Viracept (nelfinavir mesylate, AG1343): A potent, orally bioavailable inhibitor of HIV-1 protease

Article abstract of DOI:10.1021/jm9704098

Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor

Full text of DOI:10.1021/jm9704098

J . Med. Chem. 1997, 40, 3979-3985
3979
Vir a cep t (Nelfin a vir Mesyla te, AG1343): A P oten t, Or a lly Bioa va ila ble In h ibitor
of HIV-1 P r otea se
Stephen W. Kaldor,† Vincent J . Kalish,‡ J ay F. Davies, II,‡ Bhasker V. Shetty,‡ J ames E. Fritz,†
Krzysztof Appelt,‡ J effrey A. Burgess,† Kristina M. Campanale,† Nickolay Y. Chirgadze,† David K. Clawson,†
Bruce A. Dressman,† Steven D. Hatch,† Deborah A. Khalil,‡ Maha B. Kosa,‡ Penny P. Lubbehusen,†
Mark A. Muesing,† Amy K. Patick,‡ Siegfried H. Reich,*,‡ Kenneth S. Su,† and J ohn H. Tatlock‡
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121, and Lilly Research Laboratories,
Eli Lilly and Company, Indianapolis, Indiana 46285
Received J une 20, 1997X
Using a combination of iterative structure-based design and an analysis of oral pharmacoki-
netics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor
of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki ) 2 nM) and
antiviral agent (HIV-1 ED50 ) 14 nM). An X-ray cocrystal structure of the enzyme-AG1343
complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor
interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate
that AG1343 is well absorbed orally in a variety of species and possesses favorable pharma-
cokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing
for the treatment of AIDS.
In tr od u ction
The human immunodeficiency virus protease (HIVPr)
has emerged as a promising therapeutic target for the
treatment of AIDS due to the essential role this enzyme
plays in virus maturation. Inhibitors of HIVPr are
presently being used in therapy for the treatment of
AIDS.1 The best of these inhibitors combine potent
antiviral activity with good pharmacokinetics.2 Inhibi-
tor development has been facilitated by the use of
F igu r e 1. Structures of the lead HIV-1 protease inhibitor 1
HIVPr-ligand cocrystal structure information.2-4 We
and the optimized drug Viracept (nelfinavir mesylate, AG1343).
have previously described the application of protein
crystallography to the design and optimization of novel
nonpeptidic HIVPr inhibitors.4 In the course of this
structure-based work, inhibitor 1 was discovered (Figure
1). Compound 1, containing a novel 2-methyl-3-hy-
droxybenzamide group, was found to be a tight binding
inhibitor of HIVPr (Figure 1).4 While compound 1 was
considered interesting by virtue of its small nonpeptide
P2 group, it was not viewed as a viable drug candidate
due to its suboptimal antiviral activity (ED50 ) 970 nM)
and poor aqueous solubility. However, its relatively low
molecular weight (556 Da) and nonpeptidic structure
prompted further structure-activity studies in this
series. Using the crystal structure of compound 1 in
complex with HIVPr and other structural information
as a guide, we identified a compound with potent in vitro
antiviral activity and superior pharmacokinetics in a
variety of species.4,5 We report herein the identification
of 11a (Viracept, nelfinavir mesylate, AG1343), an orally
bioavailable inhibitor of HIVPr which is now being
marketed for the treatment of AIDS.6
the desired sodium aryl thiolate as previously de-
scribed.4c,7 Protected amino acids 2a -c were converted
to the chloromethyl ketones 4a -c by treatment of the
respective diazoketone intermediates with hydrochloric
acid. Sodium borohydride reduction of the ketone
yielded the corresponding chloromethyl alcohols as a
mixture of diastereomers. The desired products were
obtained as the more polar isomers by radial chroma-
tography. Base-induced cyclization of the intermediate
chloro alcohols produced the corresponding amino ep-
oxides 6a -c. Terminal opening of the epoxides with the
known secondary amine 78 afforded the N-Cbz-protected
amino alcohols. Treatment with HBr-acetic acid liber-
ated the primary amines 9a -c in high yield. 2-Methyl-
3-hydroxybenzoic acid was obtained by treatment of the
diazonium salt of 2-methyl-3-aminobenzoic acid with
aqueous sulfuric acid. Carbodiimide-mediated coupling
of amines 9a -c with the benzoic acid followed by
mesylate salt formation yielded inhibitors 11a -c.
Discu ssion
Ch em istr y
Initially, we focused on enhancing the aqueous solu-
bility and antiviral activity of 1. Superposition of the
enzyme bound conformations of 1 and Ro31-8959,2a
derived from their respective cocrystal structures, sug-
gested that the known tertiary amine-containing dipep-
tide isostere 9c8 could be combined with 2-methyl-3-
hydroxybenzoic acid to afford 11c. This resulted in a
slightly weaker enzyme inhibitor but markedly im-
proved antiviral agent when compared to compound 1
The syntheses of compounds 11a -c are summarized
in Scheme 1. The unnatural amino acids 2a and 2b
were prepared from N-Cbz-L-serine by formation of the
corresponding â-lactone followed by in situ opening with
* To whom correspondence should be addressed.
† Eli Lilly and Company.
‡ Agouron Pharmaceuticals, Inc.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00409-3 CCC: $14.00 © 1997 American Chemical Society

3980 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Notes
Sch em e 1a
a
Reaction conditions: (a) PPh3, DMAD, THF; Na-aryl thiolate, THF; (b) i-BuOCOCl, Et3N, THF; CH2N2; (c) HCl, Et2O; (d) NaBH4,
THF/H2O; (e) KOH, EtOH; (f) 7, EtOH (80 °C); (g) HBr, AcOH; (h) 3-hydroxy-2-methylbenzoic acid, DCC, HOBt, DMF; (i) MeSO3H,
CH2Cl2 (step i omitted for 11b).
Ta ble 1. Representative Biological Data for HIV-1 Protease
Inhibitors
hydrogen bonds from the two amide carbonyls of the
inhibitor into the flap region of the enzyme in a manner
analogous to that observed in other HIV-1 protease-
inhibitor complexes.2,3 The RMS deviation between the
backbone atoms of nelfinavir and Ro31-8959 was 0.49
Å, confirming that they adopt similar binding modes.
From the cocrystal structure it is not clear why the
S-phenyl substituent of nelfinavir is favored 50-fold over
the S-naphthyl in 11b, although it is apparent that the
second ring found in the S-naphthyl analog makes at
Ki
ED50
entry
R
X
(nM)a
(nM)a,b
best one favorable interaction with Val-182, the remain-
der of the S3 pocket being relatively hydrophilic in
nature.
1
3
2.0
98
21
970
14
100
33
11a (AG1343)
SPh
MeSO3H
free base
MeSO3H
11b
11c
2-S-naphthyl
Compounds 11a and 11c were potent antiviral agents
in cell culture (Table 1), with ED50 values of 14 and 33
nM, respectively, against the HIV strain IIIB in CEM
cells, and exhibited minimal cellular toxicity (TD50s
>5000 nM).9 Although both compounds showed en-
couraging oral bioavailability results in a preliminary
animal screen, nelfinavir was selected for more exten-
sive antiviral and pharmacokinetic studies due to its
superior antiviral activity. Additional antiviral testing
demonstrated that nelfinavir was effective against the
replication of several laboratory and clinical HIV-1 and
HIV-2 isolates with ED50 values ranging from 9 to 60
nM.11
Ph
a
b
Reference 9. Concentration required to inhibit 50% HIV-
induced cell death.
