Cyclic HIV-1 protease inhibitors derived from mannitol: Synthesis, inhibitory potencies, and computational predictions of binding affinities

Article abstract of DOI:10.1021/jm960728j

Ten C₂-symmetric cyclic urea and sulfamide derivatives have been synthesized from L-mannonic γ-lactone and D-mannitol. The results of experimental measurement of their inhibitory potencies against HIV-1 protease were compared to calculated free

Full text of DOI:10.1021/jm960728j

J . Med. Chem. 1997, 40, 885-897
885
Cyclic HIV-1 P r otea se In h ibitor s Der ived fr om Ma n n itol: Syn th esis, In h ibitor y
P oten cies, a n d Com p u ta tion a l P r ed iction s of Bin d in g Affin ities
J ohan Hulte´n,† Nicholas M. Bonham,† Ulrika Nillroth,‡ Tomas Hansson,§ Guido Zuccarello,|
Abderrahim Bouzide,| J ohan A° qvist,§ Bjo¨rn Classon,∇ U. Helena Danielson,‡ Anders Karle´n,†
Ingemar Kvarnstro¨m,| Bertil Samuelsson, and Anders Hallberg*,†
Department of Organic Pharmaceutical Chemistry, Uppsala Biomedical Center, Uppsala University, Box 574,
S-751 23 Uppsala, Sweden, Department of Biochemistry, Uppsala Biomedical Center, Uppsala University, Box 576,
S-751 23 Uppsala, Sweden, Department of Molecular Biology, Uppsala Biomedical Center, Uppsala University, Box 590,
S-751 24 Uppsala, Sweden, Department of Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden, Medivir AB,
Lunastigen 7, S-141 44 Huddinge, Sweden, and Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, S-106 91 Stockholm, Sweden
Received October 17, 1996X
Ten C2-symmetric cyclic urea and sulfamide derivatives have been synthesized from L-mannonic
γ-lactone and D-mannitol. The results of experimental measurement of their inhibitory
potencies against HIV-1 protease were compared to calculated free energies of binding derived
from molecular dynamics (MD) simulations. The compounds were selected, firstly, to enable
elucidation of the role of stereochemistry for binding affinity (1a -d ) and, secondly, to allow
evaluation of the effects of variation in the link to the P1 and P1′ phenyl groups on affinity (1a
and 2-5). Thirdly, compounds with hydrogen bond-accepting or -donating groups attached to
the phenyl groups in the P2 and P2′ side chains (6 and 7) were selected. Binding free energies
were estimated by a linear response method, whose predictive power for estimating binding
affinities from MD simulations was demonstrated.
In tr od u ction
Human immunodeficiency virus (HIV) is the caus-
ative agent of acquired immune deficiency syndrome,
AIDS.1 HIV protease processes the viral polyproteins
Pr55gag and Pr160gag-pol into structural proteins and
enzymes, including the protease itself. The activity of
the protease is vital for proper assembly and maturation
of infectious virons.2 Therefore HIV protease is an
important chemotherapeutic target.3
Numerous examples of potent inhibitors of HIV-1
protease have been disclosed.4 A common feature
observed in the X-ray crystal structures of linear inhibi-
tor-HIV-1 protease complexes is the presence of a
tetracoordinated structural water molecule linking the
bound inhibitors to the flexible flaps of the HIV-1
protease dimer.5 On the basis of this structural infor-
mation, Lam et al. have performed an elegant design
of nonpeptide cyclic ureas, constituting an entirely new
class of HIV-1 protease inhibitors.6 More recently,
Sham et al. have reported a series of new azacyclic ureas
possessing high potency as inhibitors of HIV-1 protease.7
The fundamental characteristic of these inhibitors, e.g.,
DMP 323 and A-98881 (Figure 1), is the cyclic urea
carbonyl oxygen mimicking the structural water mol-
ecule.6
The application of computer modeling and simulation
of molecular interactions to drug design is a field
currently in rapid growth. Methods allowing reliable
predictions of affinity and specificity of ligands would
be of paramount value in the discovery process, aiding
F igu r e 1. Structures of DMP 323 and A-98881.
in the selection of ligands with high potential before
synthesis is commenced. In cases where the three-
dimensional structure of the drug target is known,
methods for scoring putative inhibitors from single
conformations of ligand-target complexes have been
developed.8 But even with a 3D-structure of the target
available, computational predictions of binding affinity
for new ligands is difficult in the absence of accurate
3D-structures of the corresponding complexes. The
main reason for this lies in the difficulty of predicting
the exact structure of the complex. Force field-based
structural optimization and scoring has been applied
recently to HIV-1 protease inhibitors with promising
results.9 A° qvist et al. have described another approach,
which has yielded good results for several systems,10 in
which absolute binding free energies are estimated by
a linear response approximation from thermal averag-
ing of the structure and interaction energies by molec-
* Corresponding author.
† Department of Organic Pharmaceutical Chemistry.
‡ Department of Biochemistry.
§ Department of Molecular Biology.
| Linko¨ping University.
∇ Medivir AB.
Stockholm University.
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00728-5 CCC: $14.00 © 1997 American Chemical Society

886 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Hulte´n et al.
Sch em e 1a
a
(a) PhOH, NaH, THF or BnMgCl, CuI, THF or Ph(CH2)2Br, Mg, CuI, THF; (b) DEAD, DPPA, PPh3; (c) CH3CN, 3 M HCl; (d) MEMCl,
NaH, THF.
Sch em e 2a
a
(a) H2, Pd/C (10%), EtOAc; (b) CDI, CH2Cl2; (c) BnBr or methyl 4-(bromomethyl)benzoate, NaH, DMF; (d) CH3OH, concentrated HCl;
(e) CH3CN, 3 M HCl; (f) LiBH4, ether; (g) sulfamide, pyridine; (h) BnBr, NaH, DMF; (i) HCl in ether, methanol.
ular dynamics (MD) simulations. This procedure was
applied recently to DMP 323 and two linear peptide
HIV-1 protease inhibitors.11
Resu lts
Ch em istr y. The diepoxides 8a ,c were used as key
intermediates for the synthesis of 1a , 2-7, and 1c,
respectively, and the diepoxides 8b,d for the synthesis
of 1b,d , respectively (Figure 3). For the preparation of
the diepoxides 8b,d D-mannitol was used as starting
material.14 We employed this procedure for the prepa-
ration of 8a ,c from L-mannonic γ-lactone.13 The syn-
thetic methods used for the preparation of 1a -d and
2-7 are shown in Tables 1-4, and the syntheses of the
RSSR stereoisomers 1a and 2-7 are outlined in Schemes
1 and 2.
Nucleophilic ring opening of the diepoxides occurs
with high regio- and stereoselectivity,15 and accordingly,
ring opening of 8a , shown in Scheme 1, and 8b by
phenolate smoothly provided 9a ,b15c after 7 h. Ring
opening of 8c,d provided the corresponding diastereo-
mers 9c,d after 24 h reaction times, in yields ranging
of 61-72%. Standard Mitsunobu reaction16 lead to the
azides 10a -d , with inverted configuration at carbons
Several groups have used carbohydrates as chiral
precursors for the synthesis of linear HIV-1 protease
inhibitors.12 Here we describe the synthesis of 10 cyclic
C2-symmetric compounds related to DMP 323, which
were prepared from the readily available L-mannonic
γ-lactone or D-mannitol.13 For these 10 model com-
pounds, we compare our results from experimental
elucidation of their inhibitory potencies against HIV-1
protease to calculated free energies of binding derived
from MD simulations. The compounds were selected
to enable comparison of (a) the C2-symmetric diastere-
omers 1a -d , (b) the ureas 1a , 2, and 3 and the
sulfamides 4 and 5, which were modified in the link to
the P1 and P1′ phenyl groups, and (c) the compounds 6
and 7, modified in the P2 and P2′ side chains with
groups combining hydrogen bond acceptor and donor
capacity, respectively.

HIV-1 Protease Inhibitors Derived from Mannitol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 887
Ta ble 1. Structures and Preparation Methods for Mannitol
Derivatives
compda
config
R1
OPh
OPh
OPh
OPh
OPh
OPh
OPh
X
prepn methodb
9a
9b
9c
SSSS
RRRR
RSSR
SRRS
RSSR
SRRS
SSSS
RRRR
SSSS
RSSR
SSSS
RSSR
OH
OH
OH
OH
N3
N3
N3
N3
OH
N3
I
I
I
I
9d
10a
10b
10c
10d
15
16
21
22
II
II
II
II
VIII
II
IX
II
OPh
CH2Ph
CH2Ph
(CH2)2Ph
(CH2)2Ph
F igu r e 2. Structures of one urea (1a ) and one sulfamide (4)
compound with RSSR stereochemistry.
OH
N3
a
For experimental data of compounds 9b-d and 10b-d , see
b
Supporting Information. See the Experimental Section.
Ta ble 2. Structures and Preparation Methods for Mannitol
Derivatives
compda
config
R1
OPh
OPh
OPh
OPh
OPh
OPh
OPh
R2
X
prepn methodb
11a
11b
11c
11d
12a
12b
12c
12d
17
RSSR
SRRS
SSSS
RRRR
RSSR
SRRS
SSSS
RRRR
RSSR
RSSR
RSSR
RSSR
H
H
H
H
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
III
III
III
III
IV
IV
IV
IV
III
IV
III
IV
F igu r e 3. Structures of the bisepoxides 8a -d .
MEM
MEM
MEM
MEM
H
MEM
H
MEM
2 and 5. Reduction to the primary amines occurred
smoothly, but cyclization was not facile, probably due
to ring strain imposed by the trans-fused five-membered
acetonide ring.17 Deketalization of the azides furnished
11a -d . Treatment with MEM-chloride18 afforded the
bis-MEM ethers 12a -d in 83-88% isolated yields.
Hydrogenation on Pd/C provided the crude amines,
which were reacted with carbonyldiimidazole19 to give
the cyclic ureas 13a -d in 55-68% yields from the
appropriate azide. Benzylation20 to give 14a and the
enantiomer 14b was complete in 12 h, while formation
of 14c,d required 24 h reaction time for full conversion.21
Deprotection gave the target compounds 1a-d in 6-17%
overall yield from 8a -d .
OPh
CH2Ph
CH2Ph
(CH2)2Ph
(CH2)2Ph
18
23
24
a
For experimental data of compounds 11b-d and 12b-d , see
b
Supporting Information. See the Experimental Section.
HCl in ether-methanol afforded compounds 4 and 5 in
18% and 16% overall yield from the diepoxide as
depicted in Scheme 2.
In h ibition of HIV-1 P r otea se. The inhibitory effect
of the synthesized compounds was determined with
purified HIV-1 protease in a standardized assay.24 The
results are presented as IC50-values, i.e., the concentra-
tion of inhibitor resulting in 50% inhibition in this assay
(Table 5). Since some compounds were not sufficiently
potent to allow determination of IC50-values, results are
also given as percent inhibition at 10 µM (Table 5).
Compounds that showed significant inhibition were
further characterized, and Ki-values were determined
(Table 5). The model for tight-binding inhibitors used
for analysis25 was adequate except for the most potent
inhibitors, as judged by the correlation between kcat- and
Km-values, obtained when fitting data for different
inhibitors and by statistical criteria (not shown). When
the Ki-values are lower than the enzyme concentration
used, the portion of free inhibitor becomes insignificant,
since the enzyme is essentially titrated with inhibitor
under these conditions; thus, in such cases the method
For the preparation of 213 and 3 a similar reaction
sequence was adopted. Ring opening of 8a with ben-
zylmagnesium chloride or phenylethylmagnesium bro-
mide in the presence of CuI12a delivered 15 and 21,
respectively. The Mitsunobu reaction followed by de-
ketalization and reprotection with MEM-chloride pro-
vided 18 and 24. After hydrogenation, cyclization to
give the ureas 19 and 25 occurred smoothly. Benzyla-
tion and subsequent deprotection furnished 2 and 3 in
14-18% overall yield from the epoxide. Alkylation with
methyl 4-(bromomethyl)benzoate and deprotection pro-
vided the ester 6, while reduction22 before the depro-
tection furnished the hydroxymethyl derivative 7. Sulf-
amides 4 and 56b were prepared by heating the crude
amine with sulfamide in refluxing pyridine. This cy-
clization23 followed by benzylation using sodium hydride
as base in DMF and subsequent deprotection with dry

888 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Hulte´n et al.
