Inhibitors of human immunodeficiency virus type 1 protease containing 2- aminobenzyl-substituted 4-amino-3-hydroxy-5-phenylpentanoic acid: Synthesis, activity, and oral bioavailability

Article abstract of DOI:10.1021/jm9508696

Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4- [[(benzyloxycarbonyl)-L-valyl]amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5- (phenylpentanoyl)-L-valine 2-(aminomethyl)-benzimidazole amide led to a novel series of inhibitors with a shortened, modified carboxy terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported. Of particular interest are derivatives featuring the (1S,2R)-1- amino-2-hydroxyindan moiety at the P2'-position since some of them exhibit substantial oral bioavailability in mice. The influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors is discussed. Optimum enhancement of oral bioavailability was observed with L- tert-leucine in P2-position, resulting in the discovery of (2R,3S,4S)-4- [[(benzyloxycarbonyl)-L-tert-leucyl]amino]-3-hydroxy-2-[(4- methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hydroxyindan amide which combines high antiviral activity (IC₅₀ = 250 nM) with a good pharmacokinetic profile (AUC = 82.5 μM · h at a dose of 125 mg/kg po in mice).

Full text of DOI:10.1021/jm9508696

2060
J . Med. Chem. 1996, 39, 2060-2067
In h ibitor s of Hu m a n Im m u n od eficien cy Vir u s Typ e 1 P r otea se Con ta in in g
2-Am in oben zyl-Su bstitu ted 4-Am in o-3-h yd r oxy-5-p h en ylp en ta n oic Acid :
Syn th esis, Activity, a n d Or a l Bioa va ila bility
Philipp Lehr,* Andreas Billich, Brigitte Charpiot, Peter Ettmayer, Dieter Scholz, Brigitte Rosenwirth, and
Hubert Gstach
Sandoz Research Institute, Brunner Strasse 59, A-1235 Vienna, Austria
Received November 28, 1995X
Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-valyl]-
amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-(phenylpentanoyl)-L-valine 2-(aminomethyl)-
benzimidazole amide led to a novel series of inhibitors with a shortened, modified carboxy
terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported.
Of particular interest are derivatives featuring the (1S,2R)-1-amino-2-hydroxyindan moiety
at the P2′-position since some of them exhibit substantial oral bioavailability in mice. The
influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors
is discussed. Optimum enhancement of oral bioavailability was observed with L-tert-leucine
in P2-position, resulting in the discovery of (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-tert-leucyl]-
amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hy-
droxyindan amide which combines high antiviral activity (IC50 ) 250 nM) with a good
pharmacokinetic profile (AUC ) 82.5 µM‚h at a dose of 125 mg/kg po in mice).
In tr od u ction
The protease of the human immunodeficiency virus
type 1 (HIV-1 PR) is essential for replication of the
virus1 and, hence, is regarded as one of the most
promising targets for chemotherapy of HIV infection.
First reports of clinical efficacy of HIV PR inhibitors2-4
demonstrated the validity of this therapeutic principle.
High in vitro potency is readily obtained with a variety
of recently described inhibitors (see refs 5 and 6 for
reviews), but clinical efficacy, in addition, strongly
depends on pharmacological factors, such as oral bio-
availability and duration of action. Unfortunately, in
the field of peptidomimetic PR inhibitors, these factors
are currently not amenable to rational prediction. For
example, in the case of renin inhibitors no correlation
between their partition coefficients and oral absorption
or duration of action could be found.7 It is thought,
however, that lower molecular weight, reduced lipophi-
licity, diminished propensity to form hydrogen bonds,
and a smaller number of amide bonds might contribute
to improved pharmacokinetic properties of peptidic
compounds. Since these are obviously only rough
guidelines, the discovery of new chemotypes of HIV PR
inhibitors exhibiting both potent anti-HIV activity and
high oral bioavailability still remains a challenge.
In a previous report8 we described structure-activity
relationships in a series of HIV PR inhibitors containing
2-heterosubstituted 4-amino-3-hydroxy-5-phenylpen-
tanoic acid (AHPPA). This study led to compounds with
a benzimidazole group in P3′-position, e.g., compound
1 (Figure 1) which selectively inhibits HIV-1 PR (Ki )
3.4 nM) and shows potent antiviral activity in vitro (IC50
) 27 nM), establishing 2-heterosubstituted AHPPA as
a new HIV PR inhibitor core. However, these inhibitors
did not show significant oral bioavailability in mice or
rats.9 In this report, we describe a novel series of
F igu r e 1. Chemical structures of AHPPA-containing HIV PR
inhibitors 1 and 2.
derivatives with a reduced number of peptide bonds and
lower molecular weight than 1. The combination of
2-heterosubstituted AHPPA as dipeptide mimetic and
the 2-aminoindanol moiety10 in the P2′-position, as
exemplified by compound 2 (Figure 1), led to orally
bioavailable inhibitors of HIV-1 with potent antiviral
activity.
Ch em istr y
The general route for the syntheses of 2-heterosub-
stituted derivatives of AHPPA is illustrated for com-
pound 2 in Scheme 1. The key intermediate, a mixture
of the diastereomeric oxiranes 3a ,b (ratio ) 9:1), is
readily available via a three-step procedure starting
from N-(tert-butyloxycarbonyl)phenylalaninol.8 As shown
previously, this mixture can be used in the next step
without further separation. When the mixture of
epoxides 3a ,b was reacted with p-methoxybenzylamine
in ethanol, only compound 4 was obtained in a diaste-
* Corresponding author. Phone: ++43-1-86634-266. Fax: ++43-
1-86634-681.
X Abstract published in Advance ACS Abstracts, April 15, 1996.
S0022-2623(95)00869-7 CCC: $12.00 © 1996 American Chemical Society

HIV-1 PR Inhibitors Containing Substituted AHPPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2061
Inspired by the procedure of Thompson et al.,11 we
developed an alternative stereospecific approach for the
preparation of AHI (6). Instead of derivation of racemic
cis-aminoindanol with a chiral auxiliary for separation
of diastereomers at the end of the reaction sequence,
we performed the resolution of racemic trans-aminoin-
danol at an early stage of the synthesis by diastereo-
meric salt formation.12 Using (+)-dibenzoyl-D-tartrate,
only the (+)-enantiomer of trans-aminoindanol crystal-
lized from ethanolic solution. The pure enantiomer of
the free base was transformed to AHI following the
published protocol (see the Experimental Section for
details).
