Inhibitors of the C2-symmetric HIV-1 protease: Nonsymmetric binding of a symmetric cyclic sulfamide with ketoxime groups in the P2/P2' side chains

Article abstract of DOI:10.1021/jm991054q

Symmetric cyclic sulfamides, substituted in the P2/P2' position with functional groups foreseen to bind preferentially to the S2/S2' subsites of HIV-1 protease, have been prepared. Despise efforts to promote a symmetric binding, the sulfamides seemed pron

Full text of DOI:10.1021/jm991054q

4054
J . Med. Chem. 1999, 42, 4054-4061
In h ibitor s of th e C2-Sym m etr ic HIV-1 P r otea se: Non sym m etr ic Bin d in g of a
Sym m etr ic Cyclic Su lfa m id e w ith Ketoxim e Gr ou p s in th e P 2/P 2′ Sid e Ch a in s
J ohan Hulte´n,† Hans O. Andersson,‡ Wesley Schaal,† Helena U. Danielson,§ Bjo¨rn Classon,∇
Ingemar Kvarnstro¨m, Anders Karle´n,† Torsten Unge,‡ Bertil Samuelsson,| and Anders Hallberg*,†
Department of Organic Pharmaceutical Chemistry, Uppsala Biomedical Centre, Uppsala University, Box 574,
SE-751 23 Uppsala, Sweden, Department of Cell and Molecular Biology, Uppsala Biomedical Centre, Uppsala University,
Box 596, SE-751 24 Uppsala, Sweden, Department of Biochemistry, Uppsala Biomedical Centre, Uppsala University,
Box 576, SE-751 23 Uppsala, Sweden, Medivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden, Department of Chemistry,
Linko¨ping University, SE-581 83 Linko¨ping, Sweden, and Department of Organic Chemistry, Arrhenius Laboratory,
Stockholm University, SE-106 91 Stockholm, Sweden
Received April 8, 1999
Symmetric cyclic sulfamides, substituted in the P2/P2′ position with functional groups foreseen
to bind preferentially to the S2/S2′ subsites of HIV-1 protease, have been prepared. Despite
efforts to promote a symmetric binding, the sulfamides seemed prone to bind nonsymmetrically,
as deduced from X-ray crystal structure analysis of one of the most potent inhibitors, possessing
ketoxime groups in the P2/P2′ side chains. Ab initio calculations suggested that the nonsym-
metric conformation of the cyclic sulfamide scaffold had lower energy than the corresponding
symmetric, cyclic urea-like conformation.
In tr od u ction
Inhibition of HIV-1 protease in vitro results in the
production of virons that are immature and noninfec-
tious.1 HIV-1 protease inhibitors, used mainly in com-
bination with reverse transcriptase inhibitors, have
been shown to reduce the viral load and increase the
number of CD4+ lymphocytes in HIV-infected individ-
uals.2-5 The clinical emergence of resistant mutants
suggests that there will be a need for an ever increasing
armament of new drugs that will preferably have a
unique pattern of resistance development and good
bioavailability.6-9
Today there is a large abundance of structural
information on the HIV-1 protease that has been gener-
ated by X-ray crystallography10-13 and also in recent
years by 2D-NMR spectroscopy.14-18 A tetrahedrally
coordinated structural water (W301)10 is commonly
found in the 3D structures of linear peptidomimetic
HIV-1 protease inhibitors. This water is symmetrically
F igu r e 1. Urea compound 1 with symmetric binding and
sulfamide 2 with nonsymmetric binding to the HIV-1 protease.
positioned and bridges the two flaps of the dimeric
enzyme to two carbonyl groups of the inhibitors. Lam
et al. took advantage of the structural information
cyclic sulfamide scaffold adopts a nonsymmetric con-
obtained from X-ray structures in an elegant design of
formation which places the P2′ side chain into the S1′
subsite and the P1′ side chain into the S2′ subsite.21 To
guide further design prior to the initiation of a more
extensive medicinal chemistry program, based on the
core structure of 2, we were prompted to determine
whether a symmetric binding of the symmetrically
substituted cyclic sulfamides could be induced. After
some exploratory modeling, we decided to prepare six
cyclic sulfamides, 3-8, that were symmetrically sub-
stituted in the P2/P2′ positions with functional groups
foreseen to bind preferentially to the S2/S2′ subsites.
potent nonpeptidal cyclic urea inhibitors, i.e., DMP323,19
where the urea oxygen replaces and mimics the bridging
structural water.
We prepared the carbohydrate-based, C2-symmetric,
cyclic urea 1 and the cyclic sulfamide 2 (Figure 1), both
containing functionalities that mimic the tetrahedrally
coordinated structural water, and compared the X-ray
structures of the inhibitor/protease complexes.20,21 This
comparison revealed an unexpected binding of 2. The
* Corresponding author.
† Department of Organic Pharmaceutical Chemistry, UBC.
‡ Department of Cell and Molecular Biology, UBC.
§ Department of Biochemistry, UBC.
∇ Medivir AB.
Resu lts a n d Discu ssion
Syn th esis. The iodo derivative 11 served as a precur-
sor in the palladium-catalyzed reactions to provide the
Linko¨ping University.
| Stockholm University.
