New Active HIV-1 Protease Inhibitors Derived from 3-Hexanol: Conformation Study of the Free Inhibitors in Crystalline State and in Complex with the Enzyme

Article abstract of DOI:10.1111/j.1747-0285.2012.01328.x

Four novel linear non-peptidic HIV-1 protease inhibitors derived from 2,5-diamino-1,6-diphenyl-3-hexanol were synthesized and characterized. All of them exhibit tight binding to HIV-1 protease, with inhibition constants K_i in the range 20pm-5nm. The investigated inhibitors were crystallized, and their crystal structures were determined by X-ray diffraction. In all cases, the conformations found in the crystalline state differ significantly from the conformations obtained by computational docking of the inhibitor in the binding cleft of native HIV-1 protease. Owing to the prevalence of hydrophobic substituents in all these inhibitors, the conformational mobility in water solution is restricted to their compact forms. The spectrum of low-energy conformations in solution dramatically changes during the formation of inhibitor crystals (phenyl ring stacking as a leading motif) or during the formation of a complex with HIV-1 protease (elongated conformation suitable to fit the enzyme pockets as a factor responsible for tight binding). High conformational flexibility and low conformational stress in the molecules of these inhibitors most likely increase their biological activity in comparison with more rigid compounds.

Full text of DOI:10.1111/j.1747-0285.2012.01328.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01328.x
Chem Biol Drug Des 2012; 79: 798–809
Research Letter
New Active HIV-1 Protease Inhibitors Derived
from 3-Hexanol: Conformation Study of the Free
Inhibitors in Crystalline State and in Complex
with the Enzyme
Natasza E. Ziołkowska1,†, Anna Bujacz1,
Received 6 May 2011, revised 14 November 2011 and accepted for
publication 28 December 2011
´
Ramnarayan S. Randad2, John W.
3
4
´
´
ˇ
Erickson , Tereza Skalova , Jindrich
Hasek and Grzegorz Bujacz *
4
1,
ˇ
Acquired immunodeficiency syndrome (AIDS) is the final and most
serious stage of the disease caused by the human immunodefi-
ciency virus (HIV) (1–3). The main therapeutic strategies for inter-
vention in cases of HIV-1 infections involve the inhibition of
enzymes that play critical roles in the life cycle of the virus: reverse
transcriptase (RT), protease (PR), and integrase (IN) (4–6). The crys-
tal structure of HIV-1 protease was solved in 1989 (7–9). Structural
studies have become the main basis for the investigation of inhibi-
tors of this enzyme and rational drug design (10–13). Many of the
potential drugs against AIDS, including inhibitors of HIV-1 protease,
have received FDA approval; they include saquinavir (14–16), indina-
vir (17,18), ritonavir (19,20), nelfinavir (21,22), lopinavir (23,24),
amprenavir (25–27), kaletra (combination of lopinavir and ritona-
vir) (28,29), atazanavir (30,31), tipranavir (32,33), and darunavir
(34,35).
1
´
Institute of Technical Biochemistry, Technical University of Łꢀdz,
´
Stefanowskiego 4 ⁄ 10, 90-924 Łꢀdz, Poland
2Division of Chemistry I, Office of Generic Drugs, FDA, Rockville,
MD 20855, USA
3Sequoia Pharmaceuticals, Inc., 401 Professional Drive, Suite 200,
Gaithersburg, MD 20879, USA
4Institute of Macromolecular Chemistry, Czech Academy of Science,
Heyrovskeho nam. 2, 162 06 Prague 6, Czech Republic
*Corresponding author: Grzegorz Bujacz, gdbujacz@p.lodz.pl
Present address: Cell Biology and Biophysics Unit, National Institute of
Neurological Disorders and Stroke, Bethesda, MD, USA.
Four novel linear non-peptidic HIV-1 protease
inhibitors derived from 2,5-diamino-1,6-diphenyl-3-
hexanol were synthesized and characterized. All
of them exhibit tight binding to HIV-1 protease,
with inhibition constants Ki in the range 20 pM–
5 nM. The investigated inhibitors were crystallized,
and their crystal structures were determined by X-
ray diffraction. In all cases, the conformations
found in the crystalline state differ significantly
from the conformations obtained by computa-
tional docking of the inhibitor in the binding cleft
of native HIV-1 protease. Owing to the prevalence
of hydrophobic substituents in all these inhibitors,
the conformational mobility in water solution is
restricted to their compact forms. The spectrum
of low-energy conformations in solution dramati-
cally changes during the formation of inhibitor
crystals (phenyl ring stacking as a leading motif)
or during the formation of a complex with HIV-1
protease (elongated conformation suitable to fit
the enzyme pockets as a factor responsible for
tight binding). High conformational flexibility and
low conformational stress in the molecules of
these inhibitors most likely increase their biologi-
cal activity in comparison with more rigid com-
pounds.
The aim of continuing the search for new HIV-1 protease inhibitors
is to create wide-spectrum drugs, active against the mutations in
the HIV-1 genome which rapidly develop in patients under the
selection pressure of all of the clinically approved drugs (36–40).
Among the inhibitors that have been characterized to date, the sub-
strate-mimic inhibitors with hydroxyethylene (41), hydroxyethylamine
(42,43), ethylamine (44), and reduced amide (45) replacement of the
cleaved amide bond are the best known.
The inhibitors studied in this work can be classified as competitive,
substrate based but non-peptidic, with the hydroxyethylamine iso-
stere replacing the cleavable peptide bond. The structures of HIV-1
protease complexes with hydroxyethylene inhibitors form a subset
of the most frequently determined structures of the HIV-1 protease.
However, the inhibitors designed as peptide substrate analogs have
a common disadvantage of possible enzymatic cleavage and other
adverse effects in the body. The inhibitors studied here have a cen-
tral fragment (isostere of the cleavage site in substrate) resistant to
hydrolysis (hydroxyethylene) and non-peptidic substituents imitating
the side chains of the substrate. Design and synthesis of these
inhibitors were performed with the intention to optimize the inter-
actions between the P2 ⁄ P2¢ substituents of inhibitors and the
S2 ⁄ S2¢ pockets of HIV-1 protease.
Key words: acquired immunodeficiency syndrome, crystal structure,
drug design, human immunodeficiency virus, molecular structure, prote-
ase inhibitor
798

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
Despite the fact that over one hundred experimentally determined
structures of complexes of different mutants of the HIV protease
with inhibitors belonging to several different classes are already
available in the PDB (46), and over 300 in HIVdb (47), little attention
has been paid to the conformational variability in the structures of
inhibitor molecules in different environments. This study of confor-
mational behavior of inhibitors in different environments brings a
better understanding of the nature of ligand–enzyme interaction.
(TBTU) and 1-hydroxybenzo-triazole hydrate (HOBt) in the presence
of N,N-diisopropylethylamine (DIPEA) in dimethylformamide (DMF)
as a solvent. Detailed procedures for the preparation of (2S,3S,5S)-
2-N-(3-hydroxy-2-methylbenzoyl)amino-5-N-benzoylamino-1,6-diphenyl-
3-hexanol (1), (2S,3S,5S)-2,5-N,N'-bis-(2-methylbenzoyl)amino-1,6-
diphenyl-3-hexanol (2), (2S,3S,5S)-2-N-(3-hydroxy-2-methylbenzoyl)
amino-5-N-[(3S)-tetrahydrofuryloxy-carbonyl]-amino-1,6-diphenyl-3-
hexanol (3), and (2S,3S,5S)-2-N-[(2-pyridinylmethoxycarbonyl) vali-
ne]amino-5-N-(tert-butoxycarbonyl)-amino-1,6-diphenyl-3-hexanol (4)
are shown in Appendix S1.