(Table 1).9 Additional optimization work focused on the
P1 phenylalanine portion of 11c. We had previously
demonstrated that S-aryl substituents effectively span
from the S1 to S3 subsites of HIV-1 protease, providing
compounds with substantially improved enzyme inhibi-
tory activity relative to their phenylalanine counter-
parts.4c As illustrated in Table 1, the S-phenyl analog
11a (Nelfinavir) is a potent inhibitor of HIV-1 protease
(Ki ) 2.0 nM), which binds with approximately 10-fold
better affinity than phenyl analog 11c, whereas the
corresponding 2-S-naphthyl derivative 11b is a weaker
enzyme inhibitor. In an effort to understand this
unanticipated trend in binding affinities, the cocrystal
structure of nelfinavir complexed with HIV-1 protease
was studied.10 As predicted from the structure of 1,
nelfinavir binds to the enzyme in an extended confor-
mation (Figure 2).4c The tert-butylcarboxamide moiety
occupies the S2′ subsite of HIV-1 protease, the lipophilic
dodecahydroisoquinoline ring system fits into the hy-
drophobic S1′ pocket, and the central hydroxyl group
binds to the catalytic aspartates of the enzyme. The
S-phenyl group resides in the S1 site and partially
extends into the S3 region. The 2-methyl-3-hydroxy-
benzamide portion of the inhibitor occupies the S2
pocket, with the o-methyl substituent making hydro-
phobic interactions with Val-32 and Ile-84, and the
m-phenol group hydrogen bonding to the Asp-30 car-
boxylate. A tightly bound water molecule serves to relay
A summary the pharmacokinetics of nelfinavir in
different species is provided in Table 2.12 Initial inves-
tigation of the compound in fed rats indicated an oral
bioavailability of 43%. In contrast, the oral bioavail-
ability was significantly reduced in fasted rats to 29%.13
Therefore, all subsequent in vivo pharmacokinetic stud-
ies were conducted in fed animals. As shown in Table
2, nelfinavir demonstrated significant oral bioavailabil-
ity across a range of species including dogs (47%),
marmosets (17%), and cynomolgus monkeys (26%). In
addition, a single oral dose of nelfinavir exhibited
plasma levels exceeding the in vitro antiviral ED95 (58
nM, 40 ng/mL) for more than 6 h in three of the four
species (Figure 3). The observed long plasma half-life
after oral dosing is likely due to a combination of slow
dissolution and absorption. The preclinical results of
nelfinavir demonstrated potential antiviral and/or phar-
macokinetic advantages when compared to most known

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3981
F igu r e 2. View of AG1343 in complex with the HIV-1 protease. Atom colors are green (carbon) red (oxygen), blue (nitrogen), and
yellow (sulfur). A solvent accessible surface for the protein active site is shown as white dots. Water molecules are represented
as red crosses.
Ta ble 2. Meana Pharmacokinetic Parameters for AG1343 in Different Species
species
rat
rat
rat
dog
dog
monkey
monkey
marmoset
marmoset
doseb (mg/kg)
route
Cmax (µg/mL)
Tmax (h)
T1/2-el (h)
AUC0-∞ (µg h/mL)
F (%)
25
iv
12.72 ( 3.95
1.34 ( 0.63
1.47 ( 0.43
8.79, 11.92
1.18, 1.22
7.07, 7.99
0.26, 1.59
3.45, 5.67
0.38, 0.84
0.06 ( 0.01
2.82 ( 2.79
1.39 ( 1.42
0.08, 0.08
2.00, 3.00
0.08, 0.08
2.00, 3.00
0.08, 0.08
0.50, 1.00
1.26 ( 0.09
d
7.01 ( 0.89
6.09 ( 2.62e
4.04 ( 2.38
8.78, 11.23
6.19, 13.28
7.45, 10.35
1.84, 6.29
50
oral
oral
iv
oral
iv
oral
iv
oral
43
29
50c
15
1.07 ( 0.18
0.92, 1.18
1.94, 7.72
1.38, 1.47
1.58, 3.00
0.88, 1.23
d, 2.04
30
12.5
25
12.5
25
35, 59
9, 42
6.02, 6.63
1.04,e 2.89
10, 24
a
b
Values shown are mean ( SD of data from three rats and data from two animals each for dogs, monkeys, and marmosets. Delivered
in 5% dextrose to all animals except marmosets where propylene glycol:water (50:50) was used. c Animals were fasted overnight prior to
AG1343 administration. Elimination rate constant could not be determined due to prolonged absorption. e AUC0-t value presented.
d
F igu r e 3. Plasma concentrations of AG1343 (ng/mL) plotted versus time (hours) for four animal species.
HIV protease inhibitors and thus warranted additional
development of the compound for testing in humans.
Initially, the safety and pharmacokinetics of orally
administered nelfinavir were evaluated in a phase I
clinical study.14 Single doses of 100-800 mg and
multiple doses of 800-900 mg per day were well
tolerated. In addition, peak plasma levels up to 100
times the antiviral ED95 and trough levels up to 10
times the ED95 were achieved in these studies. Ad-
ditional clinical studies in HIV-infected individuals have

3982 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Notes
Ta ble 3. Meana Pharmacokinetic Parameters of AG1343 When
Administered Orally with Food to Healthy Human Volunteers
3035, 1687, 1532, 736 cm-1; MS (FD) m/e 332, 288, 271, 181.
Anal. (C17H17NO4S) C, H, N.
(R)-[3-Dia zo-2-oxo-1-[(p h en ylth io)m eth yl]p r op yl]ca r -
ba m ic Acid P h en ylm eth yl Ester (3a ). Compound 2a (12.1
g, 0.037 mol) dissolved in EtOAc (200 mL) under nitrogen was
cooled by a dry ice/CCl4 bath as triethylamine (5.09 mL, 0.037
mol) was added followed by slow addition of isobutyl chloro-
formate (7.13 mL, 0.055 mol). By long-stem funnel an ether
solution of diazomethane (0.146 mol) was added in one portion.
The diazomethane solution was prepared as follows: In a 500
mL Erlenmeyer flask free of flaws behind a blast shield was
placed diethyl ether (100 mL) and 5 N NaOH (150 mL). The
mixture was cooled by an ice bath and stirred as 1-methyl-3-
nitro-1-nitrosoguanidine (21.0 g, 0.146 mol) was carefully
added. After evolution of gas had ceased, the ether was
decanted and dried over KOH. Filtration of the ether solution
of diazomethane into the reaction flask by long-stem funnel
was made in one portion. The reaction was stirred for 30 min
before warming to room temperature for 40 min. The solution
was swept with nitrogen and evaporated to dryness. The
residue was taken up in EtOAc, washed twice with water,
twice with aqueous NaHCO3, and once with brine, and dried
over Na2SO4. A yellow oil was isolated by flash chromatog-
raphy (gradient 0-5% EtOAc in CH2Cl2) in 73% yield: 1H
NMR (300 MHz, CDCl3) δ 7.50-7.19 (m, 10H), 5.62 (d, J ) 7
Hz, 1H), 5.47 (br s, 1H), 5.11 (s, 2H), 4.50-4.32 (m, 1H), 3.33
(d, J ) 6 Hz, 1H); IR (KBr) 3012, 2115, 1720, 1501, 1367, 1228
cm-1; MS (FD) m/e 356, 328, 242.
