5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: Potent nonpeptidic inhibitors of HIV protease

Article abstract of DOI:10.1021/jm990281p

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S₃' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S₃' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

Full text of DOI:10.1021/jm990281p

J . Med. Chem. 2000, 43, 843-858
843
5,6-Dih yd r op yr a n -2-on es P ossessin g Va r iou s Su lfon yl F u n ction a lities: P oten t
Non p ep tid ic In h ibitor s of HIV P r otea se
Frederick E. Boyer,*,† J . V. N. Vara Prasad,† J ohn M. Domagala,† Edmund L. Ellsworth,† Christopher Gajda,†
Susan E. Hagen,† Larry J . Markoski,† Bradley D. Tait,† Elizabeth A. Lunney,† Alexander Palovsky,†
Donna Ferguson,‡ Neil Graham,‡ Tod Holler,‡ Donald Hupe,‡ Carolyn Nouhan,‡ Peter J . Tummino,‡ A. Urumov,‡
Eric Zeikus,‡ Greg Zeikus,‡ Stephen J . Gracheck,§ J ames M. Sanders,§ Steven VanderRoest,§
J oanne Brodfuehrer,| Krishna Iyer,| Michael Sinz,| Sergei V. Gulnik, and J ohn W. Erickson
Departments of Chemistry, Biochemistry, Infectious Diseases, and PDM, Parke-Davis Pharmaceutical Research, Division of
Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105, and Structural Biochemistry Program,
NCI-Frederick Cancer Research and Development Center, PRI/ Dyncorp, Frederick, Maryland 21702
Received J une 4, 1999
On the basis of previous SAR findings and molecular modeling studies, a series of compounds
were synthesized which possessed various sulfonyl moieties substituted at the 4-position of
the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents
were added in an attempt to fill the additional S3′ pocket and thereby produce increasingly
potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of
selected compounds were synthesized. All analogues in the study displayed decent binding
affinity to HIV protease, and several compounds were shown to possess very good antiviral
efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide
and sulfonate moieties were filling the S3′ pocket of the enzyme. However, the additional
substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to
the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not
appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested
for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of
the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors
exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the
currently marketed protease inhibitors.
In tr od u ction
droxypyran-2-one is a low-micromolar inhibitor of HIV
protease lacking antiviral activity which is presumed
to occupy two interior pockets (S1 and S1′) of the target
enzyme (Figure 1). Structure-activity relationship stud-
ies centered around the mass screening lead eventually
resulted in the 5,6-dihydro-4-hydroxy-2-pyrone II.11
Possessing one chiral center, II displayed a 300-fold
increase in activity against the enzyme over the initial
lead; however, it exhibited no antiviral activity. The lack
of efficacy against the virus displayed by II may be due
to the extensive hydrophobic nature of the compound.
Recently, we have reported further efforts at structural
modification and optimization, namely replacement of
one of the phenyl rings with alkyl groups and adding
polar substituents at both sides of the molecule, result-
ing in the identification of III (PD 178390, CI-1029) as
a clinical candidate.12 Inhibitor III exhibits subnano-
molar activity against HIV protease, excellent antiviral
efficacy and therapeutic index, favorable multispecies
pharmacokinetic profiles, and little cross-resistance with
currently marketed protease inhibitors.13 X-ray crystal
structure studies of III bound to HIV protease indicated
that the inhibitor occupies the interior four pockets of
the enzyme (S1, S2, S1′, and S2′) as shown in Figure 2.
Active site interactions include hydrogen bond formation
between the 4-hydroxyl group and the catalytic aspartic
acids Asp25 and Asp125, as well as hydrogen bonds
between the lactone moiety and NHs of the flap region
Ile50 and Ile150 amino acid residues (Figure 2). H2O-
Since first identified as the etiological agent of
acquired immune deficiency syndrome (AIDS),1 the
development of drugs to treat human immunodeficiency
virus (HIV) infection has been an area of intense
research efforts. One approach that has met with great
success is the inhibition of HIV protease,2,3 a virally
encoded aspartyl protease essential for the production
of mature, infectious virions. HIV protease is responsible
for the posttranslational processing of the Gag and Gag/
Pol polyproteins,4 proteolytic activity that cannot be
carried out by native enzymes of the infected host. The
currently marketed HIV protease inhibitors which are
peptidomimetics, though remarkably efficacious, have
serious problems associated with high costs,5 low bio-
availability,6 and significant drug interactions.3,7 Ad-
ditionally, the emergence of HIV-1 strains which are
resistant to currently available therapies will mandate
the need for new medications.2,8,9
Efforts in our laboratories to identify a potent non-
peptidic HIV protease inhibitor originated with the
high-throughput screening lead I.10 This achiral 4-hy-
* Corresponding author. Tel: 734-622-5071. Fax: 734-622-7879.
E-mail: frederick.boyer@wl.com.
† Department of Chemistry.
‡ Department of Biochemistry.
§ Department of Infectious Diseases.
| Department of PDM.
Structural Biochemistry Program.
10.1021/jm990281p CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/15/2000

844 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
ring system and possesses sulfonamide functionalities
which are seen to occupy the S3′ pocket. Recently, we
reported preliminary results of HIV protease inhibitors
possessing amide,17 ether, carbamate, and sulfamate
functionalities.18 A variety of functional groups were
investigated to better access the S3′ pocket of the
enzyme. Herein, we report inhibitors possessing sul-
fonamide, sulfonate, and sulfonylurea functionalities
with the proper orientation to fill the S3′ pocket.
F igu r e 1.
Ch em istr y
The target compounds (compounds 1-69S, Table 3)
were synthesized in a convergent manner involving the
coupling of 5,6-dihydropyran-2-one with the appropriate
thiotosylate (Table 1) in the presence of potassium
carbonate in DMF. The synthetic pathway necessitated
the synthesis of thiotosylates derived from an ap-
propriately substituted aniline or phenol. In the case
of the aniline-derived series (Scheme 1), 4-tert-butyl-1-
methyl-2-nitrobenzene19 (VI) was reduced to the aniline
with Raney nickel and treated with bromine, sodium
bromide, and sodium thiocyanide to yield the isothio-
cyanate derivative V. Protection of the amine was
accomplished with di-tert-butyl dicarbonate followed by
treatment with dithiothreitol to afford the thiol VI. The
thiol was converted to the thiotosylate by reaction with
tosyl bromide in the presence of triethylamine. The Boc
protection was then removed with HCl gas in dichlo-
romethane to yield the hydrochloride salt which was
treated with phosphate buffer (pH 7.5) to give the free
base VII. Finally, treatment with the appropriate
sulfonyl chloride in pyridine and dichloromethane gave
the desired thiotosylates VIIIa -o.
F igu r e 2. Lead compound CI-1029 occupying the interior four
active site pockets with core interactions.
In the case of the phenol-derived thiotosylates (Scheme
2), 4-tert-butyl-1-methyl-2-nitrobenzene (VI) underwent
reduction with Raney nickel followed by diazotization
with sodium nitrite to give the phenol. Treatment of this
compound with bromine, sodium bromide, and sodium
thiocyanide afforded the isothiocyanate derivative IX.
The isothiocyanate can then be subjected to dithiothrei-
tol to produce the thiol, followed by tosylation with tosyl
bromide in the presence of triethylamine to present X.
Treatment with the appropriate sulfonyl chloride in the
presence of triethylamine produces the desired thioto-
sylates VIIIp -y. Alternatively, IX can be converted to
the sulfamate XII through reaction with the corre-
sponding sulfamoyl chloride. Treatment with dithio-
threitol followed by subsequent tosylation with tosyl
bromide affords the desired thiotosylate VIIIz.
The racemic and enantiomerically resolved interme-
diate 5,6-dihydropyran-2-ones (Table 2) were synthe-
sized as previously described13 and are shown in Schemes
3 and 4, respectively. Enantiomerically pure 5,6-dihy-
dropyran-2-ones (>95% enantiomeric purity) were
synthesized with the key step being the resolution of
the corresponding â-hydroxy acids ((S)-XVII and (R)-
XVII; Scheme 4). Absolute stereochemistry was deter-
mined by small molecule X-ray of the intermediate
â-hydroxy acid (S)-XVIIb as the salt of (S)-(-) R-(naph-
thyl)ethylamine which ultimately yielded the resolved
5,6-dihydopyran-2-one (S)-XIVb. Absolute stereochem-
istry was determined in a similar fashion in a closely
related series.20 The resolved â-hydroxy acids were
elaborated to the corresponding â-keto esters which
F igu r e 3. Target 5,6-dihydropyran-2-ones occupying the five
enzymatic active site pockets.
301, commonly seen in X-ray crystallographic studies
of HIV protease, is displaced by the carbonyl of the
lactone moiety.14,15
Design
In an effort to identify more potent inhibitors of HIV
protease, a series of compounds was synthesized in
which an additional enzyme pocket could be filled in
addition to the four inner pockets occupied by III.
Molecular modeling studies indicated substitution at the
4′-position of the 3-S-(2-tert-butyl-5-methylphenyl) moi-
ety could provide access to the S3′ pocket of the HIV
protease enzyme (Figure 3). In addition, since the S3′
pocket of the enzyme is located toward the exposed
solvent region one can use various substitutions, which
might not only enhance the binding affinities of the
inhibitors but also modify the physical properties of the
inhibitors. An amino or hydroxyl group located at the
4′-position of the 3-S-(2-tert-butyl-5-methylphenyl) moi-
ety could easily be functionalized to extend into the S3′
pocket. Recently, researchers at Pharmacia and Upjohn
have reported employing a similar strategy in a related
series of compounds.16 This series of HIV protease
inhibitors is also based on the 5,6-dihydropyran-2-one

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 845
Sch em e 1. Synthesis of Thiotosylates Possessing a Sulfonamide Functionalitya
a
Reaction conditions: (a) H2, Raney nickel, MeOH; (b) NaSCN, Br2, NaBr, MeOH; (c) Boc2O, THF, 60 °C; (d) DTT, EtOH, phosphate
buffer; (e) tosyl bromide, NEt3, CCl4; (f) HCl, CH2Cl2; (g) phosphate buffer, MeOH; (h) XSO2R, pyridine, CH2Cl2.
Sch em e 2. Synthesis of Thiotosylates Possessing a Sulfonate Functionalitya
a
Reaction conditions: (a) H2, Raney nickel, MeOH; (b) NaNO3, H3O+; (c) NaSCN, Br2, NaBr, MeOH; (d) DTT, EtOH, phosphate buffer;
(e) tosyl bromide, pyridine, CCl4; (f) XSO2R, NEt3, CH2Cl2 or Et2O; (g) N,N-dimethylsulfamoyl chloride, NEt3, CH2Cl2; (h) DTT, EtOH,
phosphate buffer; (i) tosyl bromide, NEt3, CCl4.
Sch em e 3. Synthesis of Racemic Target 5,6-Dihydropyran-2-onesa
a
Reaction conditions: (a) NaH, then n-BuLi, THF; (b) R2CO(CH2)2PhR1; (c) NaOH, then H+; (d) thiotosylate, K2CO3, DMF.
upon treatment with sodium hydroxide cyclized to form
the desired 5,6-dihydropyran-2-ones.
cleavage products were separated by reverse-phase
HPLC. Absorbance was measured at 220 nm, peak
areas were determined, and % conversion to product
was used to calculate % control ([% conversion(+in-
hibitor)/% conversion(-inhibitor)] × 100).
Biologica l Assa ys
En zym e In h ibition Assa y. For determination of
IC50 values, affinity-purified HIV-1 protease (Bachem
Bioscience; 1.1 nM) was added to a solution of the fol-
lowing: the inhibitor, 40 µM peptide substrate (His-
Lys-Ala-Arg-Val-Leu-(p-NO2-Phe)-Glu-Ala-Nle-Ser-
NH2; Bachem Bioscience), and 1.0% DMSO in assay
buffer (1.0 mM dithiothreitol, 80 mM MES, 160 mM
NaCl, 1.0 mM EDTA, 0.1% poly(ethylene glycol) (MW
8000), pH 6.2 at 25 °C.) The final volume was 100 µL;
final concentration of HIV protease was 1.5 nM. The
solution was mixed, incubated for 60 min at 37 °C, and
then quenched with trifluoroacetic acid (2% final). In
this assay, the Leu-(p-NO2-Phe) bond of the substrate
was cleaved by the enzyme, and the substrate and
An ti-HIV Activity Assa y. Anti-HIV activity was
determined in an in vitro cell culture assay employing
HIV-IIIB-infected human lymphocyte-derived CEM cells
using the XTT cytopathic protocol at Southern Research
Institute.21 Antiviral activity against clinical isolates
was determined in a similar assay employing HIV-1-
infected PBMC cells. EC50 refers to the effective con-
centration at which 50% of the cells are conferred
protection against the cytopathic effects of HIV. TC50
refers to the toxic concentration of the drug that elicits
cytotoxicity in 50% of CEM cells not infected with HIV.
The probe substrates used to determine the inhibitory
activities against CYP3A4, CYP2D, and CYP2C9 were