Sch em e 1. Synthesis of HIV-1 PR Inhibitors Containing
2-Aminobenzyl-Substituted AHPPAa
Boc-tert-leucine derivative 28, synthesized according
to the general route (Scheme 1), was the starting point
for the synthesis of compounds 29-39 (Table 4). After
removal of the Boc group, the amino terminus was
acylated with various carboxylic acids. Substituted
thioacetic acids used in the preparation of derivatives
30-32 were synthesized by the method of Mikitenko et
al.13
Resu lts a n d Discu ssion
a
(a) 4-Methoxybenzylamine, EtOH, 70 °C; (b) 1 N LiOH, THF,
room temperature; (c) (1S,2R)-1-amino-2-hydroxyindan (6), HOBt,
EDC·HCl, DMF, room temperature; (d) 3 N HCl in diethyl ether,
dichloromethane, room temperature; (e) Z-tert-leucine, PyBOP,
diisopropylethylamine, DMF, room temperature.
HIV PR inhibitor 1 (Figure 1) and its derivatives
which we described previously8,9 contain two amino
14
acids filling the S2 and S2′
sites of HIV PR.15
Replacement of these amino acids by N-terminal Boc
and C-terminal benzyl groups yielded an inhibitor of
small size (compound 9, Table 1) showing weak activity
against the enzyme but, interestingly, slightly higher
bioavailability in mice (see Table 6) than the potent
inhibitor 1.
Sch em e 2. Synthesis of P1’-Modified HIV-1 PR
Inhibitors Containing 2-Aminobenzyl-Substituted
AHPPAa
Since the corresponding ethyl ester of compound 9
(data not shown) was devoid of any activity at the
enzymatic level, we investigated whether the C-terminal
benzylamino group is responsible for the observed
intrinsic activity. Compounds 12 with valine in position
P2′ and 11 with phenylglycine replacing valine in 12
were synthesized. Equal potency in enzyme inhibition
indicates that a phenyl substituent is well tolerated at
this position.
In compound 10, phenylglycinol was placed in P2′
replacing the benzylamino group in 9. The hydroxy
group of the phenylglycinol moiety was intended to
interact with the NH of Asp29 of the enzyme. An
analogous hydrogen bond is observed with the P2′-
carbonyl oxygen in previously reported HIV PR inhibi-
tors.16 However, even at 3 µM, derivative 10 did not
inhibit HIV PR. A similar result with phenylglycinol
as terminus was reported by Tucker et al. in the case of
HIV PR inhibitors containing the hydroxyethylamine
isostere.17
a
(a) R,R′-Diamino-p-xylene, EtOH, 80 °C; (b) (2-benzimida-
zolyl)propionic acid, HOBt, EDC‚HCl, DMF, room temperature;
(c) (i) 1 N LiOH, THF, room temperature, (ii) (1S,2R)-1-amino-2-
hydroxyindan (6), HOBt, EDC‚HCl, DMF, room temperature.
Incorporation of AHI10 in place of the P2′ benzylamino
moiety led to inhibitor 7 with 40-fold increased potency
relative to 9. AHI had been described previously as an
effective surrogate for the P2′ amino acid in HIV PR
inhibitors containing a hydroxyethylene isostere10 but
was not beneficial in other inhibitors.18 However,
despite the improved Ki value, compound 7 was devoid
of any antiviral activity.
In order to modify the pharmacokinetic properties,
e.g., oral uptake, we decided to elongate stepwise the
P1′ side chain and the backbone of 9. Molecular models
of HIV PR complexed with 9 suggested that elongated
moieties in the para-position of the P1′ benzylamino
reomerically pure form after chromatographic workup.
The ester group of 4 was saponified to give acid 5, which
was coupled with 1(S)-amino-2(R)-hydroxyindan (AHI,
6)10 to yield 7. Deprotection of the amino group in 7
generated 8, which was subsequently coupled with Cbz-
tert-leucine to afford inhibitor 2.
The P1′-modified inhibitors 20-23 (Table 2) were
synthesized as exemplified for compound 23 in Scheme
2: Thus, epoxide 3 was treated with 1,4-diamino-p-
xylene to produce amino derivative 15, and subsequent
acylation gave derivative 16. Selective alkaline hy-
drolysis of the ethyl ester function in 16 followed by
coupling with 6 led to inhibitor 23.

2062 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Lehr et al.
Ta ble 1. Truncated AHPPA-Containing Inhibitors: Inhibitory Activity against HIV-1 PR and Antiviral Activity against HIV-1, IIIB,
in MT4 Cells
no.
R′
R′′
OCH3
OCH3
H
OCH3
OCH3
OCH3
O(CH2)2OH
e
A
R′′′
Ki (nM)a
IC50 (nM)b
mp (°C)
formulac
C46H57N7O7
C33H41N3O6‚0.2 H2O
C30H37N3O4‚1.3 H2O
C33H43N3O6
C40H46N6O6
C37H48N6O6
C32H41N3O6‚0.5 H2O
C41H48N6O5‚1.0 H2O
1
7
9
10
11
12
13
14
Z-Val
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Val
HNCH2-2-benzimidazolyl
HN(2(R)-hydroxyindan-1(S)-yl)
HNCH2Ph
1(S)-HNCH(Ph)CH2OH
HNCH2-2-benzimidazolyl
HNCH2-2-benzimidazolyl
HNCH2Ph
3.4
65
2500
d
11
7.7
25 ( 17 (17)
231-234
183-185
50-53
bond
bond
bond
Phg
Val
bond
bond
d
d
d
65-68
380 ( 52 (4)
510 ( 163 (5)
104-109
oil
1200
680
d
d
62-64
111-115
HNCH2Ph
a
b
Usual standard deviation ( 20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
elemental analyses within ( 0.4% of the calculated value for C,H,N were obtained. No activity at 3 µM. e CH2NHCO(CH2)2-2-
benzimidazolyl.
d
Ta ble 2. Truncated Inhibitors Modified in P1′: Inhibitory Activity against HIV-1 PR and Antiviral Activity against HIV-1, IIIB,
in MT4 Cells
no.
R
Ki (nM)a
IC50 (nM)b
mp (°C)
formulac
7
17
18
19
20
21
22
23
O-CH3
O-CH2-CH3
O-CH2-CH2-OH
O-CH2-CH2(4-morpholinyl)
CH2-NH-CO-CH3
CH2-NH-CO-NH-Ph
65
92
80
130
70
33
d
d
183-185
65-69
C33H41N3O6‚0.2 H2O
C34H43N3O6
C34H43N3O7
C38H50N4O7
C35H44N4O6
C40H47N5O6‚0.2dioxane
C41H48N4O7
C43H50N6O6‚2.5H2O
4000 (1)
d
2600 (1)
d
77-82
71-76
94-98
100-104
73-76
CH2-NH-CO-O-CH2-Ph
CH2-NHCO(CH2)2-2-benzimidazolyl
19
41
d
4400 (1)
128-132
a
b
Usual standard deviation ( 20%. Number of determinations given in parentheses. c Satisfactory elemental analyses within ( 0.4%
d
of the calculated value for C,H,N were obtained. No activity observed at 10 µM.
group might reach into the S3′ pocket of the enzyme.15
Introduction of polar and charged residues in the para-
position of the P1′ residues of HIV PR inhibitors was
also used by Young et al. to enhance aqueous solubility
and hence oral bioavailability.19 Along these lines,
compounds 13 and 14 were synthesized and found to
be 2-4-fold better enzyme inhibitors than 9 (Table 1).