10.1021/jm991054q CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/14/1999

Inhibitors of the C2-Symmetric HIV-1 Protease
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4055
Sch em e 1
compounds 3-6 (Scheme 1). Compound 11 was obtained
in 80% yield after alkylation of the parent sulfamide
1020 with 3-iodobenzyl bromide in the presence of NaH
in DMF. Aryl methyl ketones can be prepared by Heck
arylation of alkyl vinyl ethers22 and with very high
regioselectivity in the presence of bidentate ligands, as
demonstrated by Cabri et al.23 In our first attempts to
prepare 3 from 11 via palladium-catalyzed arylation,
where the commonly used butyl vinyl ether was em-
ployed as olefin, a moderate yield was encountered.
However, the ligand-directed regiocontrolled R-arylation
of 2-hydroxyethyl vinyl ether with 1,3-bis(diphenylphos-
phino)propane (DPPP) as ligand24 provided 3 in 83%
yield after subsequent treatment with HCl/ether and
methanol. The ketoxime derivative 4 was prepared from
3 by treatment with hydroxylamine hydrochloride in
ethanol.25 A Heck arylation designed to promote â-aryl-
ation by palladium-nitrogen chelation control26 fur-
nished compounds 5 and 6. Thus, the selective â-aryl-
ation of [2-(dimethylamino)ethoxy]ethene and subsequent
hydrolysis in a two-phase system led to deprotection of
the vinyl ether and delivered the corresponding alde-
hyde. Reduction of the intermediate aldehyde with
NaBH4 followed by deprotection of the MEM groups
provided the hydroxyethyl derivative 5 in 19% yield over
four steps. Treatment of the â-arylated vinyl ether with
HCl/ether in methanol yielded the methyl acetal 6.
Compound 12 was obtained in 80% yield from 10 after
alkylation with methyl 4-bromomethylbenzoate in the
presence of NaH in DMF. The ester groups of 12 were
thereafter smoothly reduced27 with excess LiBH4 to
afford the hydroxymethyl derivative 7 after deprotec-
tion. Compound 12 was transformed to 8 in a mixture
of HCl/ether and methanol.
HIV P r otea se In h ibition . The HIV-1 protease was
cloned and heterologously expressed in Esherichia coli
and purified as described elsewhere.28 The Ki values for
the synthesized compounds were determined by a
fluorometric assay29 (Table 1).
Mod elin g. To determine whether a sulfamide-based
inhibitor could be expected to adopt a urea-like confor-
mation, we performed calculations on the symmetric
and nonsymmetric conformation of the models of 2
(Figure 2). Other possible ring conformations were not
explored. Ab initio calculations on the two model
conformations at the B3LYP/6-31G* level of theory
indicated that the nonsymmetric conformation was 2.4
kcal/mol lower in energy than the symmetric conforma-
tion. This penalty seemed low enough to be considered
surmountable with substituents that anchor the P2/P2′
side chains in the S2/S2′ subsites through interactions
with Asp29, Asp30, and Gly48. For information about
the interactions with these possible anchoring points in
the S2/S2′ subsite, we searched the Protein Data
Bank30,31 for coordinates for DMP323,32 a urea-based
inhibitor with hydroxymethyl substituents in the para

4056 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hulte´n et al.
Ta ble 1. Inhibitory Activity of Cyclic Sulfamide Compounds
Against HIV-1 Protease
Ch a r t 1. Urea Compound 9
crystallized and analyzed the 3D structure of the
carbohydrate-derived cyclic urea inhibitor 9 (Chart 1),
an analogue to DMP323 that had been previously
prepared. As expected, the inhibitor binds in a sym-
metric conformation and the hydroxymethyl substitu-
ents are interacting with the backbone amide NH of
Asp30/30′. Furthermore, well-ordered water molecules
tight packing to the aromatic rings of the P1/P2 and P1′/
P2′ side chains are seen distinctly.
Models for both the symmetric (urea-like) and non-
symmetric (flipped) conformations of 3-8 were made
to help anticipate their potential interactions in the
HIV-1 protease binding site. The modeling of compound
4 was complicated by the fact that the E/ Z configuration
of the ketoximes was not easily predictable. A search of
the Cambridge Structural Database33 for acetophenone
oximes revealed that the E configuration was more
frequent than the Z configuration.34
In the models of the symmetric conformations, P2 and
P2′ were directed into the S2 and S2′ pockets. The R1
and R2 functional groups (see Table 1) were generally
seen to participate in hydrogen bonding to Asp29/29′
and/or Asp30/30′. The carbonyl oxygens of 3 and 8 and
also the hydroxyl oxygens of 4 and 7 could form
hydrogen bonds with the backbone NH of either Asp29/
29′ or Asp30/30′. Furthermore the hydroxyl group of 4,
5 and 7 could form hydrogen bonds to the carboxylate
side chain of either Asp29/29′ or Asp30/30′. The acetal
of 6 and the ester group of 8 were directed toward the
solvent.
