Substituent effects on P2-cyclopentyltetrahydrofuranyl urethanes: Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.061

The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl (Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF ligand were investigated in order to maximize the li

Full text of DOI:10.1016/j.bmcl.2012.01.061

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2308–2311
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituent effects on P2-cyclopentyltetrahydrofuranyl urethanes: Design,
synthesis, and X-ray studies of potent HIV-1 protease inhibitors
Arun K. Ghosh a, , Bruno D. Chapsal a, Melinda Steffey a, Johnson Agniswamy b, Yuan-Fang Wang b,
⇑
Masayuki Amano c, Irene T. Weber b, Hiroaki Mitsuya c,d
a Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907, USA
b Department of Biology, Molecular Basis of Disease, Georgia State University, Atlanta, GA 30303, USA
c Departments of Hematology and Infectious Diseases, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences, Kumamoto 860-8556, Japan
d Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl
(Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF
ligand were investigated in order to maximize the ligand-binding site interactions in the flap region of
the protease. Inhibitors 3c and 3d have displayed the most potent enzyme inhibitory and antiviral activ-
ity. Both inhibitors have maintained impressive activity against a panel of multidrug resistant HIV-1 vari-
ants. A high-resolution X-ray crystal structure of 3c-bound HIV-1 protease revealed a number of
important molecular insights into the ligand-binding site interactions.
Received 28 December 2011
Accepted 17 January 2012
Available online 2 February 2012
Keywords:
HIV-1 protease inhibitors
P2 ligand
Ó 2012 Elsevier Ltd. All rights reserved.
Drug resistance
Design and synthesis
X-ray crystal structure
HIV-1 protease inhibitors continue to be a critical component of
frontline therapy in the treatment of HIV patients.1–3 Our contin-
uing studies on the structure-based design of inhibitors targeting
the protein backbone led to the discovery of a variety of novel
HIV-1 protease inhibitors (PIs) with broad-spectrum activity
against multidrug-resistant HIV-1 variants.4–8 We recently re-
ported various C3-functionalized cyclopentyltetrahydrofuran (Cp-
THF)-derived P2-ligands designed to specifically interact with the
flap Gly48 amide NH in the S2-subsite of the HIV-1 protease.9
One of these inhibitors, 2 (Fig. 1), containing a 3-(R)-hydroxy group
on the Cp-THF core displayed exceptionally potent enzyme inhib-
itory (Ki = 5 pM) and antiviral activity (IC50 = 2.9 nM). This inhibitor
also exhibited potent activity against a panel of multidrug-resis-
tant HIV-1 variants. The X-ray crystal structure of 2-bound HIV
protease revealed an extensive hydrogen-bonding network with
the enzyme backbone.9 Of particular interest, the 3-(R)-hydroxy
group of the Cp-THF ligand was involved in an interesting water-
mediated interaction with the backbone NH amide bond of
Gly48. This specific interaction was not present in inhibitor 1.
These additional interactions observed with 2 may have contrib-
with the addition of C3-polar substituents on the Cp-THF P2 ligand,
we subsequently speculated that N-substituted functionalities,
particularly N-acyl, N-carbamate or N-sulfonyl derivatives could
function as both a hydrogen-bonding donor and acceptor. The
NH proton could conceivably form an effective hydrogen bond
with the proximal Gly48 carbonyl while amide or urethane car-
bonyl oxygen may form an additional interaction with the protease
backbone. Indeed, our previous exploration of such hydrogen bond
donor and acceptor functionalities on P2-ligand frameworks led to
remarkably potent HIV-1 protease inhibitors with broad-spectrum
4–9
antiviral activity.
Herein, we report the design, synthesis and
biological evaluation of a series of HIV-1 protease inhibitors with
C-3 substituted Cp-THF as the P2-ligand. A number of inhibitors
exhibited exceptionally potent antiviral activity against a panel
of multidrug-resistant HIV-1 variants. A protein-ligand X-ray
crystal structure also provided important molecular insight into
the ligand-binding site interactions.
The synthesis of ligands containing various N-substituents with
either stereochemistry at C3 was accomplished starting from our
previously reported optically active ketone intermediate 410 as
shown in Scheme 1. Ketone 4 was converted to methyloxime
derivatives 5 in 96% yield. Reduction of 5 with a mixture of Pd/C
and Raney-Ni under hydrogen pressure (80 psi) provided the corre-
sponding amine as a 3:1 diastereomeric mixture.11 The amine mix-
ture was reacted with Ac2O in the presence of Et3N and a catalytic
amount of DMAP to yield a mixture of isomeric TBS-protected
9
uted toward its impressive drug resistance profile.