These promising modifications were now applied to
improve the anti-HIV activity of 7 bearing AHI in P2’.
Introduction of the ethoxy (17), hydroxyethoxy (18), or
morpholinoethoxy (19) group (Table 2) resulted in
increase of the Ki value. In contrast, with acylated
aminomethylene side chains, this activity was main-
tained (20) or, in the case of aromatic groups (21-23),
even improved. Unexpectedly, no antiviral activity was
detectable even for the best enzyme inhibitor in this
series (22) exhibiting a Ki value of 19 nM.
In another set of derivatives, compound 7 was ex-
tended in the P2/P3-positions by Z-protected amino
acids (2, 24-27, Table 3); this modification generally
yielded good enzyme inhibitors, with the best Ki values
of about 8 nM. It is quite apparent that in this series
antiviral activity does not strictly correlate with enzyme
inhibition; e.g., 24 and 25, which are equipotent at the
enzymatic level, differ in antiviral activity by a factor
of 3. Compound 2 with tert-leucine in P2 was found to
be the best inhibitor of HIV replication in this series
(IC50 ) 250 nM). The observation that the introduction
of tert-leucine while slightly reducing the Ki value of the
inhibitors leads to significantly better antiviral activity
20
had been made by us before.8 Also Schirlin et al.
found tert-leucine to be preferred in P2 of their HIV PR
inhibitor but reported cellular toxicity of their tert-
leucine-containing compounds. However, the analogues
described here do not share this property.21
Studies of the substrate specificity of HIV-1 PR22
suggested that aspartic or glutamic acid substituents
might be beneficial for good enzyme inhibition; however,
most likely poor cell penetration due to the negatively
charged aspartic acid in 26 led to a lack of antiviral
activity (Table 3).
Compound 2 was the starting point for further
derivation, first in the P3-position (Table 4) where the
Cbz group had been previously held constant. There-
fore, compounds 28-39 were synthesized in order to
evaluate the influence of heterocycles and heterosub-
stituted aromatic rings in P3 on antiviral activity. In
addition, potential effects of this change in physico-
chemical parameters on solubility and oral bioavailabil-
ity were investigated (Table 6, vide supra). Replace-
ment of the Cbz group (2) by Boc (28) led to a marked
drop in enzyme inhibition, while aromatic groups (29-
39) introduced in P3 generally yielded good inhibitors
with Ki values in the range from 6 to 18 nM. The

HIV-1 PR Inhibitors Containing Substituted AHPPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2063
Ta ble 3. Truncated Inhibitors Modified in P3-P2: Inhibitory Activity against HIV-1 PR and Antiviral Activity against HIV-1, IIIB,
in MT4 Cells
no.
R
Ki (nM)a
IC50 (nM)b
mp (°C)
formulac
7
2
24
25
26
27
Boc
65
d
183-185
146-148
81-91
112-115
111-114
158-162
C33H41N3O6‚0.2H2O
Z-Tle
Z-Val
Z-Ile
Z-Asp
Z-Asn
9.5
8.1
8.2
22
254 ( 40 (5)
344 ( 128 (5)
1102 ( 474 (5)
d
C
C
42H50N4O7‚0.3H2O
41H48N4O7
C42H50N4O7‚0.2H2O
C
C
40H44N4O9‚HCl‚1.3H2O
40H45N5O8‚0.5 H2O
15
1400 (1)
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
d
elemental analyses within (0.4% of the calculated value for C,H,N were obtained. No activity at 3 µM.
Ta ble 4. Truncated Inhibitors Modified in P3: Inhibitory Activity against HIV-1 PR and Antiviral Activity against HIV-1, IIIB, in
MT4 Cells
no.
R
Ki (nM)a
IC50 (nM)b
mp (°C)
formulac
2
28
29
30
31
32
33
34
35
36
37
38
39
Z
Boc
9.5
210
9.4
8.0
5.8
12
13
14
12
13
254 ( 40 (5)
d
146-148
180-195
130-134
88-95
95-108
98-105
102-105
115-118
110-116
90-102
117-120
121-123
95-97
C42H50N4O7‚0.3H2O
C39H52N4O7
benzimidazol-2-yl-(CH2)2CO
98 ( 49 (6)
178 ( 72 (5)
190 ( 60 (5)
180 ( 120 (5)
532 ( 105 (5)
278 ( 110 (5)
d
160 ( 65 (5)
238 ( 115 (5)
125 ( 53 (5)
88 ( 30 (5)
C44H52N6O6‚1.1H2O
C39H48N6O6S2
C39H49N7O6S
[2-(5-methyl-1,3,4-thiadiazolyl)]S-CH2-CO
[3-(4-methyl-1,2,4-triazolyl)]S-CH2-CO
[3-(2-methyl-1,2,4,5-tetrazolyl)]S-CH2-CO
(2,3-dimethoxyphenyl)NH-CO
1(R)-hydroxy-3-phenylpropionyl
2-isoquinolinyl-CO
C38H48N8O6S
C
43H53N5O8
C43H52N4O7‚0.7H2O
44H49N5O6
C
(2-hydroxyphenyl)(CH2)2CO
C43H52N4O7
C44H52N4O8
C44H52N4O8
C44H54N4O8
2-hydroxy-4-methoxycinnamoyl
3-hydroxy-4-methoxycinnamoyl
(3-hydroxy-4-methoxyphenyl)CH2-CH2-CO
18
5.8
7.7
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
d
elemental analyses within (0.4% of the calculated value for C,H,N were obtained. No activity at 3 µM.
importance of aromatic substituents in P3 for activity
has been reported by others,23 and our results with 28-
39 support this finding. Again, for this series, antiviral
activity cannot be predicted solely from the Ki values;
the most drastic example is the unexpected lack of anti-
HIV activity of compound 35 containing the isoquinolyl
moiety (Table 4). The isoquinolyl group is part of potent
inhibitors of HIV PR, eg., saquinavir,24 which contains
the hydroxyethylamine isostere. Remarkably, deriva-
tives 29 and 39 show about 2.5-fold enhancement of
antiviral activity as compared to 2. In another series
of derivatives an even more pronounced beneficial effect
of the benzimidazole and hydroxy/methoxy-substituted
phenyl moieties was observed.8,25
Finally, we tried to enhance the antiviral activity of
2 by elongation of the para-substitutent in the P1′-
position (40-46, Table 5). Improved antiviral activity
was obtained for compounds 41-46, which agrees well
with the results of Thompson et al.11 In particular the
ethoxymorpholinyl-substituted compound 42 showed
significantly increased antiviral potency in cells (IC50
) 47 nM).
saline (Table 6). While the benzimidazole inhibitor 1
did not reach the systemic circulation after oral dosage
to a significant extent (AUC ) 0.6 µM‚h), the truncated
derivative 12 showed substantial oral bioavailability in
mice (AUC ) 126.6 µM‚h, about 200-fold superior to 1).