(R)-[3-Ch lor o-2-oxo-1-[(p h en ylth io)m eth yl]p r op yl]ca r -
ba m ic Acid P h en ylm eth yl Ester (4a ). To compound 3a
(22.3 g, 0.063 mol) in diethyl ether (400 mL) at -20 °C under
nitrogen atmosphere was added HCl gas in small portions,
while the disappearance of starting material by TLC (CH2-
Cl2) was closely monitored. After 4 h, the solvent was removed
under reduced pressure, leaving a white solid (23.0 g). No
further purification was made: 1H NMR (300 MHz, CDCl3) δ
7.50-7.15 (m, 10H), 5.56 (dd, J ) 2.0, 6.7 Hz, 1H), 5.11 (s,
2H), 4.78-4.67 (m, 1H), 4.20 (d, J ) 15.9 Hz, 1H), 4.12 (d, J
) 15.9 Hz, 1H), 3.48-3.23 (m, 2H); IR (KBr) 3349, 1732, 1684,
1515, 1266; MS (FD) m/e 363. Anal. (C18H18ClNO3S) C, H,
N.
[R-(R*,S*)]-[3-Ch lor o-2-h yd r oxy-1-[(p h en ylth io)m eth -
yl]p r op yl]ca r ba m ic Acid P h en ylm eth yl Ester (5a ). Ke-
tone 4a (21 g, 0.058 mol) dissolved in THF (300 mL) was cooled
by an ice water bath as NaBH4 (2.4 g, 0.063 mol) was added
in one portion. After 30 min, TLC (5% EtOAc/CHCl3) indicated
no starting material present, but two closely moving products
at lower Rf. The reaction was quenched with aqueous NH4Cl
and the mixture extracted with ether. The organic phase was
washed sequentially with water and brine and then dried over
Na2SO4. The solvent was removed under reduced pressure
and the residue separated by two flash chromatographies
(column 1, 0-2% MeOH in CH2Cl2; column 2, 0-2% EtOAc in
CHCl3) and recrystallized from CH2Cl2 at -78 °C. The slower
moving alcohol was the major product (8.3 g, 39% yield): 1H
NMR (300 MHz, CDCl3) δ 7.47-7.19 (m, 10H), 5.22-5.03 (m,
1H), 5.09 (s, 2H), 4.01-3.89 (m, 2H), 3.75-3.58 (m, 2H), 3.32
(d, J ) 4 Hz, 2H); IR (KBr) 3321, 2951, 1688, 1542, 1246, 738
cm-1; MS (FD) m/e 366, 119. Anal. (C18H20ClNO3S) C, H, N.
[S-(R*,S*)]-[1-Oxir a n yl-2-(p h en ylth io)eth yl]ca r ba m ic
Acid P h en ylm eth yl Ester (6a ). Chloro alcohol 5a (8.3 g,
0.023 mol) was dissolved in EtOH (400 mL) and cooled by an
ice bath as KOH (1.4 g, 0.025 mol) was added. The ice water
bath was removed and the mixture allowed to warm to room
temperature. After 2 h, the solvent was removed by reduced
pressure and the residue separated by flash chromatography
(gradient, 0-2% EtOAc in CH2Cl2). Removal of solvent yielded
a white solid (6.4 g, 85% yield): 1H NMR (300 MHz,CDCl3) δ
7.45-7.15 (m, 10H), 5.12 (s, 1H), 5.08 (s, 2H), 3.77-3.62 (m,
1H), 3.21 (d, J ) 6 Hz, 2H), 2.99 (m, 1H), 2.77 (m, 2H); IR
(KBr) 3303, 3067, 1694, 1538, 1257, 741; MS (FD) m/e 329.
Anal. (C32H45N3O4S) C, H, N.
b
dose
(mg)
Cmax
(ng/mL)
Tmax
(h)
T1/2-el
(h)
AUC0-∞
(ng h/mL)
100
200
400
800
313 ( 91
439 ( 124
1577 ( 965
3163 ( 584
3.00
3.00
4.00
3.50
1.72 ( 0.35
2.21 ( 0.75
2.28 ( 0.36
3.37 ( 1.23
1250 ( 230
1828 ( 372
7555 ( 4175
22208 ( 6306
a
Values shown are mean ( SD of data from four subjects.
Median values for Tmax are presented.
b
demonstrated that nelfinavir is well absorbed in hu-
mans particularly when administered with food and
possesses minimal and easily managed side effects
(Table 3).
In conclusion, we have identified nelfinavir (AG1343),
a potent, nonpeptidic inhibitor of the HIV-1 protease
with a desirable combination of potent in vitro antiviral
activity and excellent pharmacokinetics in a variety of
species. AG1343 (Viracept) has recently been approved
for marketing for the treatment of AIDS, providing a
useful drug in the fight against this deadly disease.
Exp er im en ta l Section
3-Hyd r oxy-2-m eth ylben zoic Acid . To a cooled (-10 °C)
solution of 45 g (0.30 mol) of 3-amino-2-methylbenzoic acid and
106 g (58 mL, 1.08 mol) of concentrated sulfuric acid in 400
mL of water was added 22.6 g (0.33 mol) of sodium nitrite in
small portions so that the reaction temperatures did not exceed
7 °C. The resulting blood red solution was stirred 30 min at
-10 °C, poured into a solution of 1.2 L water and 240 mL
concentrated sulfuric acid, and then slowly heated to 80 °C
over 1 h (heavy gas evolution occurred between the temper-
atures of 40-60 °C). After gas evolution had stopped, the
reaction mixture was cooled to room temperature and ex-
tracted five times with 600 mL of ethyl acetate. The combined
organic phases were extracted four times with 500 mL of
saturated sodium carbonate, and then the combined aqueous
phases were made acidic (pH 2) by addition of concentrated
hydrochloric acid. The resulting solution was extracted four
times with 500 mL of ethyl acetate, and the combined organic
phases were extracted once with brine and dried over sodium
sulfate. Two recrystallizations from ethyl acetate/chloroform
gave 23.2 g of the product as a light orange powder: yield 52%;
1H NMR (DMSO-d6) δ 2.26 (s, 3H), 3.28-3.35 (br s, 1H), 6.92
(d, 1H, J ) 8.0 Hz), 7.02 (t, 1H, J ) 7.9 Hz), 7.14 (d, 1H, J )
7.9 Hz), 9.45-9.63 (br s, 1H); IR (CHCl3) 3600-2100 (br), 3602,
2983, 1696, 1588, 1462, 1406, 1338, 1279, 1174, 1154, 1075,
1038, 920, 892, 854, 816 cm-1; MS (FD) m/e 152 (M+, 100).
Anal. (C8H8O3) C, H.
(R)-N-[(P h en ylm et h oxy)ca r b on yl]-3-(p h en ylt h io)a la -
n in e (2a ). To a chilled (-55 °C) solution of triphenylphos-
phine (110 g, 0.418 mol) in CH2Cl2 was added dimethyl
azodicarboxylate (61.1 g, 0.418 mol) in 100 mL of CH2Cl2
dropwise by addition funnel over 15 min. After 30 min, N-Cbz-
L-serine (100 g, 0.418 mol) was introduced dropwise as a
solution in 100 mL of THF over 20 min. The reaction mixture
was slowly warmed to room temperature and stirred overnight.