In the models of the nonsymmetric conformations, P2
and P2′ were directed into the S2 and S1′ pockets,
respectively. Interactions in the S2 pockets generally
followed the pattern seen for the symmetric conforma-
tions. Interactions in the S1′ pockets focused around
Arg8. The P2 carbonyl group of 3 and 8 and the acetal
group of 6 were directed primarily into the solvent. The
P2 hydroxyl groups of 4 and 5 could form hydrogen
bonds to the carboxylate side chain of Asp30. The P2
hydroxyl group of 7 could form a hydrogen bond to the
carbonyl backbone of Asp30. The P2′ carbonyl oxygen
of 3 and 8, the acetal of 6, and the hydroxyl groups of
4, 5, and 7 could form hydrogen bonds with the guani-
dino group of Arg8. In conclusion, the modeling study
predicts that fewer hydrogen bond interactions are
available in the S1′ pocket.
a
Ki values for compounds 3-8 were determined using a
spectrofluorometric assay.29
compounds with acetyl (Ki ) 0.06 nM), ketoxime (Ki ) 0.018 nM),
and hydroxymethyl (Ki ) 0.27 nM) substituents have been
reported by DuPont-Merck.19,35
Ki values for related cyclic urea
b
Biologica l Resu lts. The Ki values of 3-8 are shown
in Table 1. The inhibitor 4, substituted with two
ketoxime functions, exhibited a Ki value of 3.4 nM as
compared to 19 nM for the parent compound 2. Oxime
functionalities when applied to the cyclic urea series
were recently found to furnish inhibitors with very high
potency, and a methyl ketoxime related to 4 had a Ki of
0.018 nM.35 Han and Lam reported that each of the
hydroxyl groups of the ketoxime in the urea derivative
donates a hydrogen bond to the carboxylate side chain
of Asp30/30′, located in the S2/S2′ subsite. Furthermore,
F igu r e 2. Superimposition of the symmetric (dark gray) and
nonsymmetric (light gray) conformation of the cyclic sulfamide
scaffold.
positions of the P2/P2′ side chains. However, at the time
of this investigation the X-ray coordinates for DMP323
in the native enzyme were not available. Therefore, we

Inhibitors of the C2-Symmetric HIV-1 Protease
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4057
each nitrogen of the ketoxime groups accepts a hydrogen
bond from the backbone amide NH of Asp30/30′ as
deduced from the X-ray crystal structures. The hy-
droxymethyl-substituted sulfamide inhibitor 7, which
exhibited an almost identical Ki (3.1 nM) as compared
to the ketoxime 4, is 10-fold less potent than the
corresponding urea analogue 9. Compound 5 was a poor
inhibitor despite modeling suggesting that the hydroxy-
ethyl substituents could interact with Asp30/30′. Com-
pounds 3 and 8 with hydrogen bond-accepting keto and
ester carbonyl groups were both weak inhibitors of the
protease and exhibited Ki values of 59 and 84 nM,
respectively. The acetal derivative 6, anticipated from
modeling experiments to allow for a favorable interac-
tion with Arg8, was in fact the worst inhibitor in the
series and found to be 20-fold less potent than the
parent sulfamide 2.
On the basis of the difference in Ki values between
the sulfamides 3, 4, 7 and the corresponding ureas, we
anticipate that the cyclic sulfamides were all binding
in an nonsymmetric conformation to the enzyme. The
difference in desolvation energy for urea and sulfamide
could account for part of this difference in affinity.
However, previous molecular dynamic calculations
showed no indication of different desolvation energy for
2 as compared to 1.20 We were encouraged to obtain a
3D structure of one of the sulfamide compounds to
elucidate this issue. Ultimately we were successful in
obtaining good crystals of the HIV protease complex
with one of the most potent inhibitors of the series, the
ketoxime 4 (Figure 3), and the 3D structure was
determined to 1.80 Å resolution with a Rcryst of 21.2%
(Rfree ) 23.6%). The analysis of the data established that
4, designed with P2/P2′ substituents anticipated to
strongly bind to the S2/S2′ subsites, in fact, adopted a
nonsymmetrical binding mode with the P2′ aryl ke-
toxime substituent positioned in the S1′ subsite (Figure
4). The ketoxime group located in the S2 subsite has
the E configuration around the double bond which
results in a hydrogen bond between the hydroxyl group
of the ketoxime and the carboxylate side chain of Asp30.
Han and Lam reported a corresponding hydrogen bond
in the cyclic urea ketoxime and in addition to this a
hydrogen bond between the nitrogen of the ketoxime
and the backbone amide NH of Asp30. The latter
interaction is however not present in the complex
between the sulfamide ketoxime 4 and the protease.
Structural water molecules with interactions similar to
the water molecules found in the complex of inhibitor 9
and the protease are also observed. The electron density
for the ketoxime group in the S1′ subsites is missing,
indicating that the ketoxime lacks specific interactions
with the protease or structural water molecules. Con-
sidering the P2′ aryl ketoxime substituent in the lipo-
philic S1′ subsite, a displacement of the structural water
seemed feasible. However, such displacement of the
structural water did not take place as deduced from the
X-ray structure (see Figure 3).36 Thus only one of the
ketoxime groups seems to interact favorably with the
protease. This leads to fewer interactions of inhibitor
4, as compared to the urea analogue, and could provide
part of the explanation for the lower activity of the
sulfamide analogue. The 10-fold lower activity of com-
pound 7 as compared to 9 can also be explained if it is
F igu r e 3. Conformation of compounds 9 (panel A) and 4
(panel B) in the HIV-1 protease active site (chain A colored
green and chain B yellow). (A) Inhibitor 9, with a Ki of 0.23
nM, adopts a symmetric conformation. Consequently, the P1/
P2 and P1′/P2′ side chains have identical interactions with the
corresponding subsites. The hydroxymethyl substituents are
interacting with the backbone amide NH of Asp30/30′. (B)
Compound 4 binds in a nonsymmetric conformation: i.e., the
P2 side chain is located in the S2 subsite, whereas the P2′
side chain is in the S1′ subsite. In the S2 subsite, hydrogen
bonds are formed between the hydroxyl group of the ketoxime
and the carboxylate side chain of Asp30 (2.9 Å) and a water
molecule (2.7 Å). In the S1 subsite there are no suitable
interactions for the ketoxime group. No electron density was
detected for the P2′ ketoxime group in the 2Fobs - Fcalc map.