Ta ble 3. Structures and Preparation Methods for Cyclic Urea
Compounds
cannot be used. The standard errors for determination
of the parameters were e10%.
The stereochemistry of the compounds was found to
be critical for their inhibitory effect. Only the RSSR
isomer 1a inhibits the enzyme significantly, Ki ) 12.2
nM. The other isomers (1b-d ) showed little (<15%) or
no inhibitory effect even at micromolar concentrations.
Small structural modifications of compound 1a gave
significant changes in the inhibitory potency. Changing
the P1/P1′ side chains from phenoxymethyl (1a ) to
phenylethyl (2) resulted in a 20-fold reduction of the Ki-
value. A 50-fold loss of inhibitory effect relative to 1a
was found when the P1/P1′ side chain was further
extended to give the phenylpropyl derivative 3. The
cyclic sulfamides 4 and 5 both showed inhibitory poten-
cies of the same order of magnitude as 1a . Interest-
ingly, exchanging the P1/P1′ ether oxygens for methyl-
ene groups in the sulfamides gave essentially equipotent
derivatives (4 vs 5), contrary to the observation with
the ureas (1a vs 2). Finally, both of the p-hydroxy-
methyl compounds (7 and DMP 323) exhibited high
potencies, while compound 6 exhibited lower binding
affinity than 1a .
prepn
compda config
R1
RSSR OPh
SRRS OPh
R2
R3
methodb
1a
1b
H
H
H
H
H
H
H
H
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
VII
VII
VII
VII
VII
VII
X
XI
V
V
1c
1d
2
3
6
7
SSSS OPh
RRRR OPh
RSSR CH2Ph
RSSR (CH2)2Ph
RSSR OPh
RSSR OPh
RSSR OPh
SRRS OPh
SSSS OPh
RRRR OPh
RSSR OPh
SRRS OPh
SSSS OPh
RRRR OPh
RSSR CH2Ph
RSSR CH2Ph
CH2-4-(CO2CH3)Ph
CH2-4-(CH2OH)Ph
13a
13b
13c
13d
14a
14b
14c
14d
19c
20
MEM
MEM
MEM
MEM
H
MEM CH2Ph
MEM CH2Ph
MEM CH2Ph
H
H
H
H
V
V
CH2Ph
VI
VI
VI
VI
V
VI
V
VI
VI
Dyn a m ics Sim u la tion s a n d F r ee En er gy Ca lcu -
la tion s. Binding free energies were estimated by an
approximation described elsewhere,10 which we will
refer to here as the linear interaction energy (LIE)
method. Two MD simulations were performed for each
inhibitor, one for the inhibitor free in water and the
other for the inhibitor bound inside the solvated protein.
The simulation results are summarized in Table 6 in
terms of the average electrostatic (polar) and van der
Waals (nonpolar) ligand interaction energies for the
bound and unbound states. Hydrogen bonds are in-
cluded implicitly in these two quantities and are mainly
reflected in the electrostatic energy. Table 6 also gives
the resulting free energies of binding obtained with the
LIE approximation. The predicted absolute binding free
energies are in reasonable agreement with the experi-
mental values (Table 5) but are generally ca. 2 kcal/
mol too negative. The simulations identify 1a as a good
inhibitor and give the correct ranking of the compounds
1a -d . In particular, the two compounds 1c,d are found
to bind poorly, while the binding energy of 1b is
somewhat overestimated. The water simulations of the
enantiomeric pairs converged as expected to give very
similar values within each pair. The average of the two
simulations is given in Table 6 for each pair. For the
compounds 2 and 3, with the ether oxygens replaced
by methylene and ethylene groups, 2 is predicted to be
MEM
MEM CH2Ph
H
25c
26
RSSR (CH2)2Ph MEM
RSSR (CH2)2Ph MEM CH2Ph
RSSR OPh MEM CH2-4-(CO2CH3)Ph
H
27d
a
For experimental data of compounds 13b-d and 14b-d , see
Supporting Information. See the Experimental Section. c 2 days
b
d
reaction time. Methyl 4-(bromomethyl)benzoate was used instead
of BnBr.
Ta ble 4. Structures and Preparation Methods for Cyclic
Sulfamide Compounds
compd
config
R1
R2
R3
prepn methoda
4
5
28
29
30
31
RSSR
RSSR
RSSR
RSSR
RSSR
RSSR
OPh
CH2Ph
OPh
OPh
CH2Ph
CH2Ph
H
H
MEM
MEM
MEM
MEM
CH2Ph
CH2Ph
H
CH2Ph
H
XIV
XIV
XII
XIII
XII
CH2Ph
XIII
a
See the Experimental Section.
Ta ble 5. Inhibitory Activity of Cyclic C2-Symmetric Urea and Sulfamide Compounds against HIV-1 Protease
Ki (nM)
∆Gbind (kcal/mol)
compd
config
inhibition (%) (at 10 µM)
IC50 (µM)
expta
calcd
expt
calcd
DMP 323
RSSR
RSSR
SRRS
SSSS
RRRR
RSSR
RSSR
RSSR
RSSR
RSSR
RSSR
nd
nd
15
10
0
nd
nd
nd
nd
nd
nd
0.015
0.08
nd
nd
nd
<1b
12.2
nd
nd
nd
0.083
1.1
15
3200
36000
7.8
0.80
0.80
0.94
9.2
<-12.8
-11.2
-14.3 ( 1.0
-12.7 ( 0.5
-11.1 ( 0.7
-7.8 ( 0.9
-6.3 ( 0.5
-11.5 ( 1.4
-12.9 ( 0.7
-12.9 ( 1.7
-12.8 ( 0.5
-11.4 ( 0.9
-14.6 ( 1.6
1a
1b
1c
1d
2
3
4
5
6
4.0
214
570
19.1
14.7
nd
-9.5
-8.9
-10.9
-11.1
10.0
0.200
0.200
8.0
7
0.010
<1b
0.051
<-12.8
a
b
The standard error was less than 10%. Ki(DMP 323) ) 0.27 nM and Ki(7) ) 0.23 nM have been determined by a different method
(Nillroth et al., to be published). nd ) not determined.

HIV-1 Protease Inhibitors Derived from Mannitol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 889
Ta ble 6. Dynamics Averages of Nonbonded Terms in the Simulationsa
∆Gbind
compd
Vvdw
Vvdw
Vel
Vel
wat
vdw
el
calcd
prot
wat
prot
DMP 323
-86.57 ( 1.17
-85.76 ( 0.17
-90.61 ( 1.08
-88.30 ( 0.70
-86.79 ( 0.52
-86.32 ( 0.21
-90.37 ( 0.23
-88.85 ( 0.03
-88.42 ( 0.04
-103.61 ( 0.10
-90.86 ( 0.43
-42.07 ( 0.19
-46.66 ( 0.88
-46.66 ( 0.88
-46.63 ( 0.12
-46.63 ( 0.12
-48.42 ( 0.25
-52.59 ( 0.44
-50.91 ( 0.09
-49.11 ( 0.04
-53.65 ( 1.39
-44.13 ( 0.53
-82.22 ( 1.08
-50.99 ( 0.10
-46.31 ( 0.20
-41.42 ( 1.30
-38.99 ( 0.65
-45.17 ( 1.66
-50.57 ( 0.21
-49.70 ( 1.91
-48.97 ( 0.22
-57.38 ( 0.63
-84.98 ( 1.33
-67.89 ( 0.48
-38.19 ( 0.44
-38.19 ( 0.44
-39.32 ( 0.12
-39.32 ( 0.12
-34.29 ( 0.97
-37.00 ( 0.96
-36.09 ( 1.31
-36.01 ( 0.57
-50.68 ( 0.51
-70.78 ( 1.34
-7.16 ( 0.2
-6.30 ( 0.2
-7.08 ( 0.3
-6.71 ( 0.2
-6.47 ( 0.1
-6.10 ( 0.1
-6.08 ( 0.1
-6.11 ( 0.1
-6.33 ( 0.1
-8.04 ( 0.3
-7.52 ( 0.2
-7.16 ( 0.8
-6.40 ( 0.3
-4.06 ( 0.4
-1.05 ( 0.7
+0.16 ( 0.4
-5.44 ( 1.3
-6.78 ( 0.6
-6.80 ( 1.6
-6.48 ( 0.4
-3.35 ( 0.6
-7.10 ( 1.4
-14.3 ( 1.0
-12.7 ( 0.5
-11.1 ( 0.7
-7.8 ( 0.9
-6.3 ( 0.5
-11.5 ( 1.4
-12.9 ( 0.7
-12.9 ( 1.7
-12.8 ( 0.5
-11.4 ( 0.9
-14.6 ( 1.6
1a
1b
1c
1d
2
3
4
5
6
7
a
Values are averages over 125 ps (250 ps for DMP 323 and 7). The error estimates are one-half the differences between the first and
last 62.5 ps (or 125 ps) and thus measure precision rather than accuracy. All values are given in kcal/mol. The contributions from the
two terms in the binding free energy approximation are also shown.
less active than 1a , in agreement with the binding
experiments. However, the calculated binding energy
for 3 is about the same as for 1a , which is a significant
overestimation compared to experimental values. Cal-
culations on the sulfamides 4 and 5 yielded binding free
energies close to those obtained for 1a , in agreement
with experimental findings. The ester 6 was predicted
to be similarly potent to 2, which is indeed also indicated
by the experimentally observed IC50-value. The calcula-
tions predict essentially equal, large negative, binding
energies for DMP 323 and 7, pointing to a negligible
effect of the added oxygen. There are, however, signifi-
cant uncertainties in this prediction, since we find that
a major part of the interaction energy of these two
inhibitors arises from interactions of the hydroxymethyl
groups with aspartates 30/230, which are mobile and
F igu r e 4. Schematic picture showing the general hydrogen-
bonding arrangements in the vicinity of the inhibitors with
RSSR stereochemistry, on the tens-of-picoseconds time scale
of the simulations. Distances in the figure are not to scale,
and all water molecules depicted are in contact with bulk
water. Dashed lines indicate that atom distances were on
average favorable for hydrogen bond formation.
in contact with bulk water, leading to large variations
during simulation time. For these two inhibitors, data
collection times for the protein simulations were there-
fore doubled. The free energy of binding obtained for
DMP 323 is more negative than has been reported
earlier11 due to the interaction with Asp 30/230, since
the aspartate side chains need to move (ø1 rotation) from
their crystallographic starting positions for this interac-
tion to occur, a conformational change that was neither
anticipated nor observed in the calculations on DMP
323.11 As noted above, exact experimental values of the
inhibition constants could not be obtained for the potent
inhibitors 7 and DMP 323 (Ki of 0.27 nM has been
reported for DMP 323).6
diequatorial hydroxyls, but this seems to be at the
expense of other favorable interactions and still results
in poor binding. The average structures for the RSSR
inhibitors are summarized in Figure 4, which sum-
marizes the average hydrogen-bonding arrangement in
the vicinity of the inhibitor on the time scale of the
simulations.