Based upon the 2-bound X-ray crystal structure of HIV-1 prote-
ase, and given the significant gain in antiviral activity observed
⇑
Corresponding author. Tel.: +1 765 494 5323; fax: +1 765 496 1612.
E-mail address: akghosh@purdue.edu (A.K. Ghosh).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.061

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2308–2311
2309
OMe
Y
Ligands
6a-g
OH
OH
H
N
H
H
O
O
N
H2N
Ph
N
H
S
S
4-NO2PhOCOCl,
pyridine
O
O
O
O
O
O
O
O
9
Ph
1
2
, X = H, Y = CH2OH
, X = OH, Y = OMe
C3 X
O
O
THF-CH3CN,
Et3N
Cp-THF
O
NO2
O
OMe
OH
H
N
H
H
8a-g
N
S
R
OMe
OH
O
O
H
N
Ph
O
N
H
S
C3 NH
O
O
3c
, 3-(S) (
GRL-0249A
)
O
O
O
3d
GRL-0289A)
, 3-(R) (
Ph
3a-g
OMe
R
Figure 1. Structure of protease inhibitors 1,2, 3c–d.
Scheme 2. Synthesis of protease inhibitors 3a–g.
Me2NH gas to provide the corresponding TBS-protected ligand. Re-
moval of the TBS-group with TBAF furnished ligand 6g in excellent
yield.
For the synthesis of HIV protease inhibitors, all ligand alcohols
6a–g were reacted with 4-nitrophenyl chloroformate in the pres-
ence of pyridine in CH2Cl2 to furnish the corresponding mixed acti-
vated carbonates 8a–g (Scheme 2).9,10 The activated carbonates
were then reacted with previously reported hydroxyethylamine
isostere 99 in the presence of Et3N in THF/CH3CN for 2–4 days to
give corresponding inhibitors 3a–g.
OTBS
OTBS
OMe
.
MeONH2 HCl
O
O
NaHCO3
4
5
O
N
1. H2, Pd/C,
Ra-Ni
2. See conditions
3. TBAF
OH
OH
Inhibitors 3a–g were initially tested in enzyme inhibitory as-
says using the method developed by Toth and Marshall,12 and then
evaluated for in vitro antiviral assays. Results are shown in Table 1.
All inhibitors displayed impressive inhibitory potency and high
antiviral activity. Inhibitors 3a and 3b that, contain a 3-(S)- or 3-
(R)-N-acetyl substituent on the Cp-THF ligand, exhibited similar
potency (7.4 and 7.5 pM, respectively). Interestingly, the stereo-
chemistry at C3 seemed to have little effect. Inhibitor 3c with a
3-(S)-N-methoxycarbonyl displayed the most impressive enzyme
and antiviral potency (Ki = 1.8 pM and IC50 = 1.6 nM). The isomeric
inhibitor 3d provided slightly lower potency. Inhibitors 3e and 3f
that contain a 3-N-mesyl on the Cp-THF ligand, showed a substan-
tial reduction in activity probably due to the increase in steric bulk
created by the N-mesyl group. Inhibitor 3g that contains an
O-substituted dimethylaminocarbamate in place of N-substituted
carbamate at C3 provided a good antiviral activity, similar to that
of 3d.
+
O
O
(S)
(R)
NH
NH
dr = 3:1
R
R
2. Conditions:
Ac2O, Et3N :
6a, R = Ac
6b, R = Ac
6d,
ClCO2Me, Py :
6c, R = CO2Me
6e, R = SO2Me
R = CO2Me
MsCl, Et3N :
6f, R = SO2Me
OTBS
OH
1. 4-NO2PhOCOCl,
pyridine
O
O
O
2. Me2NH, THF
3. TBAF, THF
OH
O
6g
N
7
Scheme 1. Synthesis of C3-substituted ligands 6a–g.