Conserved water molecules (light blue) are located between
the P1/P2 and P1′/P2′ side chains in both complexes. The figure
was drawn with the program MOLSCRIPT 2.02.50
assumed that only one of the hydroxymethyl substitu-
ents in 7 is interacting favorably with the S2/S2′
subsite.11
Con clu sion
In summary, our results suggest that the cyclic
sulfamide core structure is not prone to adopt a cyclic

4058 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hulte´n et al.
F igu r e 4. Stereoview of the X-ray structure of inhibitor 9 (top) and inhibitor 4 (bottom), showing the conformation of the cyclic
sulfamide scaffold and the direction of the P2/P2′ and P1/P1′ side chains. The position of the P2′ ketoxime of 4 was determined
by modeling.
water and diethyl ether. The solvent was removed and to the
crude product in methanol (5 mL) was added wet HCl in ether
urea conformation and consequently that the structure-
activity relationship for the two classes of compounds
(10 mL). The reaction mixture was stirred overnight at 25 °C.
should differ considerably. On the basis of ab initio
The solvent was removed and purification by flash column
calculations of a truncated form of the cyclic sulfamides,
chromatography on silica (CH2Cl2/CH3OH, 200:1) gave the
the nonsymmetric conformation was found to be 2.4
product as an oil (27 mg, 82%): IR (CHCl3) υ 1682, 1598, 1496,
1
1437 cm-1; [R]D ) +15.8° (c ) 0.36, CHCl3, 22 °C); H NMR
(270.2 MHz, CDCl3) δ 7.92 (s, 2H) 7.75 (d, J ) 7.7 Hz, 2H),
7.68 (d, J ) 7.7 Hz, 2H), 7.36 (t, J ) 7.7 Hz, 2H), 7.16 (appt,
J ) 7.8 Hz, 4H), 6.89 (t, J ) 7.2 Hz, 2H), 6.63 (d, J ) 8.4 Hz,
4H), 4.90 (m, 4H), 4.32 (m, 4H), 4.17 (dd, J ) 9.6, 5.1 Hz, 2H),
kcal/mol lower in energy than the symmetric urea-like
form. This penalty seemed low enough to be considered
surmountable with substituents that anchor the P2/P2′
side chains in the S2/S2′ subsites. However, the X-ray
crystal structure of inhibitor 4 clearly shows a nonsym-
metric mode of binding despite efforts to induce a
symmetric binding through symmetrically substituted
ketoxime groups in P2/P2′ side chains.
4.08 (dd, J ) 9.7, 6.1 Hz, 2H), 3.39 (brs, 2H), 2.47 (s, 6H); 13
C
NMR (100.2 MHz, CDCl3) δ 198.3, 157.5, 139.0, 137.2, 132.4,
129.6, 129.0, 127.6, 127.3, 121.7, 114.4, 75.1, 67.1, 55.6, 52.9,
26.7. Anal. (C36H38N2O8S‚0.5H2O) C, H, N.
(3R,4S,5S,6R)-2,7-Bis[(3-m eth yloxim op h en yl)m eth yl]-
4,5-d ih yd r oxy-3,6-b is(p h en oxym et h yl)-1,2,7-t h ia d ia ze-
p in e 1,1-Dioxid e (4). To a solution of compound 3 (43 mg,
0.065 mmol) in a mixture of ethanol (2 mL) and water (1 mL)
were added hydroxylamine hydrochloride (23 mg, 0.33 mmol)
and NaOH (41 mg, 1.0 mmol). The reaction mixture was
heated at 60 °C for 3 h. The solvent was removed and
purification by flash column chromatography on silica (CH2-
Cl2/CH3OH, 50:1) gave the product as a white solid (32 mg,
73%): 1H NMR (399.2 MHz, CD3OD) δ 7.76 (s, 2H), 7.49 (d, J
) 7.8 Hz, 2H), 7.46 (d, J ) 7.8 Hz, 2H), 7.27 (t, J ) 7.8 Hz,
2H), 7.18 (dd, J ) 8.8, 7.5 Hz, 4H), 6.87 (t, J ) 7.4 Hz, 2H),
6.73 (d, J ) 8.8 Hz, 4H), 5.05 (d, J ) 17.9 Hz, 2H), 4.92 (d, J
) 17.6 Hz, 2H), 4.41 (t, J ) 6.7 Hz, 2H), 4.13 (m, 6H), 2.19 (s,
6H); 13C NMR (67.8 MHz, acetone-d6) δ 159.1, 154.3, 141.0,
138.1, 130.1, 128.9, 128.2, 125.4, 125.0, 121.6, 115.3, 74.7, 67.1,
55.7, 52.7, 11.5. Anal. (C36H40N4O8S‚1.0H2O) C, H, N.