The MD average structures from the calculations give
a picture of binding arrangements over some tens of
picoseconds. On this time scale, all inhibitors were
found to bind somewhat asymmetrically in the binding
pocket, especially 1a ,b. The reason for this asymmetry
of binding was the protonation state chosen for the
catalytic aspartates. Asp 25 was considered to be
negatively charged, Asp 225 was protonated,26 and both
hydroxyls of the inhibitor were found to point toward
Asp 25, tilting the seven-membered ring slightly. Strong
hydrogen bonds can form between diequatorial hydroxyl
groups and the charged aspartate. Only one of the
diaxial hydroxyl groups found in relaxed conformations
of the free compounds 1c,d would be able to hydrogen
bond to Asp 25. A strong charge-dipole interaction
would then be replaced by a weaker dipole-dipole
interaction with Asp 225, leading to weaker binding. For
1c, the calculations indeed predict a conformational
change in the seven-membered cyclic urea ring, yielding
Discu ssion
High binding affinity is attained by both steric and
electrostatic complementarity between the ligand and
its target. Imperfect steric fit generally can be relaxed
by small adjustments in the protein. Electrostatic
complementarity, which involves both polar interactions
(e.g., hydrogen bonds) and nonpolar interactions (e.g.,
hydrophobic effects), usually cannot be adjusted easily
due to the long-range nature of electrostatic fields and
is therefore a major determinant of binding specificity.
These effects are apparent in the results for the stereo-
isomers 1a -d . For all four compounds, shape comple-
mentarity alone provides a basic level of affinity, as
demonstrated by the van der Waals term of the MD
simulation results, but the exact polar-hydrophobic
matching within this shape results in the observed
(Table 5) large differences, leading to high affinity in

890 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Hulte´n et al.
The asymmetric protonation state26
some cases (in the nanomolar range for 1a ). The
present results indicate that the stereochemistry of the
compounds is critical for their inhibitory potency. It is
especially important for the hydroxyl groups to be
positioned optimally, and the RSSR stereoconfiguration
of 1a enables favorable hydrogen bonds to the charged
aspartate.
of the catalytic
Asp 25/225 used in the present simulations seems to
explain the asymmetric binding mode of the inhibitors
on the short time scales of the simulations, depicted in
Figure 4. However, even if this asymmetry is correct
on the short time scale of the simulations, it need not
be observable necessarily in crystallographic studies,
since these involve implicit averaging over many protein
dimers that need not be isomorphous with respect to
the aspartate protonation throughout the crystal. In-
stead, such experiments may reflect the average of
Figure 4 and its symmetry-related counterpart, in
addition to any contributions from other protonation
states, depending on the pH of the crystal.
The MD calculations suggest the following explana-
tion for the 20-fold enhancement of binding affinity of
1a as compared 2: A dipole is formed by each partially
negative ether oxygen and its two partially positive
neighboring carbons, since the angle between these
three atoms is less than 180°. Analysis of the average
structure from the MD simulations shows that for 1a ,
these dipoles are aligned to an electrostatic field arising
from the protein environment, with the major contribu-
tion from the charged Asp 25 but also with significant
contributions from the flap peptide bonds from residues
49-50 and 249-250.
The cyclic sulfamides 4 and 5 were synthesized with
RSSR stereochemistry since this configuration was
found to be optimal for the urea compounds. The
binding energies measured for the sulfamide compounds
4 and 5 are both approximately equal to that found for
1a , in agreement with the result derived from the MD
simulations. One might expect the same electrostatic
effects in 4 and 5 as in 1a and 2, but the average
structures of 4 and 5 predict that there is not enough
space available for the ether oxygens to be aligned to
the field, due to the extra volume from the sulfonyl
group compared to the carbonyl group. Thus, no
decrease in affinity results from the substitution of the
ether oxygen in compound 4 with the methylene group
in compound 5.
Con clu sion
Synthesis of these inhibitors from readily available
L-mannonic γ-lactone or D-mannitol is relatively straight-
forward, and the ether oxygens, easily incorporated by
the synthetic methodology employed, in fact seem to
enhance binding 20-fold as compared to the correspond-
ing methylene analog (2) in the cyclic urea series.
Furthermore, the methylene analog in the sulfamide
series (5) is 15-fold more potent than the corresponding
cyclic urea derivative, but steric effects seem to preclude
addition of these two enhancement factors.
The predictive power of the LIE method for estimating
binding affinities for the ligands from MD simulations
has been demonstrated. The enzymatic measurements
give the following ranking of inhibitors (in decreasing
potency) DMP 323, 7 > 1a , 4, 5 > 2, 3, 6 > 1b-d ,
whereas the theoretical predictions are DMP 323, 7 >
1a , 3-5 > 1b, 2, 6 > 1c,d , showing that the relative
ordering of the inhibitors is well reproduced by the
calculations, with the exception of the large overestima-
tion of the potency of compound 3 and the slight
overestimation for compound 1b.
The reason for the apparently systematic overestima-
tion of the binding strength of the compounds studied
here by ca. 2 kcal/mol is not clear. It is, of course,
conceivable that our linear response formula with its
current parameterization is not accurate enough (al-
though earlier calculations suggested so). Another
possibility is that differences between the experimental
conditions and the simulation model (e.g., ionic strength)
affect the result in a systematic way. A third possibility
is that some force field deficiency is responsible for the
error. One may, for example, question the non-polar-
extended atom representation of phenyl groups in the
GROMOS force field27 in this context. This somewhat
oversimplified model could lead to an exaggeration of
the hydrophobic effect which may be especially signifi-
cant here since the compounds studied each have four
phenyl groups, in contrast to those considered in earlier
applications of the method. Nevertheless, the results
obtained here suggest that the LIE method is useful as
a tool in design projects where the structure of the
molecular target is known.
Compounds 6 and 7 were synthesized in order to
study hydrogen-bonding capacity in the S2/S2′ cavity.
As expected, 7 exhibited high binding affinity in analogy
to DMP 323. This is probably due to the ability of 7 to
interact with the negatively charged aspartates 30/230,
as discussed above. The simulations point to a large
net loss of electrostatic binding energy of 6 compared
to 1a . This illustrates a general principle of ligand
binding energetics, namely, that a comparison always
must be made to the unbound reference state, where
the ligand is free in water. Therefore, not only must
the addition of a group to the ligand lead to favorable
interactions in the bound state, but the interactions with
the receptor must be stronger than the interactions of
the same added group with surrounding water mol-
ecules of the unbound state, or no net increase in
binding energy will be achieved (Table 6).
One interesting structural feature emerging from the
simulations is that the hydrogen bonds between the
inhibitor and the flaps seem not to be as strong as pos-
sible, as reflected by the fact that only one strong hydro-
gen bond to the NH groups 50/250 is observed in the
tens-of-picoseconds average structure (Figure 4). This
could indicate that the cyclic urea carbonyl group might
not be the optimal mimic of the crystallographically
found water molecule, but this could also be due to im-
perfections in the representation of the groups involved
in the force field used in this work. Moreover it is inter-
esting to note that water molecules (in contact with bulk
water) can interact with the cylic urea ring hydroxyls
even when the inhibitor is bound to the protein.
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 polarimeter. Specific rotations ([R]D) are reported
in deg/dm, and the concentration (c) is given in g/100 mL in
the specific solvent. 1H and 13C NMR spectra were recorded

HIV-1 Protease Inhibitors Derived from Mannitol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 891
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; 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 Mikro Kemi AB,
Sweden, or Analytische Laboratorien, Germany, 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 visualized with UV
light and H2SO4 in ethanol, phosphomolybdic acid, or ninhy-
drin. Standard workup: organic layers were dried with
MgSO4 and concentrated in vacuo.
2,5-Dia zid o-3,4-b is-O-[(2-m et h oxyet h oxy)m et h yl]-1,6-
d i-O-p h en yl-2,5-d id eoxy-d -id itol (12a ). Meth od IV. Com-
pound 11a (1.39 g, 3.63 mmol) in THF (75 mL) was stirred
with NaH (0.43 g, 14.5 mmol) for 15 min. (2-Methoxyethoxy)-
methyl chloride (MEMCl) (1.81 g, 14.5 mmol) was added, and
the reaction mixture was stirred overnight. The reaction was
quenched with aqueous saturated NH4Cl; the mixture was
diluted with water and extracted with ether (2 × 100 mL).
The combined ether extracts were dried and concentrated. The
crude product was purified by flash chromatography (pentane/
CH2Cl2, 1:1, to CH2Cl2) to give the product as a colorless oil
(1.73 g, 85%): IR (film) ν 3041, 2927, 2104, 1599, 1498 cm-1
;
1
[R]D ) -29.6° (c ) 1.0, CHCl3, 25 °C); H NMR (270.2 MHz,
CDCl3) δ 7.29 (dd, J ) 8.2, 7.6 Hz, 4H, OArH[m]), 6.98 (t, J )
7.2 Hz, 2H, OArH[p]), 6.94 (d, J ) 8.2 Hz, 4H, OArH[o]), 4.9
(d, J ) 6.9 Hz, 2H, OCH2O), 4.81 (d, J ) 6.9 Hz, 2H, OCH2O),
4.31 (m, 4H, CH2OPh), 4.08 (m, 4H, CHOCH2, CHN3), 3.78
(dt, J ) 11.2, 4.3 Hz, 2H, OCH2CH2O), 3.69 (dt, J ) 11.6, 4.6
Hz, 2H, OCH2CH2O), 3.51 (t, J ) 4.5 Hz, 4H, OCH2CH2O),
3.35 (s, 6H, CH3); 13C NMR (67.8 MHz, CDCl3) δ 158.1, 129.5,
121.4, 114.6, 97.3, 77.6, 71.6, 68.3, 68, 60.6, 59. Anal.
(C26H36N6O8) C, H, N.
3,4-O-Isopr opyliden e-1,2:5,6-dian h ydr o-D-iditol (8c): mp
69-70 °C; IR (KBr) ν 2994, 1379, 1249, 1180 cm-1; [R]D
)
+17.5° (c ) 1.20, CHCl3, 24 °C); 1H NMR (270.2 MHz, CDCl3)
δ 3.85 (dd, J ) 3.1, 1.6 Hz, 2H, CHOC), 3.07 (m, 2H, CHO),
2.84 (dd, J ) 5.1, 4.1 Hz, 2H, CH2O), 2.74 (dd, J ) 5.1, 2.1 Hz,
2H, CH2O), 1.40 (s, 6H, CH3); 13C NMR (67.8 MHz, CDCl3) δ
110.8, 78.1, 51.2, 43.9, 26.8. Anal. (C9H14O4) C, H.
(4R,5S,6S,7R)-5,6-Bis[(2-m eth oxyeth oxy)m eth oxy]-4,7-
bis(p h en oxym eth yl)-1,3-d ia zep a n -2-on e (13a ). Meth od
V. To compound 12a (716 mg, 1.28 mmol) in ethyl acetate
(25 mL) was added a catalytic amount of 10% Pd/C. Hydrogen
was added to the system at atmospheric pressure, and the
reaction mixture was stirred overnight. The suspension was
filtered through Celite and concentrated to give a colorless oil.
In order to ensure high dilution conditions, the crude amine
(595 mg) was dissolved in CH2Cl2 (50 mL) and the carbonyl-
diimidazole (CDI) (230 mg, 1.41 mmol) was dissolved in CH2-
Cl2 (50 mL), respectively; this was slowly added with a syringe
pump to a reservoir of CH2Cl2 (300 mL), and the reaction
mixture was stirred overnight. The solvent was removed, and
purification by flash chromatography (CH2Cl2 to CH2Cl2/CH3-
OH, 100:1) gave the product as a white solid (426 mg, 67%):
mp 218-220 °C; IR (KBr) ν 3228, 3106, 2936, 1687, 1600, 1498,
3,4-O-Isop r op ylid en e-1,6-d i-O-p h en yl-L-m a n n itol (9a ).