The structures and biological activity of four new peptidomimetic
inhibitors of HIV-1 protease, derivatives of 1,6-diphenyl-3-hexanol
based on the hydroxyethylene isostere (48), are reported here. The
synthesized compounds were recrystallized to obtain single crystals
suitable for X-ray analysis. The conformation of free inhibitors in
crystalline state is compared with their conformation after binding
into the HIV-1 protease active site.
Biological activity
Ki values for 1–4 were determined by a fluorometric assay with
the fluorogenic substrate Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-
Val-Gln-Lys(DABCYL)-Arg and the chromogenic substrate Lys-Ala-
Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-NleONH2, as previously described
(49,50). The antiviral activity of 1–4 is expressed as 50% inhibitory
concentration IC50 (Table 1) and was measured as previously
described (50).
Methods and Materials
Chemical methods
Although the reported series includes only four inhibitors, it is not
possible to write only a single general procedure of obtaining them.
Condensation of the core unit with P2 ⁄ P2¢ substituents was carried
out using, in most cases, the standard peptide coupling reagents
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
X-ray crystallographic studies
Single crystals of compounds 1–4 were obtained by recrystalliza-
tion using vapor diffusion method. The diffraction data sets for crys-
tals of 1 and 2 were collected at room temperature on a CAD4
Table 1: Structural formulae and biological activity of 1–4
No of
inhibitor
P3
P2
P2¢
M.Wt.
522
M.p. (ꢀC)
Ki (nM)
IC50 (nM)
1.0
OH
OH
1
—
O
O
199–200
4.6
O
2
3
—
—
520
532
214–215
224–225
4.8
3.0
O
0.32
0.63
O
O
O
O
O
4
618
184–185
0.021
0.17
O
N
O
O
N
H
O
Chem Biol Drug Des 2012; 79: 798–809
799

´
Ziołkowska et al.
Figure 1: Thermal ellipsoidal view of the molecules of 1–4 with the numbering scheme of atoms and P substituents. Thermal ellipsoids
are shown with 50% probability.
diffractometer equipped with graphite monochromatized CuKa radia-
tion. An empirical absorption correction was applied by the use of
the w-scan method [EAC program (51)] for 1 and 2. The intensity
correction was applied only for 2 [DECAY program (52)]. Data correc-
tion for 1 and 2 was carried out using the Enraf-Nonius SDP crys-
tallographic computing package (52). The diffraction data sets for
crystals of 3 and 4 were collected using synchrotron radiation at
EMBL beamline BW7B (Desy, Hamburg, Germany). Diffraction
images were recorded using a Mar 165 mm CCD detector at 100 K.
Two data collection runs were made: high-resolution data run to
0.815 ꢀ and low-resolution run to 1.21 ꢀ. The data were processed
by DENZO (53) and scaled with SCALEPACK (53).
Theoretical calculations
Two experimental structures of the complexes deposited in PDB
(46), with inhibitors very similar to the investigated compounds and
with data collected at medium resolution, were used as templates:
• HIV-1 protease complex with inhibitor L700,417b, pdb code: 4PHV
(a = 58.88, b = 86.80, c = 46.79, P21212, resolution 2.1 ꢀ) (57) for
1, 2, 3;
• HIV-1 protease complex with inhibitor SB206343, pdb code: 1HPS
(a = 63.00, c = 83.30, P61, resolution 2.3 ꢀ) for 4.
The inhibitors 1–4 were docked into the protease active site
using program O (58) and minimized in DISCOVER in INSIGHTIIb using
force field cff91. Solvent was considered as a dielectric contin-
uum. Only one water molecule between inhibitor carbonyls (at P1,
P1¢ sites) and Ile49, Ile149 amines at tips of the HIV-1 protease
flaps was included into the optimization analogous with many
similar experimentally determined structures. Classical ionization
schemes of amino acids corresponding to pH 5 were used. Non-
bonded interactions were cut off at a distance of 14.00 ꢀ. Energy
and geometry optimization was carried out in three steps: (i) only
hydrogen atoms were optimized, (ii) all hydrogen atoms of prote-
All observed reflections with I > 0 were used to solve the struc-
tures of 1–4 by direct methods and to refine it by full matrix least-
squares using F's (54). Structures' solution was performed using the
program SHELXS (54), and SHELXL (55) was used for structure refine-
ment. For molecule visualization and preparing Figures 1 and 2, the
XPa program was used. Crystal data and experimental details are
shown in Table S1. The torsion angles, the dihedral angles between
planes passing through the atoms of six-membered and five-mem-
bered rings, and the lengths and angles of the hydrogen bonds for
1–4 were calculated using the program CSU (56).
800
Chem Biol Drug Des 2012; 79: 798–809

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
Figure 2: Unit cells of 2 and 3. The molecules of crystallization solvent bound into the crystal lattice are indicated by the arrows.
ase and all atoms of inhibitor were optimized, (iii) all atoms of
the whole complex were optimized. The structures were mini-
mized until maximum derivatives were <0.01 kcal ⁄ ꢀ. The torsion
angles and the dihedral angles between planes of the aromatic
rings of molecules 1–4 were calculated using CSU (56).
interacts with S3 pocket of protease. All investigated inhibitors dif-
fer in their P2¢ substituents.
The compounds discussed here were selected for crystallization
from a large group of HIV-1 protease inhibitors (59,60) (the synthe-
sis and biological activity of the inhibitors from that group will be
published elsewhere) in order to study their conformation in a crys-
tal structure. The investigated inhibitors are based on the central
fragment (2S,3S,5S)-2,5-diamino-1,6-diphenyl-3-hexanol, which was
reported previously (33). Syntheses of this series of inhibitors were
started from (2S,3S,5S)-5-amino-2-N-dibenzylamino-1,6-diphenyl-3-
hexanol after protection on 5-N by Boc group gave (2S,3S,5S)-2-
amino-5-N-(tert-butoxycarbonyl)-amino-1,6-diphenyl-3-hexanol (1s).
The procedure of obtaining the final inhibitors 1–4 is shown on the
Scheme 1, and experimental procedures are described in the
Appendix S1. All final compounds were >99.9 pure, which was
proved by standard chemical analyses, including HPLC, TLC, MS,
Results
Synthesis and design of the inhibitors
The synthesis of four protease HIV-1 inhibitors was performed:
(2S,3S,5S)-2-N-(3-hydroxy-2-methylbenzoyl)amino-5-N-benzoylamino-1,
6-diphenyl-3-hexanol (1), (2S,3S,5S)-2,5-N,N'-bis-(2-methylbenzoyl)
amino-1,6-diphenyl-3-hexanol (2), (2S,3S,5S)-2-N-(3-hydroxy-2-meth-
ylbenzoyl)amino-5-N-[(3S)-tetrahydrofuryloxy-carbonyl]-amino-1,6-diphe-
nyl-3-hexanol (3), and (2S,3S,5S)-2-N-[(2-pyridinylmethoxycarbonyl)
valine]amino-5-N-(tert-butoxycarbonyl)-amino-1,6-diphenyl-3-hexanol (4).
1
and H NMR.