Inhibitors 3c and 3d, were further evaluated against a panel of
multidrug-resistant (MDR) HIV-1 variants and their antiviral activ-
ities were compared to clinically available PI, darunavir (DRV).6,13
Results are shown in Table 2. All inhibitors exhibited low nanomo-
lar EC50 values against the wild-type HIV-1ERS104pre laboratory
strain, isolated from a drug-naïve patient.13 Inhibitor 3d had the
most potent activity (EC50 = 3 nM) similar to that of DRV. When
tested against a panel of multidrug-resistant HIV-1 strains, the
EC50 of 3d remained in the low nanomolar value range (15–
24 nM) and its fold-changes in activity were similar to those ob-
served with DRV.6,13 Interestingly, inhibitor 3c, with the opposite
(S) stereochemistry at C3, displayed slightly lower antiviral activi-
ties against all viral strains compared to 3d. However, the fold-
changes in EC50 for 3c remained low (<3) against all MDR HIV-1
viruses. The fold-changes contrasted with those of 3d and even
DRV, for which the respective EC50’s increased by a factor of at least
three against the MDR viruses examined.14
amide intermediates in 80% yield. Treatment of the respective
amides with TBAF in THF and chromatographic separation fur-
nished diastereomerically pure ligands 6a and 6b in excellent yield.
Similarly, the amine mixture was treated with methyl chlorofor-
mate and pyridine, or mesyl chloride and Et3N to afford the corre-
sponding carbamates and sulfonamides. Treatment of the
respective crude mixtures with TBAF in THF followed by chromato-
graphic separation afforded diastereoisomeric N-carbamate ligands
6c and 6d, and N-mesyl ligands 6e and 6f, respectively. The assign-
ment of stereochemistry on the ligands was carried out by NOE or
NOESY experiments of the corresponding mixed activated carbon-
ates 8a, 8c, and 8e. To probe the importance of the free NH, we have
synthesized ligand 6g containing a 3-(R)-O-dimethylaminocarba-
mate group. This was synthesized in three consecutive steps start-
ing from our previously reported optically active alcohol 7.9
Treatment of 7 with 4-nitrophenyl chloroformate in the presence
of pyridine furnished the corresponding mixed activated carbonate.
The resulting carbonate was reacted with a bubbling stream of
In order to gain molecular insights on the ligand-binding site
interactions responsible for the potent activity and excellent resis-
tance profile of 3c, we have determined the X-ray crystal structure

2310
A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2308–2311
Table 1
Table 2
Structures and potency of inhibitors 3a–ga
Comparison of the antiviral activity of 3c, 3d, and DRV against multidrug-resistant
HIV-1 variants
Entry Inhibitor structure
Ki
(pM)
IC50
(nM)b
Virusa
EC50 (l
M) SDs, (fold-change)b
3c
3d
DRV
OMe
OMe
OMe
OMe
HIV-1ERS104pre (wt) 0.029 0.002
0.003 0.001
0.004 0.001
OH
H
HIV-1MDR/B (X4)
HIV-1MDR/C (X4)
HIV-1MDR/G (X4)
HIV-1MDR/TM (X4)
0.075 0.011 (3) 0.018 0.003 (6) 0.019 0.006 (5)
0.030 0.006 (1) 0.015 0.005 (5) 0.011 0.003 (3)
0.039 0.001 (1) 0.020 0.005 (7) 0.011 0.002 (3)
0.074 0.006 (3) 0.024 0.004 (8) 0.028 0.001 (7)
O
N
N
S
O
O
O
1
2
3
7.4
7.5
1.8
25
31
1.6
O
O
O
O
O
Ph
3a
O
a
HN
HN
HN
HN
Amino acid substitutions identified in the protease-encoding region of HIV-
ERS104pre, HIV-1MDR/B, HIV-1MDR/C, HIV-1MDR/G, HIV-1MDR/TM compared to the con-
1
Me
sensus type B sequence cited from the Los Alamos database include L63P in HIV-
1ERS104pre; L10I, K14R, L33I, M36I, M46I, F53I, K55R, I62V, L63P, A71V, G73S, V82A,
L90M, I93L in HIV-1MDR/B; L10I, I15V, K20R, L24I, M36I, M46L, I54V, I62V, L63P,
K70Q, V82A, and L89M in HIV-1MDR/C; L10I, V11I, T12E, I15V, L19I, R41K, M46L,
L63P, A71T, V82A, and L90M in HIV-1MDR/G; L10I, K14R, R41K, M46L, I54V, L63P,
A71V, V82A, L90M, I93L in HIV-1MDR/TM. HIV-1ERS104pre served as a source of wild-
type HIV-1.