In a separate reaction flask, thiophenol (45.8 g, 0.417 mol) in
THF (800 mL) under nitrogen atmosphere was treated at room
temperature with 60% NaH (16.7 g of NaH, 0.417 mol). After
15 min, the N-[(benzyloxy)carbonyl]serine â-lactone solution
was added slowly by cannula over 20 min. The resulting
mixture was stirred for 2.5 h and then concentrated in vacuo
to a semisolid. This material was dissolved in water and
extracted twice with CH2Cl2. Ethyl acetate was added to the
aqueous layer, followed by sufficient 1 M NaHSO4 to adjust
to pH 2.5. After separation, the aqueous phase was extracted
once more with ethyl acetate, and the combined ethyl acetate
layers were in turn washed with brine and dried over sodium
sulfate. Upon concentration, a thick solid was obtained which
crystallized on standing (54.1 g, 39%): 1H NMR (300 MHz,
CDCl3) δ 7.55-7.18 (m, 10H), 5.55 (d, J ) 7 Hz, 1H), 5.08 (s,
2H), 4.73-4.60 (m, 1H), 3.55-3.30 (m, 2H); IR (KBr) 3304,
[R-(1R*,2R*,3′s*,4a′S*,8a′S*)]-[3-[3′-[[(1,1-Dim eth yleth yl)-
a m in o]ca r b on yl]oct a h yd r o-2′(1H )-isoq u in olin yl]-2-h y-
dr oxy-1-[(ph en ylth io)m eth yl]pr opyl]car bam ic acid P h en -
ylet h yl E st er (8a ). Compound 7 (3-[[(1,1-dimethylethyl)-
amino]carbonyl]octahydro-2′(1H)-isoquinoline, 5.00 g, 0.021

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3983
mol) was dissolved in EtOH (300 mL), and epoxide 6a (6.31 g,
0.019 mol) was added. The solution was refluxed for 8 h. The
solvent was removed under reduced pressure and the product
isolated by flash chromatography (gradient of 0-20% EtOAc
in CH2Cl2). Removal of solvent yielded 8a as a white solid
(4.3 g, 40%): 1H NMR (300 MHz, CDCl3) δ 7.41-7.11 (m, 10H),
5.90 (d, J ) 5 Hz, 1H), 5.64 (s, 1H), 5.05 (d, J ) 4 Hz, 2H),
4.08-3.90 (m, 2H), 3.40 (d, J ) 6 Hz, 2H), 3.05 (s, 1H), 2.95-
2.85 (m, 1H), 2.62-2.45 (m, 2H), 2.28-2.15 (m, 2H), 2.05-
1.88 (m, 2H), 1.78-1.10 (m, 7H), 1.29 (s, 9H); IR (KBr) 3330,
2925, 2862, 1706, 1661, 1520, 1454, 1246, 738, 694 cm-1; MS
(FD) m/e 568, 467. Anal. (C32H45N3O4S) C, H, N.
[R-(2R*,3R*,3′S*,4a ′S*,8a ′S*)]-2-[3-Am in o-2-h yd r oxy-4-
(p h en ylt h io)b u t yl]-N-(1,1-d im et h ylet h yl)d eca h yd r o-3′-
isoqu in olin eca r boxa m id e (9a ). Cbz-protected amine 8a
(1.00 g, 0.0018 mol) was dissolved in 30% HBr/AcOH (40 mL)
at room temperature. The reaction mixture was stirred for 1
h, and then the solvent was removed under reduced pressure.
The residue was azeotroped with toluene three times. The
residue was then dissolved in methanol (30 mL), diethylamine
(2 mL) and concentrated NH4OH (2 mL) were added, and the
solvent was removed under reduced pressure. The residue was
extracted from water with EtOAc. The organic phase was
washed with aqueous NaHCO3 and then with brine. The
extract was dried over Na2SO4, and the solvent was removed
under reduced pressure. The residue was separated by flash
chromatography (gradient, CHCl3 (3 drops of NH4OH/1000
mL) with 0-10% MeOH). Removal of solvent under reduced
pressure gave a white foam (0.54 g, 71% yield): 1H NMR (300
MHz, CDCl3) δ 7.41-7.16 (m, 5H), 6.07 (s, 1H), 3.78-3.70 (m,
1H),3.45-3.38 (m, 1H), 3.03-2.84 (m, 3H), 2.38-2.20 (m, 3H),
2.00-1.05 (m, 12H), 1.33 (s, 9H); IR (KBr) 2924, 2862, 1660,
1517, 1454, 1439, 737, 691; MS (FD) m/e 434, 293.
[S-(2R*,1′S*,2′s*,3′′R*,4a ′′R*,8a ′′R*]-N-[3′-[3′′-[[(1,1-Di-
m eth yleth yl)a m in o]ca r bon yl]octa h yd r o-2′′(1H)-isoqu in -
olin yl]-2′-h yd r oxy-1′-[(p h en ylth io)m eth yl]p r op yl]-2-[((2′′-
m e t h y l-3′′-h y d r o x y p h e n y l)c a r b o n y l)a m in o ]b u t a n e -
d ia m id e (10a ). To a cold (-10 °C) solution containing 70 mg
(0.16 mmol) of amine 9a , 24.6 mg (0.160 mmol) of 2-methyl-
3-hydroxybenzoic acid, and 22 mg (0.16 mmol) of 1-hydroxy-
benzotriazole hydrate in 4 mL of THF was added 33 mg (0.16
mmol) of 1,3-dicyclohexylcarbodiimide. The reaction mixture
was gradually warmed to room temperature overnight. After
a total reaction time of 48 h, the mixture was concentrated
under reduced pressure and then redissolved in ethyl acetate.
The mixture was filtered through Celite, and the resulting
clear solution was washed sequentially with saturated sodium
bicarbonate and brine, dried over sodium sulfate, filtered, and
concentrated. The crude product was purified by radial
chromatography (1 mm plate, eluent of 3% methanol in CH2-
Cl2), yielding 54 mg (59%) of 10a as a white foam: 1H NMR
(300 MHz,CDCl3) δ 7.45 (m, 2H), 7.32-7.17 (m, 4H), 7.06 (m,
2H), 6.84 (m, 1H), 5.51 (br s, 1H), 5.37 (m, 1H), 4.47 (m, 1H),
4.07 (m, 1H), 3.78 (m, 1H), 3.42 (m, 1H), 2.91 (m, 1H), 2.61-
2.43 (m, 2H), 2.31 (s, 3H), 2.30-2.17 (m, 2H), 2.02-1.93 (m,
2H), 1.79-1.12 (m, 12H), 1.09 (s, 9H); IR (KBr) 3297, 2925,
2862, 1627, 1586, 1530, 1482, 1466, 1366, 1287, 1221, 1156,
1119, 1026, 801, 735, 689 cm-1; HRMS (FAB) m/e calcd for
C32H46N3O4S 568.3209, found 568.3182.