The structural formulae of 1–4 are shown in Table 1. The substitu-
ents P1 and P1¢ of all four inhibitors investigated here are hydro-
phobic. Phenyl rings are attached to the C1 and C6 atoms of the
hexanol chain. These moieties imitate phenylalanine, which inter-
acts with S1 and S1¢ pockets of the protease. P2 substituents are
identical for both 1 and 3: 3-hydroxy-2-methylbenzoyl. 2 is a sym-
metric inhibitor with 2-methylbenzoyl group as P2 and P2¢. Inhibitor
4 additionally has a 2-pyridinylmethoxycarbonyl group as P3, which
Crystal structures of the inhibitors
Crystals suitable for data collection were obtained by the vapor dif-
fusion method. CAD4 diffractometer was used for diffraction data
collection for the crystals of 1 and 2. Synchrotron radiation was
needed to obtain useful diffraction data for 3 and 4. Thermal ellip-
soidal view of the molecules of 1–4 is shown in Figure 1.
Chem Biol Drug Des 2012; 79: 798–809
801

´
Ziołkowska et al.
Ph
Ph
Ph
O
O
H
N
H
N
H
N
H
N
1) a
2) b
c
O
O
O
H2N
N
N
H
N
H
OH
O
O
OH
O
OH
O
Ph
Ph
Ph
4
1s
2
d
Ph
O
H
N
O
HO
N
H
OH
e
O
Ph
2s
Ph
Ph
Ph
O
O
O
H
N
H
NH2
Ph
HO
HO
N
O
g
HO
f
N
H
N
H
N
H
OH
OH
O
OH
O
O
Ph
Ph
1
3
3s
Scheme 1: The procedure of obtaining inhibitors 1–4. a,e – 1 M, HCl in dioxane, b – 2-methyl-benzoic acid, c – 2-pyridinylmethoxycar-
bonyl-(S)-val, d – 2-methyl-3-hydroxybenzoic acid, f – benzoic acid, g – (S)-3-tetrahydrofurylo-N-succinimidyl carbonate, CH3CN, ET3N.
The investigated inhibitors crystallize in various space groups and
crystallographic systems (triclinic, monoclinic, orthorhombic, and
trigonal – Table S1) and show significant differences of packing
in the unit cells. The structures of all investigated compounds
reveal good agreement between the measured and calculated
data. The best structural model was obtained for 1 where Robs
is <0.05 (Table S1). Diffraction data for 2 show that for 4146
unique reflections, there are only 1966 reflections observed with
I > 2r(I), whereas the structure contains 392 refined parameters.
Additionally, the presence of a loosely bound solvent molecule in
the crystal lattice introduces a partial disorder in the structure of
2. However, the structure was solved and refined to Robs < 0.07.
p-stacking of aromatic substituents. The inhibitor molecules are
interconnected by hydrogen bonds in the crystalline state and form
molecular layers mutually connected by van der Waals interactions
and by phenyl rings stacking. In addition, the presence of planar sol-
vent molecules influences the conformation of molecules 2 and 3
(Figure 2). The network of hydrogen bonds in the crystalline state of
inhibitors 2–4 presented in Table 3 is very similar. The molecules
are bound into chains parallel to the y-axis for 2 and into chains
parallel to the z-axis for 4. A two-dimensional network of hydrogen
bonds is created for 1 and 3: a pair of bridges binds the molecules
of 1 into chains parallel to y-axis and a second pair of bridges
binds these chains in the direction of the z-axis. The molecules of 3
are bound into chains parallel to the x-axis by three hydrogen
bridges, and these chains are connected by one strong hydrogen
bond in the direction of z-axis. The details of hydrogen bonding
geometry in the crystal lattices of 1–4 are presented in Table 2.
The molecules of the inhibitors under study are generally very flexi-
ble. However, the bulky phenyl substituents have restricted mobility
and number of low-energy conformations in water, in solid state,
and also after binding to the HIV-1 protease-binding site. The con-
formation of the investigated inhibitors in crystalline state is largely
determined by crystal packing. The exact position and orientation of
the molecules is then influenced by a net of hydrogen bonds and
Molecules of benzene (crystallization solvent) are present in the
crystal lattices of 2 and 3 (Figure 2). Two locations of that mole-
cule are shown for 2 in the disordered model of solvent, with
Table 2: Hydrogen bonding geometry in the crystal lattices of inhibitors 1–4
X -H....Y
X…Y (ꢀ)
X-H…Y (ꢀ)
Symmetry codes
X -H....Y
X…Y (ꢀ)
X-H…Y (ꢀ)
Symmetry codes
1
2
O1 – H...O3
N2 – H...O2
O4 – H...O1
N1 – H...O4
2.679 (3)
2.965 (3)
2.708 (3)
3.086 (3)
170.2 (31)
158.0 (25)
179.6 (39)
159.5 (26)
1 ) x, 0.5 + y, 0.5 ) z
1 ) x, 0.5 + y, 0.5 ) z
2 ) x, )0.5 + y, 0.5 ) z
2 ) x, 0.5 + y, 0.5 ) z
O1 – H...O2
N1 – H...O2
N2 – H...O3
–
2.884 (6)
2.915 (5)
2.929 (5)
–
146.0 (38)
160.0 (5)
161.3 (5)
–
x, 1 + y, z
x, 1 + y, z
x, )1 + y, z
–
3
4
O1 – H...O2
N1 – H...O2
N2 – H...O3
O4 – H...O6
2.761 (3)
2.966 (3)
2.929 (2)
2.743 (3)
163.2 (36)
151.2 (31)
149.6 (30)
162.4 (33)
1 + x, y, z
1 + x, y, z
)1 + x, y, z
x, y, 1 + z
O1 – H...O2
N1 – H...O2
N2 – H...O3
N3 – H...O4
2.769 (3)
2.900 (2)
2.921 (2)
2.960 (3)
176.0 (2)
160.5 (2)
168.2 (2)
158.6 (2)
x, y, 1 + z
x, y, 1 + z
x, y, )1 + z
x, y, )1 + z
Only hydrogen bonds with the H…Y distance shorter than 2.80 ꢀ are listed.
802
Chem Biol Drug Des 2012; 79: 798–809

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
Table 3: Comparison of selected torsion angles (ꢀ) of 1–4 in
the crystal structures of free inhibitors (in crystal) and in the molec-
ular models of complexes with HIV-1 protease (in the complex with
HIV protease)
parameters (about 41 ꢀ) corresponds to interactions in the crystal
lattice of 4. There are hydrophobic intermolecular interactions
around the threefold symmetry axis between tert-butyl groups and
phenyl and pyridinyl rings in the unit cell of 4. There are also
p-stacking interactions between the aromatic rings of the mole-
cules from neighboring unit cells.
In the
In the
complex
complex
In
crystal
with HIV-1 In
with HIV-1
protease
protease
crystal
The values of selected torsion angles of 1–4 are shown in Table 3.