(3R,4S,5S,6R)-2,7-Bis{[3-(2-h yd r oxyeth yl)ph en yl]m eth -
yl}-3,6-bis(p h en oxym eth yl)-4,5-d ih yd r oxy-1,2,7-th ia d ia z-
ep in e 1,1-Dioxid e (5). Into the reaction vessel were added
K2CO3 (21.9 mg, 0.158 mmol), LiCl (11.2 mg, 0.264 mmol),
NaOAc (12.99 mg, 0.158 mmol), and DMF (1.0 mL). To this
mixture were added were added Pd(OAc)2 (2.96 mg, 0.0132
mmol) and compound 11 (55.5 mg, 0.055 mmol) followed by
addition of water (0.11 mL) and [2-(dimethylamino)ethoxy]-
ethene (38.0, 0.329 mmol). The reaction mixture was heated
at 80 °C overnight. After cooling, the reaction mixture was
partitioned between water and diethyl ether. The solvent was
removed, the crude product was dissolved in diethyl ether (15
mL), 3 M HCl aq (15 mL) was added, and the reaction mixture
was stirred vigorously overnight at 25 °C. The ether layer was
separated and the aqueous layer was extracted with ether (2
× 20 mL). The combined ether extracts were dried and
concentrated in vacuo. The crude aldehyde was dissolved in
methanol (3 mL) at 0 °C. To this solution was added NaBH4
Exp er im en ta l Section
Ch em istr y. Gen er a l In for m a tion . Melting points were
recorded on an Electrothermal melting point apparatus and
are uncorrected. Optical rotations were obtained on a Perkin-
Elmer 241 polarmeter. Specific rotations ([R]D) are reported
in degrees/decimeter and the concentration (c) is given in
grams/100 mL in the specific solvent. 1H and 13C NMR spectra
were recorded on a J EOL J NM-EX 270 spectrometer at 270.2
and 67.8 MHz, respectively, or on a J EOL J NM-EX 400
spectrometer at 399.8 and 100.5 MHz, respectively, and the
chemical shifts are given in ppm relative to tetramethylsilane.
Infrared spectra were recorded on a Perkin-Elmer 1600 series
FTIR instrument. Elemental analyses were performed by
MikroKemi AB, Sweden, or Analytische Laboratorien, Ger-
many, and were within (0.4% of calculated values. Mass
spectroscopy was carried out on a J EOL SX 102 instrument.
Flash column chromatography was performed on silica gel 60,
0.04-0.063 mm (E. Merck), with gradient elution unless
otherwise noted. Thin-layer chromatography was performed
on precoated silica gel F-254 plates (0.25 mm; E. Merck) and
was visualized with UV light and H2SO4 in ethanol or
phosphomolybdic acid or ninhydrin. Standard workup: organic
layers were dried with MgSO4 and concentrated in vacuo.
(3R,4S,5S,6R)-2,7-Bis[(3-a cet ylp h en yl)m et h yl]-4,5-d i-
h yd r oxy-3,6-bis(p h en oxym eth yl)-1,2,7-th ia d ia zep in e 1,1-
Dioxid e (3). Into the reaction vessel were added Pd(OAc)2
(0.81 mg, 0.0036 mmol), DPPP (2.97 mg, 0.0072 mmol), and a
small amount of DMF (1.0 mL). To this mixture were added
Et3N (18.3 mg, 0.18 mmol) and compound 11 (50 mg, 0.050
mmol) followed by addition of TlOAc (37.9 mg, 0.144 mmol),
2-hydroxyethyl vinyl ether (58.3 mg, 0.66 mmol), and DMF
(1.0 mL). The reaction mixture was heated at 80 °C overnight.
After cooling, the reaction mixture was partitioned between

Inhibitors of the C2-Symmetric HIV-1 Protease
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4059
(6.3 mg, 0.167 mmol). The reaction mixture was stirred
overnight under N2 atmosphere, slowly rising the temperature
to room temperature, and thereafter HCl in ether (5 mL) was
added and stirred overnight at room temperature. The solvent
was removed and purification by flash column chromatography
on silica (CH2Cl2/CH3OH, 50:1) gave the product as a white
solid (12.8 mg, 19%): IR (CHCl3) υ 3684, 3603, 1597, 1495
CDCl3) δ 166.9, 157.5, 143.4, 130.1, 129.7, 129.6, 127.6, 121.9,
114.5, 73.3, 67.1, 55.9, 53.2, 52.3. Anal. (C36H38N2O10S) C, H,
N.
(3R,4S,5S,6R)-2,7-Bis[(3-iod op h en yl)m eth yl]-4,5-bis[(2-
m eth oxyeth oxy)m eth oxy]-3,6-bis(p h en oxym eth yl)-1,2,7-
th ia d ia zep in e 1,1-d ioxid e (11). To a solution of 1020 (50 mg,
0.088 mmol) in DMF (2 mL) were added NaH (80% suspension,
10.5 mg, 0.35 mmol) and 3-iodobenzyl bromide (104.2, 0.35
mmol). The reaction mixture was stirred under a N2 atmo-
sphere overnight and concentrated in vacuo. Purification by
flash column chromatography on silica gel (CH2Cl2/CH3OH,
100:1) gave the product as a colorless oil (77 mg, 88%): IR
(film) υ 1674, 1599, 1566 cm-1; [R]D ) +14.1° (c ) 0.81, CHCl3,
1
cm-1; [R]D ) +5.8° (c ) 0.72, CHCl3, 22 °C); H NMR (399.8
MHz, CDCl3) δ 7.32 (m, 6H), 7.09 (d, J ) 7.3 Hz, 2H), 6.96 (t,
J ) 7.4 Hz, 2H), 6.72 (d, J ) 7.8 Hz, 4H), 4.77 (m, 4H), 4.30-
4.10 (m, 8H), 3.75 (t, J ) 6.4 Hz, 4H), 3.31 (brs, 2H), 2.77 (t,
J ) 6.4 Hz, 4H), 1.68 (brs, 2H); 13C NMR (100.2 MHz, CDCl3)
δ 157.6, 139.1, 138.2, 129.6, 128.9, 128.5, 128.4, 126.0, 121.6,
114.5, 75.1, 66.7, 63.5, 56.2, 53.0, 39.0. Anal. (C36H42N2O8S)
C, H, N.