[S-(2R*,1′S*,2′s*,3′′R*,4a ′′R*,8a ′′R*]-N-[3′-[3′′-[[(1,1-d i-
m eth yleth yl)a m in o]ca r bon yl]octa h yd r o-2′′(1H)-isoqu in -
olin yl]-2′-h yd r oxy-1′-[(p h en ylth io)m eth yl]p r op yl]-2-[((2′′-
m e t h yl-3′′-h yd r oxyp h e n yl)ca r b on yl)a m in o]b u t a n e d i-
a m id e Meth a n esu lfon a te Sa lt (11a , AG1343). To a stirring
cooled (0 °C) solution of 50 mg (0.088 mmol) of amine 10a in
anhydrous methylene chloride (4 mL) was added dropwise over
5 min 88 µL (0.088 mmol) of a 1.0 M solution of methane-
sulfonic acid in methylene chloride. Solvent was removed from
the precipitated salt by concentration under reduced pressure.
On standing overnight under high vacuum (0.2-0.1 Torr) 58
mg (99%) of salt 11a was obtained as an off white foam: 1H
NMR (DMSO-d6) δ 9.18-9.02 (br s, 1H), 8.10 (s, 2H), 7.33-
7.25 (m, 4H), 7.19-7.14 (m, 1H), 7.00-6.94 (m, 1H), 6.80 (m,
2H), 5.80-5.70 (m, 1H), 4.08-3.78 (m, 4H), 3.59-2.88 (m, 4H),
2.27 (s, 3H), 2.12 (s, 3H), 1.97-1.81 (m, 5H), 1.74-1.60 (m,
4H), 1.56-1.45 (m, 2H), 1.40-1.23 (m, 3H), 1.19 (s, 9H). Anal.
(C33H49N3O7S2) C, H, N.
(R)-N-[(P h en ylm eth oxy)ca r bon yl]-3-(2-n a p h th ylth io)-
a la n in e (2b). A 250 mL round bottom flask, equipped with
stir bar and an addition funnel, was charged with 1.28 g (8.00
mmol) of naphthalene-2-thiol and 30 mL of THF. Under a
nitrogen atmosphere, 1.77 g (8.16 g) of 60% NaH was added
in portions to produce a yellow solution. After 15 min of
stirring, the â-lactone (formed by the dehydration of Cbz-L-
serine) was added over 5 min through the addition funnel as
a solution in 20 mL of THF. The resultant solution was stirred
for an additional hour and then concentrated in vacuo. The
residue was dissolved in ethyl acetate and washed once with
0.5 N NaHSO4 and once with brine. The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo. Pu-
rification by flash chromatography provided the title compound
(2.08 g, 68%) as a pale yellow solid: 1H NMR (300 MHz, CDCl3)
δ 3.42-3.61 (br m, 2H), 5.53-5.76 (br s, 1H), 4.85-5.08 (br
m, 2H), 5.54-5.76 (br s, 1H), 7.06-7.97 (m, 12H); [R]D -55.72°
(c 1.0, MeOH); IR (KBr) 3348, 3048, 1746, 1715, 1674, 1560,
1550, 1269, 1200, 1060 cm-1; MS (FD) m/e 381 (M+), 381 (100).
Anal. (C20H19NO4S) C, H, N.
(R)-[3-Dia zo-2-oxo-1-[(2-n a p h th ylth io)m eth yl]p r op yl]-
ca r ba m ic Acid P h en ylm eth yl Ester (3b). A 1 L round
bottom flask was charged with 15.4 g (40.3 mmol) of (R)-N-
[(phenylmethoxy)carbonyl]-3-(2-naphthylthio)alanine and 230
mL of ethyl acetate. The solution was cooled to -30 °C in dry
ice/carbon tetrachloride, and 5.62 mL (4.08 g, 40.3 mmol)
triethylamine was added dropwise via syringe. Next, 7.84 mL
(8.26 g, 60.5 mmol) of isobutyl chloroformate was added by
syringe. The resulting solution was stirred under nitrogen in
the cold. As the mixed anhydride solution was stirring, a
bilayer of 170 mL of diethyl ether and 170 mL of 5 N NaOH
was prepared in a 500 mL Erlenmeyer flask. While the flask
was kept behind a blast shield, 10 g of N-methyl-N-nitro-N-
nitrosoguanidine was added carefully. When the copious gas
evolution ceased, the ether layer was decanted from the
aqueous layer onto KOH, dried for a few minutes, and poured
through a long-stemmed funnel into the mixed anhydride
solution. The above diazomethane formation and addition was
repeated an additional time using identical quantities of ether
and NaOH and 30 g of N-methyl-N-nitro-N-nitrosoguanidine.
After the solution was stirred in the cold for 20 min, nitrogen
was bubbled through the solution using a fire-polished Pasteur
pipet for approximately 2 min to remove any excess diazo-
methane, and then the solution was concentrated in vacuo.
The residue was purified by flash chromatography to yield
13.62 g (83%) of the title compound as a yellow oil: 1H NMR
(300 MHz, CDCl3) δ 3.32-3.46 (m, 2H), 4.40-4.67 (m, 1H),
5.00-5.09 (m, 2H), 5.44 (s, 1H), 5.76 (d, J ) 7.8 Hz, 1H), 7.25-
7.86 (m, 12H).
(R)-[3-Ch lor o-2-oxo-1-[(2-n aph th ylth io)m eth yl]pr opyl]-
ca r ba m ic Acid P h en ylm eth yl Ester (4b). In a 500 mL
round bottom flask was placed 13.62 g (33.59 mmol) of the
above diazo ketone in 230 mL of diethyl ether. The solution
was cooled to -20 °C in dry ice/CCl4. A short burst (about 2
s) of anhydrous HCl was passed through the solution, and gas
evolution was immediately apparent. The reaction was stirred
and monitored by TLC, care being taken not to add excess HCl.
When the reaction was complete by TLC, the solution was
concentrated in vacuo and purified by flash chromatography
to afford 12.05 g (87%) of the title compound as a pale tan
solid: 1H NMR (300 MHz, CDCl3) δ 3.41 (dd, J ) 12, 6 Hz,
1H), 3.53 (dd, J ) 12, 6 Hz, 1H), 4.18 (AB q, ∆ν ) 41.9 Hz, J
) 15.9 Hz, 2H), 4.77 (dd, J ) 9, 3 Hz, 1H), 5.04 (AB q, J ) 12
Hz, ∆ν ) 10.4 Hz, 2H), 5.59 (d, J ) 7 Hz, 1H), 7.24-7.85
(complex, 12H); [R]D -80.00° (c 1.0, MeOH); IR (CHCl3) 3426,
3031, 3012, 1717, 1502, 1340, 1230, 1228, 1045 cm-1; MS (FD)
m/e 413 (M+), 413(100). Anal. (C22H20NO3SCl) C, H, N.
[R-(R*,S*)]-[3-Ch lor o-2-h yd r oxy-1-[(2-n a p h t h ylt h io)-
m eth yl]p r op yl]ca r ba m ic Acid P h en ylm eth yl Ester (5b).