Meth od I. To phenol (8.6 g, 91.5 mmol) in THF/toluene (1:3,
500 mL) was added NaH (1.8 g, 60.8 mmol) under N2
atmosphere; this was stirred for 20 min at 25 °C. The
diepoxide 8a (2.83 g, 15.2 mmol) was added, and the temper-
ature was raised to 95 °C for 7 h. The solution was allowed
to cool and washed with 1 M NaOH (2 × 150 mL). The
combined aqueous extracts were extracted with ether (100
mL). The combined organic extracts were dried and concen-
trated. Purification by flash chromatography (CH2Cl2 to CH2-
Cl2/CH3OH, 100:1) gave the product as a white solid (4.09 g,
72%): mp 108-111 °C; IR (CH2Cl2) ν 3600-3300, 3050, 2932,
1
1594, 1492 cm-1; [R]D ) -38.0° (c ) 1.03, CHCl3, 25 °C); H
NMR (270.2 MHz, CDCl3) δ 7.3 (dd, J ) 7.5, 6.9 Hz, 4H,
OArH[m]), 6.95 (m, 6H, OArH[o + p]), 4.27 (d, J ) 7.6 Hz,
2H, CHOC), 4.05 (m, 6H, CHOH, CH2OPh), 3.6 (s, 2H, OH),
1.39 (s, 6H, CH3); 13C NMR (67.8 MHz, CDCl3) δ 158.5, 129.4,
121.1, 114.7, 109.8, 79.7, 71.7, 69.4, 26.9. Anal. (C21H26O6)
C, H.
1
1466, 1243 cm-1; [R]D ) +50.1° (c ) 1.14, CHCl3, 25 °C); H
NMR (399.8 MHz, CDCl3) δ 7.28 (dd, J ) 8.8, 7.5 Hz, 4H,
OArH[m]), 6.99 (t, J ) 7.4 Hz, 2H, OArH[p]), 6.95 (d, J ) 8.8
Hz, 4H, OArH[o]), 4.86 (d, J ) 7.1 Hz, 2H, OCH2O), 4.79 (d, J
) 7.3 Hz, 2H, OCH2O), 4.73 (br s, 2H, NH), 4.11 (app s, 6H,
CHOCH2, CH2OPh), 3.97 (br s, 2H, CHN), 3.75 (dt, J ) 11.3,
4.2 Hz, 2H, OCH2CH2O), 3.71 (dt, J ) 11.5, 4.2 Hz, 2H, OCH2-
CH2O), 3.52 (t, J ) 4.5 Hz, 4H, OCH2CH2O), 3.35 (s, 6H, CH3);
13C NMR (67.8 MHz, CDCl3) δ 164.0, 157.9, 129.5, 121.4, 114.6,
96.3, 74.6, 72.0, 67.8, 67.3, 58.9, 50.5. Anal. (C27H38N2O9) C,
H, N.
2,5-Dia zid o-3,4-O-isop r op ylid en e-1,6-d i-O-p h en yl-2,5-
d id eoxy-D-id itol (10a ). Meth od II. To a solution of com-
pound 9a (0.98 g, 2.62 mmol) and triphenylphosphine PPh3
(1.44 g, 5.5 mmol) in THF (10 mL) at -15 °C was added diethyl
azodicarboxylate (DEAD) (1.11 g, 5.5 mmol), and this was
stirred for 5 min. Diphenyl phosphorazidate (DPPA) (1.51 g,
5.5 mmol) was added, and the reaction mixture was stirred at
25 °C overnight. The solvent was removed, and the crude
residue was purified by dry flash chromatography (pentane/
CH2Cl2, 6:1) followed by flash chromatography (pentane/CH2-
Cl2, 10:1-1:1) to give the product as a white solid (0.72 g,
65%): mp 75-77 °C; IR (CH2Cl2) ν 3042, 2936, 2113, 1595,
(4R,5S,6S,7R)-1,3-Diben zyl-5,6-bis[(2-m eth oxyeth oxy)-
m e t h oxy]-4,7-b is(p h e n oxym e t h yl)-1,3-d ia ze p a n -2-on e
(14a ). Meth od VI. To compound 13a (150 mg, 0.28 mmol)
in DMF (10 mL) were added NaH (80% suspension) (50 mg,
1.68 mmol) and benzyl bromide (287 mg, 1.68 mmol). The
reaction mixture was stirred under a N2 atmosphere overnight.
Excess NaH was quenched carefully with ethanol, and the
reaction mixture was diluted with water and extracted with
ether (2 × 50 mL). The combined ether extracts were washed
with brine, dried, and concentrated. Purification by flash
chromatography (CH2Cl2/CH3OH, 100:1) gave a colorless oil
(180 mg, 89%): IR (film) ν 3062, 2888, 1653, 1599, 1496, 1471
1
1492 cm-1; [R]D ) -39.0° (c ) 1.09, CHCl3, 25 °C); H NMR
(270.2 MHz, CDCl3) δ 7.29 (dd, J ) 8.6, 7.6 Hz, 4H, OArH[m]),
6.98 (t, J ) 7.4 Hz, 2H, OArH[p]), 6.92 (d, J ) 8.6 Hz, 4H,
OArH[o]), 4.33 (m, 2H, CHOC), 4.25 (d, J ) 6.3 Hz, 4H, CH2-
OPh), 3.75 (m, 2H, CHN3), 1.46 (s, 6H, CH3); 13C NMR (67.8
MHz, CDCl3) δ 157.9, 129.6, 121.6, 114.6, 110.9, 76.9, 67.8,
59.5, 26.9. Anal. (C21H24N6O4) C, H, N.
1
cm-1; [R]D ) -56.2° (c ) 1.26, CHCl3, 25 °C); H NMR (270.2
2,5-Dia zid o-1,6-d i-O-p h en yl-2,5-d id eoxy-D-id itol (11a ).
Meth od III. Compound 10a (2.0 g, 4.71 mmol) was added to
a mixture of acetonitrile (60 mL) and 3 M HCl (15 mL). The
temperature was raised to 50 °C and maintained for 4 h. The
solvent was removed, and the crude product was purified by
flash chromatography (CH2Cl2 to CH2Cl2/CH3OH, 25:1) to give
the product as a white solid (1.45 g, 80%); IR (film) ν 3427,
3064, 2932, 2110, 1595, 1493, 1239 cm-1; [R]D ) -25.1° (c )
1.0, CHCl3, 25 °C); 1H NMR (270.2 MHz, CDCl3) δ 7.29 (dd, J
) 7.9, 6.6 Hz, 4H, OArH[m]), 7.0 (t, J ) 7.3 Hz, 2H, OArH[p]),
6.92 (d, J ) 7.9 Hz, 4H, OArH[o]), 4.29 (m, 4H, CH2OPh), 3.96
(m, 4H, CHOH, CHN3), 2.91 (s, 2H, OH); 13C NMR (67.8 MHz,
CDCl3) δ 157.9, 129.6, 121.7, 114.6, 70.9, 68, 62.4; HRMS calcd
for C18H20N6O4 384.1546, found 384.1544.
MHz, CDCl3) δ 7.25 (m, 14H, ArH), 6.95 (t, J ) 7.2 Hz, 2H,
OArH[p]), 6.82 (d, J ) 7.9 Hz, 4H, OArH[o]), 5.16 (d, J ) 14.2
Hz, 2H, CH2Ph), 4.58 (d, J ) 7.0 Hz, 2H, OCH2O), 4.49 (d, J
) 6.9 Hz, 2H, OCH2O), 4.32 (app t, J ) 9.8 Hz, 2H, CH2OPh),
4.22 (dd, J ) 9.9, 2.6 Hz, 2H, CH2OPh), 4.09 (d, J ) 14.1 Hz,
2H, CH2Ph), 3.89 (br d, J ) 9.6, 2H, CHN), 3.45-3.3 (m, 10H,
CHOCH2, OCH2CH2O), 3.28 (s, 6H, CH3); 13C NMR (67.8 MHz,
CDCl3) δ 161.8, 158.6, 138.5, 129.6 (2 C), 128.9, 127.7, 121.2,
114.9, 96.5, 77.0, 71.7, 67.8, 65.7, 59.9, 59.1, 55.9. Anal.
(C41H50N2O9) C, H, N.
(4R,5S,6S,7R)-1,3-Dib en zyl-4,7-b is(p h en oxym et h yl)-
5,6-d ih yd r oxy-1,3-d ia zep a n -2-on e (1a ). Meth od VII. To
a solution of acetonitrile (4 mL) and 3 M HCl (1 mL) was added
14a (100 mg, 0.14 mmol). The reaction mixture was heated

892 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Hulte´n et al.
to 50 °C for 5 h. The solvent was removed, and purification
by flash chromatography (CH2Cl2 to CH2Cl2/CH3OH, 100:1)
gave a white solid (67.2 mg, 89%): mp 218-221 °C; IR (KBr)
ν 3600-3300, 3026, 2892, 1586, 1496 cm-1; [R]D ) -29.5° (c )
1.42, CHCl3, 25 °C); 1H NMR (399.8 MHz, CDCl3) δ 7.34-7.18
(m, 10H, ArH), 4.08 (app s, 2H, CHOC), 2.93 (dd, J ) 9.8, 4.4
Hz, 2H, CHN3), 2.85 (ddd, J ) 13.9, 9.1, 5.4 Hz, 2H, CH2Ph),
2.73 (ddd, J ) 13.9, 8.8, 7.3 Hz, 2H, CH2Ph), 2.14 (dddd, J )
14.4, 9.0, 9.0, 5.4 Hz, 2H, CH2CH2Ph), 1.93 (dddd, J ) 14.2,
9.3, 7.3, 4.6 Hz, 2H, CH2CH2Ph), 1.48 (s, 6H, CH3); 13C NMR
(67.8 MHz, CDCl3) δ 140.4, 128.6, 128.4, 126.2, 110.4, 79.7,
1
0.43, DMSO, 25 °C); H NMR (270.2 MHz, DMSO-d6) δ 7.31
(m, 14H, ArH), 6.96 (t, J ) 7.3 Hz, 2H, OArH[p]), 6.86 (d, J )
8.1 Hz, 4H, OArH[o]), 5.36 (br s, 2H, OH), 4.96 (d, J ) 14.3
Hz, 2H, CH2Ph), 4.29 (m, 4H, CH2OPh), 4.00 (d, J ) 14.4 Hz,
2H, CH2Ph), 3.70 (br m, 2H, CHN), 3.42 (m, 2H, CHOH); 13C
NMR (67.8 MHz, DMSO-d6) δ 161.8, 158.9, 139.4, 130.2, 129.5,
129.2, 127.9, 121.5, 115.3, 70.8, 66.0, 62.2, 56.2. Anal.
(C33H34N2O5) C, H, N.
59.3, 32.4, 26.8. Anal. (C
23H28N6O2) C, H, N.
2,5-Dia zid o-1,6-d iben zyl-1,2,5,6-tetr a d eoxy-D-id itol(17).
Compound 17 was synthesized from 16 according to method
III in 86% yield: mp 72-73 °C; IR (KBr) ν 3537, 3500-3200,
3030, 2941, 2103, 1602, 1495, 1454, 1280 cm-1; [R]D ) +6.3°
(c ) 1.09, CHCl3, 25 °C); 1H NMR (270.2 MHz, CDCl3) δ 7.33-
7.25 (m, 4H, ArH), 7.23-7.15 (m, 6H, ArH), 3.61 (dt, J ) 7.2,
4.0 Hz, 2H, CHOH), 3.29 (ddd, J ) 9.5, 5.6, 3.9 Hz, 2H, CHN3),
2.79 (ddd, J ) 13.9, 11.2, 6.6 Hz, 2H, CH2Ph), 2.72 (ddd, J )
13.9, 11.9, 7.6 Hz, 2H, CH2Ph), 2.56 (d, J ) 5.6 Hz, 2H, OH),
1.95 (m, 4H, CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ 140.5,
128.6, 128.3, 126.2, 73.4, 62.9, 32.1, 32.0. Anal. (C20H24N6O2)
C, H, N.
