Structure-activity relationship of HIV-1 protease inhibitors containing α-hydroxy-β-amino acids. Detailed study of P1 site

Article abstract of DOI:10.1016/S0968-0896(99)00127-3

The structure-activity relationship of HIV-1 protease (HIV-1 PR) inhibitors containing α-hydroxy-β-amino acids is discussed. We demonstrated that substituent groups on the P₁ aromatic rings of the inhibitors exert significant influence on their biological activity. Inhibitors bearing an alkyl or a fluorine atom at the meta and para position on their P₁ benzene ring were found to be good inhibitors. We also discovered that the substitution positions of the P₂ benzamides were crucial for good antiviral potency. In this study, inhibitor 48 was the most potent {IC₉₀ (CEM/HIV-1 IIIB) 27 nM} and showed good pharmacokinetics in rats.

Full text of DOI:10.1016/S0968-0896(99)00127-3

Bioorganic & Medicinal Chemistry 7 (1999) 2063±2072
Structure±Activity Relationship of HIV-1 Protease Inhibitors
Containing ꢀ-Hydroxy-ꢁ-amino Acids. Detailed Study of P1 Site
Eiji Takashiro, a,* Ichiro Hayakawa, a Tamayo Nitta, a Atsushi Kasuya, a
Shuichi Miyamoto, a Yuji Ozawa, b Ryuichi Yagi, b Ikue Yamamoto, b
Takahiro Shibayama, c Akihiko Nakagawa c and Yuichiro Yabe a,*
aExploratory Chemistry Research Laboratory, Sankyo Co. Ltd. 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
bBiological Research Laboratory, Sankyo Co. Ltd. 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
cAnalytical and Metabolic Research Laboratories, Sankyo Co. Ltd. 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 26 March 1999; accepted 6 May 1999
AbstractÐThe structure±activity relationship of HIV-1 protease (HIV-1 PR) inhibitors containing a-hydroxy-b-amino acids is
discussed. We demonstrated that substituent groups on the P1 aromatic rings of the inhibitors exert signi®cant in¯uence on their
biological activity. Inhibitors bearing an alkyl or a ¯uorine atom at the meta and para position on their P1 benzene ring were found
to be good inhibitors. We also discovered that the substitution positions of the P2 benzamides were crucial for good antiviral
potency. In this study, inhibitor 48 was the most potent {IC90 (CEM/HIV-1 IIIB) 27 nM} and showed good pharmacokinetics in
rats. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
aromatic ring system which ®ts into the S1 hydrophobic
pocket is very important. Therefore, investigating the
structure±activity relationship of the P1 site became of
interest to us. To the best of our knowledge there has
been no previous e€ort carried out to discuss the
importance of substitution positions on the P1 benzene
rings. In developing more active compounds, additional
optimization work was focused on the P1 phenylalanine
portion (Fig. 1).
The importance of HIV-1 protease inhibitors in the
treatment of AIDS is now well established. (for selected
reviews on HIV-1 protease inhibitors, see refs 1 and 2)
More recent advances in inhibitor design have focused
on structure modi®cation to improve potency, oral
bioavailability3,4 and to circumvent drug resistance.5±11
Despite its success, it is nearly impossible to treat all of
the growing world-wide AIDS population based on
currently available drugs. Therefore, the need for inex-
pensive and widely available HIV protease inhibitors
still exists.
Results and Discussion
Chemistry
Pioneering work by Kiso et al. demonstrated that
AHPBA (3(S)-amino-2(S)-hydroxy-4-phenylbutanoic
acid) has great potential as a transition-state mimic for
HIV-1 protease.12±16 In previous papers, we also repor-
ted that AHPBA-derived HIV-1 PR inhibitors having
All commercially available benzoic acids were pur-
chased, and others were prepared as follows. 3-Hy-
droxy-2-methylbenzoic acid,22 2,4-dimethyl-3-hydroxy-
benzoic acid23 and 2,6-dimethyl-3-hydroxybenzoic acid24
were prepared according to known methods. The prep-
aration of 3-hydroxy-2,5-dimethylbenzoic acid was
carried out by Charlesworth's procedure.25 5-Fluoro-3-
hydroxy-2-methylbenzoic acid 1 was synthesized from
5-¯uoro-2-methylbenzoic acid by nitration, reduction,
diazotization and hydrolysis.
0
4(S)-Cl-Pro at the P1 site showed potent inhibitory
activity against HIV-1 PR, and some of them also
showed good anti-HIV activity.17±19 Kaldor et al.
reported that Nel®navir with an S-phenyl (thiophenyl)
group at the P1 site was a potent inhibitor, which bound
with an approximately 10-fold greater anity than a
phenyl analogue.20,21 This shows that the lipophilic
To enhance the interaction between inhibitor and protease
at the P1 site we chose AHPPA (3-amino-2-hydroxy-5-
phenylpentanoic acid) as our ®rst target molecule.
*
Corresponding authors.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00127-3