A 25 mL round bottom flask was charged with 530 mg (1.28
mmol) of the above chloro ketone, 10 mL of THF, and 1 mL of
water. The solution was cooled in an ice bath, and 73 mg (1.92
mmol) of NaBH4 was added in one portion. The reaction was
stirred in the cold, and was found to be complete by TLC in
30 min. The pH was carefully adjusted to 3 with about 10
mL of saturated NH4Cl and 500 µL of 5 N HCl. The acidic
solution was extracted twice with methylene chloride. The

3984 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Notes
combined organic layers were washed once with water, dried
over Na2SO4, filtered, and concentrated in vacuo. TLC analy-
sis showed the presence of two diastereomers, with the lower
Rf compound being the desired product. Purification was
accomplished by radial chromatography using 100% methylene
chloride and provided 212 mg (40%) of the title compound as
a tan solid: 1H NMR (300 MHz, CDCl3) δ 3.40 (apparent s,
2H), 3.61-3.71 (m, 2H), 3.97-3.99 (m, 2H), 4.99 (apparent s,
2H), 5.16 (br s, 1H), 7.21-7.83 (complex, 12H); FDMS m/e 415
(M+), 415 (100); [R]D -47.67° (c 0.86, MeOH); IR (CHCl3) 3630,
3412, 3011, 1720, 1502, 1236, 1044 cm-1. Anal. (C22H22NO3-
ClS) C, H, N.
[S-(R*,S*)]-[1-Oxir a n yl-2-(2-n a p h t h ylt h io)et h yl]ca r -
ba m ic Acid P h en ylm eth yl Ester (6b). A mixture of 31 mg
(0.55 mmol) of KOH in 1 mL of ethanol was prepared in a 10
mL round bottom flask and allowed to stir until all of the KOH
was in solution. Next, a solution of 190 mg (0.46 mmol) of
the above chloro alcohol in 4 mL of ethyl acetate and 2 mL of
ethanol was added via Pasteur pipet. The resultant solution
was stirred at room temperature and followed by TLC. The
reaction was complete in 15 min, at which time the reaction
mixture was poured into a bilayer of water and methylene
chloride. The layers were separated, and the aqueous layer
was extracted once more with methylene chloride. The
combined organic layers were washed once with water, dried
over Na2SO4, and concentrated in vacuo. The product was
purified by radial chromatography to provide 172 mg (99%) of
the title compound as a light tan solid: 1H NMR (300 MHz,
CDCl3) δ 2.76 (br s, 2H) 3.01 (br s, 1H), 3.31 (d, J ) 5 Hz, 2H),
3.77 (br s, 1H), 5.05 (s, 2H), 5.22 (d, J ) 6 Hz, 1H), 7.25-7.85
(complex, 12H); [R]D -125.42° (c 0.59, MeOH); FDMS m/e 379
(M+), 379 (100); IR (CHCl3) 3640, 3022, 2976, 1720, 1502, 1235,
1045 cm-1. Anal. (C22H21NO3S) C, H, N.
[R -(1R *,2R *,3′s*,4a ′S *,8a ′S *)]-[3-[3′-[[(1,1-d im e t h yl-
eth yl)a m in o]ca r bon yl]octa h yd r o-2′(1H)-isoqu in olin yl]-
2-h yd r oxy-1-[(2-n a p h t h ylt h io)m et h yl]p r op yl]ca r b a m ic
Acid P h en yleth yl Ester (8b). A 10 mL round bottom flask
was charged with 165 mg (0.40 mmol) of the above epoxide,
94 mg (0.43 mmol) of 3-[[(1,1-dimethylethyl)amino]carbonyl]-
octahydro-2′(1H)-isoquinoline, and 5 mL of ethanol. The flask
was equipped with a reflux condenser and heated to 80 °C for
19 h. The solution was then cooled to room temperature and
concentrated in vacuo. Radial chromatography of the residue
afforded 103 mg (42%) of the title compound as an off-white
foam: 1H NMR (300 MHz, CDCl3) δ 1.098-1.732 (m, 20H),
2.13-2.31 (m, 2H), 2.44-2.53 (m, 1H), 2.56-2.68 (m, 1H),
2.86-2.97 (m, 1H), 3.52 (br s, 2H), 4.02 (br s, 2H), 4.98
(apparent s, 2H), 5.65 (s, 1H), 5.94 (s, 1H), 7.25-7.83 (complex,
13H); FDMS m/e 629 (M+), 138 (100); [R]D -92.45° (c 1.06,
MeOH); IR (CHCl3) 3429, 3010, 2929, 1713, 1670, 1514, 1455,
1047 cm-1. Anal. (C35H47N3O4S) C, H, N.
[R-(2R*,3R*,3′S*,4a ′S*,8a ′S*)]-2-[3-Am in o-2-h yd r oxy-4-
(2-n a p h th ylth io)bu tyl]-N-(1,1--d im eth yleth yl)d eca h yd r o-
3′-isoqu in olin eca r boxa m id e (9b). In a 10 mL round bottom
flask under nitrogen were placed 50 mg (0.081 mmol) of the
above Cbz-amino alcohol and 1 mL of 38% HBr in acetic acid.
The red solution was stirred at room temperature for 1 h and
concentrated in vacuo. The residue was azeotroped once with
toluene and dried on the vacuum pump. The residue (61 mg,
quantitative) was used crude in the next coupling: 1H NMR
(300 MHz, CDCl3) δ 1.14 (s, 1H), 1.17-2.07 (complex, 15H),
2.66, 2.87 (m, 2H), 3.21-3.25 (m, 2H), 3.75 (d, J ) 12 Hz, 1H),
3.85 (d, J ) 6 Hz, 1H), 4.36-4.47 (m, 1H), 6.73 (s, 1H), 7.39-
7.90 (complex, 7H); FDMS 483 (M+), 483 (100).
the resulting clear solution was washed sequentially with
saturated sodium bicarbonate and brine, dried over sodium
sulfate, filtered, and concentrated. The crude product was
purified by radial chromatography (2 mm plate, gradient
eluent of 5-15% acetone in CH2Cl2), yielding 65 mg (73%) of
10b as a white solid: 1H NMR (300 MHz,CDCl3) δ 7.90 (s,
1H), 7.73 (m, 3H), 7.53-7.40 (m, 3H), 7.21 (d, J ) 8.8 Hz, 1H),
6.93 (m, 2H), 6.79 (m, 1H), 5.53 (br s, 1H), 5.36 (br s, 1H),
4.10 (m, 1H), 3.92-3.83 (m, 1H), 3.51 (m, 1H), 2.94 (d, 1H),
2.61-2.42 (m, 2H), 2.29 (s, 3H), 2.28-2.17 (m, 2H), 2.06-1.93
(m, 1H), 1.80-1.15 (m, 12H), 1.10 (s, 9H); IR (KBr) 3427, 3311
(br), 2929, 2864, 1703, 1661, 1587, 1514, 1456, 1393, 1366,
1276, 1200, 1177, 1146, 1119, 1070, 1042 cm-1; [R]D -112° (c
0.25, MeOH). MS(FD) m/e 618 (M
+ 1, 100). Anal.
(C36H47N3O4S) C, H, N.
[3S -(3R *,4a R *,8a R *,2′S *,3′R *)]-2-[2′-H y d r o x y -3′-
(p h en ylm et h yl)-4′-a za -5′-oxo-5′-(2′′-m et h yl-3′′-h yd r oxy-
p h en yl)p en tyl]d eca h yd r oisoqu in olin e-3-N-ter t-bu tylca r -
boxa m id e (10c). To a cold (-10 °C) solution containing 261
mg (0.65 mmol) of amine 9c, 100 mg (0.65 mmol) of 2-methyl-
3-hydroxybenzoic acid, and 88 mg (0.65 mmol) of 1-hydroxy-
benzotriazole hydrate in 6 mL of THF and 0.2 mL of DMF
was added 134 mg (0.65 mmol) of 1,3-dicyclohexylcarbodiimide.
