not improved
over inhibitor 5a. We have also prepared inhibitor 5d
incorporating
a
(S)-pyrrolidinone derivative. It showed
significant reduction of enzyme Ki as well as antiviral activity.
We previously observed that macrocyclic inhibitors with 13- and
14- membered rings are nicely accommodated by the S1-S2′
subsites. In an effort to promote hydrogen bonding interactions in
this region, we incorporated N-methylsulfonamide functionality.
The corresponding 13- and 14-membered macrocyclic inhibitors
5e and 5f (entries 5 and 6, Table 1) showed reduced enzyme
inhibitory activity compared to the corresponding inhibitors with
carbon chains. The corresponding oxocyclic inhibitors 5g and 5h
(entries 7 and 8, Table 1) showed improved enzyme inhibitory
activity, however, inhibitor 5h did not show appreciable antiviral
activity (IC50 > 1 µM).
The X-ray crystal structure of the inhibitor 3-bound HIV-1
protease revealed that the phenolic oxygen on the macrocycle do
not form any hydrogen bonds in the S1′-subsite. Based upon this
X-ray structure, we envisioned that the corresponding positional
isomer, particularly incorporation of oxygen at the benzylic
position may lead to improved potency as this oxygen would be
within proximity to interact with backbone atoms in the S2′-
subsite. Inhibitors 5i and 5j incorporated the benzyl ether oxygen
within the 13-membered ring cycle with both Z- and E-olefin
(entries 1 and 2, Table 2). Both inhibitors showed excellent
enzyme inhibitory activity. However, the antiviral activity of
both compounds was significantly reduced compared to the
corresponding phenolic ether derivatives 3 and 4. In contrast, the
Scheme 3. Reagents and conditions: (a) HSO3Cl, CH2Cl2, 0 °C, 20 min; (b)
cyanuric chloride, Et3N, dry acetone, 23 °C to 60 °C, 24 h, 37% over 2 steps;
(c) TBSCl, NaH (60% susp. in mineral oil), TBAI, dry THF, 0 °C, 1 h, 90%;
(d) Na2S2 in EtOH, NaOH, EtOH, reflux, 2 h; (e) NCS, 2M HCl in MeCN, -
10 °C to 20 °C, 30 min, 35% over 2 steps; (f) 9, iPrOH, 56 °C, 14 h, 82% for
n = 1, 63% for n = 2; (g) 27 or 29, aqueous NaHCO3, CH2Cl2, 23 °C, 18 h,
85-92%; (h) TBAF, dry THF,
0 °C, 15 min, 78%; (o) allyl-tert-
butylcarbonate, Pd(PPh3)4, dry THF, 60 °C, 3 h; (j) Grubbs II, dry CH2Cl2, 40
°C, 3-6 h, 85-93%; (k) TFA, CH2Cl2, 23 °C, 3 h; (l) 14, DIPEA, MeCN, 23
°C, 8 days, 34-60%; (m) H2, 10% Pd/C, EtOAc, 23 °C, 12 h, 87-90%.
providing epoxide opening products 32 and 33, respectively in
excellent yield. Reaction of these amines with sulfonyl chloride

Inhibitor 5o with
showed excellent enzyme inhibitory potency as well as antiviral
activity (entry 7, Table 2).
a para-aminosulfonamide derivative
Table 1. Structures and activity of inhibitors 5a-h
We selected inhibitors 5m and 5o for further evaluation
against a DRV-resistant HIV-1 variants. These DRV-resistant
R
HIV-1DRV variants are highly resistant to all current clinically
used PIs including DRV and nucleoside/nucleotide reverse
transcriptase inhibitors such as tenofovir. In these assay, MT-4
cells (1 × 104) were exposed to wild-type HIV-1 and a DRV-
R
resistant variant (HIV-1DRV
) and subjected to various
P20
concentrations of each PI. IC50 values were determined using p24
assay.32,33 The results are shown in Table 3.
Inhibitor 5o potently blocked the replication of wild-type
HIV-1NL4 showing improved antiviral activity compared to
3
inhibitor 5m and DRV. Furthermore, this inhibitor suppressed the
R
replication of HIV-1 DRV P20-resistent variant. As can be seen, the
Table 2. Structures and activity of inhibitors 5i-o
14-membered macrocyclic inhibitors with E- and Z-derivatives
(compounds 5k and 5l, entries 3 and 4, Table 2) showed greater
than 20-fold reduction of enzyme inhibitory activity over
inhibitors 5i and 5j. Interestingly, both 13- and 14-membered
saturated macrocyclic inhibitors 5m and 5n showed much
improved enzyme inhibitory activity. Also, inhibitor 5m with 13-
membered macrocyclic e exhibited antiviral IC50 of 5.3 nM.