846 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
Sch em e 4. Synthesis of Enantiomerically Pure Target 5,6-Dihydropyran-2-onesa
a
Reaction conditions: (a) LDA, benzyl acetate, THF; (b) H2, 5% Pd/C, MeOH; (c) (S)-R-methylbenzylamine, EtOAc; (d) 1 N HCl, ETOAc;
(e) CDI, THF, then Mg(O2CCH2CO2Et)2; (f) 1 N NaOH, then 1 N HCl; (g) thiotosylate, K2CO3, DMF. Note: The resolved (R)-enantiomer
of â-hydroxy acid (XIV) is obtained by utilizing (R)-R-methylbenzylamine in the resolution step (c) leading to the (R)-5,6-dihydro-4-
hydroxy-2-pyranone (XVI).
Ta ble 1. Method of Preparation of Thiotosylate Intermediates
general method
entry
R
of preparation
method of purification
% yield
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
NHSO2Me
NHSO2Ph
NHSO2Ph(4-F)
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
triturated: EtOAc/hexanes
triturated: hexanes
76
58
92
85
67
66
57
79
90
96
78
78
48
76
77
65
70
72
78
73
87
68
66
98
chromatographed (CHCl3)
chromatographed (CHCl3:EtOAc 9:1)
chromatographed (CHCl3:EtOAc 9:1)
chromatographed (CHCl3:EtOAc 19:1)
triturated: EtO2/hexanes
triturated: EtOAc/hexanes
triturated: EtOAc/hexanes
triturated: hexanes
triturated: EtOAc/CH2Cl2/hexanes
triturated: EtOAc/hexanes
chromatographed (CHCl3:EtOAc 9:1)
chromatographed (CHCl3:EtOAc 32:1)
chromatographed (CHCl3/EtOAc 9:1)
chromatographed (CHCl3)
triturated: EtO2
chromatographed (CHCl3)
chromatographed (CHCl3)
triturated: EtO2/hexanes
triturated: EtO2
triturated: EtO2
triturated: EtO2/hexanes
none
NHSO2Ph(4-Cl)
NHSO2Ph(4-CF3)
NHSO2Ph(4-CN)
NHSO2Ph(3-CN)
NHSO2Ph(4-NHCOMe)
NHSO2Ph(2-thiophene)
NHSO2(N-methylimidazole)
NHSO2(2-pyridyl)
NHSO2(3-pyridyl)
NHSO2[2-(5-CF3)pyridyl]
NHSO2NHCH2CH3
NHSO2N(CH3)2
OSO2Me
OSO2Ph
OSO2Ph(4-F)
OSO2Ph(4-CN)
OSO2(2-thiophene)
OSO2(N-methylimidazole)
OSO2(2-pyridyl)
OSO2(3-pyridyl)
OSO2NHCH2CH3
s
t
u
v
w
x
testosterone, bufurolol, and tolbutamide, respectively.
The samples were analyzed by HPLC-UV or fluores-
cence.
varied the substituents that would fill the S3′ pocket.
Though the steric bulk of the substituent occupying the
S3′ pocket was varied, all the analogues in the study
displayed decent binding affinity to HIV protease, with
IC50 or Ki in the low-nanomolar to subnanomolar range.
The presence or absence of a polar group on the C-6
phenethyl occupying the S2 pocket did not have a great
effect on enzymatic activity. The biological activities
associated with the target inhibitors are shown in
Tables 4-6.
Resu lts a n d Discu ssion
In our previously reported SAR, which led to the
identification of the lead compound CI-1029 (Figure 2),
we found that to best fill the S1 pocket of HIV protease,
an alkyl substituent at the C-6 position of the 5,6-
dihydropyran-2-one ring system was preferred. A p-
(hydroxy or amino)phenethyl was found to be optimal
for occupying the S2 pocket.12,13 In this study we initially
held isopropyl and p-hydroxyphenethyl groups constant
for occupying the S1 and S2 pockets, respectively, and
I. Su lfon a m id es. A. C-6 Isop r op yl Ser ies. In this
series of inhibitors (compounds 2-12), increasing the
steric bulk from a methyl (2) to a benzenesulfonamide
(3) does not produce any significant increase in enzyme

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 847
Ta ble 2. 5,6-Dihydropyran-2-one Intermediates Used for This
or antiviral activities. A C-6 isopropyl group is seen to
be optimal for producing favorable biological activity.
Studya
C. P ola r F u n ction a lity on th e P h en eth yl Gr ou p .
Since changing the C-6 alkyl group or varying the
sulfonamido moiety did not result in substantial in-
creases in desired activity, we decided to examine what
effect polarity might have on activity. In our previous
studies leading to CI-1029, the presence of a polar group
on the 4-position of the phenethyl moiety was found to
produce compounds with better overall therapeutic
profiles though there was no influence on HIV protease
binding affinity. This can be seen by examining the
unsubstituted aniline derivatives with and without the
phenolic moiety (compounds 1 and 25, respectively).
While both derivatives display similar inhibitory activity
against the enzyme, the derivative possessing a p-
hydroxy group on the phenethyl moiety exhibits better
antiviral activity (1.0 µM vs 1.9 µM) and decreased
cellular toxicity (92 µM vs 69 µM) as compared to the
deshydroxy compound.
chirality entry R1
R2
chirality entry R1
R2
R/ S
S
R
R/ S
S
R
a
b
c
d
e
f
OH isopropyl
OH isopropyl
OH isopropyl
H
H
H
R/ S
R/ S
R/ S
R/ S
R/ S
R/ S
h
i
j
k
l
m
OH methyl
OH n-propyl
OH isobutyl
OH cyclopropyl
OH cyclopentyl
OH cyclohexyl
isopropyl
isopropyl
isopropyl
R/ S
g
NH2 isopropyl
a
Syntheses of 5,6-dihydropyran-2-ones are shown in Schemes
3 and 4 and described in ref 13.
binding antiviral activity; however cytotoxicity is slightly
higher. While para-substitution on the phenyl ring is
allowed (4-6, 8), it does not lead to any significant
changes in cellular activity or cytotoxicity. Substitution
in the meta-position with a nitrile group (7) leads to a
decrease in antiviral activity by approximately 3-fold
as compared to the p-cyano (8) derivative although they
display similar Ki’s. Replacement of the phenyl by a
comparable isostere such as a thiophene (9) leads to a
significant decrease in enzymatic binding; however, the
antiviral activity is unaffected and the cytotoxicity is
slightly lower. Increasing the size of the heterocycle to
a 2-pyridyl (10) or 3-pyridyl (11) group results in a slight
loss of cellular activity. Para-substitution on the 2-py-
ridyl (12) does not affect activity; however the associated
toxicity is increased. The best compound in this series
based on EC50 and TI was compound 6 possessing
4-trifluoromethylphenylsulfonamide. However, the un-
substituted aniline (1) displayed better activity than any
of the sulfonamides with an EC50 of 1.0 µM and TC50 of
92 µM.
B. Oth er C-6 Alk yl Gr ou p s. The C-6 isopropyl group
series did not provide any analogues with greatly
enhanced biological activity as compared to the parent
unsubstituted aniline (1). To fully investigate the effect
of the alkyl substituent on biological activity, other
groups possessing varied steric bulk were also placed
at the C-6 position. In general, the methyl group at the
C-6 position (compounds 14-22) resulted in a consistent
drop in antiviral activity when compared to the analo-
gous isopropyl derivatives. This trend can be seen by
comparing the activities associated with several of the
inhibitors (2 vs 14, 3 vs 15, 4 vs 16, etc.). The best
derivative in this series is the 4-fluorophenylsulfon-
amide (16) with an EC50 of 5.6 µM and a therapeutic
index of 39.
In this series (compounds 26-34), a number of
compounds were synthesized with various substitutions
on the 4-position of the 3-S-(2-tert-butyl-5-methylphenyl)
moiety while keeping the isopropyl group at the C-6
position constant and the phenethyl moiety with and
without a polar substituent at the para-position. In this
series of inhibitors, the presence of the p-hydroxy group
on the phenethyl moiety had no significant effect on
enzymatic activity. However, compounds that do not
contain the p-hydroxyl on the phenethyl group generally
displayed increased antiviral activity and similar toxic-
ity as compared to inhibitors possessing the phenolic
group. The increase in antiviral activity was seen to be
from 3f7-fold. The compounds with a heterocyclic
sulfonamide seemed to be affected most by the removal
of the phenol. Compound 31 possessing a 2-pyridylsul-
fonamide exhibits an approximately 6-fold increase in
activity against HIV and greater cytotoxicity as com-
pared to the hydroxylated derivative 10 (66 µM vs >100
µM). The greatest increase in activity was found to be
with the compound possessing the 2-(5-CF3-pyridyl)-
sulfonamide moiety (34) which exhibited an EC50 of 0.6
µM and a TI of 100, a greater than 7-fold potency
improvement over the corresponding p-hydroxyphen-
ethyl derivative (12).
Since analogous compounds that have a phenethyl or
p-hydroxyphenethyl group as a C-6 substituent on the
dihydropyran-2-one ring system display comparable
enzymatic inhibitory activities, the differences in anti-
viral activities associated with equivalent derivatives
may be related to changes in physical property. The
sulfonamido moieties may confer additional hydrophilic
properties to the molecule that necessitate the removal
of a polar functionality for favorable antiviral activity.
The overall polarity associated with the various inhibi-
tors is important for antiviral activity.12 It appears the
amount of polarity required for favorable antiviral
activity may be obtained through appropriate substitu-
tion on the C-6 or C-3 portion of the molecules.
To further analyze what effect the C-6 alkyl group
has on activity, derivatives were synthesized where the
sulfonyl moiety was held constant and the C-6 alkyl
group was varied. In the sulfonamide series, the ben-
zenesulfonamide was held constant while the C-6 alkyl
group was varied from methyl (15), to isopropyl (3), to
cyclopropyl (23), and to cyclopentyl (24). Reducing or
increasing the size of the alkyl substituent from an
isopropyl group does not enhance enzymatic inhibitory
II. Su lfon a tes. A. C-6 Isop r op yl Ser ies. In this
series (compounds 36-43) the nitrogen group of the
sulfonamide was replaced with an oxygen. The meth-
anesulfonate (36) possesses an IC50 of 2.2 nM and EC50
of 0.6 µM with cellular toxicity of >100 µM for a TI of