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E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
Figure 1. Design of HIV PR inhibitors with AHPPA (3-amino-2-hydroxy-5-phenylpentanoic acid) or substituted AHPBA (3-amino-2-hydroxy-4-
phenylbutanoic acid).
Unfortunately, AHPPA could not be synthesized by the
well-known Reetz's procedure,26 so we had to develop a
new synthetic route toward AHPPA (Scheme 1).
available optically active amino acids were purchased
and others were prepared by resolution of N-acyl dl-
amino acids.39 (For resolution of N-acyl dl-amino
acids, see refs 37 and 38.)
One of the most versatile synthetic methods of peptide
isosteres involves the use of chiral amino aldehydes or
Weinreb amides. Several approaches using the addition
of organometallics have also appeared.27 For our study,
we used vinyl magnesium bromide.28±31 N,N-Diethyl-
The linkage of the above b-amino-a-hydroxy carboxylic
acids and 4(S)-Cl-proline-tert-butylamide was carried
out using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
.
imide hydrochloride (EDCI) and 1-hydroxybenzo-
amide32
2
derived from Boc-l-homophenylalanine
triazole (HOBt) in dimethyformamide (DMF). These
dipeptides were coupled with substituted benzoic acids
by the EDCI-HOBt method in good yield.
(Boc=tert-butoxycarbonyl) was converted into the cor-
responding vinyl ketone 3 in 58% yield. Reduction with
.
NaBH4/CeCl3 7H2O gave a 3:1 mixture of diastereo-
mers 4.33 Ozonolysis of 4, followed by reduction with
methylsul®de gave aldehyde, which was oxidized by
Structure±activity relationship
34
NaClO2 to give an acid. This acid was then converted
Table 1 shows the protease inhibitory activity of 3-hy-
droxy-2-methylbenzamide containing compounds.18
into a methyl ester 5 in 52% yield from compound 4. 5
was hydrolyzed in alkaline condition to a€ord diastereo-
merically pure acid 6.
Because compound 8 showed good protease inhibitory
activity (IC50=1.4 nM) but low cellular IC90 value (as
shown in Table 3), we next turned attention to sys-
tematic replacement on P1 benzene rings in AHPBA.
Among compounds 9 to 27, inhibitors 10, 11, 14, 19,
All other 3(S)-amino-2(S)-hydroxy-4-arylbutanoic acids
were prepared by Dondoni's method35 or Ajinomoto's
procedure36 from l-amino acids. All commercially
.
Scheme 1. (a) Vinyl magnesium bromide/THF 58%; (b) NaBH4, CeCI3 7H2O/MeOH 90%; (C) O3, then methylsul®de; (d) NaClO2, NaH2PO4, 2-
.
methyl-2-butene/t-BuOH-H2O; (e) TMSCHN2/benzene±MeOH 52% (3 steps); (f) 1N-NaOHaq/MeOH; (g) 4(S)-Cl-Pro-tert-butylamide, EDCl HCl,
.
HOBt/DMF 78% (2 steps); (h) 4N-HCl/1,4-dioxane; (i) 3-hydroxy-2-methylbenzoic acid, EDCl HCL HOBt/THF 60% (2 steps).