(4S,5R,6R,7S)-1,3-Dib en zyl-4,7-b is(p h en oxym et h yl)-
5,6-d ih yd r oxy-1,3-d ia zep a n -2-on e (1b). Compound 1b was
synthesized from 14b according to method VII in 72% yield:
mp 219-223 °C; IR (CHCl3) ν 3585, 3056, 2937, 1645, 1596,
1463, 1449, 1269, 1237 cm-1; [R]D ) +23.3° (c ) 0.30, DMSO,
1
25 °C); H NMR (270.2 MHz, DMSO-d6) δ 7.3 (m, 14H, ArH),
6.95 (t, J ) 7.3 Hz, 2H, OArH[p]), 6.85 (d, J ) 8.1 Hz, 4H,
OArH[o]), 5.39 (br s, 2H, OH), 4.97 (d, J ) 14.2 Hz, 2H, CH2-
Ph), 4.3 (m, 4H, CH2OPh), 4.0 (d, J ) 15.0 Hz, 2H, CH2Ph),
3.75 (br m, 2H, CHN), 3.37 (m, 2H, CHOH); 13C NMR (67.5
MHz, DMSO-d6) δ 160.9, 158.0, 138.5, 129.4, 128.7, 128.4,
127.1, 120.7, 114.4, 69.8, 65.0, 61.3, 55.3. Anal. (C33H34N2O5)
C, H, N.
2,5-Dia zid o-1,6-d iben zyl-3,4-bis-O-[(2-m eth oxyeth oxy)-
m eth yl]-1,2,5,6-tetr a d eoxy-D-id itol (18). Compound 18 was
synthesized from 17 according to method IV in 70% yield: IR
(film) ν 3026, 2926, 2103, 1603, 1496, 1453 cm-1; [R]D ) -47.8°
1
(c ) 1.32, CHCl3, 25 °C); H NMR (399.8 MHz, CDCl3) δ 7.3
(4S,5S,6S,7S)-1,3-Diben zyl-4,7-bis(p h en oxym eth yl)-5,6-
d ih yd r oxy-1,3-d ia zep a n -2-on e (1c). Compound 1c was
synthesized from 14c according to method VII in 76% yield:
IR (KBr) ν 3600-3200, 3030, 2927, 1621, 1598, 1495, 1470,
(m, 4H, ArH), 7.21 (m, 6H, ArH), 4.90 (d, J ) 7.1 Hz, 2H,
OCH2O), 4.81 (d, J ) 6.8 Hz, 2H, OCH2O), 3.82 (m, 2H,
CHOCH2), 3.76 (ddd, J ) 11.0, 5.1, 3.6 Hz, 2H, OCH2CH2O),
3.64 (ddd, J ) 10.7, 5.9, 3.7 Hz, 2H, OCH2CH2O), 3.46 (m, 4H,
OCH2CH2O), 3.33 (s, 6H, CH3), 3.18 (m, 2H, CHN3), 2.80 (ddd,
J ) 13.9, 9.3, 5.6 Hz, 2H, CH2Ph), 2.71 (ddd, J ) 13.7, 9.3, 6.8
Hz, 2H, CH2Ph), 2.1-1.93 (m, 4H, CH2CH2Ph); 13C NMR (67.8
MHz, CDCl3) δ 140.7, 128.5, 128.4, 126.1, 97.3, 81.2, 71.5, 68.0,
60.7, 58.9, 32.4, 32.3. Anal. (C28H40N6O6) C, H, N.
1
1241 cm-1; [R]D ) +47.3° (c ) 0.69, CHCl3, 25 °C); H NMR
(399.8 MHz, CDCl3) δ 7.35 (m, 4H, ArH), 7.28 (m, 10H, ArH),
7.00 (t, J ) 7.3 Hz, 2H, OArH[p]), 6.85 (d, J ) 8.8 Hz, 4H,
OArH[o]), 4.98 (d, J ) 14.4 Hz, 2H, CH2Ph), 4.13 (dd, J ) 9.7,
5.3 Hz, 2H, CH2OPh), 4.08 (dd, J ) 9.7, 5.3 Hz, 2H, CH2OPh),
4.02 (d, J ) 14.4 Hz, 2H, CH2Ph), 3.93 (m, 2H, CHOH), 3.42
(m, 2H, CHN), 2.62 (br s, 2H, OH); 13C NMR (67.5 MHz, CDCl3)
δ 163.3, 158.2, 137.8, 129.5, 129.2, 128.7, 127.7, 121.4, 114.6,
71.0, 66.9, 63.2, 52.9. Anal. (C33H34N2O5) C, H, N.
(4R,5S,6S,7R)-5,6-Bis[(2-m et h oxyet h oxy)m et h yl]-4,7-
bis-(2-p h en yleth yl)-1,3-d ia zep a n -2-on e (19). Compound 19
was synthesized from 18 according to method V in 66% yield:
mp 147-150 °C; IR (KBr) ν 3264, 3025, 2926, 1677, 1454, 1361
(4R,5R,6R,7R)-1,3-Dib en zyl-4,7-b is(p h en oxym et h yl)-
5,6-d ih yd r oxy-1,3-d ia zep a n -2-on e (1d ). Compound 1d was
synthesized from 14d according to method VII in 70% yield:
IR (KBr) ν 3600-3200, 3030, 2926, 1621, 1598, 1494, 1469
1
cm-1; [R]D ) +30.9° (c ) 1.25, CHCl3, 25 °C); H NMR (270.2
MHz, CDCl3) δ 7.26 (m, 4H, ArH), 7.16 (m, 6H, ArH), 4.76 (d,
J ) 6.9 Hz, 2H, OCH2O) 4.67 (d, J ) 7.3 Hz, 2H, OCH2O),
4.17 (br s, 2H, NH), 3.66 (br s, 2H, CHOCH2), 3.53 (m, 6H,
CHN, OCH2CH2O), 3.42 (m, 4H, OCH2CH2O), 3.31 (s, 6H,
CH3), 2.72 (m, 4H, CH2Ph), 1.87 (m, 4H, CH2CH2Ph); 13C NMR
(67.8 MHz, CDCl3) δ 163.7, 140.6, 128.6, 128.4, 126.2, 96.3,
77.2, 71.5, 67.6, 59.0, 51.1, 33.9, 32.4. Anal. (C29H42N2O7) C,
H, N.
1
cm-1; [R]D ) -43.3° (c ) 0.51, CHCl3, 25 °C); H NMR (399.8
MHz, CDCl3) δ 7.37-7.25 (m, 14H, ArH), 7.00 (t, J ) 7.3 Hz,
2H, OArH[p]), 6.85 (d, J ) 7.8 Hz, 4H, OArH[o]), 4.99 (d, J )
14.4 Hz, 2H, CH2Ph), 4.12 (dd, J ) 9.6, 5.1 Hz, 2H, CH2OPh),
4.09 (dd, J ) 9.6, 5.1 Hz, 2H, CH2OPh), 4.03 (d, J ) 14.2 Hz,
2H, CH2Ph), 3.95 (m, 2H, CHOH), 3.42 (m, 2H, CHN), 2.55
(br s, 2H, OH); 13C NMR (67.5 MHz, CDCl3) δ 163.2, 158.2,
137.8, 129.5, 129.3, 128.7, 127.7, 121.4, 114.6, 71.0, 66.9, 63.2,
52.9. Anal. (C33H34N2O5) C, H, N.
(4R,5S,6S,7R)-1,3-Diben zyl-5,6-bis[(2-m eth oxyeth oxy)-
m eth oxy]-4,7-bis(2-p h en yleth yl)-1,3-d ia zep a n -2-on e (20).
Compound 20 was synthesized from 19 according to method
VI in 86% yield: IR (film) ν 3061, 2926, 1641, 1495, 1453, 1357
1,6-Diben zyl-3,4-O-isop r op ylid en e-1,6-d id eoxy-L-m a n -
n itol (15). Meth od VIII. To a stirred suspension of CuI (6.1
g, 32 mmol) in THF (50 mL) at -20 °C was added benzylmag-
nesium chloride (32 mL of a 2.0 M solution, 64 mmol). After
1 h at 0 °C, the diepoxide 8a (2.0 g, 10.75 mmol) was added
and the reaction mixture was stirred for 30 min at 0 °C. The
reaction was quenched with saturated aqueous NH4Cl; the
mixture was diluted with water (50 mL) and extracted with
ether (3 × 150 mL). The combined ether extracts were washed
with brine (50 mL), dried, and concentrated. Purification by
flash chromatography (CH2Cl2 to CH2Cl2/CH3OH, 100:1) gave
a white solid (3.47 g, 87%): mp 80-83 °C; IR (KBr) ν 3600-
3100, 2922, 1600, 1494, 1453, 1374, 1069 cm-1; [R]D ) -34.6°
1
cm-1; [R]D ) -60.9° (c ) 1.17, CHCl3, 25 °C); H NMR (270.2
MHz, CDCl3) δ 7.22 (m, 16H, ArH), 7.07 (m, 4H, ArH), 5.09
(d, J ) 14.2 Hz, 2H, NCH2Ph), 4.50 (d, J ) 6.9 Hz, 2H,
OCH2O), 4.41 (d, J ) 6.6 Hz, 2H, OCH2O), 3.92 (d, J ) 13.9
Hz, 2H, NCH2Ph), 3.39 (m, 12H, CHOCH2, CHN, OCH2CH2O),
3.32 (s, 6H, CH3), 2.68 (ddd, J ) 14.2, 9.2, 5.9 Hz, 2H, CH2CH2-
Ph), 2.46 (dt, J ) 14.2, 8.2 Hz, 2H, CH2CH2Ph), 1.96 (m, 4H,
CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ 126.3, 141.5, 138.1,
129.5, 128.6, 128.4, 128.3, 127.5, 125.9, 96.2, 77.1, 71.6, 67.1,
60.3, 59.0, 56.0, 33.0, 28.6. Anal. (C43H54N2O7) C, H, N.
(4R,5S,6S,7R)-1,3-Diben zyl-4,7-bis(2-p h en yleth yl)-5,6-
d ih yd r oxy-1,3-d ia zep a n -2-on e (2).13 Compound 2 was syn-
thesized from 20 according to method VII in 91% yield: mp
210-212 °C; IR (KBr) ν 3600-3100, 3026, 2910, 1579, 1483,
1
(c ) 0.786, CHCl3, 25 °C); H NMR (270.2 MHz, CDCl3) δ 7.2
(m, 10H, ArH), 3.67 (m, 4H, CHOH, CHOC), 3.24 (s, 2H, OH),
2.87 (ddd, J ) 13.5, 10.2, 5.6 Hz, 2H, CH2Ph), 2.74 (ddd, J )
13.9, 9.6, 6.6 Hz, 2H, CH2Ph), 2.11 (dddd, J ) 13.9, 10.2, 5.9,
4.0 Hz, 2H, CH2CH2Ph), 1.78 (dddd, J ) 13.9, 9.3, 5.3, 5.3 Hz,
2H, CH2CH2Ph), 1.35 (s, 6H, CH3); 13C NMR (67.8 MHz,
CDCl3) δ 141.9, 128.5, 128.4, 125.8, 108.9, 82.9, 72.5, 35.7, 31.3,
26.8. Anal. (C23H30O4) C, H.
1
1451, 1233 cm-1; [R]D ) -22.6° (c ) 1.17, CHCl3, 23 °C); H
NMR (399.8 MHz, CDCl3) δ 7.33-7.26 (m, 4H, ArH), 7.25-
7.17 (m, 12H, ArH), 7.13 (m, 4H, ArH), 5.13 (d, J ) 14.1 Hz,
2H, NCH2Ph), 3.91 (d, J ) 14.2 Hz, 2H, NCH2Ph), 3.48 (s, 2H,
CHOH), 3.35 (m, 2H, CHN), 2.78 (ddd, J ) 13.2, 9.3, 6.1 Hz,
2H, CH2CH2Ph), 2.56 (ddd, J ) 14.0, 9.8, 7.1 Hz, 2H, CH2CH2-
Ph), 2.18 (s, 2H, OH), 2.10-1.96 (m, 4H, CH2CH2Ph); 13C NMR
(67.8 MHz, CDCl3) δ 162.4, 141.3, 137.9, 129.3, 128.5, 128.3,
128.2, 127.5, 125.9, 71.1, 60.7, 55.7, 33.1, 28.1. Anal.
(C35H38N2O3) C, H, N.