848 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
Ta ble 3. Dihydropyran-2-one Analogues and Their Physical Properties
entry R1
R2
R3
method of purification
% yield
56
mp (°C)
molecular formulaa
anal.b
CHN
1
OH isopropyl
OH isopropyl
OH isopropyl
NH2
NH2
NH2
chromatographed; CHCl3:MeOH (9:1)
125-127
C27H35N1O4S1‚
0.83H2O
1S
1R
chromatographed; EtOAc
57
51
147-149 d C27H35N1O4S1‚
CHN
CHN
0.31H2O
chromatographed; CH2Cl2:EtOAc (3:1)
147-149 d C27H35N1O4S1‚
0.45H2O
2
3
4
OH isopropyl
OH isopropyl
OH isopropyl
NHSO2CH3
NHSO2Ph
NHSO2Ph(4-F)
chromatographed; EtOAc:hexanes (9:1)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; EtOAc:hexanes (7:3)
68
63
66
124 effer
200-203 d C33H39N1O6S2
123-125
C28H37N1O6S2
CHN
CHN
CHN
C33H38F1N1O6S2‚
0.34H2O
5
6
OH isopropyl
OH isopropyl
NHSO2Ph(4-Cl)
NHSO2Ph(4-CF3)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; EtOAc:hexanes (3:1)
70
61
214-218 d C33H38Cl1N1O6S2
227-229 d C34H38F3N1O6S2‚
0.47H2O
CHN
CHN
7
8
OH isopropyl
OH isopropyl
NHSO2Ph(3-CN)
NHSO2Ph(4-CN)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; EtOAc:hexanes (7:3)
51
65
>132 effer
C34H38N2O6S2
CHN
CHN
217-220 d C34H38N2O6S2‚
0.37H2O
9
10
11
OH isopropyl
OH isopropyl
OH isopropyl
NHSO2(2-thiophene)
NHSO2(2-pyridyl)
NHSO2(3-pyridyl)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; EtOAc
chromatographed; CHCl3:MeOH (19:1)
72
68
33
207-210 d C31H37N1O6S3
CHN
CHN
CHN
>136 effer
>135 effer
C32H38N2O6S2
C32H38N2O6S2‚
0.84H2O
12
OH isopropyl
NHSO2[2-(5-CF3)-
pyridyl]
NHSO2[2-(5-CF3)-
pyridyl]
NHSO2[2-(5-CF3)-
pyridyl]
NH2
chromatographed; CH2Cl2:MeOH (18:1)
chromatographed; CH2Cl2:MeOH (18:1)
72
38
42
70
57
55
50
52
>129 effer
>142 effer
>134 effer
158-168
133 effer
C33H37F3N2O6S2‚
CHN
0.29H2O
12S OH isopropyl
12R OH isopropyl
C33H37F3N206S2‚
0.53H2O
C33H37F3N206S2‚
0.49H2O
C25H31N1O4S1‚
0.65H2O‚0.98HCl
C26H33N1O6S2‚
0.51H2O
C31H35N1O6S2‚
0.70H2O
CHN
chromatographed; CH2Cl2:MeOH (18:1)
then EtOAc:hexanes (7:3)
chromatographed; CHCl3:MeOH (19:1)
CHN
13
14
15
16
17
OH methyl
OH methyl
OH methyl
OH methyl
OH methyl
CHN,Cl-
CHN
NHSO2CH3
chromatographed; CHCl3:MeOH (99:1)
chromatographed; CHCl3:MeOH (9:1)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; EtOAc:hexanes (4:1)
NHSO2Ph
136 effer
CHN
NHSO2Ph(4-F)
NHSO2Ph(4-Cl)
122 effer
C31H34F1N1O6S2‚
0.58H2O
C31H34Cl1N1O6S2‚
0.33H2O
CHN
>160 effer
CHN
18
19
OH methyl
OH methyl
NHSO2Ph(4-CF3)
NHSO2Ph(4-CN)
chromatographed; EtOAc:hexanes (3:1)
chromatographed; EtOAc:EtOH (49:1)
55
41
128 effer
153 effer
C32H34F3N1O6S2
C32H34N2O6S2‚
1.09H2O
CHN
CHN
20
21
22
23
24
25
26
27
28
OH methyl
OH methyl
OH methyl
NHSO2Ph(4-NHCOMe) chromatographed; EtOAc:EtOH (49:1)
NHSO2Ph(2-thiophene) chromatographed; CHCl3:MeOH (9:1)
27
66
28
70
62
52
62
51
55
185 effer
>132 effer
164 effer
123-125
125-127
104-105
88
C33H38N2O7S2‚
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
CHN
1.16H2O
C29H33N1O6S3‚
0.67H2O
C29H35N3O6S2‚
0.87H2O
C33H37N1O6S2‚
1.0H2O
C35H41N1O6S2‚
0.40H2O
NHSO2(N-methyl-
imidazole)
triturated: Et2O/hexanes
OH cyclopropyl NHSO2Ph
chromatographed; hexanes:EtOAc (3:1)
then EtOAc
chromatographed; hexanes:EtOAc (3:1)
then EtOAc
OH cyclopentyl NHSO2Ph
H
H
H
H
isopropyl
isopropyl
isopropyl
isopropyl
NH2
chromatographed; EtOAc:hexanes
(starting at 1:4 gradient to 4:1)
chromatographed; EtOAc:hexanes (4:1)
C27H35N1O3S1
NHSO2CH3
NHSO2Ph(4-CF3)
NHSO2Ph(3-CN)
C28H37N1O5S2‚
0.72H2O
C34H38F3N1O5S2‚
0.60H2O
C34H38N2O5S2‚
0.40H2O
chromatographed; CHCl3:MeOH (19:1)
chromatographed; EtOAc:hexanes (7:3)
188-192
>175 effer
29
29S
30
H
H
H
H
isopropyl
isopropyl
isopropyl
isopropyl
NHSO2Ph(4-CN)
NHSO2Ph(4-CN)
NHSO2(2-thiophene)
NHSO2(2-pyridyl)
chromatographed; EtOAc:hexanes (7:3)
chromatographed; CH2Cl2:MeOH (24:1)
chromatographed; EtOAc:hexanes (2:1)
chromatographed; EtOAc:hexanes (9:1)
65
89
50
64
>125 effer
>130 effer
96-116
C34H38N2O5S2
C34H38N205S2
C31H37N1O5S3
C32H38N2O5S2‚
0.51H2O
CHN
CHN
CHN
CHN
31
>134 effer
32
H
H
H
H
H
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
NHSO2(3-pyridyl)
chromatographed; CHCl3:MeOH (18:1)
chromatographed; CHCl3:MeOH (18:1)
chromatographed; CH2Cl2:MeOH (24:1)
chromatographed; CH2Cl2:EtOAc (3:1)
chromatographed; CH2Cl2:MeOH (48:1)
44
36
46
75
46
63
193-194 d C32H38N2O5S2‚
CHN
CHN
CHN
CHN
CHN
CH
0.40H2O
33
NHSO2(N-methyl-
imidazole)
NHSO2[2-(5-CF3)-
pyridyl]
NHSO2[2-(5-CF3)-
pyridyl]
NHSO2[2-(5-CF3)-
pyridyl]
>146 effer
>143 effer
>162 effer
>145 effer
117-118
C31H39N3O5S2‚
1.04H2O
34
C33H37F3N2O5S2
34S
34R
35
C33H37F3N2O5S2‚
0.33H2O
C33H37F3N2O5S2
OH isopropyl
OH
chromatographed; hexanes:EtOAc (4:1)
to EtOAc (100%) gradient
C27H34O5S1
36
OH isopropyl
OSO2CH3
OSO2 CH3
chromatographed; EtOAc
chromatographed; EtOAc (100%)
then EtOAc:hexanes (3:1)
81
45
>180 d
>205 d
C28H36O7S2
C28H36O7S2
CH
CH
36S OH isopropyl

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 849
Ta ble 3 (Continued)
entry R1
R2
R3
method of purification
% yield
mp (°C)
molecular formulaa
anal.b
CH
CH
CHN
37
38
39
OH isopropyl OSO2Ph
OH isopropyl OSO2Ph(4-F)
OH isopropyl OSO2Ph(4-CN)
chromatographed; EtOAc
chromatographed; EtOAc
chromatographed; EtOAc
77
75
80
>180 d
C33H38O7S2
C33H37F1O7S2
C34H36N107S2‚
0.40H2O
>180 d
200 d
40
41
OH isopropyl OSO2(2-thiophene)
OH isopropyl OSO2(N-methyl-
imidazole)
chromatographed; EtOAc
chromatographed; EtOAc
75
67
>180 d
>170 effer
C31H36O7S3
C31H38N2O7S2‚
0.45H2O
CHN
CHN
42
OH isopropyl OSO2(2-pyridyl)
chromatographed; EtOAc
86
>180 d
C32H37N1O7S2‚
0.25H2O
CHN
43
44
45
46
OH isopropyl OSO2(3-pyridyl)
chromatographed; EtOAc
chromatographed; EtOAc
chromatographed; EtOAc:hexanes (7:3)
triturated: CHCl3/Et2O
64
64
70
60
>180 d
C32H37N1O7S2
C31H34O7S2
C31H33F1O7S2
C29H34N2O7S2
CHN
CH
CH
OH methyl
OH methyl
OH methyl
OSO2Ph
>180 d
OSO2Ph(4-F)
OSO2(N-methyl-
imidazole)
>170 d
>160 effer
HPLC
47
48
49
50
51
52
OH cyclohexyl OSO2Ph
chromatographed; EtOAc
chromatographed; EtOAc
chromatographed; EtOAc
chromatographed; EtOAc
58
74
75
58
72
62
>180 d
C36H42O7S2‚
0.50H2O
C37H41N1O7S2‚
0.65H2O
C36H41F1O7S2‚
0.65H2O
C34H42N2O7S2‚
0.94H2O
CH
OH cyclohexyl OSO2Ph(4-CN)
OH cyclohexyl OSO2Ph(4-F)
>200 d
CHN
CH
>180 d
OH cyclohexyl OSO2(N-methyl-
>153 effer
169-171
CHN
CHN
HPLC
imidazole)
OH n-propyl
OSO2(N-methyl-
imidazole)
chromatographed; hexanes:EtOAc (3:1)
to EtOAc (100%) gradient
chromatographed; hexanes:EtOAc (3:1)
to EtOAc:hexanes (1:1) gradient
then EtOAc:MeOH (19:1)
C31H38N2O7S2‚
2.8H2O
C31H38N2O7S2
OH isobutyl
OSO2(N-methyl-
imidazole)
53
54
55
56
57
58
59
H
H
H
H
isopropyl OH
chromatographed; hexanes:EtOAc (4:1)
70
63
82
55
71
65
57
87-89
120-122
>180 d
>180 d
185 d
C
27H34O4S1‚
CH
to EtOAc (100%) gradient
0.70H2O
isopropyl OSO2(N-methyl-
imidazole)
isopropyl OSO2(2-pyridyl)
chromatographed; hexanes:EtOAc (3:1)
to EtOAc:hexanes (4:1) gradient
chromatographed; EtOAc
C31H38N2O6S2‚
CHN
0.50H2O
C32H37N1O6S2‚
0.26H2O
C32H37N1O6S2‚
CHN
isopropyl OSO2(3-pyridyl)
chromatographed; EtOAc
CHN
0.40H2O
NH2 isopropyl OSO2Ph(4-CN)
none
none
none
C34H38N2O6S2‚
1.0HCl‚0.46H2O
C32H38N2O6S2‚
1.0HCl‚0.32H2O
C31H39N3O6S2‚
0.9HCl‚0.50H2O
C31H37N1O6S3
CHN,Cl-
CHN,Cl-
CHN,Cl-
NH2 isopropyl OSO2Ph(3-pyridyl)
>180 d
>180 d
>150 d
NH2 isopropyl OSO2(N-methyl-
imidazole)
60
61
62
63
NH2 isopropyl OSO2Ph(2-thiophene) chromatographed; CH2Cl2:MeOH (9:1)
OH isopropyl NHSO2N(CH3)2
OH isopropyl NHSO2NHEt
39
52
52
72
CHN
CHN
CHN
CHN
chromatographed; EtOAC:hexanes (3:1)
chromatographed; CHCl3:MeOH (19:1)
chromatographed; EtOAc:hexanes (9:1)
187-189 d C29H40N2O6S2
>152 effer
136 effer
C29H40N2O6S2
C29H40N2O5S2‚
0.20H2O
H
isopropyl NHSO2NHEt
64
65
OH isopropyl OSO2N(CH3)2
OH isopropyl OSO2NHEt
chromatographed; EtOAc
chromatographed; hexanes:EtOAc (4:1)
to EtOAc (100%) gradient
triturated: Et2O
86
59
>180 d
97-99
C29H39N1O7S2
C29H39N1O7S2
CHN
CHN
66
67
68
NH2 isopropyl OSO2N(CH3)2
NH2 isopropyl OSO2NHEt
52
78
68
59
>205 d
C29H40N1O6S2‚
0.60H2O
C29H40N1O6S2‚
1.0HCl‚1.0H2O
C29H39N1O6S2
CHN,Cl-
CHN,Cl-
CHN
triturated: Et2O
185-188
78-79
H
H
isopropyl OSO2NHEt
chromatographed; hexanes:EtOAc (4:1)
to EtOAc (100%) gradient
chromatographed; CH2Cl2:MeOH (48:1)
69S
isopropyl OSO2Ph(4-CN)
>85 effer
C34H37N1O6S2
CHN
a
b
Water content was not experimentally determined. All compounds were with in (0.4% of the theoretical values.
>175. Contrary to what is observed with the sulfon-
amides, increasing the steric bulk of the sulfonate group
from a methyl to a phenyl group (37) results in a greater
than 4-fold drop in activity against the enzyme. Ad-
ditionally, there is a modest decrease in antiviral
activity, with an increase in toxicity as well, resulting
in a TI of approximately 41. Substitution on the para-
position of the phenyl ring (compounds 38 and 39) has
no significant effect on cellular activity. Replacement
of the phenyl ring by a heterocyclic ring (inhibitors 40-
43) provided no substantial change in antiviral activity.
In this series of inhibitors, compound 36 possessing a
methanesulfonate moiety displays the best overall
therapeutic profile. Compound 36 exhibits modestly
better antiviral activity and is less cytotoxic than the
unsubstituted phenol 35.
B. Oth er C-6 Alk yl Gr ou p s. As in the sulfonamide
series, to fully investigate the effect of the alkyl sub-
stituent on biological activity, other groups possessing
varied steric bulk were also placed at the C-6 position.
In general, a C-6 methyl group results in inhibitors
(compounds 44-46) that have similar enzymatic inhibi-
tory activities as compared to the C-6 isopropyl ana-
logues. The methyl analogues display approximately
3-4-fold less antiviral activity than the analogous
isopropyl derivatives.