1
21 °C); H NMR (270 MHz, CDCl3) δ 7.73 (s, 2H), 7.51 (appt,
4H), 7.23 (m, 4H), 7.02 (t, J ) 7.7 Hz, 2H), 6.93 (t, J ) 7.3 Hz,
2H), 6.70 (d, J ) 8.9 Hz, 4H), 4.92 (d, J ) 17.2 Hz, 2H), 4.84
(d, J ) 7.1 Hz, 2H), 4.79 (d, J ) 6.8 Hz, 2H), 4.76 (d, J ) 17.4
Hz, 2H), 4.43 (t, J ) 6.9 Hz, 2H), 4.21 (s, 2H), 4.00 (m, 4H),
3.74 (ddd, J ) 11.0, 5.4, 4.1 Hz, 2H), 3.64 (ddd, J ) 10.9, 4.9,
4.1 Hz, 2H), 3.42 (m, 4H), 3.27 (s, 6H); 13C NMR (67.8 MHz,
CDCl3) δ 157.8, 142.0, 136.3, 136.0, 130.5, 129.7, 126.5, 121.5,
114.5, 96.3, 94.5, 77.3, 71.6, 68.2, 66.1, 59.1, 53.5, 51.2. Anal.
(C40H48I2N2O10S) C, H, N.
(3R,4S,5S,6R)-2,7-Bis{[3-(2,2-d im eth oxyeth yl)p h en yl]-
m eth yl}-4,5-d ih yd r oxy-3,6-bis(p h en oxym eth yl)-1,2,7-th i-
a d ia zep in e 1,1-Dioxid e (6). Into the reaction vessel were
added K2CO3 (21.9 mg, 0.158 mmol), LiCl (11.2 mg, 0.264
mmol), NaOAc (12.99 mg, 0.158 mmol), and DMF (1.0 mL).
To this mixture were added Pd(OAc)2 (2.96 mg, 0.0132 mmol)
and compound 11 (55.5 mg, 0.055 mmol) followed by addition
of water (0.11 mL) and [2-(dimethylamino)ethoxy]ethene (38.0,
0.329 mmol). The reaction mixture was heated at 80 °C
overnight. After cooling, the reaction mixture was partitioned
between water and diethyl ether. The solvent was removed,
the crude product was dissolved in methanol (5 mL), and
saturated HCl in ether (10 mL) was added. The reaction
mixture was stirred overnight and concentrated in vacuo.
Purification by flash column chromatography on silica gel
(CH2Cl2, 200:1) gave a colorless oil (18.8 mg, 39%): IR (film)
(3R,4S,5S,6R)-2,7-Bis[[4-(methoxycarbonyl)phenyl]meth-
yl]-4,5-bis[(2-methoxyethoxy)methoxy]-3,6-bis(phenoxymethyl)-
1,2,7-th ia d ia zep in e 1,1-Dioxid e (12). To a solution of 10 (50
mg, 0.088 mmol) in DMF (2 mL) were added NaH (80%
suspension, 10.5 mg, 0.35 mmol) and methyl 4-bromomethyl-
benzoate (80 mg, 0.35 mmol). The reaction mixture was stirred
under a N2 atmosphere overnight and concentrated in vacuo.
Purification by flash column chromatography on silica gel
(CH2Cl2/CH3OH, 100:1) gave the product as a colorless oil (61
mg, 80%): IR (film) υ 1716, 1600, 1496, 1436 cm-1; [R]D ) -1.0°
1
υ 3600-3200, 1701, 1599, 1496 cm-1; H NMR (270.2 MHz,
CDCl3) δ 7.29-7.11 (m, 12H), 6.93 (t, J ) 7.4 Hz, 2H), 6.68 (d,
J ) 7.7 Hz, 4H), 4.80 (d, J ) 15.8 Hz, 2H), 4.69 (d, J ) 15.7
Hz, 2H), 4.46 (t, J ) 5.6 Hz, 2H), 4.24 (m, 6H), 4.08 (m, 2H),
3.28 (s, 12H), 3.22 (brd, 2H), 2.83 (d, J ) 5.6 Hz, 4H); 13C NMR
(67.8 MHz, CDCl3) δ 157.8, 137.9, 137.5, 129.6, 128.9 (2C),
128.8, 126.2, 121.6, 114.6, 105.2, 75.3, 66.7, 53.5, 53.4, 39.5.
Anal. (C40H50N2O10S) C, H, N.