In contrast to our initial assumption,25 this finding
indicates that the low oral adsorption of 1 cannot be
attributed solely to the presence of the benzimidazole
group since this structural feature is shared by 12. The
AHI-containing compounds 2 and 24, too, were orally
bioavailable. The absolute oral bioavailability of 2 in
mice was determined to be 47%. Thus, the inhibitor
levels after oral administration remained far above the
concentrations needed to efficiently block HIV replica-
tion in vitro for a prolonged time.21 Introduction of the
benzimidazole group in P3 which markedly increased
anti-HIV activity (compound 29) led to a complete loss
of oral bioavailability. Introduction of other P3 residues
also resulted in significantly reduced oral uptake or even
abolished it completely (30-39). The 3-hydroxy-4-
methoxyphenyl group in 39, which had led to enhanced
antiviral activity and oral uptake in another series of
inhibitors,25 in the present case only led to an increase
in the anti-HIV effect but concomitantly to loss of oral
Selected compounds were tested for oral bioavailabil-
ity in mice and for solubility in phosphate-buffered

2064 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Lehr et al.
Ta ble 5. Truncated Inhibitors Modified in P1′: Inhibitory Activity against HIV-1 PR and Antiviral Activity against HIV-1, IIIB, in
MT4 Cells
no.
R
Ki (nM)a
IC50 (nM)b
mp (°C)
formulac
2
40
41
42
43
44
45
46
O-CH3
O-CH2-CH3
O-CH2-CH2-OH
O-CH2-CH2-4-morpholinyl
CH2-NHCO-CH3
CH2-NHCO-NH-Ph
9.5
9.9
16
9.5
5.7
3.3
9.9
4.1
254 ( 40 (5)
256 ( 109 (5)
155 ( 60 (5)
47 ( 20 (5)
99 ( 68 (5)
175 ( 80 (5)
142 ( 63 (5)
161 ( 66 (5)
146-148
104-109
109-113
oil
C42H50N4O7‚0.3H2O
C43H52N4O7‚1.0H2O
C43H52N4O8‚1.9H2O
C47H59N5O8‚1.5H2O
C44H53N5O7
C49H56N6O7
C50H57N5O8
C52H59N7O7‚0.9H2O
96-99
106-108
94-97
CH2-NHCO-O-CH2-Ph
CH2-NHCO(CH2)2-2-benzimidazolyl
115-118
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
elemental analyses within (0.4% of the calculated value for C,H,N were obtained.
Ta ble 6. Oral Bioavailability of AHPPA-Containing HIV PR
Inhibitors in Mice
triazolylthioacetic acid (31), was also correlated with
lower systemic uptake.
aqueous
solubilityc
(µM)
oral bioavailabilitya
AUC (0 f ∞) (µM‚h) Cmax (µM)
To summarize the results, systematic structural
modifications of lead structure 1 led to compounds of
different size and polarity and with a varying number
of peptide bonds. From the effect of the structural
modifications on oral bioavailability, the following con-
clusions may be drawn. 1. Reduced size of an inhibitor
alone will not lead to enhanced oral bioavailability. 2.
Structural features regarded as beneficial for oral
uptake in one series of compounds may not enhance
bioavailability in other types of inhibitors. 3. Only a
very narrow band of tolerated modifications exists in
the case of compound 2. 4. In our series of compounds,
there is no correlation between solubility and oral
bioavailability. Due to the poor predictability of the
pharmacokinetic behavior, only the testing of extended
series of compounds will allow the identification of drug
candidates with optimal combination of the desired
properties.
no.
1
2
9
0.6
82.5
0.8
126.6
136.9
b
60.3
1.6
7.4
11.8
b
0.2
23.9
5.9
b
b
b
0.2
16.5
0.4
9.1
12.3
b
20.0
0.7
2.8
1.8
b
<1
<1
<1
9
2.9
12.2
39
12
24
29
30
31
32
36
38
39
40
41
42
43
44
45
46
260
78
0.25
4.3
2.6
b
b
b
<1
16.5
14.5
25.3
<1
b
b
b
b
<1
<1
In conclusion, we have synthesized a series of AH-
PPA-containing HIV PR inhibitors in a search to
adequately balance antiviral activity and oral bioavail-
ability, since only compounds which sufficiently fulfill
both criteria are expected to be effective antiviral drugs
in vivo. Compound 2 was chosen for further develop-
ment due to its good pharmacokinetic profile, although
it is not the most potent antiviral derivative of the
present study. Additional antiviral and pharmacoki-
netic characterization of 2 has been already published
elsewhere.21
a
Compounds were administered in a liquid formulation to mice
with an oral dose of 125 mg/kg. Concentration of compounds in
whole blood were determined at various points of time. Maximal
observed concentration (Cmax) and area-under-the-curve (AUC) are
b
given. No compound detectable in blood above the detection limit
of 0.1 µM. c Solubility in aqueous phosphate-buffered saline (pH
7.4).
uptake. Obviously, structural elements which seem to
favor oral uptake cannot be exchanged between different
groups of HIV PR inhibitors. Our findings indicate that
rather the overall structural features of a compound
govern the oral bioavailability.
Exp er im en ta l Section
Even more striking are the observed effects of subtle
variations of the para substituents of the P1′ residue of
compound 2: Replacing the methoxy substituent by
ethoxy (40) or an hydroxyethoxy group (41) reduced oral
bioavailability by a factor of 4 and 14, respectively. All
other variations in P1′ (42-46) abolished oral bioavail-
ability totally. Whereas the aqueous solubility in-
creased slightly by introduction of more polar substit-
uents in the 4-position of the P1’ benzylamine residue
(CH2NHCOCH3 (43) > O(CH2)2-(4-morpholinyl) (42) >
O(CH2)2-OH (41) > OCH3 (2)), the oral bioavailability
was lowered. Enhancement of aqueous solubility due
to the introduction of polar substituents in P3, e.g.,
Ch em istr y. 1H-NMR spectra were recorded with a Bruker
WC-250 or AMX-500 spectrometer; chemical shifts are given
in ppm (δ) relative to Me4Si as internal standard. J values
are given in hertz (Hz). Elemental analyses were performed
by the Analytical Department, Sandoz Basle, Switzerland, and
Mikroanalytisches Laboratorium, Institut fu¨r Physikalische
Chemie, Universita¨t Wien, and are within (0.4% of the
theoretical value. Optical rotations were measured on a
Perkin-Elmer 141 polarimeter. Analytical thin-layer chroma-
tography was performed on silica gel 60 F254 glass plates
(HPTLC, E. Merck). Preparative column chromatography was
performed on silica gel (40-63 µm) under pressure (∼0.2 mPa).