OH
OH
OH
H
N
O
N
S
O
O
Ph
3b
O
b
EC50 values were determined by using PHA-PBMs as target cells and the inhi-
bition of p24 Gag protein production for each drug was used as an endpoint. The
numbers in parentheses represent the fold-change in EC50 values for each isolate
compared to the EC50 values for the wild-type HIV-1ERS104pre. All assays were con-
ducted in duplicate, and the data shown represent mean values ( 1 standard
deviations) derived from the results of two or three independent experiments. PHA-
PBMs were derived from a single donor in each independent experiment. DRV
(darunavir).
Me
H
N
O
N
S
O
O
O
Ph
3c
O
1.23 Å. The inhibitor is bound to the protease dimer in two orien-
tations related by a 180° rotation, with a 50/50 relative occupancy.
The protease backbone structure showed a very low rms deviation
OMe
H
of 0.11 Å for all Ca carbons compared to the protease complexes of
O
O
N
N
29 or DRV.21 As shown in Figure 2, the inhibitor interactions in the
protease binding site extend from S2 to S20 protease subsites and
consist of a series of strong hydrogen bonds and weaker C–Hꢀ ꢀ ꢀO
S
O
O
O
4
5
6
7
4.0
4.6
O
Ph
3d
and C–Hꢀ ꢀ ꢀ
p interactions similar to those previously described
for DRV,21 or inhibitor 2.9 The Cp-THF cyclic oxygen forms a strong
hydrogen bond with the backbone amide NH of Asp29 in the
protease S2-binding site, similar to that previously observed with
other Cp-THF-based inhibitors 15 and 2.9 Critical differences, how-
ever, are observed with the additional interactions that the 3-(S)-
N-methoxycarbonyl amino substituent on the Cp-THF makes
throughout the S2–S3 subsites. As shown in Figure 2, the carba-
mate NH forms a strong hydrogen bond with the Gly48 backbone
carbonyl. The carbamate carbonyl is observed to interact with
OMe
OMe
OMe
OMe
OH
OH
OH
H
N
O
O
N
S
O
32
28
O
O
O
Ph
3e
O
S
O
HN
HN
O
Me
the Arg80 guanidine side chain through
a conserved water
H
N
molecule. The methyl of the methoxy group then fits within the
S3-hydrophobic pocket. The C3-N-methoxycarbonyl amino group
creates a network of tight hydrogen bonds that literally links the
protease flap region to the S2–S3 subsites’ dimer interface. These
new interactions and enthalpic nature of the additional hydrogen
bonding created by the P2-ligand may certainly exert an enhanced
anchoring effect of the inhibitor into the S2-subsite and further
stabilize the closed conformation of the protease–ligand complex.
In conclusion, we have reported the structure-based design of a
series of highly potent HIV-1 protease inhibitors incorporating C3-
substituted cyclopentyltetrahydrofuranyl urethanes as P2-ligands.
Various C3-N-substituents were investigated in order to create
multiple interactions in the S2-subsite of the protease and specifi-
cally with the protease flap region. Inhibitors 3c and 3d displayed
remarkable inhibitory potency and antiviral activity. When tested
against a panel of MDR HIV-1 strains, inhibitor 3d, with a 3-(R)-
methoxycarbonyl on the Cp-THF ligand, provided the most impres-
sive EC50s and fold-changes in activity which are comparable to
those observed with clinically available DRV. Isomeric inhibitor
3c displayed lower antiviral activity. However, it exhibited strik-
ingly low fold-changes of antiviral activity when tested against
MDR HIV-1 viruses. An X-ray crystal structure of the protease–3c
O
N
S
O
O
180
50
O
Ph
3f
O
S
O
Me
H
O
N
N
S
O
O
20
4.7
O
Ph
3g
O
NMe2
a
Values are the mean value of at least two experiments.