The reaction mixture was gradually warmed to room temper-
ature overnight. After a total reaction time of 48 h, the
mixture was concentrated under reduced pressure and then
redissolved in ethyl acetate. The mixture was filtered through
Celite, and the resulting clear solution was washed sequen-
tially with saturated sodium bicarbonate and brine, dried over
sodium sulfate, filtered, and concentrated. Purification of the
crude product by radial chromatography (2 mm plate, gradient
eluent of 1%-5% methanol/methylene chloride) gave 304 mg
of a white solid: yield 87%; [R]D -75.00° (c ) 2.00, MeOH);
1H NMR (CDCl3) δ 1.18 (s, 9H), 1.19-2.05 (m, 18H), 2.20-
2.35 (m, 2H), 2.50-2.70 (m, 2H), 2.90-3.05 (m, 2H), 3.22-
3.35 (m, 1H), 3.96-4.05 (m, 1H) 4.45-4.55 (m, 1H), 5.77 (s,
1H), 6.53 (d, J ) 7.4 Hz, 2H), 6.75 (d, J ) 7.8 Hz, 1H), 6.85-
6.90 (m, 1H), 7.15-7.35 (m, 6H); IR (CDCl3) 3606, 3600-3100
(br), 3429, 3011, 2929, 2865, 1663, 1604, 1587, 1514, 1455,
1367, 1277, 1200, 1156, 1046, 910 cm-1; HRMS (FAB) m/e calcd
for C32H46N3O4 536.3488, found 536.3488.
[3S -(3R *,4a R *,8a R *,2′S *,3′R *)]-2-[2′-H y d r o x y -3′-
(p h en ylm et h yl)-4′-a za -5′-oxo-5′-(2′′-m et h yl-3′′-h yd r oxy-
p h en yl)p en tyl]d eca h yd r oisoqu in olin e-3-N-ter t-bu tylca r -
boxa m id e Mon om eth a n esu lfon a te Sa lt (11c). To a stir-
ring cooled (0 °C) solution of 125 mg (0.23 mmol) of 3S-
(3R*,4aR*,8aR*,2′S*,3′R*)]-2-[2′-hydroxy-3′-(phenylmethyl)-4′-
a za -5′-oxo-5′-(2′′-m et h yl-3′′-h yd r oxyp h en yl)p en t yl]-
decahydroisoquinoline-3-N-tert-butylcarboxamide in anhy-
drous methylene chloride (5 mL) was added dropwise over 5
min 240 µL (0.24 mmol) of a 1.0 M solution of methane sulfonic
acid in methylene chloride. After the mixture was pumped
overnight under high vacuum (0.2-0.1 torr) 136 mg of the
1
crude salt was obtained as an off-white foam: yield 95%; H
NMR (methanol-d4) δ 1.12 (s, 9H), 1.10-2.20 (m, 16H), 2.60-
2.75 (m, 4H), 3.10-3.50 (m, 6H), 3.60-3.70 (m, 1H), 3.90-
4.30 (m, 3H), 6.53 (d, J ) 7.35 Hz, 1H), 6.55 (d, J ) 7.87 Hz,
1H), 6.89 (t, J ) 7.82 Hz, 1H). Anal. (C33H49N3O7S) C, H, N.
Ack n ow led gm en t. We thank the HIV Protease
groups at Agouron and Lilly for their contributions to
this project, and acknowledge Peter J ohnson, Robert
J ackson, Michael Varney, Carlos Lopez, and Richard
J askunas for their encouragement and support of this
effort.
[S-(2R*,1′S*,2′s*,3′′R*,4a ′′R*,8a ′′R*]-N-[3′-[3′′-[[(1,1-Di-
m eth yleth yl)a m in o]ca r bon yl]octa h yd r o-2′′(1H)-isoqu in -
olin yl]-2′-h yd r oxy-1′-[(2-n a p h yth ylth io)m eth yl]p r op yl]-
2-[((2′′-m e t h y l-3′′-h y d r o x y p h e n y l)c a r b o n y l)a m in o ]-
bu ta n ed ia m id e (10b). To a cold (-10 °C) solution containing
70 mg (0.145 mmol) of amine 9b, 22 mg (0.145 mmol) of
2-methyl-3-hydroxybenzoic acid, and 19 mg (0.145 mmol) of
1-hydroxybenzotriazole hydrate in 4 mL of THF was added
29 mg (0.145 mmol) of 1,3-dicyclohexylcarbodiimide. The
reaction mixture was gradually warmed to room temperature
overnight. After a total reaction time of 48 h, the mixture was
concentrated under reduced pressure and then redissolved in
ethyl acetate. The mixture was filtered through Celite, and
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(4) (a) Kalish, V. J .; Tatlock, J . H.; Davies, J . F., II; Kaldor, S. W.;
Dressman, B. A.; Reich, S.; Pino, M.; Nyugen, D.; Appelt, K.;
Musick, L.; Wu, B. Structure-based Design of Nonpeptidic P2
Substituents for HIV-1 Protease Inhibitors. Bioorg. Med. Chem.
Lett. 1995, 5, 727-732. (b) Kaldor, S. W.; Dressman, B. A.;
Hammond, M.; Appelt, K.; Burgess, J . A.; Lubbehusen, P. P.;
Muesing, M. A.; Hatch, S. D.; Wiskerchen, M.; Baxter, A. J .
Isophthalic Acid Derivatives: Amino Acid Surrogates for the
Inhibition of HIV-1 Protease. Bioorg. Med. Chem. Lett. 1995, 5,
721-726. (c) Kaldor, S. W.; Appelt, K.; Fritz, J . E.; Hammond,
M.; Crowell, T. A.; Baxter, A. J .; Hatch, S. D.; Wiskerchen, M.;
Muesing, M. A. A Systematic Study of P1-P3 Spanning Sidechains
for the Inhibition of HIV-1 Protease. Bioorg. Med. Chem. Lett.
1995, 5, 715-720. (d) Kaldor, S. W.; Hammond, M.; Dressman,
B. A.; Fritz, J . E.; Crowell, T. A.; Hermann, R. A. New Dipeptide
Isosteres Useful for the Inhibition of HIV-1 Protease. Bioorg.
Med. Chem. Lett. 1994, 4, 1385-1390. (e) Reich, S. H.; Melnick,
M.; Davies, J .; Appelt, K.; Lewis, K.; Fuhry, M. M.; Pino, M.; et
al. Protein Structure-Based Design of Potent Orally Bioavailable,
Nonpeptide Inhibitors of Human Immunodeficiency Virus Pro-
tease Proc. Natl. Acad. Sci. U.S.A. 1995 92, 8, 3298. (f) Reich,
S. H.; Melnick, M.; Pino, M.; Fuhry, M. M.; Trippe, A.; Appelt,
K.; Davies, J . F., II; Wu, B.; Musick, L. Structure-Based Design
and Synthesis of Substituted 2-Butanols as Nonpeptidic Inhibi-
tors of HIV Protease: Secondary Amide Series. J . Med. Chem.
1996, 39, 2781-2794. (g) Melnick, M.; Reich, S. H.; Lewis, K.