1
(c ) 1.05, CHCl3, 21 °C); H NMR (270.2 MHz, CDCl3) δ 7.97
(d, J ) 8.6 Hz, 4H), 7.52 (d, J ) 8.6 Hz, 4H), 7.20 (dd, J ) 7.7,
7.3 Hz, 4H), 6.91 (t, J ) 7.3 Hz, 2H), 6.67 (d, J ) 7.6 Hz, 4H),
5.00 (d, J ) 18 Hz, 2H), 4.90 (d, J ) 17.5 Hz, 2H), 4.85 (d, J
) 6.8 Hz, 2H), 4.81 (d, J ) 7.1 Hz, 2H), 4.47 (t, J ) 6.9 Hz,
2H), 4.25 (apps, 2H), 4.02 (d, J ) 7.1 Hz, 4H), 3.88 (s, 6H),
3,68 (m, 4H), 3.40 (m, 4H), 3.25 (s, 6H); 13C NMR (67.8 MHz,
CDCl3) δ 167.0, 157.8, 144.9, 130.0, 129.7, 129.0, 126.9, 121.6,
114.4, 96.2, 77.2, 71.6, 68.2, 66.0, 59.1, 53.5, 52.2, 51.9. Anal.
(C44H54N2O14S) C, H, N.
(3R,4S,5S,6R)-2,7-Bis{[4-(h ydr oxym eth yl)ph en yl]m eth -
yl}-3,6-bis(p h en oxym eth yl)-4,5-d ih yd r oxy-1,2,7-th ia d ia z-
ep in e 1,1-Dioxid e (7). To a solution of 12 (105.8 mg, 0.122
mmol) in ether (10 mL) was added LiBH4 (16 mg, 0.733 mmol)
followed by the addition of toluene (5 mL) and the reaction
was refluxed for 2 h at 80 °C. The reaction was quenched with
water (10 mL) and the water phase was extracted with 2 ×
10 mL of ether. The solvent was removed, the crude product
was dissolved in methanol (5 mL), and saturated HCl in ether
(10 mL) was added. This was stirred overnight at room
temperature. The solvent was removed and purification by
flash column chromatography on silica (CH2Cl2/CH3OH, 100:
1-25:1) gave compound 7 (33.2 mg, 83%): IR (KBr) υ 3600-
3200, 2927, 1598, 1496 cm-1; [R]D ) +14.4° (c ) 0.52, CHCl3,
22 °C); 1H NMR (270.2 MHz, CDCl3) δ 7.48 (d, J ) 8.2 Hz,
4H), 7.29 (d, J ) 8.2 Hz, 4H), 7.24 (dd, J ) 8.7, 7.4 Hz, 4H),
6.91 (t, J ) 7.4 Hz, 2H), 6.81 (d, J ) 8.7 Hz, 4H), 4.96 (m,
4H), 4.92 (brs, 2H), 4.58 (d, J ) 5.6 Hz, 4H), 4.43 (t, J ) 6.8
Hz, 2H), 4.32-4.10 (m, 8H); 13C NMR (100.2 MHz, CDCl3) δ
159.3, 142.0, 139.5, 130.3, 128.0, 127.4, 121.8, 115.4, 74.8, 67.1,
64.5, 56.0, 52.7. Anal. (C34H38N2O8S) C, H, N.
Mod elin g. Sym m etr ic ver su s Non sym m etr ic Con for -
m a tion of 2. A model for the urea-like conformation of a cyclic
sulfamide was obtained by fitting 2 to the X-ray coordinates
of the inhibitor of the HIV-1 protease complex of 1. A model
of the observed conformation of 2 was taken from the X-ray
coordinates of the inhibitor of the HIV-1 protease complex of
2. The above models were simplified by truncation of the P1/
P1′ and P2/P2′ groups to methyls (see Figure 2). Relaxation
of the models to the nearest local minima was accomplished
with MacroModel 5.537 using the MMFF38 force field. Ab initio
optimizations at the B3LYP/6-31G* level with Gaussian9439
were performed to further refine the geometries. The energy
difference between the resulting structures was used to
estimate the accessibility of the urea-like ring conformation
as compared to the nonsymmetrical “flipped” conformation.
Mod elin g of th e Ketoxim e 4. Acetophenone oxime was
used as a model of the ketoxime function of 4. The model was
relaxed in vacuo with MacroModel 5.5 using the MMFF force
field. The model was then subjected to a 1000-step Monte Carlo
run allowing rotation of the phenyl group and the CdN double
bond. Two minima were identified: the E and Z isomers. Ab
initio optimizations at the B3LYP/6-31G* level with Gaussi-
an94 were performed to further refine the geometries.
(3R, 4S, 5S, 6R)-2,7-Bis{[4-(m et h oxyca r b on yl)p h en yl]-
m ethyl}-3,6-bis(ph en oxym eth yl)-4,5-dih ydr oxy-1,2,7-thiadi-
a zep in e 1,1-Dioxid e (8). To a solution of compound 12 in
methanol (2.5 mL) was added HCl in ether (5 mL). The
reaction mixture was stirred overnight and concentrated in
vacuo. Purification by flash column chromatography on silica
(CH2Cl2/CH3OH, 200:1) gave the product as a colorless oil (9.8
Mod elin g of 3-8. The symmetric and nonsymmetric
models of 2 were augmented with the R1 and R2 groups shown
in Table 1 to produce the starting models for the urea-like and
nonsymmetrical conformations of 3 and 5-8. The models were
further refined in MacroModel 5.5 by substructure minimiza-
tion of the P2/P2′ groups under the AMBER*40 force field (5.5
Å shell from the P2/P2′atoms of the enzyme portion of the
HIV-1 protease complex of 2). Since we were unable to predict
mg, 83%): IR (CHCl3) υ 1717, 1599, 1496, 1436 cm-1; [R]D
)
1
+6.6° (c ) 0.98, CHCl3, 22 °C); H NMR (270.2 MHz, CDCl3)
δ 7.94 (d, J ) 8.4 Hz, 4H), 7.46 (d, J ) 8.4 Hz, 4H), 7.20 (dd,
J ) 8.5, 7.4 Hz, 4H), 6.93 (t, J ) 7.4 Hz, 2H), 6.66 (d, J ) 8.7
Hz, 4H), 4.86 (m, 4H), 4.34-4.16 (m, 6H), 4.08 (dd, J ) 9.4,
5.4 Hz, 2H), 3.88 (s, 6H), 3.08 (s, 2H); 13C NMR (67.8 MHz,

4060 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ta ble 2. Data Processing Statistics
Hulte´n et al.