2,5-Dia zid o-1,6-d iben zyl-3,4-O-isop r op ylid en e-1,2,5,6-
t et r a d eoxy-D-id it ol (16). Compound 16 was synthesized
from 15 according to method II in 70% yield: IR (CH2Cl2) ν
3073, 2937, 2107, 1603, 1496, 1234 cm-1; [R]D ) -47.7° (c )

HIV-1 Protease Inhibitors Derived from Mannitol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 893
1,6-Bis(2-ph en yleth yl)-3,4-O-isopr opyliden e-1,6-dideoxy-
L-m a n n itol (21). Meth od IX. To Mg (763 mg, 31.8 mmol),
ether (10 mL), and a small amount of iodine was added
phenylethyl bromide (5.88 g, 31.8) under mild reflux. The
phenylethylmagnesium bromide was added to a suspension
of CuI (3.07 g, 16.1 mmol) in dry THF (35 mL) at -40 °C. The
temperature was slowly raised to -20 °C over 60 min and then
lowered to -40 °C followed by addition of the diepoxide 8a
(1.0 g, 5.3 mmol). The temperature was once again slowly
raised to -20 °C. The reaction was quenched with saturated
aqueous NH4Cl; the mixture was diluted with water (40 mL)
and extracted with ether (3 × 150 mL). The combined ether
extracts were washed with brine (40 mL), dried, and concen-
trated. Purification by flash chromatography (isohexane/ethyl
acetate, 10:1-6:1) gave a white solid (1.37 g, 64%): IR (CHCl3)
ν 3600-3200, 3026, 2860, 1603, 1496, 1453 cm-1; [R]D ) -26.9°
(c ) 0.81, CHCl3, 25 °C); 1H NMR (270.2 MHz, CDCl3) δ 7.3-
7.1 (m, 10H, ArH), 3.7-3.5 (m, 4H, CHOC, CHOH), 3.28 (br
s, 2H, OH), 2.7-2.5 (m, 4H, CH2CH2Ph), 1.9-1.4 (m, 8H,
CH2CH2CH2Ph), 1.34 (s, 6H, CH3); 13C NMR (67.8 MHz,
CDCl3) δ 142.3, 128.3, 128.2, 125.7, 108.7, 83.0, 72.9, 35.8, 33.8,
26.8. Anal. (C25H34O4) C, H.
2.42 (m, 4H, CH2CH2Ph), 1.80-1.33 (m, 8H, CH2CH2CH2Ph);
13C NMR (67.8 MHz, CDCl3) δ 162.0, 141.9, 138.3, 129.6, 128.5,
128.3, 128.2, 127.4, 125.8, 96.3, 76.5, 71.6, 67.0, 61.3, 58.9, 56.0,
35.8, 28.9, 26.9. Anal. (C45H58N2O7) C, H, N.
(4R,5S,6S,7R)-1,3-Diben zyl-4,7-bis(3-p h en ylp r op yl)-5,6-
d ih yd r oxy-1,3-d ia zep a n -2-on e (3). Compound 3 was syn-
thesized from 26 according to method VII in 74% yield: IR
(film) ν 3600-3200, 3026, 2923, 1602, 1495, 1475, 1453 cm-1
;
1
[R]D ) -3.8° (c ) 0.476, CHCl3, 25 °C); H NMR (270.2 MHz,
CDCl3) δ 7.25 (m, 10H, ArH), 7.15 (m, 10H, ArH), 5.01 (d, J )
14.2 Hz, 2H, NCH2Ph), 3.82 (d, J ) 14.2 Hz, 2H, NCH2Ph),
3.39 (br s, 2H, CHOH), 3.24 (br d, J ) 9.9 Hz, 2H, CHN), 2.49
(m, 4H, CH2CH2Ph), 1.95 (br s, 2H, OH), 1.85-1.42 (m, 8H,
CH2CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ 162.2, 141.8,
138.1, 129.5, 128.6, 128.4, 128.3, 127.6, 125.9, 71.2, 61.8, 56.1,
35.9, 29.1, 26.5. Anal. (C37H42N2O3) C, H, N.
(4R,5S,6S,7R)-1,3-Bis{[4-m e t h oxyca r b on yl)p h e n yl]-
m eth yl}-5,6-bis[(2-m eth oxyeth oxy)m eth oxy]-4,7-bis(p h e-
n oxym eth yl)-1,3-d ia zep a n -2-on e (27). Compound 27 was
synthesized from 13a according to method VI in 72% yield,
with methyl 4-(bromomethyl)benzoate as alkylating agent: IR
(film) ν 3040, 2890, 1720, 1655, 1598, 1467 cm-1; [R]D ) -46.4°
1
2,5-Diazido-1,6-bis(2-ph en yleth yl)-3,4-O-isopr opyliden e-
1,2,5,6-tetr a d eoxy-D-id itol (22). Compound 22 was synthe-
sized from 21 according to method II in 67% yield: IR (CHCl3)
ν 3050, 2974, 2111, 1602, 1496, 1453 cm-1; [R]D ) -46.2° (c )
(c ) 1.21, CHCl3, 25 °C); H NMR (399.8 MHz, CDCl3) δ 7.93
(d, J ) 8.3 Hz, 4H, CH2ArH[o]), 7.29 (m, 8H, ArH), 6.99 (t, J
) 7.3 Hz, 2H, OArH[p]), 6.79 (d, J ) 8.0 Hz, 4H, OArH[o]),
5.25 (d, J ) 14.2 Hz, 2H, NCH2Ar), 4.66 (d, J ) 6.9 Hz, 2H,
OCH2O), 4.59 (d, J ) 7.1 Hz, 2H, OCH2O) 4.23 (d, J ) 6.3 Hz,
4H, CH2Ph), 4.18 (d, J ) 14.2 Hz, 2H, NCH2Ar), 3.93 (app s,
8H, CHN, CH3OOC), 3.51-3.34 (m, 10H, CHOCH2, OCH2-
CH2O), 3.33 (s, 6H, CH3); 13C NMR (67.8 MHz, CDCl3) δ 166.9,
161.4, 158.4, 143.7, 130.1, 129.6, 129.5, 129.4, 121.3, 114.8,
96.7, 71.7, 67.7, 65.5, 61.0, 59.1, 56.1, 52.2. Anal. (C45H54N2O13)
C, H, N.
1
1.05, CHCl3, 25 °C); H NMR (270.2 MHz, CDCl3) δ 7.28 (m,
4H, ArH), 7.18 (m, 6H, ArH), 3.99 (m, 2H, CHOC), 2.93 (m,
2H, CHN3), 2.66 (t, J ) 7.2 Hz, 4H, CH2CH2Ph), 1.93-1.55
(m, 8H, CH2CH2CH2Ph), 1.44 (s, 6H, CH3); 13C NMR (67.8
MHz, CDCl3) δ 141.5, 128.4, 128.3, 126.0, 110.3, 79.8, 60.5,
35.4, 30.2, 28.0, 26.8. Anal. (C25H32N6O2) C, H, N.
2,5-Dia zid o-1,6-b is(2-p h en ylet h yl)-1,2,5,6-t et r a d eoxy-
D-id itol (23). Compound 23 was synthesized from 22 accord-
ing to method III in 91% yield: mp 130-131 °C; [R]D ) +4.7°
(c ) 0.51, DMSO, 25 °C); IR (KBr) ν 3600-3200, 3026, 2936,
(4R,5S,6S,7R)-1,3-Bis{[4-(m et h oxyca r b on yl)p h en yl]-
m et h yl}-4,7-b is(p h en oxym et h yl)-5,6-d ih yd r oxy-1,3-d i-
a zep a n -2-on e (6). Meth od X. To compound 27 (37 mg, 0.044
mmol) in methanol (4 mL) was added concentrated HCl (0.16
mL). The reaction mixture was heated to 40 °C for 5 h. The
solvent was removed. Purification by flash chromatography
(CH2Cl2 to CH2Cl2/CH3OH, 100:1) gave the product as a white
solid (26.4 mg, 92%): mp 187-189 °C; IR (KBr) ν 3600-3300,
3040, 2950, 1720, 1604, 1471 cm-1; [R]D ) +3.2° (c ) 1.43,
CHCl3, 25 °C); 1H NMR (399.8 MHz, CDCl3) δ 7.95 (d, J ) 8.3
Hz, 4H, CH2ArH[o]), 7.31 (m, 8H, ArH), 7.02 (t, J ) 7.3 Hz,
2H, OArH[p]), 6.8 (d, J ) 7.8 Hz, 4H, OArH[o]), 5.15 (br d, J
) 11.9 Hz, 2H, NCH2Ar), 4.39 (m, 2H, CH2OPh), 4.20 (m, 4H,
NCH2Ar, CH2OPh), 3.92 (s, 6H, CH3OOC), 3.80 (m, 2H, CHN),
3.59 (br s, 2H, CHOH), 2.8 (br s, 2H, OH); 13C NMR (100.2
MHz, CDCl3) δ 166.8, 161.5, 157.8, 143.3, 130.1, 129.7, 129.6,
129.2, 121.7, 114.6, 71.5, 65.1, 60.0, 55.8, 52.2. Anal.
(C37H38N2O9) C, H, N.
(4R,5S,6S,7R)-1,3-Bis{[4-(h ydr oxym eth yl)ph en yl]m eth -
yl}-4,7-bis(p h en oxym eth yl)-5,6-d ih yd r oxy-1,3-d ia zep a n -
2-on e (7). Meth od XI. To compound 27 (60 mg, 0.072 mmol)
in ether (2 mL) was added LiBH4 (4.70 mg, 0.216 mmol). The
reaction mixture was heated to reflux for 12 h. Excess LiBH4
was quenched carefully with 1 M HCl (20 mL), and ether (20
mL) was added. The layers were separated, and the water
phase was extracted with ether (2 × 20 mL). The combined
ether extracts was washed with saturated NaHCO3 (20 mL),
dried, and concentrated to yield 45 mg of the crude product,
which was used without further purification. To a solution of
the crude diol in methanol (3 mL) was added concentrated HCl
(1 mL); this was stirred overnight at 25 °C. The solvent was
removed, and the crude product was purified by flash chro-
matography (CH2Cl2/CH3OH, 50:1-20:1) to give a white solid
(27.1 mg, 63%): mp 161-163 °C; IR (KBr) ν 3600-3200, 3040,
2923, 1600, 1473, 1301 cm-1; [R]D ) -10.6° (c ) 0.491, CH3-
OH, 25 °C); 1H NMR (399.8 MHz, CD3OD) δ 7.34-7.18 (m,
12H, ArH), 6.98 (t, J ) 7.4 Hz, 2H, OArH[p]), 6.90 (d, J ) 8.0
Hz, 4H, OArH[o]), 5.12 (d, J ) 13.9 Hz, 2H, NCH2Ar), 4.58 (s,
4H, CH2OH), 4.34 (m, 4H, CH2OPh), 4.06 (d, J ) 14.2 Hz, 2H,
NCH2Ar), 3.88 (m, 2H, CHN), 3.36 (br s, 2H, CHOH); 13C NMR
(100.2 MHz, CD3OD) δ 163.4, 159.5, 141.8, 138.0, 130.1, 130.0,
128.0, 121.7, 115.4, 71.1, 65.8, 64.5, 62.3, 56.3. Anal.
(C35H38N2O7) C, H, N.
1
2110, 1495 cm-1; H NMR (399.8 MHz, DMSO-d6) δ 7.28 (m,
4H, ArH), 7.17 (m, 6H, ArH), 5.29 (d, J ) 7.8 Hz, 2H, OH),
3.45 (m, 2H, CHOH), 3.32 (m, 2H, CHN3), 2.58 (m, 4H,
CH2CH2Ph), 1.74-1.42 (m, 8H, CH2CH2CH2Ph); 13C NMR
(67.8 MHz, DMSO-d6) δ 141.8, 128.2, 128.1, 125.6, 73.3, 62.5,
34.8, 29.2, 27.6. Anal. (C22H28N6O2) C, H, N.