850 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
Ta ble 4. Dihydropyran-2-one Analogues and Associated Biological Activities
entry
R1
R2
R3
IC50 (Ki)a (nM)
EC50b (µM)
TC50c (µM)
TId
1
2
3
4
5
6
7
8
9
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
H
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
NH2
NHSO2Me
NHSO2Ph
2.7 (0.67)
4.0
1.8 (0.53)
4.1
5.3
18.3
(0.21)
2.2 (0.41)
8.7
2.6
2.6
1.0
5.3
1.4
1.8
1.6
1.1
5.3
1.6
1.8
4.1
5.8
4.3
3.7
19
5.2
2.4
3.9
3.5
5.0
55
92
>100
74
92
>19
53
37
41
60
>19
53
>56
>24
>17
16
>27
>5
>19
39
NHSO2Ph(4-F)
66
66
66
NHSO2Ph(4-Cl)
NHSO2Ph(4-CF3)
NHSO2Ph(3-CN)
NHSO2Ph(4-CN)
NHSO2(2-thiophene)
NHSO2(2-pyridyl)
NHSO2(3-pyridyl)
NHSO2[2-(5-CF3)pyridyl]
NH2
NHSO2Me
NHSO2Ph
NHSO2Ph(4-F)
NHSO2Ph(4-Cl)
NHSO2Ph(4-CF3)
NHSO2Ph(4-CN)
NHSO2Ph(4-NHCOMe)
NHSO2Ph(2-thiophene)
NHSO2(N-methylimidazole)
NHSO2Ph
>100
85
>100
>100
>100
68
>100
>100
>100
93
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13.0
9.7
1.5
3.7
3.5
71
79
18
23
12.3
2.1
>100
>100
>100
>100
76
67
69
>100
>20
>2
>18
>2
32
27
36
>63
34
44
132
29
96
48
>23
100
41
6.3
1.9
3.6
8.6
3.6
(0.83)
(27.0)
(1.4)
9.1
3.6
3.0
5.6
58
methyl
cyclopropyl
cyclopentyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
methyl
2.4
2.5
1.9
1.6
0.7
1.5
0.5
2.1
0.7
1.6
4.2
0.6
1.6
0.6
1.6
0.8
0.6
2.4
4.3
0.7
1.6
12
NHSO2Ph
NH2
NHSO2Me
H
H
H
H
H
H
H
H
NHSO2Ph(4-CF3)
NHSO2Ph(3-CN)
NHSO2Ph(4-CN)
NHSO2(2-thiophene)
NHSO2(2-pyridyl)
NHSO2(3-pyridyl)
NHSO2(N-methylimidazole)
NHSO2[2-(5-CF3)pyridyl]
OH
OSO2Me
OSO2Ph
OSO2Ph(4-F)
OSO2Ph(4-CN)
OSO2(2-thiophene)
OSO2(N-methylimidazole)
OSO2(2-pyridyl)
OSO2(3-pyridyl)
OSO2Ph
OSO2Ph(4-F)
OSO2(N-methylimidazole)
OSO2Ph
24
66
66
61
67
76
3.5
1.9
>100
H
(1.04)
(0.03)
2.2
10.4
9.6
4.5
21.8
2.4
5.0
3.5
9.8
16.9
2.0
131
21.0
29.0
8.0
2.2
3.6
(1.1)
3.0
6.7
14.0
1.3
4.1
66
66
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-H
>100
>175
66
66
67
72
41
83
122
30
>23
120
48
7
16
>6
<1
11
7
63
54
23
37
60
46
14
>100
84
76
82
methyl
methyl
6.3
16
100
>100
59
29
66
>100
>100
>100
66
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
n-propyl
isobutyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
>59
2.6
9.5
1.6
1.9
4.4
1.8
1.1
1.4
4.6
0.4
2.2
1.8
6.0
OSO2Ph(4-CN)
OSO2Ph(4-F)
OSO2(N-methylimidazole)
OSO2(N-methylimidazole)
OSO2(N-methylimidazole)
OH
OSO2(N-methylimidazole)
OSO2(2-pyridyl)
OSO2(3-pyridyl)
OSO2Ph(4-CN)
4-H
4-H
4-H
4-NH2
4-NH2
4-NH2
4-NH2
66
65
65
53
151
30
>56
11
OSO2Ph(3-pyridyl)
OSO2(N-methylimidazole)
OSO2Ph(2-thiophene)
66
2.2
18.5
>100
66
a
b
Enzyme inhibition was determined at pH 6.2 as previously described.12 EC50 is the effective concentration at which 50% of the cells
are protected from HIV infection and is the average of at least two runs. c TC50 is the concentration which elicits cytotoxicity in 50% of
uninfected CEM cells. TI, therapeutic index ) TC50/EC50
d
.
Several analogues were synthesized with a C-6 cy-
clohexyl group to examine its utility in providing desir-
able activity against HIV protease (47-50). Increasing
the size of the C-6 substituent from an isopropyl to a
cyclohexyl leads to a significant loss in enzymatic and
antiviral activities. Compound 47, which possesses a

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 851
Ta ble 5. Dihydropyran-2-one Analogues Possessing a
Sulfonylurea or Sulfamate Functionality and Their Chemical
Data and Biological Activities
arylsulfonyl groups with sulfamoyl moieties to produce
sulfonylurea and sulfamate derivatives (Table 5). While
a number of various sulfonylureas and sulfamates were
synthesized,17,18 the most promising were derivatives
with small alkyl groups on the sulfamoyl nitrogen. The
dimethylsulfonylurea analogue (61) exhibits an EC50 of
1.7 µM and TC50 of >100 µM for a TI of >59. The
monoethyl derivative (62) is less potent against the
virus displaying an EC50 of 3.3 µM. Removal of the
p-hydroxyl group from the C-6 phenethyl substituent
slightly increases antiviral activity and cytotoxicity. The
monoethylsulfamate 65 displays antiviral activity in the
same range as dimethylsulfamate compound 64 (1.0 and
0.7 µM, respectively) and is less toxic in the cellular
assay (>100 µM vs 70 µM). Replacement of the p-
hydroxy group on the C-6 phenethyl substituent with a
primary amine does not affect antiviral activity but
modestly increases cytotoxicity in both the dimethyl-
(66) and monoethylsulfamates (67). Removal of the
polar substituent entirely on the phenethyl moiety
(compound 68) has similar effects on antiviral activity
and cytotoxicity.
IC50 (Ki) EC50 TC50
entry
R1
R2
(nM)
(µM) (µM)
TI
61
62
63
64
65
66
67
68
4-OH
4-OH
4-H
4-OH
4-OH
NHSO2N(Me)2
NHSO2NHEt
NHSO2NHEt
OSO2N(Me)2
OSO2NHEt
3.6
1.7
3.3
3.4
4.9
3.9
4.3
8.9
1.7
3.3
1.2
0.7
1.0
0.8
0.7
0.8
>100
>100
77
>59
>30
64
70
100
>100 >100
4-NH2 OSO2N(Me)2
4-NH2 OSO2NHEt
4-H
64
83
67
80
128
84
OSO2NHEt
phenylsulfonate, displays approximately 13-fold less
inhibitory activity against HIV protease than the analo-
gous isopropyl derivative 37 and exhibits very little
cellular activity. While para-substitution on the phen-
ylsulfonate (compounds 48 and 49) restores some of the
desirable activity to the cyclohexyl analogues, they still
show much less activity than the comparable isopropyl
derivatives (38 and 39).
As in the sulfonamide series, to further investigate
the effect of the C-6 alkyl substituent on biological
activity, derivatives were synthesized where the sulfonyl
moiety was held constant and the C-6 alkyl group was
varied. In the sulfonate series, a N-methylimidazole-
sulfonate moiety was held constant while the C-6 alkyl
group was varied (i.e., methyl (46), n-propyl (51),
isopropyl (41), isobutyl (52), cyclohexyl (50)). Except for
the cyclohexyl analogue, which was approximately 3-4-
fold less active, all analogues displayed similar activity
against the enzyme. Except for the methyl analogue
which displayed less antiviral activity, the other ana-
logues displayed a similar level of activity against the
virus as the isopropyl derivative. This finding indicated
that in this series of compounds, though a C-6 alkyl
substituent is important for activity, the steric bulk of
this alkyl substituent can vary greatly and still provide
favorable activity.
En a n tiom er ica lly Resolved 5,6-Dih yd r op yr a n -2-
on es. As a result of the promising biological activities
and therapeutic indices of a number of the racemic
compounds, several enantiomerically pure dihydropy-
ran-2-ones were synthesized (Scheme 4). The associated
biological activities of these optically pure compounds
are displayed in Table 6. The (S)-enantiomers 1S, 12S,
and 34S possess substantially increased binding affinity
to HIV protease relative to the (R)-enantiomers 1R,
12R, and 34R (19-, 24-, and 21-fold increased binding
affinity, respectively). The (S)-enantiomers display
greater antiviral activity as compared to the corre-
sponding (R)-enantiomers, the greatest difference being
in the case of the unsubstituted aniline compound 1S,
which displayed 0.49 µM antiviral activity while the (R)-
enantiomer 1R was essentially inactive against HIV.
The (S)-enantiomers generally exhibited approximately
2-fold increases in activity against HIV relative to the
racemic analogues. Only the (S)-enantiomer of the
sulfonate compound 69S was synthesized and was seen
to possess similar activity against HIV as the compa-
rable sulfonamide analogue 29S.
X-r a y Cr ysta l Str u ctu r es of An a logu es 3 a n d 50
Bou n d to Wild -Typ e HIV P r otea se. The X-ray crystal
structures of 3 and 50 bound to HIV protease both show
a binding mode similar to that previously observed with
this series of inhibitors. That is, the 5,6-dihydropyran-
2-one ring spans the active site with the 4-hydroxyl
group binding at the catalytic dyad, Asp25/125, and the
carbonyl group interacting with Ile50 and/or Ile150 in
the flap region. In addition, the substituents at the C-3
and C-6 positions of the dihydropyranone ring occupy
the four pockets surrounding the catalytic site of the
enzyme.
C. p-P h en eth yl P ola r Gr ou p . In the sulfonate
series, the presence of the p-hydroxyl on the C-6
phenethyl group was generally found to enhance anti-
viral activity. Only in the case of the N-methylimida-
zolesulfonate derivative (54) was the antiviral activity
seen to be better for the comparable analogue that did
not possess the p-hydroxyl on the phenethyl moiety. The
presence or absence of the polar group on the phenethyl
moiety did not have any significant effect on enzymatic
inhibitory activity in these compounds.
Previous SAR studies indicated that the optimal
p-phenethyl polar substituent for the C-6 phenethyl was
found to be a primary amine. To examine if this was
the case in this series, several compounds were synthe-
sized in which the phenolic group was replaced with an
anilino group (57-60). The replacement of the hydroxyl
group with an amino group has no significant effect on
enzymatic inhibition or antiviral activity.
An overlay of the X-ray crystal structures of 3, 50,
and CI-1029 bound to HIV protease is shown in Figure
4. This figure highlights the similarity in the binding
of the closely related substituted dihydropyranone core
for 3 and CI-1029. A variation in binding is observed at
the P2 site, where the phenol group in 3 binds through
a water molecule to Asp30, while the aniline group in
CI-1029 has a direct contact with the Asp30 side chain.
III. Su lfon ylu r ea s a n d Su lfa m a tes. A logical ex-
tension of our SAR is the replacement of the alkyl or