For all the investigated compounds, there are very similar torsion
angles in the rotation around the external bonds of the peptidomi-
metic inhibitor core chain: N1-C2, N1-C19, C2-C3, N2-C5, and N2-
C26(C31). The conformation of 1 varies considerably in comparison
with the other compounds, where the substituents P1¢ and P2¢ have
different orientation. A noticeable difference is observed for the tor-
sion angles of rotation around bonds C3-C4 and C4-C5 (in the cen-
tral part of inhibitor) and for the torsion angles of rotation around
the bonds C1–C2 and C5–C6. Contrarily, these torsion angles vary
only by a few degrees for inhibitors 2, 3, and 4. Analysis of the
X-ray structures presented in Table 4 shows that the dihedral
angles between planes 1 and 1¢ (passing through the atoms of P1,
P1¢ substituents) are very similar for compounds 2 and 3. The loca-
tion and orientation of these substituents is mostly determined by
the presence of solvent molecules in the crystal lattices. Dihedral
angles between the planes 1¢ ⁄ 2 for 2, 3 and the planes 1¢ ⁄ 3 for 4
are in the narrow range of about 71ꢀ–80ꢀ. This fact suggests that
attaching another substituent interacting with S3 pocket of the
enzyme, when P2 has rather small size (L-valine), does not affect
the mutual location of the aromatic rings of the considered chemi-
cal groups.
Inhibitor
1
2
C19 N1
C2
C1
97.7
3.6
66.3
150.0
0.3
174.4
)61.5
115.9
5.9
83.4
)0.8
172.0
)66.4
)167.2
66.4
C2
C7
N1
O1
C3
C4
N1
C1
C2
C3
C4
C5
C19 O2
C2
C3
C4
C5
C6
C5
C3
O1
C5
N2
C13
C4
166.6
)74.4
)60.4
162.9
173.5
)65.3
)176.2 )163.9
54.8
62.9
)126.6 )95.1
)7.7
3
77.7
)98.2
)168.1
C26 N2
C5 N2
Inhibitor
)120.0 )95.9
C26 O3
4.8
5.0
4.1
4
C19 N1
C2
C1
121.6
0.3
164.1
)76.1
)55.2
160.5
156.4
)8.3
)167.3
)53.0
)167.9
54.8
120.6
0.2
90.2
13.8
167.7
)69.9
)149.6
55.3
C2
C7
N1
O1
C3
C4
N1
C1
C2
C3
C4
C5
C19 O2
C2
C3
C4
C5
C6
C5
C5
C3
O1
C5
N2
170.5
)80.6
)56.6
162.5
179.1
—
C13 170.0
175.4
168.3
—
C26 N2
C31 N2
C4
C4
)129.3 )145.4
—
—
22.7
—
—
—
)119.9 )124.3
—
C5
C5
N3
N2
N2
C24 O5
C26 O3
C31 O3
6.9
—
—
—
—
4.5
)2.2
167.9
165.6
C25
172.5
)84.4
C24 O5
C25 C26
Inhibitor conformations in crystalline state and
in the complex with HIV-1 protease
Based on experience in X-ray structure determination of several
HIV-1 protease complexes with substrate-analog inhibitors (41–
43,61–65), we docked the 1–4 inhibitors into the binding site of
HIV-1 protease and optimized their geometry as described in
Methods and Materials. The X-ray structures of 1–4 were used
as input data for molecular modeling of their complexes with
occupation refined to 56% and 44%. The presence of a large
solvent molecule (the benzene ring) is an important factor for
determining the conformation and crystal packing of 2 and 3,
especially for the location of P1, P1¢, P2, and P2¢. A very small
unit cell parameter c (about 5 ꢀ) in comparison with a and b
Table 4: Orientation of planar substituents of inhibitors 1–4 in crystal and after binding to HIV protease
Inhibitor
Planes
In crystal
In the complex with HIV-1 protease
Differences
1
2
3
4
1
2
3
4
1
2
3
4
3 ⁄ 1¢
3 ⁄ 1
2 ⁄ 2¢
2 ⁄ 1
2 ⁄ 1¢
1 ⁄ 1¢
1 ⁄ 2¢
1¢ ⁄ 2¢
–
–
–
–
–
–
70.7
33.4
–
–
–
88.3
–
–
–
–
–
–
–
–
74.7
75.9
–
–
–
72.7
–
–
–
–
–
–
–
–
)4.0
)42.5
–
–
–
15.6
–
–
55.6
67.8
30.0
38.4
14.9
25.2
59.9
14.8
79.6
87.7
73.7
21.4
81.5
10.0
74.2
83.6
85.2
47.2
28.3
79.6
88.5
19.6
63.9
79.6
26.9
38.2
72.1
39.8
52.4
70.7
44.4
88.4
74.4
43.6
60.7
78.7
27.3
)11.8
)58.5
18.8
)49.0
)54.4
33.0
)23.4
7.5
47.9
21.3
)49.3
37.1
)78.4
0.2
40.0
24.5
)31.5
The table lists dihedral angles (ꢀ) between the mean planes of aromatic substituents of inhibitors 1–4 in the crystal structures (in crystal) and after binding to
HIV-1 protease (in the complex with HIV protease). The shadowed areas in columns (differences) show the rotation of the dihedral angles between the crystal
and protein-bound conformations more than 30ꢀ. The mean planes were fitted through six atoms of aromatic substituents or five atoms of tetrahydrofurane
ring. Planes 1, 1¢, 2, 2¢, 3 correspond to substituents P1, P1¢, P2, P2¢, P3, respectively.
Chem Biol Drug Des 2012; 79: 798–809
803

´
Ziołkowska et al.
HIV-1 protease. The hydroxyl group of each inhibitor forms hydro-
gen bonds to catalytic aspartates Asp 25A and Asp 25B. The side
chains of inhibitors fill the respective HIV-1 protease pockets S1
and S2. The geometry of complexes was optimized using the pro-
gram INSIGHTII.b
Figure 3 [prepared using Pymol (66)] shows a comparison of confor-
mations of inhibitors in the forms of complexes with HIV-1
protease. The selected torsion angles and dihedral angles between
the planes of the aromatic rings of 1–4 before and after docking
of an inhibitor are presented in Tables 3 and 4. The best similarity
of these structures is observed for 1 where the atoms of the main
chain and the P2, P2¢ substituents of the inhibitor are mostly in the
same positions as in the crystal structures and only the phenyl rings
of P1, P1¢ are rotated around the bonds C1–C2 and C5–C6. The dif-
ference in the position of P1¢ and P2¢ is significant for 2–4 where
the largest disagreement is noticeable for rotation around the
bonds C3–C4 and C4–C5 (about 100ꢀ). Additional differences
between the X-ray structures and the modeled structures in com-
plex are the orientation of P1¢ phenyl ring of 2 (rotation round the
bond C5–C6), the orientation of P1 phenyl ring of 3 (rotation round
the bond C1–C2), and the orientation of P3 pyridinyl ring of 4 (rota-
tion around the bond O5–C25). The dihedral angles of the planes of
the aromatic rings of 1–3 vary considerably between the crystal
structures and the molecular models. Similarities are observed only
for the angles between the planes 1 and 2 of 1 (describing mutual
orientation of rings of P1, P2) and the planes 1¢ and 2 of 2 and 3
(showing mutual orientation of rings of P1¢, P2). The angles
between the planes of the aromatic rings of 4 for the free inhibitor
in the crystal structure and in the molecular model of the enzyme-
inhibitor complex are very similar except for the angle between the
Figure 3: The top and side views of the inhibitors 1–4 modeled
into the native HIV-1 protease [carbon atoms colored blue (1), light
gray (2), yellow (3), green (4)]. The inhibitor oxygens are red, nitro-
gens are dark blue). The side chains of the Asp25 and Asp25¢ in
the active site are shown in thin lines. The side view is rotated
about 20ꢀ around y-axis to make a view more transparent.