E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
2065
Table 1. HIV-1 PR inhibitory activity of 3-hydroxy-2-methylbenzamide bearing compounds
No.
Ar
IC50 (nM)
1.4
No.
Ar
IC50 (nM)
0.7
No.
Ar
IC50 (nM)
1.3
8
15
22
9
0.9
0.9
16
17
0.8
0.8
23
24
0.9
1.2
10
11
12
0.6
4.5
18
19
1.6
0.6
25
26
1.4
1.5
13
14
0.9
0.6
20
21
0.9
0.5
27
1.0
and 23 were very potent; however, 12 was not a good
inhibitor. The superior potency of the former com-
pounds showed that meta- and para-substitutions were
favourable. These results suggest that aromatic rings
with meta- and para-substitutions at the P1 site may
bind favourably in inhibitor±protease complexes. Com-
paring 12 with 15, the latter with an ortho-¯uorine atom
was more potent as a protease inhibitor. It was con-
cluded that only smaller substituents were acceptable at
the ortho position.
It is well known that protease inhibition is not always
compatible with anti-HIV activity. Compound 8 with
AHPPA showed good protease inhibition but a poor
cellular IC90 value (IC90=730 nM). Although com-
pounds 45 and 48 with a 3-hydroxy-2,5-dimethylben-
zoyl group were of similar potency to 9 with a 3-
hydroxy-2-methylbenzoyl group, 45 and 48 showed
more potency than 9 in a cell-based assay. Although
protease inhibition of 50 was equipotent to the other
compounds, it was much more potent in terms of its
anti-HIV activity. These results suggest that the 5-¯uoro-
3-hydroxy-2-methylbenzoyl or the 3-hydroxy-2,5-di-
methylbenzoyl moieties are important not only for
interaction with protease, but also for cellular penetration.
In our previous paper,19 we found that the substitution
positions of P2 benzamides signi®cantly in¯uenced
inhibitory activity, (for the studies of P2 benzamides, see
refs 40±42) but the best combination of P1 and P2
structures remained unknown. Therefore we were inter-
ested in optimizing P1 and P2 structures (Table 2).
Compound 48 was a good candidate for anti-AIDS
drugs among these inhibitors due to its good pharmaco-
kinetic parameters. When 48 (10 mg/kg) was admini-
stered orally to rats, the AUC, Cmax, Tmax, T1/2 and
It was clearly shown that inhibitors having a methyl
group or a ¯uorine atom at the 5-position were potent.
On the other hand, substitutions at the 4- and 6-position
decreased their inhibitory activity (9 versus 28 versus 29
versus 30 versus 31) (10 versus 41 versus 42) (19 versus
36 versus 37) (21 versus 32 versus 33 versus 34 versus
35). Because we succeeded in developing highly active
protease inhibitors, our interest was now in their anti-
HIV activity. Table 3 summarizes the anti-HIV activity
of selected inhibitors.18
.
bioavailability were as follows: 3.10 mg h/mL, 1.28 mg/
mL, 0.33 h, 1.79 h and 48%, respectively.
Conclusion
Our study revealed that substitution positions of the P1
benzene rings was crucial for their inhibitory activity.
Meta- and para-substitutions on the P1 benzene rings

2066
E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
Table 2. HIV-1 PR inhibitory activity of substituted benzamide bearing compounds
No.
Ar
R1
R2
H
R3
H
IC50 (nM)
8.2
No.
Ar
R1
R2
H
R3
H
IC50 (nM)
8.6
28
Me
41
Me
29
30
H
H
Me
F
H
H
1.3
1.7
42
43
H
H
H
Me
H
3.6
Me
49.9
31
H
H
Me
2.5
44
H
H
Me
34.3
32
33
Me
H
H
H
H
3.5
1.9
45
46
H
H
Me
Me
H
H
1.3
1.2
Me
34
35
H
H
F
H
2.1
4.6
47
48
Me
H
H
H
H
3.2
0.8
H
Me
Me
36
37
38
39
40
Me
H
H
H
H
Me
H
6.3
3.9
2.5
1.3
3.4
49
50
51
52
H
H
H
H
H
F
Me
H
2.8
1.2
1.5
1.9
Me
H
H
Me
F
H
Me
H
H
H
H
Me
increased their biological activity. We also found that
methyl or ¯uorine groups at the 5-positon of P2 benza-
mide moiety signi®cantly contributed to their anti-HIV
activity. Further work to develop more active com-
pounds is now in progress.
N2. Melting points (mp) were determined with a
Yanaco melting point apparatus and are not corrected.
Infrared (IR) spectra were measured with a Nic 5SXC
FT-IR spectrophotometer. 1H NMR spectra were
recorded on a JEOL JNM-GX 270 FT-NMR or a
Varian Mercury 400 spectrometer. Chemical shifts
were expressed in d ppm from the internal standard tet-
ramethylsilane (TMS). Splitting patterns are indicated
as follows: s, singlet; d, doublet; t, triplet; m, multiplet;
br, broad peak. EI- and FAB-MS were taken on a
JEOL JMS-D 300 mass spectrometer and relevant data
Experimental
All experiments dealing with air- and moisture-sensitive
compounds were conducted under an atmosphere of dry