852 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
Ta ble 6. Enantiomerically Resolved Dihydropyran-2-onesa and Associated Biological Activities
entry
R1
R2
IC50 (Ki) (nM)
EC50 (µM)
TC50 (µM)
TI
1
1S
1R
12
OH
OH
OH
OH
OH
OH
H
H
H
H
H
NH2
NH2
NH2
2.7 (0.67)
(0.07)
(13.0)
1.0
0.49
91
4.3
1.8
18.0
0.5
0.22
0.6
0.45
5.9
0.6
92
215
211
68
66
66
66
31
66
65
92
439
2
16
37
NHSO2[2-(5-CF3)pyridyl]
NHSO2[2-(5-CF3)pyridyl]
NHSO2[2-(5-CF3)pyridyl]
NHSO2Ph(4-CN)
12S
12R
29
29S
34
34S
34R
36
36S
69S
(0.39)
(9.54)
9.1
(0.20)
(1.04)
(0.31)
(6.40)
2.2
4
132
141
100
144
11
>175
151
23
NHSO2Ph(4-CN)
NHSO2[2-(5-CF3)pyridyl]
NHSO2[2-(5-CF3)pyridyl]
NHSO2[2-(5-CF3)pyridyl]
OSO2CH3
OSO2CH3
OSO2Ph(4-CN)
66
OH
OH
H
>100
(0.03)
(0.70)
0.53
1.1
80
25
a
The ratio of enantiomers is g95:5.
F igu r e 4. Overlay of the X-ray crystal structures of 3 (blue), 50 (red), and CI-1029 (white) bound to HIV protease. The enzyme
sites are labeled in yellow.
In 3, one oxygen of the sulfonamide group forms a
hydrogen bond with Asp129(NH). This interaction is
analogous to the hydrogen bond between the methyl-
enehydroxy group in CI-1029 and Asp129(NH).
chain then rotates from a -gauche orientation as
observed in both the 3 and CI-1029 structures to
+gauche in accommodating the binding of the methyl
substituent (Figure 5).
The benzenesulfonamido group in 3 occupies an
additional site of the enzyme, the S3′ pocket, with the
phenyl ring stacking against the Arg8/Asp129 ionic pair.
However, this site of interaction does not increase the
binding affinity of 3 relative to that of CI-1029. Any gain
in enthalpy of binding is at least partially counterbal-
anced by the unfavorable entropic effect due to the
addition of flexibility to the molecule.
Compound 50 has a cyclohexyl group at the P1 site
in place of the isopropyl moiety found in both 3 and CI-
1029. Although the overall binding mode of the substi-
tuted core remains the same, a shift in orientation is
observed (Figure 4). The bulky size of the cyclohexyl
group may prevent the inhibitor from probing as deeply
into the S1 pocket, coming much closer to the flap region
of the enzyme than the analogues with the isopropyl
substituent at P1. This results in the methyl-substituted
S-phenyl ring at C-3 being drawn further into the
binding cavity. Interestingly, the proximal Ile184 side
At the P2 site, the phenol group in 50 interacts
directly with Asp30 as is observed with the CI-1029
aniline. In the prime side, 50 contains a N-methylimi-
dazolesulfonate at the 4-position of the C-3 S-phenyl
ring. The imidazole sits lower in the S3′ pocket than does
the phenyl group of the benzenesulfonamido moiety in
3. The orientation of the imidazole ring in the pocket
eliminates any possibility of π system interactions with
the side chain residues as is seen with compound 3. The
position of the imidazole ring does however allow one
of the ring nitrogen atoms to form a hydrogen bond with
Asp129(NH). The observed shift in binding also results
in the P1′ tert-butyl group orienting lower in the S1′
region. The overall alteration in binding appears to
result in nonoptimal occupancy of subsites and could
contribute to the 4-fold drop in potency for 50 relative
to 3. It also illustrates, however, that while optimal
binding may not be realized, inhibitors can still retain
potent activity versus the enzyme. This characteristic

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 853
F igu r e 5. Overlay of the X-ray crystal structures of 3 (yellow) and 50 (red) bound to HIV protease. The inhibitors and Ile184 are
shown with capped sticks representation.
Ta ble 7. Pharmacokinetic Parameters of Selected
Dihydropyran-2-one Analogues in Micea
Cmax
AUC
Cmax
AUC
entry vehicleb (µM) (µg‚h/mL) entry vehicleb (µM) (µg‚h/mL)
1
B
B
B
B
B
B
B
B
21.0
21.7
1.27
2.00
10.3
1.14
0.54
9.2
44.82
46.31
0.98
ND
ND
ND
36S
43
60
68
69
70
71
B
B
B
A
B
A
B
6.54
0.63
0.22
0.62
1.60
0.50
0.93
5.4
1S
12
29
29S
31
34
34S
ND
ND
ND
ND
ND
1.58
0.56
22.0
F igu r e 6. PNU-140690 illustrating the five enzymatic pockets
binding mode.
a
b
Dose: 25 mg/kg. A: 20% 0.1 N NaOH/80% PEG 400. B: 20%
0.1 N NaOH/80% 0.5% methylcellulose. ND, not determined.
of this series of inhibitors may prove extremely advan-
tageous in combating mutant proteases.
tors, a finding that would not have been apparent
without X-ray crystallography studies.23 In both PNU-
140690 and the current series of inhibitors, the sulfonyl
moiety on the C-3 phenyl ring substituent of the 5,6-
dihydropyran-2-one system is seen to occupy the S3′
pocket of the enzyme as anticipated.
Com p a r ison of In h ibitor s 3 a n d 50 to P NU-
140690. The structurally similar 5,6-dihydropyran-2-
one HIV protease inhibitor PNU-140690 was recently
identified by Pharmacia and Upjohn as a clinical can-
didate.16,22 PNU-140690 in X-ray crystallographic stud-
ies has been shown to occupy five pockets of HIV
protease (Figure 6). PNU-140690 and the current series
of inhibitors share the 5,6-dihydropyran-2-one core
template and display similar core binding interactions.
PNU-140690 and inhibitors 3 and 50 interact through
hydrogen bonds between the 4-hydroxyl group and the
catalytic amino acids Asp25 and Asp125. Also similar
to inhibitors 3 and 50, PNU-140690 forms hydrogen
bond interactions with NH groups of the flap region
Ile50 and Ile150 amino acid residues. However, differ-
ences arise as to the method by which PNU-140690 and
the inhibitors in this study fill the interior four enzy-
matic pockets that surround the catalytic site. Whereas
the C-6 phenethyl substituent of the 5,6-dihydropyran-
2-one ring system of PNU-140690 occupies the S1 pocket
of the enzyme, the 4-hydroxyphenethyl substituents of
inhibitors 3 and 50 are seen to fill the S2 pocket of the
enzyme. The C-6 n-propyl substituent of PNU-140690
occupies the S2 pocket of the target enzyme, in contrast
to inhibitors 3 and 50 in which the C-6 alkyl moieties
fill the S1 pocket in the X-ray crystal structures. These
results clearly illustrate that different enantiomers are
the more active enantiomer in the two series of inhibi-
P h a r m a cok in etic Stu d ies. Several of the 5,6-dihy-
dropyran-2-ones were dosed in mice to determine the
effects of the various substitutions on bioavailability
(Table 7). The mice were given a 25 mg/kg dose orally
by gavage. The vehicle used for dosing was in most cases
20% 0.1 N NaOH/80% 0.5% methylcellulose. In two
cases the vehicle used was 20% 0.1 N NaOH/80% PEG
400. The results of the mouse pharmacokinetic studies
illustrate that substitution on the 3-S-(2-tert-butyl-5-
methylphenyl) moiety with a sulfonyl-containing group
greatly reduces plasma levels. The most favorable
pharmacokinetic profile belongs to the unsubstituted
aniline, compound 1. Substitution on the aniline only
served to produce low Cmax levels and AUC values. In
many cases the plasma Cmax values were so low that
adequate AUC values could not be determined. Inter-
estingly, in the two cases where the racemic compounds
(29 and 34) and the resolved (S)-enantiomers (29S and
34S) of the sulfonyl compound were dosed, the (S)-
enantiomer displayed significantly higher Cmax levels
(5- and 17-fold increases in Cmax values, respectively).
The increase in the level of Cmax for the (S)-enantiomer
compared to racemic analogue is not a constant phe-