Figure 4: Comparison of conformations of free inhibitors 1–4 in crystalline state (black) and conformations of inhibitors after docking in
the complexes with HIV-1 protease (gray).
804
Chem Biol Drug Des 2012; 79: 798–809

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
Figure 5: The network of hydrogen bonds (within 3.6 ꢀ) and non-bonded interactions (within 3.8 ꢀ) formed between protease HIV-1 and
the inhibitors 1–4 in the enzyme–inhibitor complexes.
planes 1 and 3 (passing through the atoms of rings of P1 and P3,
respectively).
of the inhibitors' main chains and in the non-peptidic nature of
inhibitors' side chains. The starting geometry of the HIV-1 protease
used for molecular modeling of enzyme–inhibitor complexes was
taken from the structures deposited in PDB (46) (pdb codes: 4PHV,
1HPS).
Inhibitor binding
The binding scheme of hydroxyethylene isostere inhibitors is well
known [see for example several structures of complexes with HIV-
1 protease (1FQX, 1IIQ, 1LZQ, 1M0B, 1Z8C, 1ZBG, 1ZJ7, 1ZLF,
1ZPK, 1ZSF, 1ZSR) (41–43,62–65)]. A significant difference between
conformation of those inhibitors and compounds 1–4 is in the
antiparallel orientation of the amide groups at the opposite sides
The modeled inhibitors 1–4 are connected with the HIV-1 prote-
ase-binding site by networks of hydrogen bonds with lengths in the
range 2.66–3.65 ꢀ [see Figure 4 prepared using LIGPLOT (67)]. There
are two hydrogen bridges mediated by the buried water molecule
connecting Ile 50A and Ile 50B and the O1, O2 atoms of the inhibi-
Chem Biol Drug Des 2012; 79: 798–809
805

´
Ziołkowska et al.
tors 1–4. The hydroxyethylene isostere binds to the catalytic resi-
dues Asp 25A and Asp 25B via three hydrogen bonds in 1 and 3
and via four hydrogen bonds in 2 and 4. Additionally, the isostere
hydroxyl group creates a hydrogen bond to Ala 28B for 3. The
hydrogen bridges formed by the other main chain atoms of the
investigated inhibitors are between N1 and Gly 27A (1); N2 and
Gly 27B (2); and N1 and Asp 25B, N1 and Gly 27B, N2 and Gly
27A, N3 and Gly48B, O4 and Arg8A, O4 and Asp29B (4). Hydrogen
bonds also exist between the P substituents and the protease resi-
dues: the hydroxyl group of P2 and Asp 30A (1), the hydroxyl group
of P2 and Asp 29B (3), and N4 atom of pyridinyl ring of P3 and Gly
48B (4).
a correlation with Ki (20ꢀ – ꢀ5 nM, 40ꢀ – ꢀ5 nM, 61ꢀ – 0.32 nM,
73ꢀ – 0.02 nM) for inhibitors 1–4.
Discussion
Crystal and molecular structures of the crystalline inhibitors are
good basis for a glimpse on inhibitor flexibility, factors responsible
for their conformation, and the subsequent modeling of HIV-1 prote-
ase–inhibitor interactions. Comparison of the structures of the mol-
ecules of free inhibitors in the crystal and models of the inhibitors
in complex with HIV-1 protease is presented here. It provides
detailed information about conformational changes caused by the
influence of external forces invoked by interactions with the sur-
rounding molecules in the inhibitor crystals and after creating the
complex with HIV-1 protease with moieties creating enzyme-binding
sides. The water interaction with inhibitor plays an important role
in changing of conformation upon complex formation. The hydropho-
bicity of mimicking side chains substituents force compact 'spheri-
cal' conformation in crystal form and in aqueous fluids is
compensated by favorable interaction with HIV-1 protease after
conformational fitting. All of these interactions have to be taken
into account in in silico modeling of HIV-1 protease–inhibitor com-
plexes.
Figure 5 shows the superposition of inhibitors 1–4 docked into the
native HIV-1 protease. The overlap is based on fitting of all the pro-
tease Ca atoms [calculated by program O (58)]. It is evident and not
surprising that the chemical differences in inhibitors are reflected
by conformational changes of their molecules and also by the con-
formation of protease side chains in the neighborhood of the inhibi-
tors. HIV-1 protease dimer has twofold symmetry but the
asymmetry of inhibitors (the central group –COH-CH2– and in the
inhibitor side chains) induces asymmetry of the inhibitor–protease
complexes and thus the direction is important. Respecting the used
nomenclature, the shift in direction from P1 to P1¢ is called here
the shift to the right and vice versa.
1, 3, and 4 exhibit very good biological activity (Table 1) because
of the possibility of creating many favorable contacts between sub-
stituents P, P' and the S, S' pockets of the enzyme (Figure 4). Com-
pound 3, with (S)-3-tetrahydrofurane ring as P2¢, combining
hydrophobic and hydrophilic properties, is a better inhibitor of HIV-1
protease (Ki = 0.32 nM) than 1 and 2, with phenyl and 2-methyl-
The differences in inhibitors' conformation in the complex with HIV-1
protease shown in the Figure 5 are to some degree compensated by
small rotational movements of the protease side chains and by the
movement of water molecule trapped usually between the P1 and
P1¢ carbonyls and the amine groups at the tips of the protease flaps.
It means that the atom positions of the overlapped inhibitors should
not be compared absolutely but should be evaluated for each inhibi-
tor individually. In this sense, the most important differences influ-
encing biological activity of inhibitors could be as follows:
phenyl rings as P2¢, respectively. The best inhibitor is
4
(Ki = 0.02 nM), with L-valine as P2, and 2-pyridinylmethoxy moiety
as P3; these groups form a large number of hydrogen bonds in the
binding site of the protease.
The difficulties in crystallization suggested significant conforma-
tional flexibility of the molecules of the investigated compounds.
The use of synchrotron radiation (not standard for diffraction data
collection for crystals of small molecules) was necessary to obtain
useful data for 3 and 4. The unit cell parameters for crystals of 4
were relatively large (Table 2), comparable with the parameters typ-
ical for protein crystals. X-ray structural data reveal that in the crys-
talline state, none of the 1–4 inhibitors is in the b-extended
conformation – the conformation characteristic for inhibitors bound
in their complexes with the enzyme. Two main factors, a network
of hydrogen bonds and the hydrophobic interactions, determine the
conformation of the inhibitor both in its crystalline state and in
complex with HIV-1 protease. Because the compounds contain only
a small number of potential hydrogen bond donors, only a few such
bonds are seen in the crystal structures, mostly between symmetri-
cally related molecules.
• a shift of the molecule of the most active inhibitor 4 to the
right, invoking a shift of the hydrophobic side chains in P2, P1, and
P1¢ positions by almost 2 ꢀ,
• different conformation of the central part of inhibitor 4 between
the P2 and P2¢ sites in comparison with 1–3,
• the right shift of the Ile in 4 with respect to the 2-methylphenol
moieties in P2 position of 1–3,
• the aromatic residue or purely hydrophobic residue in the P2
position seems to play a negative role.