Solvents were AR grade and used without further purification.
Evaporations were carried out in vacuo with a rotary evapora-

HIV-1 PR Inhibitors Containing Substituted AHPPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2065
tor (Bu¨chi). Melting points were determined with a thermovar
apparatus (Reichert-J ung) and are not corrected. Representa-
tive methods for all compounds synthesized according to
Schemes 1 and 2 are described.
amino]-5-phenylpentan(2′-hydroxy-1′-indanyl)amide dihydro-
chloride (8), and 940 mg (1.81 mmol) of PyBOP was dissolved
in 100 mL of dimethylformamide. Diisopropylethylamine (930
mg, 7.3 mmol) was added at room temperature, and the
mixture was stirred for 12 h. The solvent was evaporated,
and the residue was dissolved in ethyl acetate (140 mL). The
solution was washed with 1 N HCl, saturated NaHCO3
solution, and brine (60 mL each), concentrated in vacuo to 20
mL, and filtered over silica gel (40 g of silica gel 60, glass frit
G3, eluent 300 mL of ethyl acetate). The filtrate was evapo-
rated in vacuo, and the residue was crystallized from 60 mL
of toluene/tert-butyl methyl ether (1/4): yield 932 mg of 2
(2R,3S,4S)-4-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-h ydr oxy-2-[(4-m eth oxyben zyl)am in o]-5-ph en ylpen tan o-
ic Acid Eth yl Ester (4). To a solution of 20 g (59.6 mmol) of
4(S)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,3-epoxy-5-
phenylpentanoic acid ethyl ester (3a ,b)8 in 250 mL of ethanol
was added 16.4 g (119.5 mmol) of 4-methoxybenzylamine. The
mixture was stirred at 70 °C for 12 h. The solvent was distilled
off in vacuo, and the residue was dissolved in 400 mL of ethyl
acetate, washed with 1 N HCl, saturated NaHCO3 solution,
and brine, and dried over MgSO4. The solvent was evaporated
in vacuo, and the residue was chromatographed on silica gel
(solvent: cyclohexane/ethyl acetate ) 3/1): yield 24.9 g of 4
(71%); mp 146-148 °C; [R]20 ) -26.1° (c ) 1, methanol); 1H-
D
NMR (CDCl3) δ 0.89 (s, 9 H), 2.58 (bs, 1 H), 2.91-3.10 (m, 3
H), 3.17 (AX of ABX, J AX ) 5 Hz, J AB ) 15 Hz, 1 H), 3.35 (d,
J ) 8 Hz, 1 H), 3.61 (s, 2 H), 3.70 (d, J ) 10 Hz, 1 H), 3.75 (s,
3 H), 3.89 (d, J ) 8 Hz, 1 H), 4.38 (q, J ) 7 Hz, 1 H), 4.62 (bs,
1 H), 4.69 (bs, 1 H), 4.90 and 5.02 (AB, J AB ) 12 Hz, 2 H), 5.14
(d, J ) 8 Hz, 1 H), 5.40 (dd, J ) 5, 8 Hz, 1 H), 6.36 (d, J ) 9
Hz, 1 H), 6.78 (d, J ) 9 Hz, 2 H), 7.12-7.36 (m, 16 H), 7.94 (d,
J ) 9 Hz, 1 H). Anal. (C42H50N4O7) C,H,N.
(88%) as an oil which crystallized upon prolonged storage; mp
1
73-77 °C; [R]20 ) -15.4° (c ) 1, CH3OH); H-NMR (CDCl3)
D
δ 1.26 (t, J ) 7 Hz, 3 H), 1.37 (s, 9 H), 2.78-3.01 (m, 2 H),
3.30 (d, J ) 7 Hz, 1 H), 3.53 and 3.73 (AB, J ) 12.5 Hz, 2 H),
3.66-3.71 (m, 1 H), 3.80 (s, 3 H), 4.02 (q, J ) 8 Hz, 1 H), 4.20
(q, J ) 7 Hz, 2 H), 4.79 (d, J ) 9 Hz, 1 H), 6.82 (s, 1 H), 6.85
(s, 1 H), 7.15-7.34 (m, 7 H). Anal. (C26H36N2O6) C,H,N.
(2R,3S,4S)-4-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-h ydr oxy-2-[(4-m eth oxyben zyl)am in o]-5-ph en ylpen tan o-
ic Acid (5). To a solution of 42.6 g (90.1 mmol) of 4 in 500
mL of tetrahydrofuran was added 99 mL (99 mmol) of 1 N
LiOH, and the reaction mixture was stirred for 12 h at room
temperature. Neutralization with 1 N HCl led to a precipitate,
which was filtered off and dried: yield 33.3 g of 5 (83%); mp
203-206 °C; 1H-NMR (CDCl3/DMSO-d6 ) 4/1) δ 1.38 (s, 9 H),
2.85-2.98 (m, 2 H), 3.34 (d, 1 H), 3.80 (s, 3 H), 3.85 (d, 2 H),
3.94 (d, 1 H), 4.02 (q, 1 H), 5.75 (bs, 1 H), 6.86 (d, 2 H), 7.15-
7.40 (m, 7 H). Anal. (C24H32N2O6) C,H,N.