854 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
Ta ble 8. Pharmacokinetic Parameters of Selected
at 100 µM drug concentration, particularly against
CYP2C9. Compound 39, which possesses a p-cyanophen-
ylsulfonate moiety, displays notable inhibition at a
concentration as low as 1 µM. Essentially complete
inhibition of CYP3A4 and CYP2C9 occurs at 100 µM
inhibitor concentration. The inhibition potential of
compound 39 against CYP2D6 was not determined.
These results suggest that the free aniline may possess
less of a potential for drug interactions as a result of
metabolic enzyme inhibition. On the other hand, com-
pounds such as inhibitor 39, which possesses the larger
sulfonyl moiety, may have the potential for drug-drug
interactions, similar to marketed protease inhibitors.
Indinavir displays substantial inhibition of CYP3A4 at
drug concentration of 1 µM and almost complete inhibi-
tion at 10 µM concentration. Indinavir has an inhibition
profile similar to that of inhibitor 1S against CYP2C9
and CYP2D6 with some differences at 100 µM drug
concentrations.
Dihydropyran-2-ones in Ratsa
entry
Cmax (µM)
T1/2 (h)
AUC (µg‚h/mL)
29S
34S
36S
3.96
3.50
6.54
5.7
2.3
0.94
10.0
6.7
5.39
a
Dose: 10 mg/kg, NaOH solution buffered to pH 7.4.
nomenon as this trend is not observed in the unsubsti-
tuted aniline (compounds 1 and 1S).
The pharmacokinetic profiles of several compounds
were also examined in rats (Table 8). Rats were given
a 10 mg/kg dose orally with a NaOH solution buffered
to pH 7.4 as the vehicle. The results of this study were
similar to what was observed in mice: very low Cmax
and AUC values. Except for compound 36S, the T1/2
values were longer in the rat than in the mouse. The
reason the substituted derivatives possess lesser phar-
macokinetic parameters relative to the unsubstituted
aniline (compounds 1 and 1S) is unclear. The lower Cmax
and AUC values may be due to the higher molecular
weight or lower solubilities of these compounds. The
pharmacokinetics in rodents reported for PNU-140690
are similar to those for the compounds described here.
An t ivir a l Act ivit y in Clin ica l Isola t es. A major
obstacle in the effective treatment of AIDS is the
emergence of drug-resistance strains of HIV-1.24,25 To
assess their effectiveness against various strains, the
anti-HIV activities of selected inhibitors were deter-
mined against a series of HIV-1 strains obtained from
clinical isolates. The antiviral activity was measured in
an in vitro assay employing HIV-1-infected PBMC cells.
The antiviral activities of the inhibitors in this assay
are shown in Table 9. Generally, each of the inhibitors
displayed very good antiviral activity in the series of
clinical isolates tested. Except for one instance (com-
pound 40 against strain 1026-60), the antiviral activities
of compounds 1S, 29S, 34, and 40 against the selected
HIV-1 strains were in the submicromolar range. Anti-
HIV activities against the clinical isolates were within
2-fold of the associated antiviral activities of the inhibi-
tors in the CEM cell assay (Table 2). Except against the
protease inhibitor-resistant strain 144-44, inhibitor 29S
was the most antivirally potent against the clinically
isolated strains that were tested. Of the five inhibitors
tested in this assay, only compound 36 exhibited a
significant decrease in antiviral activity against the
clinical isolates as compared to CEM cells (approxi-
mately 2-3-fold less antiviral activity). All inhibitors
possess very good activity against HIV-1 strains 1002-
60, 1026-60, and 144-44 which exhibit resistance to the
marketed protease inhibitors Indinavir and Saquinavir.
Cytoch r om e P 450 In h ibition . One of the primary
problems encountered with the currently marketed
protease inhibitors is the occurrence of significant side
effects and numerous drug interactions.3,5b,7 These
impediments to effective therapy are in many instances
due to the inhibitory effects of the protease inhibitors
on the cytochrome P450 system, particularly the CYP3A4
isozyme.26,27 To assess their potential for metabolic
liabilities, compounds 1S and 39 were tested against
several cytochrome P450 isozymes, and the results are
shown in Table 10. The unsubstituted aniline analogue
(1S) does not inhibit any of the three isozymes tested
up to a concentration of 10 µM. Inhibition is observed
Con clu sion s
On the basis of previous SAR and insights from X-ray
crystallographic structures, a series of compounds was
synthesized that possessed various sulfonyl moieties at
the 4-position of the C-3 phenyl ring substituent of the
dihydropyran-2-one ring system. The sulfonyl substit-
uents were added in an attempt to fill the additional
S3′ pocket and thereby produce more potent inhibitors
of the target enzyme. The various sulfonyl moieties
synthesized included sulfonamides, sulfonates, sulfo-
nylureas, and sulfamates. Racemic and enantiomerically
resolved varieties of selected compounds were synthe-
sized with the (S)-enantiomer being the more potent
enantiomer. All analogues in the study displayed decent
binding affinity to HIV protease, with IC50 or Ki in the
double-digit nanomolar to subnanomolar range. Several
compounds from each series were shown to possess very
good antiviral activities, with EC50’s of 1 µM and TI’s
of >100. Generally, in the sulfonamide and sulfonylurea
series, compounds that did not possess a polar group
on the C-6 phenethyl moiety provided better anti-HIV
activities. In the sulfonate and sulfamate series, the
presence of the p-hydroxyl on the C-6 phenethyl group
was generally seen to enhance antiviral activity. X-ray
crystallographic structures of two inhibitors confirmed
that the sulfonamide and sulfonate moieties were filling
the S3′ pocket of the enzyme as anticipated. However,
the added sulfonyl substitution did not provide improved
enzymatic inhibitory or antiviral activity as compared
to the resolved unsubstituted aniline 1S. Inhibitor 1S,
which occupies only the four interior pockets of the
enzyme, displays an EC50 of 0.49 µM antiviral activity
and a TI of greater than 400. The addition of the
sulfonyl moiety substitution does not appear favorable
when examining pharamacokinetic parameters. Com-
pounds that possessed the sulfonyl substitution exhib-
ited lower plasma Cmax levels and half-lives in all species
tested as compared to the unsubstituted aniline deriva-
tive. Selected inhibitors exhibited similar antiviral
activity against all of the HIV-1 clinical isolates tested,
including highly resistant strains, as they did against
the wild-type HIV-1. To assess their potential for
inhibition of the cytochrome P450 system, selected
compounds were tested against several cytochrome P450
isozymes. The unsubstituted aniline analogue (1S) does

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 855
Ta ble 9. Selected Dihydropyran-2-ones and Their Associated Antiviral Activitya against Clinically Isolated HIV-1 Strains
entry
HIV-1 strain
EC50 (µM)
TC50 (µM)
TI
entry
HIV-1 strain
EC50 (µM)
TC50 (µM)
TI
1S
WeJ o
RoJ o
0.25
0.20
0.22
0.59
0.74
0.31
0.24
0.11
0.12
0.12
0.10
ND
>100
>100
>100
>100
>100
>100
>100
55.6
>400.0
>500.0
>454.5
>169.5
>135.1
>322.6
>416.7
506
36
40
WeJ o
RoJ o
2.05
1.24
1.79
1.93
1.84
1.12
0.73
0.39
0.70
0.50
0.78
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
>48.8
>80.6
SLKA
TEKI
1002-60
1026-60
144-44
WeJ o
RoJ o
SLKA
TEKI
1002-60
1026-60
144-44
WeJ o
RoJ o
SLKA
TEKI
SLKA
TEKI
1002-60
1026-60
144-44
WeJ o
RoJ o
SLKA
TEKI
1002-60
1026-60
144-44
1002-60
1026-60
144-44
1002-60
1026-60
144-44
>55.9
>51.8
>54.3
>89.3
>137.0
>259.1
>143.3
>200.0
>128.7
29S
55.6
55.6
55.6
ND
ND
55.6
464
464
556
ND
1.42
0.54
>100
16.55
3.0
>100
>100
>100
>100
>100
>50
>70.32
>185.5
1
0.58
0.69
0.68
0.71
0.81
0.61
0.27
0.26
96
34
>100
>100
>100
>100
>100
>100
>100
>144.9
>147.1
>140.8
>123.5
>163.9
>370.4
>384.6
Indinavir
>6.0
>33.3
>3.4
Saquinavir
14.54
19.45
11.06
1002-60
1026-60
144-44
>50
>50
>2.6
>4.5
a
Antiviral activity measured in HIV-1-infected PBMC cells. ND, not determined.
Ta ble 10. Inhibition of Cytochrome P450 Activity by Selected Dihydropran-2-onesa
3A4
2C9
2D6
entry
1S
39
Indinavir
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
103.0
79.0
27.0
96.8
34.6
5.0
41.1
6.86
BLQ (0)
93.2
83.5
93.0
75.5
18.1
89.0
21.6
BLD (0)
68.0
105.0
ND
100.0
95.0
ND
82.0
56.7
ND
31.0
a
Percent cytochrome P450 activity remaining in the presence of HIV protease inhibitors. BLD, below levels of detection; ND, not
determined.
300 and 400 spectrometers and chemical shifts are reported
in δ units relative to internal standard, trimethylsilane. All
mass spectra were obtained on a Finnigan 4500GCMS or VG
analytical 7070E/F spectrometer. Elemental analyses were
performed by Robertson Microlit Laboratories, Inc. or Quan-
titative Technologies, Inc. and all compounds gave analytical
results of (0.4% of theorectical values. All starting materials
were obtained from commercial sources unless otherwise
specified in the experimental. Flash chromatography was
performed using silica gel 60, 230-400 mesh, purchased from
Mallinckrodt.
5-Tr iflu or om eth yl-2-p yr id in esu lfon yl Ch lor id e. 2-Mer-
capto-5-(trifluoromethyl)pyridine (5.0 g, 27.91 mmol), CCl4 (100
mL) and H2O (20 mL) were cooled to 0 °C and Cl2 gas bubbled
through via pipet with stirring for 1 h. CH2Cl2 was added and
the organic phase separated and washed with brine, dried over
Na2SO4, filtered and concentrated to produce the title com-
pound as a clear oil which solidified to a white solid upon
storage in a freezer. The isolated product was used without
further purification. Isolated yield: 5.9 g (86%). 1H NMR
(CDCl3): δ 8.26 (d, 1H), 8.33 (m, 1H), 9.08 (m, 1H).
2-P yr id in esu lfon yl Ch lor id e. 2-Mercaptopyridine (5.0 g,
27.91 mmol), CCl4 (100 mL) and H2O (20 mL) were cooled to
0 °C and Cl2 gas bubbled through via pipet with stirring for 1
h. CH2Cl2 was added and the organic phase separated and
washed with brine, dried over Na2SO4, filtered and concen-
trated to produce the title compound as a tinted oil which
solidified upon storage in a freezer. The isolated product was
used without further purification. Isolated yield: 5.0 g (63%).
1H NMR (CDCl3): δ 7.70 (m, 1H), 8.03-8.11 (m, 2H), 8.84 (m,
1H).
5-ter t-Bu tyl-2-m eth ylp h en yla m in e. A solution of 4-tert-
butyl-1-methyl-2-nitrobenzene17(VI) in MeOH (1.0 L) was
treated with Raney nickel (25 g) in a Parr shaker. The
apparatus was sealed under hydrogen pressure to 52 psi,
heated at 35 °C for 15 h. The apparatus was then cooled to
room temperature, vented and the contents filtered. The
filtrate was concentrated in vacuo and distilled, collecting the
major fraction (75-85 °C, 1.0 Torr) to afford the title com-
not inhibit any of the three isozymes tested up to a
concentration of 10 µM. In particular, inhibitor 1S does
not display substantial inhibition of the CYP3A4 isozyme
activity up to concentrations of 100 µM.
Exp er im en ta l Section
En zym e In h ibition Assa y. For determination of IC50
values, affinity-purified HIV-1 protease (Bachem Bioscience;
1.1 nM) was added to a solution of the following: the inhibitor,
40 µM peptide substrate (His-Lys-Ala-Arg-Val-Leu-(p-NO2-
Phe)-Glu-Ala-Nle-Ser-NH2; Bachem Bioscience), and 1.0%
DMSO in assay buffer (1.0 mM dithiothreitol, 80 mM MES,
160 mM NaCl, 1.0 mM EDTA, 0.1% poly(ethylene glycol) (MW
8000), pH 6.2 at 25 °C). The final volume was 100 µL; final
concentration of HIV protease was 1.5 nM. The solution was
mixed, incubated for 60 min at 37 °C, and then quenched with
trifluoroacetic acid (2% final.) In this assay, the Leu-(p-NO2-
Phe) bond of the substrate was cleaved by the enzyme, and
the substrate and cleavage products were separated by reverse-
phase HPLC. Absorbance was measured at 220 nm, peak areas
were determined, and % conversion to product was used to
calculate % control ([% conversion(+inhibitor)/% conversion-
(-inhibitor)] × 100).
An ti-HIV Activity Assa y. Anti-HIV activity was deter-
mined in an in vitro cell culture assay employing HIV-IIIB-
infected human lymphocyte-derived CEM cells using the XTT
cytopathic protocol at Southern Research Institute.20 EC50
refers to the effective concentration at which 50% of the cells
are conferred protection against the cytopathic effects of HIV.
TC50 refers to the toxic concentration of the drug that elicits
cytotoxicity in 50% of CEM cells not infected with HIV.
Antiviral activity against clinical isolates was determined in
a similar assay employing HIV-1-infected PBMC cells.
Ch em ica l Syn th esis. Melting points were determined in
open capillary tubes on a Hoover melting point apparatus and
are uncorrected. Infrared (IR) spectra were determined in KBr
pellets on a Nicolet FT IR SX-20 spectrophotometer. Proton
magnetic resonance (NMR) spectra were recorded on Varian