than 50 Å2 were accepted. The Rcryst and Rfree factors were used
to monitor the refinement.49 The refinement statistics are
shown in Table 3.
HIV-1 PR/4
HIV-1 PR/9
space group
wavelength (Å)
no. of crystals
P21212
0.958
5
P21212
0.920
2
Ack n ow led gm en t. We thank the Swedish Founda-
tion for Strategic Research (SSF), the Swedish Medici-
nal Research Council (MFR), and Medivir AB for
financial support.
cell dimensions a, b, c (Å)
58.85, 86.68,
46.97
1.80
59.55, 87.68,
47.29
1.92
dmin (Å)
no. of observations
no. of unique reflections
completeness (%)
Rmerge (%)a
40892
18434
80.6
28401
15933
81.4
Refer en ces
12.4
5.5
(1) Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J .; Heimbach,
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Immunodeficiency Virus Protease is Required for Viral Infectiv-
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Hahn, B. H.; Saag, M. S.; Shaw, G. M. Viral Dynamics in Human
Immunodeficiency Virus Type 1 Infection. Nature 1995, 273,
117-122.
reflections I > 3σ (%)
reflections I > 3σ (%) in
highest resolution shell
B in resolution (Å)
52.9
17.0
60.0
32.0
1.86-1.80
1.99-1.92
a
Rmerge ) ∑|Ii
-
I |/∑ Ii, where Ii is an observation of the
intensity of an individual reflection and I is the average intensity
over symmetry equivalents.
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J . M.; Markowitz, M. Rapid Turnover of Plasma Virons and CD4
Lymphocytes in HIV-1 Infection. Nature 1995, 373, 123-126.
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and Resistance Studies of AG1343, an Orally Bioavailable
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crob. Agents Chemother. 1996, 40, 292-297.
Ta ble 3. Refinement Statistics of Final Models
HIV-1 PR/4
HIV-1 PR/9
resolution of data (Å)
cryst (%)
24.0-1.80
21.2
8.0-1.92
19.8
25.4
R
Rfree (%)
23.6
(5) Palella, F. J ., J r.; Delaney, K. M.; Moorman, A. C.; Loveless, M.
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no. of atoms
1667
96
23.3
1628
78
27.4
no. of water molecules
mean B factor, protein (Å2)
mean B factor, inhibitor (Å2)
deviation from ideality
bond lengths (Å)
17.4
13.9
0.007
1.261
25.420
0.801
0.011
1.729
25.453
1.390
bond angles (deg)
dihedrals (deg)
impropers (deg)
the position of the structural waters for the models of 3-8,
we instead used a GB/SA41 solvation model. Short Monte Carlo
runs exploring 4-6 rotatable bonds were performed on each
model under the same conditions to find reasonable positioning
of the P2/P2′ groups.
Cr yst a llogr a p h y. HIV-1 protease was expressed in E.
coli.42 The enzyme was isolated from inclusion bodies through
a slightly modified version of the method by Taylor et al.43
Briefly, the inclution body fraction was disolved in 8 M urea,
separated on a Poros Q column (Roche), refolded by dialysis
at 4 °C, and finally purified on a Poros S column. The material
was concentrated by ammonium sulfate precipitation, desalted,
and finally concentrated on Centricon membrane centrifuga-
tion tubes (Amicon).
Crystallization was performed by the vapor diffusion method.
Inhibitor (40 mM, disolved in DMSO) was added to a 2 mg/
mL protease solution giving a final molar ratio of 2:1 and
incubated for 30 min on ice. Drops consisting of 5 µL of the
protease-inhibitor mixture plus 5 µL of the crystallization
buffer (50 mM MES, pH 5.5, 0.4 M NaCl, and 0.02% (w/v)
NaN3) were equilibrated against the same buffer at 4 °C.
Crystals grew to a size of 0.27 mm × 0.12 mm × 0.10 mm
within 2 weeks.
X-ray data were recorded at 4 °C on MAR imaging plates
on the synchrotron beam lines I711 at MAX-lab Lund, Sweden
(compound 4), and 9.5 DRAL Daresbury, England (compound
9). The programs DENZO and SCALEPACK were used for
processing and scaling.44,45 A summary of data collection
statistics is given in Table 2. The protease model coordinates
from 1AJ V were used for molecular replacement calculations.
Refinement was performed using the program packages
X-PLOR (complex 9) and CNS (complex 4).46,47 The difference
Fourier map (Fo-Fc) clearly showed the position and orienta-
tion of the inhibitor together with the positions of a large
number of water molecules. The inhibitor was built manually
into the electron density using the program O.48 Water
molecules were added to the structure of HIV-1 protease/4
determined from the difference Fourier maps at chemically
acceptable sites. Only solvent molecules with B values less
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J M991054Q