(2R,3S,4S)-2-[[(4-Am in om eth yl)ben zyl]a m in o]-4-[[(1,1-
d im et h ylet h oxy)ca r b on yl]a m in o]-3-h yd r oxy-5-p h en yl-
p en ta n oic Acid Eth yl Ester (15). A solution of 38 (10 g, 30
mmol) and R,R-diamino-p-xylene (12.2 g, 90 mmol) in 600 mL
of ethanol was kept at 80 °C for 20 h. The solvent was
evaporated, and the residue was chromatographed on silica
gel (ethyl acetate/methanol/aqueous ammonia ) 10/10/1): yield
1
8.6 g of 15 (61%); H-NMR (CDCl3) δ 1.25 (t, J ) 7 Hz, 3 H),
1.37 (s, 9 H), 2.45 (bs, 4 H), 2.78-2.90 (m, 2 H), 3.3 (d, J ) 8
Hz, 1 H), 3.58 and 3.74 (AB, J ) 13 Hz, 2 H), 3.66 (d, J ) 8
Hz, 2 H), 3.8 (bs, 1 H), 4.00-4.23 (m, 3 H), 5.04 (d, J ) 9 Hz,
1 H), 7.10-7.33 (m, 9 H). Anal. (C26H37N3O5) C,H,N.
(2R,3S,4S)-2-[[4-[[[2-(Ben zim id a zol-2-yl)p r op ion yl]a m i-
n o]m et h yl]b en zyl]a m in o]-4-[[(1,1-d im et h ylet h oxy)ca r -
bon yl]a m in o]-3-h yd r oxy-5-p h en ylp en ta n oic Acid Eth yl
Ester (16). A solution of 15 (660 mg, 1.4 mmol) and 2-(benz-
imidazol-2-yl)propionic acid (266 mg, 1.4 mmol) in a mixture
of dimethylformamide (5 mL) and 1,4-dioxane (5 mL) was
treated with 1-hydroxybenzotriazole (229 mg, 1.4 mmol) and
N-ethyl-N′-[3-(dimethylamino)propyl]carbodiimide hydrochlo-
ride (268 mg, 1.4 mmol) at 0 °C. The reaction mixture was
stirred for 3 days at room temperature. After evaporation of
the solvents the residue was chromatographed on silica gel
(ethyl acetate/methanol ) 20/1): yield 598 mg of 16 (76%); mp
(2R,3S,4S,1′S,2′R)-4-[[(1,1-Dim eth yleth oxy)ca r bon yl]-
a m i n o ]-3-h y d r o x y -2-[(4-m e t h o x y b e n z y l)a m i n o ]-5-
p h en ylp en ta n (2′-h yd r oxy-1′-in d a n yl)a m id e (7). 5 (11.9 g,
26.7 mmol) was dissolved in 1.3 L of dimethylformamide; 4.0
g (26.8 mmol) of 6, 3.60 g (26.6 mmol) of 1-hydroxybenzotri-
azole, and 5.20 g (27.0 mmol) of N-ethyl-N′-[3-(dimethylamino)-
propyl]carbodiimide hydrochloride were added at room tem-
perature. The reaction mixture was stirred for 3 days at room
temperature. The solvent was evaporated, and ethyl acetate
was added. The solution was washed with 1 N HCl, saturated
NaHCO3 solution, and brine and dried over MgSO4, and the
solvent was evaporated; 70 mL of a mixture of cyclohexane/
ethyl acetate (1/2) was added to the oily residue. The title
compound crystallized overnight. The crystals were filtered
off, washed with cold cyclohexane/ethyl acetate (1/2), and dried
1
85-90 °C; H-NMR (CDCl3) δ 1.22 (t, J ) 7 Hz, 3 H), 1.35 (s,
9 H), 2.72 (t, J ) 7 Hz, 2 H), 2.87 (d, J ) 7 Hz, 2 H), 3.12 (t,
J ) 7 Hz, 2 H), 3.30 (d, J ) 8 Hz, 1 H), 3.47 and 3.64 (AB, J
) 13 Hz, 2 H), 3.65-3.78 (m, 4 H), 4.00-4.20 (m, 3 H), 4.28
(bs, 2 H), 5.14 (d, J ) 9 Hz, 1 H), 6.95 and 7.04 (2 d, J ) 8 Hz,
4 H), 7.10-7.30 (m, 8 H), 7.40-7.60 (bs, 2 H). Anal.
(C36H45N5O6) C,H,N.
in vacuo: yield 8.45 g of 7 (55%); mp 183-185 °C; [R]20
)
D
-6.7° (c ) 1, methanol); 1H-NMR (CDCl3) δ 1.36 (s, 9 H), 1.91
(d, J ) 7.5 Hz, 2 H), 2.99 and 3.09 (AB of ABX, J AX ) 5.4 Hz,
J BX ) 2.2 Hz, J AB ) 16 Hz, 2 H), 3.38 (d, J ) 8 Hz, 1 H), 3.56
and 3.61 (AB, J AB ) 12 Hz, 2 H), 3.79 (s, 3 H), 3.88 (d, J ) 7
Hz, 1 H), 4.03-4.15 (m, 1 H), 4.60-4.68 (m, 1 H), 5.06 (d, J )
10 Hz, 1 H), 5.37 (dd, J ) 7, 9.5 Hz, 1 H), 6.79 (d, J ) 8.5 Hz,
2 H), 7.15 (d, J ) 8.5 Hz, 2 H), 7.10-7.24 (m, 9 H), 8.22 (d, J
) 9 Hz, 1 H). Anal. (C33H41N3O6) C,H,N.
(2R,3S,4S,1′S,2′R)-2-[[4-[[[2-(Ben zim id a zol-2-yl)p r op io-
n yl]a m in o]m eth yl]ben zyl]a m in o]-4-[[(1,1-dim eth yleth ox-
y)car bon yl]am in o]-3-h ydr oxy-5-ph en ylpen tan (2′-h ydr oxy-
1′-in d a n yl)a m id e (23). Saponification of 16 was carried out
following the procedure given for 5. Coupling of the free acid
to 6 was done as described for 7: yield 72%; mp 128-132 °C;
1H-NMR (CDCl3) δ 1.35 (s, 9 H), 2.18 (bs, 1 H), 2.44 and 2.62
(2 m, 2 H), 2.55 (bs, 3 H), 2.83 (m, 1 H), 2.95 and 3.02 (AB
part of ABX, J ) 15, 10, 2 Hz, 2 H), 3.05 and 3.17 (AB part of
ABX, J ) 16, 6 Hz, 2 H), 3.43 (d, J ) 5 Hz, 1 H), 3.47 and 3.75
(AB, J ) 15 Hz, 2 H), 3.94 and 4.43 (AB part of ABX, J ) 15,
8, 6 Hz, 2 H), 4.18 (m, 2 H), 4.73 (bs, 1 H), 5.26 (d, J ) 10 Hz,
1 H), 5.44 (dd, J ) 5, 9 Hz, 1 H), 6.65 and 6.83 (2 d, J ) 8 Hz,
4 H), 7.03 and 7.13 (2 t, J ) 7 Hz, 2 H), 7.15-7.30 (m, 9 H),
7.44 (bs, 2 H), 8.28 (d, J ) 9 Hz, 1 H), 8.73 (bs, 1 H). Anal.
(C40H46N6O6‚2.5H2O) C,H,N.