856 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Boyer et al.
pound. 1H NMR (CDCl3): δ 1.26 (s, 9 H), 2.11 (s, 3 H), 3.54
NMR (DMSO-d6): δ 1.16 (s, 9 H), 1.96 (s, 3 H), 2.38 (s, 3 H),
5.88 (br s, 3 H), 6.83 (s, 1 H), 6.95 (s, 1 H), 7.39 (d, 2 H), 7.45
(d, 2 H).
(br s, 3 H), 6.69 (d, 1 H), 6.72 (dd, 1 H), 6.96 (d, 1 H).
5-ter t-Bu tyl-2-m eth yl-4-th iocya n a top h en yla m in e (V).
A solution of 5-tert-butyl-2-methylphenylamine (1.0 g, 6.1
mmol), sodium thiocyanide (1.62 g, 20.0 mmol) and MeOH (4.0
mL) was cooled to 0 °C and treated via addition funnel with a
solution of bromine (0.35 mL, 6.7 mmol), sodium bromide (0.63
g, 6.13 mmol), and MeOH (5.0 mL). The mixture was stirred
for 30 min and then carefully diluted with saturated NaHCO3
solution. The mixture was extracted twice with CH2Cl2, the
organic phases combined, dried (Na2SO4) and the solvent
evaporated. The resulting residue was subjected to flash silica
gel chromatography, eluting with 4:1 CH2Cl2:hexanes to 19:1
CH2Cl2:hexanes + 1% MeOH to provide the title compound.
1H NMR (CDCl3): δ 1.42 (s, 9 H), 2.09 (s, 3 H), 3.80 (bs, 2 H),
6.69 (s, 1 H), 6.89,7.35 (s, 1 H).
5-ter t-Bu tyl-2-m eth ylp h en ol. 5-tert-Butyl-2-methylphen-
ylamine (3.0 g, 18.4 mmol) was taken in 15% aqueous H2SO4
(110 mL) and heated to 70 °C. Sodium nitrite (1.41 g, 20.4
mmol) in H2O (20 mL) was added dropwise the reaction
mixture and mixture stirred an additional 30 min following
complete addition. The reaction mixture was allowed to cool
and extracted with EtOAc. The organic phase was washed with
H2O, followed by brine, dried over MgSO4, filtered and the
solvent removed in vacuo to afforded the title compound which
was utilized without further purification. Isolated yield: 2.85
g (94%). 1H NMR (CDCl3): δ 1.45 (s, 9H), 2.22 (s, 3H), 5.30
(br s, 1H), 7.05 (s, 1H), 7.11 (d, 1H), 7.28 (d, 1H).
5-ter t-Bu tyl-2-m eth yl-4-th iocya n a top h en ol (IX). A solu-
tion of 5-tert-butyl-2-methylphenol (350 g, 2.13 mol) and
sodium thiocyanate (555 g, 6.85 mol) in MeOH (1400 mL) was
cooled to 5 °C. A solution of sodium bromide (214 g, 2.08 mol),
bromine (126 mL, 2.38 mol), and MeOH (1800 mL) was added
slowly and the temperature raised to 40 °C over a period of
30 min. The precipitate was filtered and the filtrate concen-
trated by half. Saturated sodium carbonate (3.5 L) and H2O
(4.5 L) were added. The mixture was extracted several times
with EtOAc (total 3 L), and the organic layers were combined
and concentrated. To the resulting residue were added tert-
butylmethyl ether (500 mL) and hexanes (500 mL) and the
mixture was allowed to stand for 17 h at -5 °C. The resulting
precipitate was filtered to give the title compound. Mp: 99-
[4-(Cya n oth io)-5-(1,1-d im eth yleth yl)-2-m eth ylp h en yl]-
im id od ica r bon ic Acid Bis(1,1-d im eth yleth yl) Ester . A
solution of 5-tert-butyl-2-methyl-4-thiocyanatophenylamine
(8.0 g, 36.36 mmol) and di-tert-butyl dicarbonate (31.7 g, 145.3
mmol) in THF (80 mL) was stirred at 60 °C under N2 for 72 h.
Solvent was then removed in vacuo and Et2O added; the
solution was washed with saturated NaHCO3 solution, fol-
lowed by brine, dried (Na2SO4), filtered and concentrated. The
material was purified by flash chromatography on silica gel,
eluting with 4:1 hexanes:EtOAc to 1:1 hexanes:EtOAc to afford
the title compound. Isolated yield: 14.0 g (91%). 1H NMR
(CDCl3): δ 1.41 (s, 9 H), 1.43 (s, 3 H), 1.52 (s, 18 H), 7.14 (s,
1 H), 7.58 (s, 1 H).
1
102 °C. H NMR (CDCl3): δ 1.46 (s, 9H), 2.22 (s, 3H), 5.01 (s,
1H), 6.89 (s, 1H), 7.49 (s, 1H).
[5-(1,1-Dim eth yleth yl)-4-m er ca p to-2-m eth ylp h en yl]im -
id od ica r bon ic Acid Bis(1,1-d im eth yleth yl) Ester (VI). To
a solution of [4-(cyanothio)-5-(1,1-dimethylethyl)-2-methyl-
phenyl]imidodicarbonic acid bis(1,1-dimethylethyl) ester (11.65
g, 27.7 mmol) in denatured EtOH (160 mL) were added
dithiothreitol (16.8 g, 109.1 mmol) and 0.2 M KH2PO4 solution
(40 mL) and the mixture was stirred overnight at 50 °C. The
solvent was evaporated, H2O was added and extracted with
CHCl3, the organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated. The resulting residue was
subjected to flash silica gel chromatography, eluting with 1:1
CHCl3:hexanes switching to 19:1 CHCl3:EtOAc, to afford the
title compound. Isolated yield: 10.95 g (quantitative yield).
1H NMR (CDCl3): δ 1.40-1.46 (m, 27 H), 2.07 (s, 3 H), 7.00
(s, 1 H), 7.05 (s, 1 H).
5-ter t-Bu tyl-4-m er ca p to-2-m eth ylp h en ol. To a round-
bottom flask (500 mL) equipped with magnetic stir and
condenser were added 5-tert-butyl-2-methyl-4-thiocyanatophe-
nol (7.35 g, 158 mmol), dithiothreitol (25.61 g, 166 mmol),
EtOH (250 mL), and 0.02 M KH2PO4 buffer solution (25 mL)
and the reaction was heated to reflux. In 2 h the reaction was
not complete by TLC, additional dithiothreitol (5.0 g 32.4
mmol) and 0.02 M KH2PO4 buffer solution (25 mL) was added
and the reaction mixture refluxed an additional 2 h. The EtOH
was removed under reduced pressure and the reaction quenched
with brine and extracted with Et2O:hexanes (1:1). The organic
layer was washed with brine (3×), dried over MgSO4 and the
solvent removed in vacuo. The resulting residue submitted to
flash silica gel column chromatography, eluting with CHCl3
1
to afford the title compound. Isolated yield: 24.3 g (79%). H
[5-(1,1-Dim eth yleth yl)-2-m eth yl-4-[[(4-m eth ylp h en yl)-
su lfon yl]t h io]p h en yl]im id od ica r b on ic Acid Bis(1,1-d i-
m eth yleth yl) Ester . To a solution of tosyl bromide (17.1 g,
72.73 mmol) and NEt3 (7.36 g, 72.73 mmol) in CCl4 (250 mL)
at 0 °C was added a solution of [5-(1,1-dimethylethyl)-4-
mercapto-2-methylphenyl]imidodicarbonic acid bis(1,1-di-
methylethyl) ester (10.7 g, 27.05 mmol) in CCl4 (250 mL)
dropwise over an 8 h period. The mixture was then allowed to
warm to room temperature overnight. Chloroform was added,
and the solution was washed with H2O, followed by brine, dried
over Na2SO4, filtered and concentrated. The resulting material
was submitted to flash silica gel chromatography, eluting with
4:1 CHCl3:hexanes to afford the title compound. Isolated
NMR (CDCl3): δ 1.45 (s, 9H), 2.15 (s, 3H), 3.43 (s, 1H), 4.56
(br s, 1H), 6.82 (s, 1H), 7.05 (s, 1H).
Tolu en e-4-th iosu lfon ic Acid S-(2-ter t-Bu tyl-4-h yd r oxy-
5-m eth ylp h en yl) Ester (X). To a solution of tosyl bromide
(30.55 g, 130 mmol) and pyridine (10.51 mL, 130 mmol) in CCl4
(50 mL) cooled to 0 °C was added a solution of 5-tert-butyl-4-
mercapto-2-methylphenol (24.3 g, 123.8 mmol) in CCl4 (150
mL) dropwise over a 30 min period. Chloroform was then
added, and the solution was washed with H2O, followed by
brine, dried over MgSO4, filtered and concentrated. The
resulting solid was washed with hexanes:Et2O (4:1) and
filtered to present the title compound. Isolated yield: 40.0 g
1
(91%). H NMR (CDCl3): δ 1.22 (s, 9H), 2.11 (s, 3H), 2.42 (s,
1
yield: 11.5 g (77%). H NMR (CDCl3): δ 1.16 (s, 9 H), 1.40 (s,
18 H), 2.10 (s, 3 H), 2.40 (s, 3 H), 7.07 (s, 1 H), 7.19 (d, 2 H),
7.40 (s, 1 H), 7.44 (d, 2 H).
3H), 6.87 (s, 1H), 7.16 (s, 1H), 7.26 (d, 2H), 7.49 (d, 2H).
Syn th esis of th e Th iotosyla tes. Gen er a l Meth od A: To
a solution of toluene-4-thiosulfonic acid S-(4-amino-2-tert-
butyl-5-methylphenyl) ester in neat pyridine or a pyridine/CH2-
Cl2 mixture was added the corresponding chloro derivative
(1.2-1.5 equiv) and the mixture stirred under nitrogen at room
temperature overnight. Generally, 1 N HCl was then added
and the mixture extracted with EtOAc. The organic phase was
again washed with 1 N HCl, then washed with brine:H2O
(1:1), followed by twice with brine, dried over MgSO4 or Na2-
SO4, filtered and concentrated. The isolated material was
generally purified by flash silica gel chromatography unless
otherwise specified.
Tolu en e-4-th iosu lfon ic Acid S-(4-Am in o-2-ter t-bu tyl-
5-m eth ylp h en yl) Ester (VII). [5-(1,1-Dimethylethyl)-2-meth-
yl-4-[[(4-methylphenyl)sulfonyl]thio]phenyl]imidodicarbonic acid
bis(1,1-dimethylethyl) ester (10.95 g, 19.92 mmol) was dis-
solved in CH2Cl2 (200 mL) and HCl gas bubbled in while
stirring at room temperature for 40 min. The solvent was
evaporated, and the isolated residue was triturated with Et2O
to afford the title compound as the hydrochloride salt. Isolated
yield: 7.0 g (91%). The free base was liberated by dissolving
the isolated solid in MeOH, and pH 7.5 K2HPO4/KH2PO4 buffer
was added until a precipitate formed. The resulting precipitate
Tolu en e-4-t h iosu lfon ic Acid S-(2-ter t-Bu t yl-4-m et h -
a n esu lfon yla m in o-5-m eth ylp h en yl) Ester (VIIIa ). The
1
was filtered off and washed with H2O and then hexanes. H