Both Figure 5 comparing different inhibitors in the active site and
Figure 3 showing large differences in inhibitor conformations in the
inhibitor crystals and in the protease active site evidence that the
stacking of aromatic substituents and their conformational mobility
play an important role, especially the perpendicular orientation of
phenyl ring in P1 position in 4. It is related also to different torsion
angle C7-C1-C2-C3 for the inhibitor 4 (Table 3) and to the dihedral
angle between phenyl planes in P1 and P1¢ (see Table 4), showing
The known macromolecular structure of a protease–inhibitor com-
plex (37) deposited in PDB (46) and the crystal structures of free
inhibitors were used to model the interactions in the active site of
HIV-1 protease. The investigated compounds acting as competitive
inhibitors bind tightly to the enzyme's active site. In the enzyme–
806
Chem Biol Drug Des 2012; 79: 798–809

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
inhibitor complex, the chemical groups of the inhibitor molecule can
serve as hydrogen bond donors and hydrogen bond acceptors in
interactions with the amino acids creating the binding pocket.
References
1. Gallo R.C., Sarin P.S., Gelmann E.P., Robert-Guroff M., Richardson
E., Kalyanaraman V.S., Mann D., Sidhu G.D., Stahl R.E., Zolla-
Pazner S., Leibowitch J., Popovic M. (1983) Isolation of human
T-cell leukemia virus in acquired immunodeficiency syndrome
(AIDS). Science;220:865–867.
2. Barre-Sinoussi F., Chermann J.C., Rey F., Nugeyre M.T., Chamaret
S., Gruest J., Dauguet C., Axler-Blin C., Vezinet-Brun F., Rouzioux
C., Rozenbaum W., Montagnier L. (1983) Isolation of a T-lympho-
tropic retrovirus from a patient at risk for acquired immunodefi-
ciency syndrome (AIDS). Science;220:868–871.
3. Coffin J., Hasse A., Levy J.A., Montagnier L., Oroszlan S., Teich
N., Temin H., Toyoshima K., Varmus H., Vogt P., Weiss R. (1986)
Human immunodeficiency viruses (Letter). Science;232:697.
4. Johnston M.I., Hoth D.F. (1993) Present status and future pros-
pects for HIV therapies. Science;260:1286–1293.
5. De Clerq E. (1995) Toward improved anti-HIV chemotherapy: ther-
apeutic strategies for intervention with HIV infections. J Med
Chem;38:2491–2517.
The entropy terms seem to be related to rather flexible aromatic
substituents in P1, P1¢ positions. Dihedral angle between the P1
and P1¢ phenyl rings is correlated with the measured inhibition con-
stants. The optimized models show that hydrophobic interactions
with the enzyme are crucial not only in the S1 and S1¢ pockets but
also in the S2 and S2¢ pockets. The flexibility of the central part of
inhibitors (i.e., rotations around the bonds C2–C3 and C3–C4) deter-
mines the elasticity of the inhibitor and fitting to the HIV-1 PR
active site.
In spite of the fact that all four inhibitors fit virtually identical
positions in the HIV-1 protease-binding site, the networks of
hydrogen bonds, the strain in the main chain, the mobility of
side chains, and the van der Waals interactions vary significantly.
The resulting affinity of these inhibitors depends mainly on fit-
ting the hydrophobic side chains into the binding pockets of HIV-
1 protease with an excessive strain in the inhibitor main chain
and the reduction in the side chains' mobility. The binding site
of HIV-1 protease, rich in hydrogen bond donors and acceptors,
is able to create many types of networks that stabilize the exact
position(s) of inhibitors under the HIV-1 protease flaps. Thus, the
numerous hydrogen bonds are important for binding affinity, but
not decisive.
6. Fauci A.S. (1993) Multifactorial nature of human immunodefi-
ciency virus disease: implications for therapy. Science;262:1011–
1017.
7. Lapatto R., Blundell T., Hemmings A., Overington J., Wilderspin
A.F., 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. (1989) X-ray
analysis of HIV-1 proteinase at 2.7 ꢀ resolution confirms structural
homology among retroviral enzymes. Nature;342:299–302.
8. Navia M.A., Fitzgerald P.M., McKeever B.M., Leu C.T., Heimbach
J.C., Herber W.K., Sigal I.S., Darke P.L., Springer J.P. (1989)
Three-dimensional structure of aspartyl protease from human
immunodeficiency virus HIV-1. Nature;337:615–620.
9. Wlodawer A., Miller M., Jaskolski M., Sathyanarayana B.K.,
Baldwin E., Weber I.T., Selk L.M., Clawson L., Schneider J., Kent
S.B. (1989) Conserved folding in retroviral proteases: crystal
structure of a synthetic HIV-1 protease. Science;245:616–621.
10. Velazquez-Campoy A., Muzammil S., Ohtaka H., Schçn A., Vega
S., Freire E. (2003) Structural and thermodynamic basis of resis-
tance to HIV-1 protease inhibition: implications for inhibitor
design. Curr Drug Targets Infect Disord;3:311–328.
Conclusions and Future Directions
The experimental data reported here show that the molecules of
the inhibitors under study are very flexible. Large rotations around
the bonds of the main chain and the side chains of the inhibitors
are necessary to bind into the enzyme and ⁄ or to form the crystal-
line state. Thus, the flexible nature of the inhibitors and their ability
to adopt an energetically most favorable conformation while binding
to the enzyme increase their biological activity in comparison with
more rigid compounds. The structural data presented here extend
the knowledge of HIV-1 protease inhibitors and are a good basis
for the design of more active compounds.
11. Rodrꢁguez-Barrios F., Gago F. (2004) HIV protease inhibition: lim-
ited recent progress and advances in understanding current pit-
falls. Curr Top Med Chem;4:991–1007.
Acknowledgments
12. Kaushik-Basu N., Basu A., Harris D. (2008) Peptide inhibition of
HIV-1: current status and future potential. BioDrugs;22:161–175.
13. Mastrolorenzo A., Rusconi S., Scozzafava A., Barbaro G., Supu-
ran C.T. (2007) Inhibitors of HIV-1 protease: current state of the
art 10 years after their introduction. From antiretroviral drugs to
antifungal, antibacterial and antitumor agents based on aspartic
protease inhibitors. Curr Med Chem;14:2734–2748.
14. Roberts N.A., Martin J.A., Kinchington D., Broadhurst A.V., Craig
J.C., Duncan I.B., Galpin S.A. et al. (1990) Rational design of
peptide-based HIV proteinase inhibitors. Science;248:358–361.
15. Ohta Y., Shinkai I. (1997) Saquinavir. Biorg Med Chem;5:465–
466.
The crystallographic work was supported by the Polish Committee
for Scientific Research, KBN (Grant No 4P05F 00819), Czech-Polish
ˇ
bilateral program CONTAC (Grant No 8 ⁄ 2002-2003) and GA CR
ˇ
305 ⁄ 07 ⁄ 1073 and GA AV CR IAA500500701. The authors are
grateful to Wiesław Majzner for his assistance in the data collec-
tion on CAD4 and to Dr Alexander Wlodawer and Jerry Alexandra-
tos for editorial suggestions. The EMBL – Hamburg sponsored the
data collection on DESY synchrotron. The experimental parts
including chemistry and biological tests were performed in the
Structural Biochemistry Program, National Cancer Institute – Fred-
erick Cancer Research and Development Center, SAIC, Frederick,
MD, USA.
16. FDA approves SQV ⁄ r. IAPAC Mon. 10, 39, 2004.
Chem Biol Drug Des 2012; 79: 798–809
807

´
Ziołkowska et al.
17. Vacca J.P., Dorsey B.D., Schleif W.A., Levin R.B., McDaniel S.L.,
Darke P.L., Zugay J. et al. (1994) L-735,524: an orally bioavailable
HIV-1 protease inhibitor. Proc Natl Acad Sci USA;91:4096–4100.