Ster eospecific Syn th esis of (1S,2R)-1-Am in o-2-h ydr oxy-
in d a n (6). (1S,2S)-1-Amino-2-hydroxyindan Dibenzoyl-D-
tartrate. A solution of 145 g (0.97 mol) of racemic trans-1-
amino-2-hydroxyindan17 in 3.5 L of 95% ethanol was added to
a stirred solution of 348.4 g (0.97 mol) of (+)-dibenzoyl-D-
tartaric acid monohydrate in 1.36 L of 95% ethanol at room
temperature. The mixture was allowed to stir for an additional
5 min. After 3 h the precipitate was filtered off, washed with
(2R,3S,4S,1′S,2′R)-4-[[[N-[(Ben zyloxy)ca r bon yl]-L-ter t-
leu cyl]a m in o]-3-h yd r oxy-2-[(4-m eth oxyben zyl)a m in o]-5-
p h en ylp en ta n (2′-h yd r oxy-1′-in d a n yl)a m id e (2). 7 (6 g,
10.4 mmol) was dissolved in a mixture of 20 mL of dichlo-
romethane and 4 mL of methanol; 300 mL of 3 N hydrochloric
acid in ether was added, and the mixture was stirred for 3 h
at room temperature. The precipitate was filtered off, washed
with ether, and dried in vacuo to yield 5.48 g of (2R,3S,4S,-
1′S,2′R)-4-amino-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phe-
nylpentan(2′-hydroxy-1′-indanyl)amide dihydrochloride (8)
(96%), mp 147-149 °C, an aliquot of which was used in the
next step without further purification: [R]20 ) -8.1° (c ) 1,
D
methanol); 1H-NMR (DMSO-d6) δ 2.80-3.20 (m, 4 H), 3.80 (s,
3 H), 3.90-4.43 (m, 5 H), 4.50-4.60 (m, 1 H), 5.18-5.38 (m, 3
H), 6.70-7.05 (m, 3 H), 7.10-7.58 (m, 10 H), 8.08 (bs, 2 H),
8.45-8.60 (m, 1 H). Anal. (C28H35Cl2N3O4) C,H,N.
Z-tert-Leucine (480 mg, 1.81 mmol), 1.0 g (1.82 mmol) of
(2R,3S,4S,1′S,2′R)-4-amino-3-hydroxy-2-[(4-methoxybenzyl)-

2066 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Lehr et al.
900 mL of ethanol, transferred in a round-bottom flask, and
refluxed for 3 h with 4.9 L of ethanol. After standing for 1
day at room temperature, the precipitate was filtered off,
washed with 900 mL of ethanol, and dried in a vacuum oven
at 50 °C. A recrystallized sample was kept as seed crystals:
yield 167 g of (1S,2S)-1-amino-2-hydroxyindan dibenzoyl-D-
of the substrate H-Lys-Ala-Arg-Val-Leu-pNph-Glu-Ala-Nle-
NH2, originally described by Richards et al.28 Briefly, HIV-
PR was incubated at 37 °C in 0.1 M morpholinoethanesulfonic
acid, 0.37 M NaCl, and 4 mM EDTA, pH 6.25, with 280 µM
substrate in the presence and absence of inhibitors. From the
decrease of absorbance at 298 nm, initial reaction rates were
calculated.
IC50 values for test compounds were obtained by fitting the
initial velocity data (V) from the inhibition of substrate
hydrolysis to the equation V ) V0‚IC50/(I + IC50), where I
denotes the inhibitor concentration and V0 the velocity of the
uninhibited reaction. Kinetic constants Ki were calculated
from IC50 values by using the equation IC50 ) Et/2 + Ki(1 +
S/Km), where Et is the total enzyme concentration, S is the
substrate concentration, and Km is the Michaelis constant for
the substrate.29 Ki values reported here are the mean of two
determinations, which usually yielded the same result within
limits of (20%.
tartrate (34%); mp 209-210 °C; [R]20 ) + 100.9° (methanol,
D
c ) 1.11). Anal. (C27H25NO9) C,H,N.
(1S,2S)-1-Am in o-2-h yd r oxyin d a n . A suspension of 166.9
g (329 mmol) of (1S,2S)-1-amino-2-hydroxyindan dibenzoyl-
D-tartrate in 2 L of ethyl acetate and 600 mL (900 mmol) of
1.5 N NaOH was extracted in a perforator for 4 h. The organic
layer was filtered, the solvent was evaporated, and the residue
was dried in vacuo at 50 °C to give 47.3 g of (1S,2S)-1-amino-
2-hydroxyindan (96%): mp 144-145 °C; [R]20 ) + 22.8°
D
1
(methanol, c ) 1.12); H-NMR (CDCl3) δ 2.30 (bs, 3 H), 2.95
and 3.10 (AB of ABX, J AB ) 16 Hz, J AX ) 3 Hz, J BX ) 5 Hz, 2
H), 4.3-4.5 (m, 2 H), 7.2-7.4 (m, 4 H). Anal. (C9H11NO)
C,H,N.
In h ibition of HIV-1-In d u ced Cytop a th ic Effect in MT4
Cells. The assay procedure described by Pauwels et al.30 was
used with minor modifications. The HTLV I-transformed cell
line MT4 was used as the target cell. Inhibition of HIV-1,
strain IIIB,31 induced cytopathic effect was determined by
measuring the viability of both HIV- and mock-infected cells.
Viability was assessed spectrophotometrically via in situ
reaction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT). Virus-infected and uninfected cultures with-
out compound were included as controls as were uninfected
cells treated with compound. The cell concentration was
chosen so that the number of cells/mL increased by a factor of
10 during the 5 days of incubation in mock-infected cultures.
Virus inoculum was adjusted such to cause cell death in 90%
of the target cells after 5 days of incubation. The virus was
adsorbed to a cell suspension containing 1 × 106 cells/mL at
37 °C for 1 h. Then, the infected cells were added to microtiter
plates containing the test compounds to give 1 × 105 cells/
mL. Thus, compounds were added postadsorption.
P h a r m a cok in etic Stu d ies. Female Balb/c mice were
used. For oral administration (by gavage) the animals were
fasted for 24 h prior to the start of and throughout the
experiment; water was given ad libitum. For analysis, blood
was collected in heparinized tubes. Samples (typically 0.4-
1.0 mL) were frozen in liquid nitrogen and stored at -70 °C.
After thawing, 200 µL of a suitable internal standard (0.01
mg/mL in methanol) and 4 vol of methanol were added. For
calibration, blood samples were spiked with 10-10 000 ng of
test compound, and then internal standard and methanol were
added as above. Samples were mixed for 10 min and then
centrifuged at 4000 rpm, 4 °C, for 10 min. The supernatants
were removed and dried in vacuo.
For solid-phase extraction of the compounds, 1 mL dispos-
able sulfobenzyl extraction columns (Baker) were used. The
dried supernatant of the methanol extraction, resuspended in
10% methanol in 10 mM NH4Ac, pH 4.0, was applied followed
by successive washes (1 mL each) with 10 mM NH4Ac, pH 4.0
(twice), methanol, hexane, and 10% trifluoroacetic acid. Com-
pounds were eluted with 5% ammonia in methanol. Eluates
were dried in vacuo.