Potent Nonpeptidic Inhibitors of HIV Protease
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 857
title compound was prepared according to general method A
using toluene-4-thiosulfonic acid S-(4-amino-2-tert-butyl-5-
methylphenyl) ester (0.30 g, 0.86 mmol) and methanesulfonyl
chloride (0.15 g, 1.29 mmol) in pyridine (8 mL) and CH2Cl2 (1
mL). The isolated material was triturated with EtOAc and
hexanes to afford the title compound. Isolated yield: 0.28 g
allowed to stir at room temperature overnight. CH2Cl2 was
then added, and the solution was washed with H2O, followed
by brine, dried over MgSO4, filtered and concentrated. The
resulting residue taken up in a small amount of EtOAc and
hexanes added to present a solid which was filtered and dried
under high vacuum to afford the title compound. Isolated
1
1
(76%). H NMR (DMSO-d6): δ 1.18 (s, 9H), 2.14 (s, 3H), 2.38
yield: 1.09 g (68%). H NMR (CDCl3): δ 1.15 (s, 9H), 2.23 (s,
(s, 3H), 3.02 (s, 3H), 7.09 (s, 1H), 7.39 (m, 3H), 7.47 (d, 2H),
9.29 (br s, 1H).
3H), 2.37 (s, 3H), 3.02 (s, 6H), 7.19 (d, 2H), 7.22 (s, 1H) 7.33
(s, 1H), 7.40 (d, 2H).
Syn th esis of Ta r get 4-Hyd r oxy-5,6-d ih yd r op yr a n -2-
on es. Gen er a l Meth od C: The appropriate dihydropyrone (1
equiv) in DMF (1-12 mL per mmol of dihydropyrone) was
treated with K2CO3 (2.2-8 equiv) followed by the appropriate
thiotosylate reagent (1.0-1.2 equiv). The reaction was stirred
at room temperature (2.5 h to overnight). The reaction was
then taken in EtOAc and treated with either 1 N HCl or
saturated aqueous NH4Cl. The layers were separated and the
aqueous layer was extracted with EtOAc. The combined
organic extracts were washed with brine:H2O (1:1), then with
brine, dried over MgSO4, filtered and concentrated.
3-(4-Am in o-2-ter t-bu tyl-5-m eth ylp h en ylsu lfa n yl)-4-h y-
d r oxy-6-[2-(4-h yd r oxyp h en yl)eth yl]-6-isop r op yl-5,6-d ih y-
d r op yr a n -2-on e (1). The title compound was prepared ac-
cording to general method C using 4-hydroxy-6-[2-(4-hy-
droxyphenyl)ethyl]-6-isopropyl-5,6-dihydropyran-2-one (0.60 g,
2.17 mmol), toluene-4-thiosulfonic acid S-(4-amino-2-tert-butyl-
5-methylphenyl) ester (0.84 g, 2.40 mmol), K2CO3 (0.90 g, 6.51
mmol) and DMF (12 mL). The isolated material was subjected
to silica gel flash chromatography, eluting with CHCl3:MeOH
(9:1) to afford the pure product. Isolated yield: 0.57 g (56%).
1H NMR (DMSO-d6): δ 0.89 (d, 3H), 0.93 (d, 3H), 1.42 (s, 9H),
1.71 (s, 3H), 1.88-1.92 (m, 2H), 2.15 (m, 1H), 2.46-2.51 (m,
partially obscured by DMSO, 2H), 2.67 (d of ABX q, 1H), 2.90
(d of ABX q, 1H), 6.55 (s, 1H), 6.61 (s, 1H), 6.64 (d, 2H), 6.93
(d, 2H), 9.13 (br s, 1H). IR (KBr): 3382, 2963, 2933, 2877, 1669,
1613, 1515, 1481, 1452, 1388, 1375, 1268, 1239, 1050, 911, 833,
763, 698 cm-1. MS-APCI (m/z+): 470.2. Anal. (C27H35N104S2‚
0.83H2O) C, H, N.
General method A was also used to synthesis intermediates
VIIIb-o (see Table 1).
Gen er a l Meth od B: To a solution of toluene-4-thiosulfonic
acid S-(2-tert-butyl-4-hydroxy-5-methyl-phenyl) ester in CH2-
Cl2 or Et2O was added triethylamine (1.0-3.0 equiv) followed
by the corresponding chloro derivative (1.0-2.0 equiv) neat
or dissolved in CH2Cl2 or Et2O and the mixture stirred under
nitrogen at room temperature from 10 min to overnight,
monitoring the reaction by thin-layer chromatography. Gener-
ally, 1 N HCl was then added and the mixture extracted with
EtOAc. The organic phase was washed with brine, dried over
MgSO4 or Na2SO4, filtered and concentrated. The isolated
material was generally purified by flash silica gel chromatog-
raphy unless otherwise specified.
Meth an esu lfon ic Acid 5-ter t-Bu tyl-2-m eth yl-4-(tolu en e-
4-su lfon ylsu lfa n yl)p h en yl Ester (VIIIp ). The title com-
pound was prepared according to general method B using
toluene-4-thiosulfonic acid S-(2-tert-butyl-4-hydroxy-5-meth-
ylphenyl) ester (1.50 g, 4.30 mmol), methanesulfonyl chloride
(0.49 g, 4.30 mmol) and triethylamine (0.43 g, 4.30 mmol) in
CH2Cl2 (30 mL). The isolated material was submitted to flash
silica gel chromatography, eluting with CHCl3 to afford the
1
title compound. Isolated yield: 1.2 g (65%). H NMR (DMSO-
d6): δ 1.18 (s, 9H), 2.14 (s, 3H), 2.38 (s, 3H), 3.02 (s, 3H), 7.09
(s, 1H), 7.39 (m, 3H), 7.47 (d, 2H), 9.29 (br s, 1H).
General method B was also used to synthesis intermediates
VIIIq-x (see Table 1).
Dim eth ylsu lfa m ic Acid 5-ter t-Bu tyl-2-m eth yl-4-th io-
cya n a top h en yl Ester (XI). To a round-bottom flask (250 mL)
equipped with condenser, magnetic stir, and nitrogen purge
were charged 5-tert-butyl-2-methyl-4-thiocyanatophenol (1.5
g, 6.80 mmol), cesium carbonate (2.44 g, 7.49 mmol), and
acetonitrile (30 mL) and the slurry was brought to reflux for
15 min. Dimethylsulfamoyl chloride (0.80 mL, 7.49 mmol) was
then added via syringe. A precipitate formed immediately and
the reaction was monitored by TLC until complete. The
reaction was quenched with brine and extracted with EtOAc.
The organic layer was dried over MgSO4 and the solvent
removed under reduced pressure. The resulting residue was
submitted to flash silica gel column chromatography, eluting
with hexanes:EtOAc (7:3) to afford the title compound. Isolated
General method C was also used to synthesize final com-
pounds 1S,R-69S. Further experimental details are given in
Table 2.
(S )-N -(5-t er t -B u t y l-4-{4-h y d r o x y -6-[2-(4-h y d r o x y -
p h en yl)eth yl]-6-isop r op yl-2-oxo-5,6-d ih yd r o-2H-p yr a n -3-
ylsu lfa n yl}-2-m e t h ylp h e n yl)-2-(5-t r iflu or om e t h yl)p y-
r id in esu lfon a m id e (12S). To (S)-3-(4-amino-2-tert-butyl-5-
methylphenylsulfanyl)-4-hydroxy-6-[2-(4-hydroxyphenyl)ethyl]-
6-isopropyl-5,6-dihydropyran-2-one (1S) (0.22 g, 0.47 mmol) in
pyridine (5 mL) and CH2Cl2 (2 mL) was added 5-trifluorom-
ethyl-2-pyridinesulfonyl chloride (0.17 g, 0.70 mmol) and the
reaction mixture stirred at room temperature overnight. 1 N
HCl was then added and the mixture extracted with EtOAc,
the organic phase was again washed with 1 N HCl, then
washed with brine:H2O (1:1), followed by twice with brine,
dried over MgSO4, filtered and concentrated. The resulting
residue was subjected to silica gel flash chromatography,
eluting with CH2Cl2:MeOH (18:1) to afford the title compound.
1
yield: 1.1 g (49%). H NMR (CDCl3): δ 1.46 (s, 9H), 2.22 (s,
3H), 3.03 (s, 6H), 7.30 (s, 1H), 7.47 (s, 1H).
Dim eth ylsu lfam ic Acid 5-ter t-Bu tyl-4-m er capto-2-m eth -
ylp h en yl Ester (XII). Dimethylsulfamic acid 5-tert-butyl-2-
methyl-4-thiocyanatophenyl ester (1.1 g, 3.3 mmol) dissolved
in EtOH (40 mL) and 0.02 M KH2PO4 buffer solution (5 mL)
was treated with dithiothreitol (1.29 g, 8.35 mmol). The
reaction was heated to reflux and allowed to stir overnight.
The reaction was then quenched with H2O (250 mL) and
extracted with a 1:1 mixture of hexanes:Et2O. The organic
layer was again washed with H2O (250 mL), followed by brine
(250 mL). The organic layer was dried over MgSO4, filtered
and concentrated. The resulting residue was dried under high
vacuum to present the title compound. Isolated yield: 1.07 g
1
Isolated yield: 0.13 g (38%). H NMR (DMSO-d6): δ 0.92 (d,
3H), 0.95 (d, 3H), 1.23 (s, 9H), 1.89-1.97 (m, 5H), 2.19 (m,
1H), 2.48-2.52 (m, partially obscured by DMSO, 2H), 2.73 (d
of ABX q, 1H), 2.95 (d of ABX q, 1H), 6.50 (s, 1H), 6.65-6.69
(m, 3H), 6.96 (d, 2H), 7.99 (d, 1H), 8.49 (d, 1H), 9.17 (s, 1H),
9.28 (s, 1H), 9.97 (s, 1H). IR (KBr): 3430, 3411, 2966, 2929,
1679, 1611, 1597, 1515, 1387, 1368, 1328, 1262, 1235, 1177,
1147, 1110, 1074, 911, 720, 697, 622, 547 cm-1. MS-APCI (m/
z+): 679.2, 373.0. Anal. (C33H37F3N206S2‚0.53H2O) C, H, N.
1
(quantitative). H NMR (CDCl3): δ 1.45 (s, 9H), 2.25 (s, 3H),
Su p p or tin g In for m a tion Ava ila ble: Additional experi-
mental details and elemental analyses. This material is
available free of charge via the Internet at http://pubs.acs.org.
3.02 (s, 6H), 3.57 (s, 1H), 7.01 (s, 1H), 7.28 (s, 1H).
Tolu en e-4-th iosu lfon ic Acid S-(2-ter t-Bu tyl-4-d im eth -
ylsu lfa m oyloxy-5-m eth ylp h en yl) Ester (VIIIz). To a solu-
tion of tosyl bromide (1.64 g, 7.0 mmol) and triethylamine (0.98
mL, 7.0 mmol) in CCl4 (25 mL) cooled to 0 °C in an ice water
bath was added a solution of dimethylsulfamic acid 5-tert-
butyl-4-mercapto-2-methylphenyl ester (1.07 g. 3.5 mmol) in
CCl4 (25 mL) dropwise over a 1 h period. After addition was
complete, the ice bath was removed and the reaction was
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