18. Cressey T.R., Plipat N., Fregonese F., Chokephaibulkit K. (2007)
Indinavir ⁄ ritonavir remains an important component of HAART
for the treatment of HIV ⁄AIDS, particularly in resource-limited
settings. Expert Opin Drug Metab Toxicol;3:347–361.
19. Kempf D.J., Sham H.L., Marsh K.C., Flentge C.A., Betebenner D.,
Green B.E., McDonald E. et al. (1998) Discovery of Ritonavir, a
potent inhibitor of HIV protease with high oral bioavailability
and clinical efficacy. J Med Chem;41:602–617.
20. Gehlhaar D.K., Verkhivker G.M., Rejto P.A., Sherman C.J., Fogel
D.B., Fogel L.J., Freer S.T. (1995) Molecular recognition of the
inhibitor AG-1343 by HIV-1 protease: conformationally flexible
docking by evolutionary programming. Chem Biol;2:317–324.
21. Shetty B.V., Kosa M.B., Khalil D.A., Webber S. (1996) Preclinical
pharmacokinetics and distribution to tissue of AG1343, an inhibi-
tor of human immunodeficiency virus type 1 protease. Antimicrob
Agents Chemother;40:110–114.
22. Patick A.K., Markowitz M., Appelt K., Wu B., Musick L., Kalish
V., Kaldor S., Reich S., Ho D., Webber S. (1996) Antiviral and
resistance studies of AG1343, an orally bioavailable inhibitor of
human immunodeficiency virus protease. Antimicrob Agents Che-
mother;40:292–297.
23. Sham H.L., Kempf D.J., Molla A., Marsh K.C., Kumar G.N., Chen
C.-M., Kati W. et al. (1998) ABT-378, a highly potent inhibitor of
the human immunodeficiency virus protease. Antimicrob Agents
Chemother;42:3218–3224.
24. Lyons F., Lechelt M., De Ruiter A. (2007) Steady-state lopinavir
levels in third trimester of pregnancy. AIDS;21:1053–1054.
25. Navia M.A., Sato V.L., Tung R.D. (1995) Design of VX-478, a
potent inhibitor of HIV protease. Int Antiviral News;3:143–145.
26. Kim E.E., Baker C.T., Dwyer M.D., Murcko M.A., Rao B.G., Tung
R.D., Navia M.A. (1995) Crystal structure of HIV-1 protease in
complex with VX- 478, a potent and orally bioavailable inhibitor
of the enzyme. J Am Chem Soc;117:1181–1182.
treatment of antiretroviral-experienced HIV-infected patients.
Expert Opin Pharmacother;8:839–850.
34. De Meyer S., Azijn H., Surleraux D., Jochmans D., Tahri A.,
Pauwels R., Wigerinck P., de Bethune M.P. (2005) 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 Chemo-
ther;49:2314–2321.
35. MacArthur R.D. (2007) Darunavir: promising initial results. Lan-
cet;369:1143–1144.
36. Rosin C.D., Belew R.K., Morris G.M., Olson A.J., Goodsell D.S.
(1999) Coevolutionary analysis of resistance-evading peptidomi-
metic inhibitors of HIV-1 protease. Proc Natl Acad Sci
USA;96:1369–1374.
37. Wang W., Kollman P.A. (2001) Computational study of protein
specificity: the molecular basis of HIV-1 protease drug resis-
tance. Proc Natl Acad Sci USA;98:14937–14942.
38. Velazquez-Campoy A., Muzammil S., Ohtaka H., Schçn A., Vega
S., Freire E. (2003) Structural and thermodynamic basis of resis-
tance to HIV-1 protease inhibition: implications for inhibitor
design. Curr Drug Targets Infect Disord;3:311–328.
39. Ghosh A.K., Chapsal B.D., Weber I.T., Mitsuya H. (2008) Design of
HIV protease inhibitors targeting protein backbone: an effective
strategy for combating drug resistance. Acc Chem Res;41:78–86.
40. Broglia R., Levy Y., Tiana G. (2008) HIV-1 protease folding and
the design of drugs which do not create resistance. Curr Opin
Struct Biol;18:60–66.
41. Jaskolski M., Tomasselli A.G., Sawyer T.K., Staples D.G., Heinrik-
son R.L., Schneider J., Kent S.B., Wlodawer A. (1991) Structure
at 2.5-ꢀ resolution of chemically synthesized human immunodefi-
ciency virus type 1 protease complexed with a hydroxyethylene-
based inhibitor. Biochemistry;30:1600–1609.
42. Dohnꢂlek J., HaÐek J., DuÐkovꢂ J., Petrokovꢂ H., Hradilek M.,
ˇ
ˇ
Soucek M., Konvalinka J., Brynda J., Sedlꢂcek J., Fꢂbry M.
(2001) A distinct binding mode of a hydroxyethylamine isostere
inhibitor of HIV-1 protease. Acta Crystalogr;D57:472–476.
27. Hou T., Yu R. (2007) Molecular dynamics and free energy studies
on the wild-type and double mutant HIV-1 protease complexed
with amprenavir and two amprenavir-related inhibitors: mecha-
nism for binding and drug resistance. J Med Chem;50:1177–1188.
28. Qazi N.A., Morlese J.F., Pozniak A.L. (2002) Lopinavir ⁄ ritonavir
(ABT-378 ⁄ r). Expert Opin Pharmacother;3:315–327.
29. Schechter M., Nunes E.P. (2007) Monotherapy with lopinavir ⁄ ri-
tonavir. Expert Opin Investig Drugs;16:735–741.
30. Piliero P.J. (2002) Atazanavir: a novel HIV-1 protease inhibitor.
Expert Opin Investig Drugs;11:1295–1301.
43. Dohnꢂlek J., HaÐek J., DuÐkovꢂ J., Petrokovꢂ H., Hradilek M.,
ˇ
ˇ
Soucek M., Konvalinka J., Brynda J., Sedlꢂcek J., Fꢂbry M.
(2002) Hydroxyethylamine isostere of an HIV-1 protease inhibitor
prefers its amine to the hydroxyl group in binding to catalytic
aspartates. J Med Chem;45:1432–1438.
44. Skꢂlovꢂ T., HaÐek J., Dohnꢂlek J., Petrokovꢂ H., Buchtelovꢂ E.,
ˇ
DuÐkovꢂ J., Soucek M., Majer P., Uhlꢁkovꢂ T., Konvalinka J.
(2003) An ethylenamine inhibitor binds tightly to both wild type
and mutant HIV-1 proteases. Structure and energy study. J Med
Chem;46:1636–1644.
31. Klei H.E., Kish K., Lin P.F., Guo Q., Friborg J., Rose R.E., Zhang
Y., Goldfarb V., Langley D.R., Wittekind M., Sheriff S. (2007) X-
ray crystal structures of HIV-1 protease mutants complexed with
atazanavir. J Virol 2007 Sep;81:9525–9535.
45. Miller M., Schneider J., Sathyanarayana B.K., Toth M.V., Marshall
G.R., Clawson L., Selk L., Kent S.B., Wlodawer A. (1989) Structure
of complex of synthetic HIV-1 protease with a substrate-based
inhibitor at 2.3 ꢀ resolution. Science;246:1149–1152.