2,5-Dia zid o-3,4-O-b is[(2-m et h oxyet h oxy)m et h yl]-1,6-
bis(2-p h en yleth yl)-1,2,5,6-tetr a d eoxy-D-id itol (24). Com-
pound 24 was synthesized from 23 according to method IV in
78% yield: IR (film) ν 3026, 2929, 2107, 1603, 1496, 1454 cm-1
;
1
[R]D ) -39.2° (c ) 0.63, CHCl3, 25 °C); H NMR (270.2 MHz,
CDCl3) δ 7.3-7.1 (m, 10H, ArH), 4.85 (d, J ) 6.9 Hz, 2H,
OCH2O), 4.75 (d, J ) 6.9 Hz, 2H, OCH2O), 3.72 (m, 4H, OCH2-
CH2O), 3.61 (m, 2H, CHOCH2), 3.48 (m, 4H, OCH2CH2O), 3.35
(s, 6H, CH3), 3.17 (m, 2H, CHN3), 2.65 (m, 4H, CH2CH2Ph),
1.72 (m, 8H, CH2CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ
141.5, 128.3, 128.2, 125.8, 97.3, 81.1, 71.5, 68.0, 61.3, 58.9, 35.4,
30.0, 27.9. Anal. (C30H44N6O6) C, H, N.
(4R,5S,6S,7R)-5,6-Bis[(2-m eth oxyeth oxy)m eth oxy]-4,7-
bis(3-p h en ylp r op yl)-1,3-d ia zep a n -2-on e (25). Compound
25 was synthesized from 24 according to method V in 78%
yield: IR (CHCl3) ν 3421, 3042, 2934, 1672, 1453 cm-1; [R]D
)
+18.6° (c ) 1.09, CHCl3, 25 °C); 1H NMR (270.2 MHz, CDCl3)
δ 7.25 (m, 4H, ArH), 7.15 (m, 6H, ArH), 4.77 (d, J ) 7.6 Hz,
2H, OCH2O), 4.68 (d, J ) 7.6 Hz, 2H, OCH2O), 4.03 (br s, 2H,
NH), 3.62 (m, 6H, CHOCH2, OCH2CH2O), 3.5 (br t, J ) 6.5
Hz, 2H, CHNH), 3.43 (m, 4H, OCH2CH2O), 3.31 (s, 6H, CH3),
2.63 (t, J ) 6.9 Hz, 4H, CH2CH2Ph), 1.82-1.50 (m, 8H,
CH2CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ 164.0, 141.4,
128.4, 128.3, 126.0, 96.1, 76.6, 71.5, 67.7, 59.0, 51.5, 35.4, 32.0,
28.0. Anal. (C31H46N2O7) C, H, N.
(4R,5S,6S,7R)-1,3-Diben zyl-5,6-bis[(2-m eth oxyeth oxy)-
m eth oxy]-4,7-bis(3-ph en ylpr opyl)-1,3-diazepan -2-on e (26).
Compound 26 was synthesized from 25 according to method
VI in 78% yield: IR (film) ν 2927, 1641, 1495, 1453, 1110, 1035
1
cm-1; [R]D ) -55.7° (c ) 0.67, CHCl3, 25 °C); H NMR (270.2
MHz, CDCl3) δ 7.25 (m, 14H, ArH), 7.12 (m, 6H, ArH), 4.96
(d, J ) 13.9 Hz, 2H, NCH2Ph), 4.50 (d, J ) 6.9 Hz, 2H,
OCH2O), 4.42 (d, J ) 6.9 Hz, 2H, OCH2O), 3.83 (d, J ) 14.2
Hz, 2H, NCH2Ph), 3.37 (m, 12H, CHOCH2, CHN, OCH2CH2O),

894 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Hulte´n et al.
(3R,4S,5S,6R)-4,5-Bis[(2-m et h oxyet h yl)m et h oxy]-3,6-
bis(ph en oxym eth yl)-1,2,7-th iadiazepan e 1,1-Dioxide (28).
Meth od XII. To compound 12a (225 mg, 0.4 mmol) in ethyl
acetate (5 mL) was added a catalytic amount of 10% Pd/C.
Hydrogen was added to the system at atmospheric pressure,
and the reaction mixture was stirred overnight. The suspen-
sion was filtered through Celite and concentrated to give a
colorless oil. The crude amine (180 mg) was dissolved in dry
pyridine (2.5 mL) under a N2 atmosphere, and sulfamide (36
mg, 0.37 mmol) was added.23 The reaction mixture was heated
to reflux for 16 h. After cooling, the solution was diluted with
ether (20 mL). The solution was washed with water (2 × 20
mL), then with 1 M aqueous HCl (2 × 10 mL), and finally with
10% aqueous Na2CO3 (10 mL). The ether solution was dried
and concentrated. The residue was purified by flash chroma-
tography (CH2Cl2 to CH2Cl2/CH3OH, 19:1). This gave 29 as a
pure white solid (138 mg, 60%): mp 72-74 °C; IR (mineral
oil) ν 3356, 3278, 1587, 1490, 1350, 1251, 1226, 1162, 1125
ArH), 5.08 (d, J ) 11.2 Hz, 2H, NH), 4.75 (d, J ) 7.4 Hz, 2H,
OCH2O), 4.70 (d, J ) 7.0 Hz, 2H, OCH2O), 3.72 (app s, 2H,
CHOCH2), 3.6 (ddd, J ) 11.0, 11.0, 3.9 Hz, 2H, CHN), 3.48
(m, 4H, OCH2CH2O), 3.41 (m, 4H, OCH2CH2O), 3.33 (s, 6H,
CH3), 2.88 (ddd, J ) 10.2, 8.6, 5.2 Hz, 2H, CH2CH2Ph), 2.72
(ddd, J ) 14.0, 8.0, 8.0 Hz, 2H, CH2CH2Ph), 1.89 (dddd, J )
13.9, 11.0, 8.8, 5.2 Hz, 2H, CH2CH2Ph), 1.66 (dddd, J ) 12.2,
8.3, 8.3, 3.9 Hz, 2H, CH2CH2Ph); 13C NMR (100.2 MHz, CDCl3)
δ 141.6, 128.8, 128.5, 126.0, 96.8, 80.4, 71.6, 67.8, 59.0, 50.2,
35.4, 32.0. Anal. (C28H42N2O8S) C, H, N.
(3R,4S,5S,6R)-2,7-Diben zyl-4,5-bis[(2-m eth oxyeth oxy)-
m eth oxy]-3,6-bis(2-p h en yleth yl)-1,2,7-th ia d ia zep a n e 1,1-
Dioxid e (31). Compound 31 was synthesized from 30 accord-
ing to method XIII in 88% yield: IR (KBr) ν 3062, 2924, 1603,
1496, 1452, 1308 cm-1; [R]D ) -3.5° (c ) 1.27, CHCl3, 23 °C);
1H NMR (399.8 MHz, DMSO-d6) δ 7.45 (d, J ) 7.1 Hz, 4H,
ArH), 7.36 (app t, J ) 7.8, 7.1 Hz, 4H, ArH), 7.30 (t, J ) 7.3
Hz, 2H, ArH), 7.18 (app t, J ) 7.5, 6.9 Hz, 4H, ArH), 7.11 (t,
J ) 7.3 Hz, 2H, ArH), 6.90 (d, J ) 7.0 Hz, 4H, ArH), 4.82 (d,
J ) 16.6 Hz, 2H, NCH2Ph), 4.73 (d, J ) 6.9 Hz, 2H, OCH2O),
4.69 (d, J ) 6.6 Hz, 2H, OCH2O), 5.53 (br m, 2H, NCH2Ph),
3.81 (br s, 2H, CHOCH2), 3.70 (br s, 2H, CHN), 3.52 (br m,
4H, OCH2CH2O), 3.39 (t, J ) 4.6 Hz, 4H, OCH2CH2O), 3.19
(s, 6H, CH3), 2.55 (br m, 2H, CH2CH2Ph), 2.33 (br m, 2H,
CH2CH2Ph), 1.96 (br m, 2H, CH2CH2Ph), 1.85 (br m, 2H, CH2-
CH2Ph); 13C NMR (100.2 MHz, DMSO-d6) δ 143.0, 140.8, 129.9,
129.8, 129.7, 129.4, 128.8, 127.4, 97.6, 81.1, 72.8, 68.8, 59.7,
58.2, 53.6, 34.2, 32.7. Anal. (C42H54N2O8S) C, H, N.
1
cm-1; [R]D ) -15.3° (c ) 1.0, CHCl3, 25 °C); H NMR (270.2
MHz, CDCl3) δ 7.29 (dd, J ) 8.2, 7.9 Hz, 4H, OArH[m]), 6.98
(t, J ) 7.3 Hz, 2H, OArH[p]), 6.88 (d, J ) 8.3 Hz, 4H, OArH[o]),
5.35 (d, J ) 10.9 Hz, 2H, NH), 4.83 (d, J ) 7.3 Hz, 2H, OCH2O),
4.78 (d, J ) 7.3 Hz, 2H, OCH2O), 4.3 (s, 2H, CHOCH2), 4.13
(m, 2H, CH2OPh), 4.04 (m, 2H, CH2OPh), 3.90 (ddd, J ) 8.6,
8.6, 5.5 Hz, 2H, CHN), 3.77 (m, 2H, OCH2CH2O), 3.61 (m, 2H,
OCH2CH2O), 3.46 (m, 4H, OCH2CH2O), 3.32 (s, 6H, CH3); 13
C
NMR (67.8 MHz, CDCl3) δ 157.8, 129.6, 121.5, 114.5, 96.4,
74.1, 71.6, 68, 66.1, 59, 49.6. Anal. (C26H38N2O10S) C, H, N.
(3R,4S,5S,6R)-2,7-Diben zyl-4,5-bis[(2-m eth oxyeth oxy)-
m eth oxy]-3,6-bis(ph en oxym eth yl)-1,2,7-th iadiazepan e 1,1-
Dioxid e (29). Meth od XIII. To compound 28 (30 mg, 0.053
mmol) in DMF (0.75 mL) was added NaH (80% suspension)
(6.3 mg, 0.21 mmol). The reaction mixture was stirred at room
temperature under a N2 atmosphere for 30 min. Benzyl
bromide (36 mg, 0.21 mmol) was added, and the reaction was
allowed to proceed overnight. Excess NaH was quenched by
dilution with water (5 mL), and the reaction mixture was
extracted with ether (2 × 10 mL). The combined ether extracts
were dried and concentrated. Purification by flash chroma-
tography (CH2Cl2 to CH2Cl2/CH3OH, 99:1) gave a colorless oil
(39 mg, 99%): IR (film) ν 3052, 2927, 2886, 1600, 1497, 1329,
1243, 1157, 1112, 1036 cm-1; [R]D ) -27.6° (c ) 1.0, CHCl3,
25 °C); 1H NMR (270.2 MHz, CDCl3) δ 7.48 (d, J ) 6.9 Hz,
4H, ArH), 7.33 (dd, J ) 7.6, 6.9 Hz, 4H, ArH), 7.22 (m, 6H,
OArH[m], ArH), 6.93 (t, J ) 7.3 Hz, 2H, OArH[p]), 6.68 (d, J
) 8.6 Hz, 4H, OArH[o]), 5.03 (d, J ) 17.2 Hz, 2H, NCH2Ph),
4.84 (m, 6H, NCH2Ph, OCH2O), 4.47 (dd, J ) 7.3, 6.6 Hz, 2H,
CHN), 4.27 (s, 2H, CHOCH2), 4.01 (m, 4H, CH2OPh), 3.74 (m,
2H, OCH2CH2O), 3.67 (m, 2H, OCH2CH2O), 3.4 (m, 4H,
OCH2CH2O), 3.28 (s, 6H, CH3); 13C NMR (67.8 MHz, CDCl3) δ
157.9, 139.6, 129.5, 128.6, 127.1, 127, 121.3, 114.4, 96, 76.8,
71.5, 67.9, 65.9, 59, 53.5, 52. Anal. (C40H50N2O10S) C, H, N.
(3R,4S,5S,6R)-2,7-Dib en zyl-3,6-b is(p h en oxym et h yl)-
4,5-dih ydr oxy-1,2,7-th iadiazepan e 1,1-Dioxide (4). Meth od
XIV. To a solution of compound 29 (14 mg, 0.019 mmol) in
methanol (1 mL) was added saturated HCl in ether (2 mL).