Human T-lymphoid (MT-2) cells (2 ꢁ 103) were exposed to 100 TCID50 of HIV-
b
1
LAI and cultured in the presence of each PI, and IC50 values were determined using
the MTT assay. The IC50 values of amprenavir (APV), saquinavir (SQV), indinavir
(IDV), and darunavir (DRV) were 0.03, 0.015, 0.03, and 0.003 M, respectively.
l
of the HIV wild-type protease complexed with 3c (Fig. 2).15 The
structure was refined to an R-factor of 14.9% and a resolution of

A. K. Ghosh et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2308–2311
2311
Figure 2. Stereoview of the X-ray structure of inhibitor 3c (green)-bound HIV-1 protease (PDB code: 4DFG). All strong hydrogen bonding interactions are shown as dotted
lines.
8. Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Acc. Chem. Res. 2008, 41,
78.
extensive interactions throughout the protease binding site. The
9. Ghosh, A. K.; Chapsal, B. D.; Parham, G. L.; Steffey, M.; Agniswamy, J.; Wang, Y.-
complex was determined at a 1.23 Å resolution. Inhibitor 3c made
complex network of hydrogen-bonding interactions created by
the N-methyl carbamate substituent in addition to those created
by the isostere in the protease active site may account for the
impressive antiviral activity and superb resistance profile observed
with inhibitor 3c. Further designs along this line and ligand optimi-
zation are currently underway.
F.; Amano, M.; Weber, I. T.; Mitsuya, H. J. Med. Chem. 2011, 54, 5890.
10. Ghosh, A. K.; Chapsal, B. D.; Baldridge, A.; Ide, K.; Koh, Y.; Mitsuya, H. Org. Lett.
2008, 10, 5135.
11. Li, J. J.; Sutton, J. C.; Nirschl, A.; Zou, Y.; Wang, H.; Sun, C.; Pi, Z.; Johnson, R.;
Krystek, S. R.; Seethala, R.; Golla, R.; Sleph, P. G.; Beehler, B. C.; Grover, G. J.;
Fura, A.; Vyas, V. P.; Li, C. Y.; Gougoutas, J. Z.; Galella, M. A.; Zahler, R.;
Ostrowski, J.; Hamann, L. G. J. Med. Chem. 2007, 50, 3015.
12. Toth, M. V.; Marshall, G. R. Int. J. Pept. Protein Res. 1990, 36, 544.
13. Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, 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. Antimicrob. Agents Chemother. 2003, 47,
3123.
Acknowledgements
This research was supported by the National Institutes of
Health (Grant GM53386 to A.K.G. and Grant GM62920 to I.T.W.).
This work was also supported by the Intramural Research Program
of the Center for Cancer Research, National Cancer Institute, Na-
tional Institutes of Health, and in part by a Grant-in-Aid for Scien-
tific Research (Priority Areas) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (Monbu Kagaku-
sho), a Grant for Promotion of AIDS Research from the Ministry of
Health, Welfare, and Labor of Japan, and the Grant to the Coopera-
tive Research Project on Clinical and Epidemiological Studies of
Emerging and Reemerging Infectious Diseases (Renkei Jigyo) of
Monbu-Kagakusho.
14. Ghosh, A. K.; Chapsal, B. Mitsuya, H. In Aspartic Acid Proteases as Therapeutic
Targets; Ghosh, A., Ed.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2010;
pp 245–262.
15. The protein-ligand X-ray structure of 3c-bound HIV-1 protease will be
deposited in PDB (PDB ID: 4DFG). The HIV-1 protease was expressed and
purified as previously described.16 The protease–inhibitor complex was
crystallized at room temperature by the hanging drop vapor diffusion
method with well solutions of 1.2 M ammonium chloride and 0.1 M sodium
acetate buffer (pH 4.8). Diffraction data were collected on a single crystal
cooled to 90 K at SER-CAT BM beamline 22, Advanced Photon Source, Argonne
National Laboratory (Chicago, IL, U.S.), with an X-ray wavelength of 1.0 Å and
processed by HKL-2000 with Rmerge of 7.2%. The PR structure was used in
molecular replacement by PHASER17 in the CCP4i suite18 and refined to 1.45 Å
resolution using SHELX-97 and COOT19 for manual modification. PRODRG-220
was used to construct the inhibitor and the restraints for refinement.
Alternative conformations were modeled, anisotropic atomic displacement
parameters (B factors) were applied for all atoms including solvent molecules,
and hydrogen atoms were added in the final round of refinement. The final
refined solvent structure comprised two Clꢂ ions and 142 water molecules.
16. Mahalingam, B.; Louis, J. M.; Hung, J.; Harrison, R. W.; Weber, I. T. Proteins
2001, 43, 455.
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