32. Poppe S.M., Slade D.E., Chong K.T., Hinshaw R.R., Pagano P.J.,
Markowitz M., Ho D.D., Mo H., Gorman R.R. 3rd, Dueweke T.J.,
Thaisrivongs S., Tarpley W.G. (1997) Antiviral activity of the di-
hydropyrone PNU-140690, a new nonpeptidic human immunode-
ficiency virus protease inhibitor. Antimicrob Agents
Chemother;41:1058–1063.
46. Berman H.M., Battistuz T., Bhat T.N., Bluhm W.F., Bourne P.E.,
Burkhardt K., Feng Z. et al. (2002) The protein data bank. Acta
Crystallogr;D58:899–907.
47. Vondrasek J., Wlodawer A. (2002) HIVdb – a database of the
structures of human immunodeficiency virus protease. Proteins:
Struct Funct Genet;49:429–431.
33. De Mendoza C., Morello J., Garcia-Gasco P., Rodriguez-Novoa S.,
Soriano V. (2007) Tipranavir: a new protease inhibitor for the
48. Stuk T.L., Haight A.R., Scarpetti D., Allen M.S., Menzia J.A.,
Robbins T.A., Parekh S.I., Langridge D.C., Tien J.-H.J., Pariza
808
Chem Biol Drug Des 2012; 79: 798–809

Active HIV-1 Protease Inhibitors Derived from 3-Hexanol
R.J., Kerdesky F.A. (1994) An efficient stereocontrolled strategy
for the synthesis of hydroxyethylene dipeptide isosters. J Org
Chem;59:4040–4041.
63. Petrokovꢂ H., Skꢂlovꢂ T., Dohnꢂlek J., DuÐkovꢂ J., Buchtelovꢂ E.,
ˇ
ˇ
Soucek M., Konvalinka J., Brynda J., Fꢂbry J.M., Sedlꢂcek J.,
HaÐek J. (2004) Role of hydroxyl group and R ⁄ S configuration of
isostere in binding properties of HIV-1 protease inhibitors. Eur J
Biochem;271:4451–4461.
49. Kageyama S., Mimoto T., Murakawa Y., Nomizu M., Ford H. Jr,
Shirasaka T., Gulnik S., Erickson J.W., Takada K., Hayashi H.,
Broder S., Kiso Y., Mitsuya H. (1993) In vitro anti-human immu-
nodeficiency virus (HIV) activities of transition state mimetic HIV
protease inhibitors containing allophenylnorstatine. Antimicrob
Agents Chemother;37:810–817.
50. Kuzmic P., Garcia-Echeveria C., Rich D.H. (1993) Stabilization of
HIV proteinase dimer by bound substrate. Biochem Biophys Res
Commun;194:301–305.
64. Skꢂlovꢂ T., Dohnꢂlek J., DuÐkovꢂ J., Petrokovꢂ H.,
ˇ
Hradꢁlek M., Soucek M., Konvalinka J., HaÐek J. (2006) HIV-1
protease mutations and inhibitor modifications monitored on a
series of complexes. Structural basis for the effect of
the A71V mutation on the active site. J Med Chem;49:5777–
5784.
ˇ
65. DuÐkovꢂ J., Dohnꢂlek J., Skꢂlovꢂ T., Petrokovꢂ H., Vondrꢂckova
ˇ
E., Hradꢁlek M., Konvalinka J., Soucek M., Brynda J., Fꢂbry M.,
51. North A.C.T., Philips D.C., Mathews F.S. (1968) A semi-empirical
method of absorption correction. Acta Crystallogr;A24:351.
52. Frenz B.A. (1984) SDP-Structure Determination Package. Delft,
Holland: Enraf-Nonius.
53. Otwinowski Z., Minor W. (1997) Processing of X-ray diffraction
data collected in oscillation mode. Methods Enzymol;276:307–326.
54. Sheldrick G.M. (2008) A short history of SHELX. Acta Crystallogr
A;A64:112–122.
ˇ
Sedlꢂcek J., HaÐek J. (2006) On the role of R-configuration of
the reaction intermediate isostere in HIV-1 protease inhibitor
binding X-ray structure at 2.0 ꢀ resolution. Acta Crystallogr D
Biol Crystallogr;D62:489–497.
66. DeLano W.L. (2002) The PyMOL Molecular Graphics System DeL-
ano Scientific. San Carlos, CA, USA. Available at: http://
www.pymol.org.
55. Sheldrick G.M. (1993) SHELXL-93. Program for Crystal Structure
Refinement. Germany: University of Gçttingen.
67. Wallace A.C., Laskowski R.A., Thornton J.M. (1995) LIGPLOT: a
program to generate schematic diagrams of protein-ligand inter-
actions. Prot Eng;8:127–134.
56. Vickovic J. (1988) Crystal structure utility, a highly automatic and
selective program for the calculation and presentation of geomet-
rical parameters and their e.s.d.`s. J Appl Crystallogr;21:987–990.
57. Bone R., Vacca J.P., Anderson P.S., Holloway M.K. (1991) X-ray crystal
structure of the HIV protease complex with L-700, 417 an inhibitor
with pseudo C2 symmetry. J Am Chem Soc;113:9382–9384.
58. Jones T.A., Kjeldgaard M. (1993) O-the Manual. Version 5.9. Swe-
den: Department of Molecular Biology, BMC, Uppsala University
and Denmark: Department of Chemistry, Aarhus University.
59. Randad R.S., Lubkowska L., Silva A.M., Guerin D.M.A., Gulnik
S.V., Yu B., Erickson J.W. (1996) Structure-based design of achi-
ral, nonpeptidic hydroxybenzamide as a novel P2 ⁄ P2¢ replace-
ment for the symmetry-based HIV protease inhibitors. Bioorg
Med Chem;4:1471–1480.
60. Randad R.S., Lubkowska L, Bujacz A., Gulnik S.V. Yu B., Silva A.,
Munshi S., Lynch T.M., Clanton D.J., Bhat T.N., Erickson J.W.
(1995) Structure-based design of achiral anthranilamides as a
P2/P2¢ surrogates for symmetry-based HIV protease inhibitors:
design, synthesis, X-ray structure, enzyme inhibition and antiviral
activity. Bioorg.Med.Chem.Letters;5:2557–2562.
Notes
aXP-Interactive Molecular Grafics (1992) Siemens Analytical Xray Inst.
b
INSIGHTII. (2000) Accelrys, Molecular Simulations Inc, available at: http://
www.accelrys.com/academic.
Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Table S1. Crystal data and experimental details of 1–4.
Appendix S1. Synthesis of compounds 1–4.
Appendix S2. CCDC-212633 (1), CCDC-212634 (2), CCDC-212635
(3), and CCDC-212636 (4) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre (Cam-
bridge, UK: Cambridge Crystallographic Data Centre, available at:
http://www.ccdc.cam.ac.uk/).
61. Veerepandian B., Cooper J.B., Sali A., Blundell T.L., Rosati R.L.,
Dominy B.W., Damon D.B., Hoover D.J. (1992) Direct observation
by X-ray analysis of the tetrahedral intermediate of aspartic pro-
teinases. Protein Sci;1:322–328.
62. Skꢂlovꢂ T., HaÐek J., Dohnꢂlek J., Petrokovꢂ H., Buchtelovꢂ E.
(2002) Mutant HIV-1 protease complexed with tetrapeptide inhib-
itor. Acta Physica Polonica A;101:659–663.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
Chem Biol Drug Des 2012; 79: 798–809
809