The reaction mixture was stirred overnight at room temper-
ature. The solvent was removed, and purification by flash
chromatography (CH2Cl2 to CH2Cl2/CH3OH, 49:1) gave a white
solid (10 mg, 93%): mp 194-195 °C; IR (mineral oil) ν 3546,
3436, 1600, 1497, 1325, 1248, 1146 cm-1; [R]D ) +7.4° (c )
1.0, CHCl3, 25 °C); 1H NMR (270.2 MHz, acetone-d6) δ 7.54
(d, J ) 7.2 Hz, 4H, ArH), 7.34-7.18 (m, 10H, ArH, OArH[m]),
6.91 (t, J ) 7.3 Hz, 2H, OArH[p]), 6.79 (d, J ) 7.9 Hz, 4H,
OArH[o]), 5.05 (d, J ) 17.5 Hz, 2H, NCH2Ph), 4.96 (m, 4H,
NCH2Ph, OH), 4.45 (t, J ) 6.6 Hz, 2H, CHN), 4.27 (m, 4H,
(3R,4S,5S,6R)-2,7-Diben zyl-3,6-bis(2-p h en yleth yl)-4,5-
d ih yd r oxy-1,2,7-t h ia d ia zep a n e 1,1-Dioxid e (5). Com-
pound 5 was synthesized from 31 according to method XIV in
77% yield: IR (KBr) ν 3600-3200, 3027, 2924, 1603, 1495,
1
1453, 1295 cm-1; [R]D ) +47.9° (c ) 1.21, CHCl3, 23 °C); H
NMR (399.8 MHz, CDCl3) δ 7.33 (d, J ) 7.8 Hz, 4H, ArH),
7.23 (m, 6H, ArH), 7.11 (app t, J ) 7.3 Hz, 4H, ArH), 7.06 (t,
J ) 7.3 Hz, 2H, ArH), 6.88 (d, J ) 6.9 Hz, 4H, ArH), 4.68 (d,
J ) 15.6 Hz, 2H, NCH2Ph), 4.28 (d, J ) 15.6 Hz, 2H, NCH2-
Ph), 3.70 (s, 2H, CHOH), 3.47 (d, J ) 8.8 Hz, 2H, CHN), 2.68
(ddd, J ) 13.2, 10.8, 4.9 Hz, 2H, CH2CH2Ph), 2.28 (br s, 2H,
OH), 2.15 (ddd, J ) 13.1, 9.8, 6.4 Hz, 2H, CH2CH2Ph), 1.98
(dddd, J ) 14.6, 10.2, 10.2, 4.9 Hz, 2H, CH2CH2Ph), 1.85 (m,
2H, CH2CH2Ph); 13C NMR (67.8 MHz, CDCl3) δ 141.8, 137.6,
128.85, 128.81, 128.7, 128.5, 128.1, 126.0, 74.1, 59.9, 53.9, 33.1,
29.4. Anal. (C34H38N2O4S) C, H, N.
Exp r ession a n d P u r ifica tion of HIV-1 P r otea se. HIV-1
protease was cloned and heterologously expressed in Escheri-
chia coli and purified as described elsewhere.28
In h ibition Stu d ies. The degree of inhibition of HIV-1
protease was determined essentially as described previously24
in a spectrophotometric assay using a chromophoric peptide,
HIV substrate III (Bachem Feinchemicalien AG, Bubendorf,
Switzerland). The measurements were performed in a total
volume of 120 µL by preincubating 60.8 µM substrate and
inhibitor in 50 mM glycine, 50 mM sodium acetate, 50 mM
MES, 50 mM Tris-HCl, 1.0 M NaCl, 1 mM EDTA, and 1 mM
DTT at pH 5.5 and 37 °C for 5 min. The reaction was started
by addition of enzyme (30 nM), and the rate of cleavage was
followed by continuously registering the change in absorbance
at 300 nm. Inhibitors were dissolved in DMSO; the final
concentration of DMSO was kept below 2.5%. Solubility was
limited: high concentrations resulted in cloudiness in the
assay cuvette and nonlinear traces of absorbance versus time.
Inhibition was determined at 10 µM for compounds not potent
enough for IC50-value determination. The IC50-value is taken
as the inhibitor concentration that results in 50% activity. It
is estimated by plotting activity versus the logarithmic inhibi-
tor concentration.
CHOH, CH2OPh), 4.16 (dd, J ) 9.4, 6.3 Hz, 2H, CH2OPh); 13
C
NMR (67.8 MHz, acetone-d6) δ 159.8, 141.6, 130.8, 129.6,
128.5, 128.1, 122.3, 115.9, 75.3, 67.6, 56.4, 53.4. Anal.
(C32H34N2O6S) C, H, N.
(3R,4S,5S,6R)-4,5-Bis[(2-m eth oxyeth oxy)m eth oxy]-3,6-
bis(2-p h en yleth yl)-1,2,7-th ia d ia zep a n e 1,1-Dioxid e (30).
Compound 30 was synthesized from 18 according to method
XII in 77% yield: mp 98-99 °C; IR (KBr) ν 3208, 3029, 2917,
1604, 1495, 1456, 1418 cm-1; [R]D ) +55.3° (c ) 0.64, CHCl3,
23 °C); 1H NMR (399.8 MHz, CDCl3) δ 7.30-7.16 (m, 10H,
Determination of Ki-values was carried out by measuring
the activity of 20 nM HIV-1 protease at different substrate
and inhibitor concentrations. The mathematical analysis
required an extensive data set comprising a wide range of
substrate and inhibitor concentrations. The lowest substrate
concentration that gave reproducible results and a detectable
absorbance change was used (10 µM). Due to limited solubility
the highest concentration of substrate that could be used was
100 µM. Two intermediate concentrations were also used (20

HIV-1 Protease Inhibitors Derived from Mannitol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 895
and 50 µM). The inhibitor concentration was chosen to be of
the order of an initially estimated Ki-value and 2-4 times this
concentration. At least three concentrations were used. Nano-
molar concentrations of enzyme were used in order to obtain
sufficiently rapid and accurate estimates of reaction velocities.
Autoproteolysis of the enzyme and the sensitivity, which is
limited by absorbance change upon cleavage of substrate, limit
the time that can be used for the experiment, precluding the
possibility of compensating lower rates achieved by decreasing
the enzyme concentration with longer reactions. The total
enzyme concentration is therefore of the same order as the
concentration of inhibitor and requires the use of a rate
equation that accounts for the presence of an enzyme-
inhibitor complex for determination of Ki.25 A rate equation
for tight-binding inhibitors was fitted to the data by nonlinear
regression analysis using QNFIT in SIMFIT.29
time needed for the aspartates to swing around to this
equilibrium position from the crystallographic starting coor-
dinates was very long. The data collected in inhibitor-water
and inhibitor-protein-water simulations were the nonbonded
force field interaction energies for inhibitor-surrounding in-
teractions (where ‘surrounding’ refers to water or water +
protein, respectively), as required by the LIE procedure10 used
to evaluate the free energy (see below). Other details were as
in the recently reported similar calculations on other HIV-1
proteinase inhibitors.11
F r ee En er gy Ca lcu la tion s. The LIE method is based on
a linear response model of binding energetics. The simulation
averages of the interaction energies between the inhibitor and
its surroundings (no interactions within the inhibitor or within
the surroundings are included in the average) are assumed to
be linearly related to the free energy of solvation in that type
of surrounding. The interaction energies are divided into
electrostatic and van der Waals parts, with different propor-
tionality constants for the different terms in the final expres-
sion for the binding energy below. As described elsewhere,10,35
these considerations result in the following semiempirical
formula, where < > denotes MD time averages:
Molecu la r Dyn a m ics Sim u la tion s. MD simulations were
performed with a modified version (A° qvist, unpublished) of the
ENZYMIX program,30 using the GROMOS force field27 with
modifications reported elsewhere.31,10 Two spherical simula-
tion systems of radius 16 Å were set up for each inhibitor. In
one, the inhibitor was placed in the center of the sphere, and
SPC water molecules32 were added to fill the sphere. For the
other simulation, the inhibitor was docked into the HIV
proteinase, the simulation sphere was centered on the inhibi-
tor, and water molecules were added to fill the nonprotein
parts of the sphere. Protein atoms outside the simulation
sphere were strongly restrained to their crystallographic
positions and only interacted through bonds over the sphere
boundary. The 16 Å radius sphere used in this work in fact
contains most of the proteinase. MD simulations were then
performed, first to allow the system to attain equilibrium and
thereafter to collect data for the free energy calculations. For
inhibitor-water simulations, the inhibitor was kept in the
center of the sphere by restraining the carbonyl carbon. The
simulation temperature was 300 K, the MD time step was 1
fs, and SCAAS surface restraints33 were employed to hold the
water surface at the desired radius. A nonbonded interaction
cutoff radius of 10 Å was used for interactions between neutral
noninhibitor groups, but the inhibitor and all charged groups
interacted with everything inside the simulation sphere.
Initial protein coordinates were from Brookhaven Protein Data
bank entry 5HVP,5g conformation 1. Net charges were left
unmodified for the residues Asp 25, Arg 8, Arg 208, Asp 29,
Asp 229, Asp 30, Asp 230, Arg 87, and Arg 287, while all other
charged residues (far from the inhibitor) were replaced from
the alternate set of GROMOS27 residues with neutral dipolar
groups. Asp 225 was considered to be protonated. Tetra-
methyl-substituted RSSR and RRRR cyclic urea rings were
energy minimized by repeated simulated annealing using
InsightII and Discover (Biosym/MSI, San Diego, CA), and the
resulting minimum energy rings and their mirror images were
used for modeling the ligands. The ligands were graphically
docked into the protein, without manipulation of protein
coordinates, to yield the starting models. We used Insight II
(Biosym/MSI) for docking except for 3, where the ligand was
docked into the protein by a conformational search with the
Macro Model program.34
The systems consisting of the inhibitor in water were
equilibrated for 190 ps at 300 K and then run for 125 ps,
collecting data every 5 fs. The protein-inhibitor-water
systems were heated under decreasing restraints in a 15 ps
stepwise heating scheme, equilibrated for 175 ps at 300 K, and
then run for 125 ps for data collection (250 ps for DMP 323
and 7). Additional harmonic distance restraints of 4 kcal/mol
Å2 were applied during the heating and the first 25 ps of the
equilibration to ensure correct initial geometries of the system.
These were applied between the inhibitor carbonyl oxygen and
the nitrogens of the peptide NH groups of Ile 50 and Ile 250
and between the inhibitor carbonyl carbon and the side chain
carboxylate carbon of Asp 25 and Asp 225. For DMP 323 and
7, similar additional restraints of 5 kcal/mol were applied
between one carboxylate oxygen of aspartates 30 and 230 and
the appropriate inhibitor hydroxyl group oxygen and hydrogen
atoms, to ensure that the aspartates would find the possibility
of hydrogen bonding to these hydroxyl groups. Otherwise, the
∆Gbind ) ∆Gel + ∆Gvdw ≈ R(<Vvdw
>
- <Vvdw>wat) +
prot
â(<Vel>prot - <Vel>wat) (1)
Here, R is an empirically determined constant, and â is
1
predicted to be
/ by the electrostatic linear response ap-
2
proximation.10,35 As in earlier work, we here use R ) 0.161
and â ) /2 to estimate the binding free energies.
1
Ack n ow led gm en t. We gratefully acknowledge sup-
port from the Swedish Natural Science Research Coun-
cil (NFR), Medivir AB, Huddinge, Sweden, and the
Swedish National Board for Industrial and Technical
Development (NUTEK). We wish to express our ap-
preciation to Torleif Ha¨rd for valuable discussions.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR
spectral data, IR, optical rotations, and elemental analyses for
compounds 9b-14b, 9c-14c, and 9d -14d (6 pages). Order-
ing information is given on any current masthead page.
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