Table 3. Antiviral activity of inhibitors 5m and 5o against HIV-
1DRV P20 resistant HIV-1 variant.
protease-inhibitor complex reveals interesting new water-
mediated hydrogen bonding interactions of the oxymethyl
oxygen with backbone atoms at the S2′-site. The ring oxygen
makes water-mediated hydrogen bonds with backbone NH of
Asp29′ as well as with the carboxyl oxygen of Gly27′, with
distances ranging from 2.9-3.4 Å. The new backbone binding
with the main chain atoms may be responsible for its ability to
maintain high potency against multidrug-resistant HIV-1
variants.12,17 Inhibitor maintains the water-mediated hydrogen
bonding interactions with Ile50 and Ile50′ amide NHs that are
conserved in the majority of inhibitor-protease complexes.35,36
Furthermore, inhibitor 5j makes weaker C—H···O interactions
throughout the active site of HIV-1 protease.37,38
In summary, we have designed novel HIV-1 protease
inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers
for the HIV-1 protease active site and investigated their
biological activity. We based our rational design upon the
premise that P1′-P2′ tethers in the contrast of a flexible
macrocycle enable effective inhibitor adaptation across a range of
side chain mutations. With the aim of developing broad-spectrum
inhibitors we also planned to establish additional contacts with
key backbone residues. Accordingly, a series of pyrrolidinone-
fused macrocyclic inhibitors have been designed and synthesized,
leading to the identification of inhibitor 5a endowed with a
favorable enzyme inhibitory profile and relevant antiviral
R
mean IC50 in nM (fold-change)a
Cpd
R
HIV-1DRV
HIV-1NL4
P20
3
3.6
1.5
1.8
240 (67)
20 (13)
87 (48)
5m
5o
DRV (1)
aMT-4 cells (1 × 104) were exposed to 50 TCID50 of wild-type HIV-1NL4 3 or
R
HIV-1DRV P20, and cultured in the presence of various concentrations of each
PI, and the IC50 values were determined using the p24 assay. The amino acid
R
substitutions identified in protease of HIV-1DRV P20, compared to HIV-1NL4
3
include L10I/I15V/K20R/L24I/V32I/M36I/M46L/L63P/V82A/L89M. All
assays were conducted in triplicate, and the data shown represent mean values
derived from the results of three independent experiments.
R
fold-difference in the IC50 values of 5o against HIV-1DRV P20
compared to wild-type HIV-1NL4 was 13-fold, while the fold-
3
differences for DRV (1) and inhibitor 5m were 48- and 67-fold,
respectively.
To gain molecular insight into the binding properties of
macrocyclic inhibitors containing benzyl ether functionality on
the macrocycle, we determined the X-ray crystal structure of
inhibitor 5j and wild-type HIV-1 protease complex. The structure
was refined at 1.27 Å resolution to give R-value of 15.9%. The
X-ray structure contains a protease dimer and inhibitor 5j in two
orientations related by a 180° rotation with relative occupancies
of 65/35%. A stereoview of the active site interactions is shown
in Figure 2. The X-ray showed similarity to the structure of
darunavir-bound HIV-1 protease complex33 with root mean
square difference of 0.16Å for Cα atoms. Larger differences
between the corresponding Cα atoms are less than 0.5Å. Inhibitor
5j shares identical P1 and P2 ligands like darunavir. However,
the major difference is in the P1′-P2′ regions where a 13-
membered macrocycle linking P1′ and P2′ ligands have been
incorporated. Interestingly, this macrocycle differs from
previously reported macrocyclic ligands with respect to a specific
oxymethyl functionality on the macrocycle.21 Most of the
interaction of the bis-THF P2 ligand, phenylmethyl P1-ligand as
well as the transition state hydroxyl group are comparable to
those of the HIV-1 protease-darunavir complex.34 The flexible
P1′-P2′ macrocyclic ring, containing an E-olefin, nicely packs
between the S1′-S2′ subsites in a zigzag crown-like shape. The
activity. We subsequently performed
a systematic study
involving the strategic placement of oxygen (and nitrogen)
heteroatoms along the macrocycle skeleton which led to
identification of derivative 5m and its aniline analogue 5o as
potent inhibitors of HIV-1 protease in the picomolar range. Of
particular importance, inhibitor 5o remained highly potent
against a DRV-resistant HIV-1 variant. The flexible P1′-P2′
macrocyclic nicely packs between the S1′-S2′ subsites in a zigzag
crown-like shape. To obtain molecular insight into the ligand-
binding site interactions, we determined X-ray crystal structure of
inhibitor 5j-bound HIV-1 protease.
The structure shows
interesting new water-mediated hydrogen bonding interactions of
the macrocyclic ring oxygen with backbone atoms at the S2′-site.
This may be responsible for inhibitor’s high affinity. Further
design and improvement of inhibitor properties are currently in
progress in our laboratory.
Figure 2. Stereoview of the X-ray structure of inhibitor 5j (turquoise)-bound HIV-1 protease (PDB code: 5WLO). All strong active site
hydrogen bonding interactions of inhibitor 5j with HIV-1 protease are shown as dotted lines.

A. K.; Mitsuya, H. Antimicrob. Agents Chemother 2003, 47, 3123-
3129.
Acknowledgments
19. de Béthune, M. P.; Sekar, V.; Spinosa-Guzman, S.; Vanstockem,
M.; De Meyer, S.; Wigerinck, P.; Lefebvre, E. ʻʻDarunavir
(Prezista, TMC114): From bench to clinic, improving treatment
options for HIV-infected patients” Antiviral drugs: From basic
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31-45.
20. De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.;
Pauwels, R.; Wigerinck, P.; de Béthune, M. P. Antimicrob. Agents
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This research was supported by the National Institutes of Health
(Grant GM53386, AKG and Grant GM62920, ITW). The X-ray
data was collected at the Southeast Regional Collaborative
Access Team (SER-CAT) beamline 22ID at the Advanced
Photon Source, Argonne National Laboratory. This work was
also supported by the Intramural Research Program of the Center
for Cancer Research, National Cancer Institute, National
Institutes of Health, and in part by a Grant-in-Aid for Scientific
Research (Priority Areas) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (Monbu
Kagakusho), a Grant for Promotion of AIDS Research from the
Ministry of Health, Welfare, and Labor of Japan, and the Grant to
the Cooperative Research Project on Clinical and
Epidemiological Studies of Emerging and Reemerging Infectious
Diseases (Renkei Jigyo) of Monbu-Kagakusho. The authors
would like to thank the Purdue University Center for Cancer
Research, which supports the shared NMR and mass
spectrometry facilities.
Supplementary Material
27. 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. J. Med. Chem. 2009, 52, 3902-
3914.
Supplementary data associated with this article can be found in
the online version.
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