For HPLC analysis, residues were taken up in 100 µL of
0.1% trifluoroacetic acid and 10% methanol in water. Aliquots
(20-100 µL) were injected into the HPLC system (HP1500;
column, Vydac RP-C18, 5 µm, 4 × 250 mm; isocratic elution
with 50% or 60% acetonitrile in 10 mM NH4Ac, pH 4.0; flow,
1 mL/min; 25 °C; UV detection at 200 nm). Drug concentration
in the samples was calculated by least-squares linear regres-
sion analysis of the peak area ratio (inhibitor/internal stan-
dard) of the spiked blood standards versus concentration.
(1S,2S)-1-(Ben zoyla m in o)-2-h yd r oxyin d a n . A solution
of 46.9 g (314 mmol) of (1S,2S)-1-amino-2-hydroxyindan and
65.3 mL (510 mmol) of triethylamine in 745 mL of DMF was
cooled to 5 °C; 44.2 g (314 mmol) of benzoyl chloride was added
via a dropping funnel within 30 min. The ice bath was
removed, and the mixture was allowed to stir for 1 h at room
temperature. The reaction mixture was poured on 2 L of ice
water; the product was collected by filtration, washed with 2
× 200 mL of H2O, and dried in a vacuum oven overnight: yield
72.5 g of (1S,2S)-1-(benzoylamino)-2-hydroxyindan (91%); mp
228-230 °C; [R]20 ) + 117.4° (methanol, c ) 0.57); 1H-NMR
D
(DMSO-d6) δ 2.75 and 3.17 (AB of ABX, J AB ) 15 Hz, J AX ) 7
Hz, J BX ) 8 Hz, 2 H), 4.35 -4.52 (m, 1 H), 5.24-5.38 (m, 1 H),
7.03-7.21, 7.40-7.60, 7.90-7.98 (m, 9 H), 8.76 (d, J ) 9 Hz,
1 H). Anal. (C16H15NO2) C,H,N.
(4S,5R)-2-P h en yl-3a ,8a -d ih yd r o-8H -in d en o-[1,2-d ]ox-
a zole. (1S,2S)-1-(Benzoylamino)-2-hydroxyindan (71.8 g, 283
mmol) was suspended in 950 mL of dichloromethane and
cooled in an ice bath; 60 mL of thionyl chloride was added via
a dropping funnel. The ice bath was removed, and the mixture
was allowed to stir at room temperature for 3 h and then
concentrated to dryness. The residue was suspended in 200
mL of saturated NaHCO3 and extracted into 4 × 250 mL of
ethyl acetate. The combined organic layers were dried over
MgSO4 and filtered through Celite. The solvent was evapo-
rated in vacuo to yield 66.0 g of (4S,5R)-2-phenyl-3a,8a-
dihydro-8H-indeno[1,2-d]oxazole (92%): mp 113-115 °C; [R]20
D
1
) -177.1° (methanol, c ) 0.99); H-NMR (DMSO-d6) δ 3.38
and 3.55 (AB of ABX, J AB ) 16 Hz, J AX ) 8 Hz, J BX ) 8 Hz, 2
H), 5.50 (t, J ) 8 Hz, 1 H), 5.78 (d, J ) 8 Hz, 1 H), 7.20-7.50
(m, 6 H), 7.60 (m, 1 H), 7.95 (m, 2 H). Anal. (C16H13NO)
C,H,N.
(1S,2R)-1-Am in o-2-h yd r oxyin d a n (6). A suspension of
66 g (280 mmol) of (4S,5R)-2-phenyl-3a,8a-dihydro-8H-indeno-
[1,2-d]oxazole in 600 mL of 20% H2SO4 was heated to reflux
for 10 h. The mixture was cooled in an ice bath; benzoic acid
was filtered off. The filtrate was adjusted to pH >12 with 10
N NaOH and extracted with 2 × 500 mL of dichloromethane.
The combined organic layers were washed with brine and dried
over MgSO4. The solvent was evaporated in vacuo to yield
37.7 g of 6 (92%): mp 115-117 °C; [R]20D ) -38.0° (methanol,
1
c ) 1.11) (lit.17 [R]20 ) -62.0° (methanol, c ) 1.0)); H-NMR
D
(DMSO-d6) δ 2.75 and 2.83 (AB of ABX, J AB) 16 Hz, J AX) 5
Hz, J BX ) 2 Hz, 2 H), 4.03 (d, J ) 5 Hz, 1 H), 4.21 (dt, J ) 2,
5 Hz, 1 H), 7.20 (m, 3 H), 7.35 (m, 1 H). Anal. (C9H11NO)
C,H,N.
Biology. All derivatives were dissolved in dimethyl sulf-
oxide to 10 mM. For cellular assays, these stock solutions were
diluted into cell culture medium containing 10% fetal bovine
serum and for enzyme assays into appropriate buffer (see
below); the concentration of dimethyl sulfoxide did not exceed
0.3% in cellular assays and 5% in enzymatic assays.
HIV P R In h ibition Assa y. HIV-1 PR was expressed in
Escherichia coli, strain J M 105, using the expressor plasmid
pTZprt+ 26 and purified to homogeneity as described before.27
Enzymatic activity was measured by following the cleavage
Deter m in ation of Aqu eou s Solu bility. A weighed amount
(about 1 mg) of inhibitor in amorphous form (lyophilized out
of dioxane to constant weight) was combined with 1 mL of PBS.
Samples were vortexed vigorously for 1 min and sonicated at
37 °C for 30 min. The suspensions were filtered through a
G3 sinter funnel (or alternatively a Pasteur pipet filled with
a cotton plug), and the filtrate was centrifuged at 100 000 rpm
for 30 min. The supernatant was analyzed by HPLC (column,

HIV-1 PR Inhibitors Containing Substituted AHPPA
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 2067
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of 2-Heterosubstituted Statine Derivatives - Implications for the
Design of Novel Inhibitors. J . Med. Chem. 1995, 38, 4917.
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RP-C18; isocratic elution with 10 mM aqueous NH4OAc/CH3-
CN ) 50/50; flow rate, 1 mL/min; UV detection at 210 nm;
room temperature). The concentration of the compound was
calculated by external standardization of peak area versus
concentration for each sample: minimal detectable concentra-
tion, 0.1 µg/mL; standard deviation, (10%.
Ack n ow led gm en t. We thank C. Aberham, R. Cson-
ga, M. Hu¨bner, M. Kern, L. Malacek, E. Mlynar, G.
Polzer, A. Pruckner, H. Schmidt, L. Strommer, K.
Wagner, S. Weber-Roth, and G. Winkler for technical
assistance.
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