K.; Mitchell, L. J .; Nguyen, D.; Trippe, A. J .; Dawson, H.; Davies,
J . F., II; Appelt, K.; Wu, B.; Musick, L.; Gehlhaar, D. K.; Webber,
S.; Shetty, B.; Kosa, M.; Andrada, D. Bis Tertiary Amide
Inhibitors of the HIV-1 Protease Generated via Protein Structure-
Based Iterative Design J . Med. Chem. 1996, 39, 2795-2811.
(5) Appelt, K.; Davies, J ., Unpublished results.
(8) See Parkes, K. E. B.; Bushnell, D. J .; Crackett, P. H.; Dunsdon,
S. J .; Freeman, A. C.; Gunn, M. P.; Hopkins, R. A.; Lambert, R.
W.; Martin, J . A.; Merrett, J . H.; Redshaw, S.; Spurden, W. C.;
Thomas, G. J . Studies Toward the Large-Scale Synthesis of the
HIV Proteinase Inhibitor Ro 31-8959. J . Org. Chem. 1994, 59,
3656-3664 and references cited therein.
(9) Ki values were measured using the chromogenic assay developed
by Richards et al. using His-Lys-Arg-Val-Leu-Phe(p-NO2)-Glu-
Ala-Nle-Ser-NH2 (American Peptide, Santa Clara, CA) as the
substrate at pH 5.6: Richards, A. D.; Phylip, L. H.; Farmerie,
W. G.; Scarborough, P.; Pavlickova, L.; Kostka, V.; Kay, J .
Sensitive, Soluble Chromogenic Substrates for HIV-1 Proteinase.
J . Biol. Chem. 1990, 265, 7733-7736. Inhibition constants were
determined using the method of Morrison and applying nonlin-
ear curve-fitting methods: Morrison, J . F. Kinetics of the
Reversible Inhibition of Enzyme-Catalysed Reactions of Tight-
Binding Inhibitors. Biochim. Biophys. Acta 1969, 185, 269-286.
For comparison, Ro 31-8959, prepared in-house, exhibited a Ki
of 0.99 ( 0.31 nM in this assay. Antiviral testing was performed
according to the method of Weislow et al.: Weislow, O. S.; Kiser,
R.; Fine, D. L.; Bader, J .: Shoemaker, R. H.; Boyd, M. R. New
Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Applica-
tion to High-Flux Screening of Synthetic and Natural Products
for AIDS-Antiviral Activity. J . Natl. Cancer Inst. 1989, 81 (8),
577-586. For comparison, Ro 31-8959, prepared in-house,
exhibited an ED50 of 2-10 nM in this assay.
(10) The HIV-1 protease cloning, expression, and purification has
been described previously (Hostomsky, Z.; Appelt, K.; Ogden,
R. C. Biochem. Biophys. Res. Commun. 1989, 161, 1056-1063).
Crystals of the inhibited enzyme were grown by the hanging
drop vapor diffusion method from 1.2-1.6 M ammonium sulfate,
5% (vol/vol) dimethyl sulfoxide, 3% (vol/vol) isopropyl alcohol,
and 0.05 M citrate-tartrate buffered at pH ) 5.8. The enzyme
crystallized in space group P2(1)2(1)2(1) with cell dimensions
of a ) 52 Å, b ) 59 Å, and c ) 62 Å. A 2.1 Å X-ray diffraction
data set was collected from one crystal using dual area detectors
from Area Detector Systems, Inc. Refinement of the AG1343
complex was initiated using the crystal structure of a previously
solved inhibited complex as a starting point. Using the X-PLOR
program (Brunger, A. T.; Kuriyan, J .; Karplus, M. Science 1987,
235, 458-460), the structure was refined to a crystallographic
R-factor of 0.20 using all data between 6 and 2.1 Å. Details of
the crystallographic work will be published elsewhere (J . F.
Davies and K. Appelt). Atomic coordinates have been deposited
in the Protein Data Bank, Chemistry Department, Brookhaven
National Laboratories, Long Island, NY 11973. Accession num-
ber: 10HR.
(11) For comparative purposes Ro 31-8959 afforded an ED50 range
of 2-30 nM in the same assays. Details of antiviral and
resistance data: Patick, A. K.; Mo, H.; Markowitz, M.; Appelt,
K.; Wu, B.; Musick, L.; Kalish, V.; Kaldor, S.; Reich, S.; Ho, D.;
Webber, S. Antiviral and Resistance Studies of AG1343, an
Orally Bioavailable Inhibitor of Human Immunodeficiency Virus
Protease. Antimicrob. Agents Chemother. 1996, 40 (2), 292-297.
(12) Animal studies: Pharmacokinetics of AG1343 were determined
after intravenous and oral administration of AG1343 to male
Sprague-Dawley rats (n ) 3), beagle dogs (one male and one
female), female cynomolgus monkeys, and marmosets (one male
and one female). Intravenous dose of AG1343 ranged from 12.5
to 25 mg/kg, and oral dose of AG1343 ranged from 25 to 50 mg/
kg. AG1343 was delivered as a solution in 5% dextrose to rats,
dogs, and monkeys or in propylene glycol:water (50:50) to
marmosets. Blood (0.25-2 mL) was sampled from the jugular,
femoral, or cephalic vein before dosing and at time points up to
48 h after dosing. Plasma was separated immediately after
sampling by centrifugation and stored at -20 °C until analyzed.
AG1343 was extracted from plasma and quantified using high
performance liquid chromatography (HPLC) and UV detection.
Details of preclinical pharmacokinetics will be published in
appropriate forum (B. Shetty).
(6) Initial disclosures on AG1343: (a) Shetty, B.; Kaldor, S.; Kalish,
V.; Reich, S.; Webber, S. AG1343, An Orally Bioavailable Non-
peptidic HIV-1 Protease Inhibitor. Presented at the 10th Inter-
national AIDS Conference, Yokohama, J apan, August 1994;
paper 321A. (b) Kalish, V.; Kaldor, S.; Shetty, B.; Tatlock, J .;
Davies, J .; Hammond, M.; Dressman, B.; Fritz, J .; Appelt, K.;
Reich, S.; Musick, L.; Wu, B.; Su, K. Iterative Protein Structure-
Based Design and Synthesis of HIV Protease Inhibitors. Pro-
ceedings of the XIIIth International Symposium on Medicinal
Chemistry, Paris, France, September 1994, p 201s. In early
reports, AG1343 was also called LY312857.
(13) Muirhead, G. J .; Williams, P. E. O.; Shaw, T. M.; McClellend,
G. R. Investigations of the Effect of Food on the Pharmacoki-
netics of the HIV Proteinase Inhibitor Ro 31-8959 in Healthy
Volunteers. AIDS 1992, 6 (Suppl. 1):Abs P74.
(14) Initial presentation of these results: Quart, B. D.; Chapman,
S. K.; Peterkin, J .; Webber, S.; Oliver, S. Phase I Safety,
Tolerance, Pharmacokinetics, and Food Effect Studies of AG1343-
A Novel HIV Protease Inhibitor. Proceedings of the 2nd National
Conference on Human Retroviruses and Related Infections,
Washington, DC, J anuary 1995; Abstract LB3, p 167.
(7) Arnold, L. D.; Kalantar, T. H.; Vederas, J . C. Conversion of
J M9704098