E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
Table 3. Anti-HIV activity of selected inhibitors
2067
concentrated in vacuo. The residue was puri®ed by
¯ash column chromatography (hexane:EtOAc=3:1) to
a€ord 5-¯uoro-2-methyl-3-nitrobenzoic acid benzy-
lester (780 mg, 41%) as a colourless crystalline. 1H
NMR (CDCl3) d 2.58 (s, 3H), 5.03 (s, 2H), 7.34±7.45
(m, 5H), 7.60 (dd, 1H, J1=8.2, J2=2.8 Hz), 7.75 (dd,
1H, J1=8.2, J2=2.8 Hz).
No.
Ar
R1
H
R2
H
R3
H
CEM/HIV-1 IIIB
IC90 (nM)
A suspension of 5-¯uoro-2-methyl-3-nitrobenzoic acid
benzylester (498 mg, 1.72 mmol) and 10% Pd±C
(250 mg) was stirred under H2 (1 atm) at room tem-
perature for 20 h. After changing the atmosphere to N2,
the reaction mixture was diluted with EtOAc. The mix-
ture was ®ltered through a Celite pad (washed with
EtOAc) and concentrated in vacuo. The resulting crude
product, 3-amino-5-¯uoro-2-methylbenzoic acid (257 mg),
was used in the next step without further puri®cation.
1H NMR (CD3OD) d 2.25 (s, 3H), 6.58 (dd, 1H,
J1=9.5, J2=2.6 Hz), 6.75 (dd, 1H, J1=9.5, J2=2.6 Hz).
8
730
9
H
H
H
H
H
H
110
51
24
29
45
H
H
Me
Me
H
H
115
48
To the stirred solution of the above product (245 mg) in
H2SO4 (98%, 3.0 mL) and H2O (1.5 mL) was added a
solution of NaNO2 (105 mg, 1.52 mmol) in H2O (2 mL)
at 0ꢀC and stirred for 2 h at this temperature. The mix-
ture was added dropwise to almost boiling solution of
50% H2SO4 (4 mL). After cooling to room temperature,
the mixture was diluted with Et2O, washed (brine), dried
(Na2SO4) and concentrated in vacuo. Crystallization
from hexane:Et2O gave 187 mg of 5-¯uoro-3-hydroxy-2-
48
50
51
52
H
H
H
H
Me
F
H
H
H
H
27
31
58
80
1
methylbenzoic acid (64%). H NMR (CD3OD) d 2.33
(s, 3H), 6.67 (dd, 1H, J1=9.4, J2=2.7 Hz), 6.98 (dd, 1H,
J1=9.4, J2=2.7 Hz).
Me
F
(tert-Butoxycarbonyl)-homophenylalanyl-diethylamide (2).
To the stirred solution of boc-homophenylalanine
(3.73 g, 13.4 mmol) and NEt3 (3.7 mL, 26 mmol) were
added isobutyl chloroformate (1.8 mL, 14 mmol) and
diethylamine (1.4 mL, 14 mmol) successively at 0ꢀC.
After stirring the mixture for 5 h at this temperature, the
reaction was stopped by adding brine and extracted
with EtOAc. The combined organic extracts were
washed successively with 5% aq citric acid, satd aq
NaHCO3 and brine. The organic extracts were dried
(Na2SO4) and concentrated in vacuo. The residue was
puri®ed by ¯ash column chromatography (hexane:
CH2Cl2:Et2O=4:3:3) to a€ord 2 (2.45 g, 50%) as a col-
ourless oil. 1H NMR (CDCl3) d 1.07±1.15 (m, 6H), 1.49
(s, 9H), 1.85±1.94 (m, 2H), 2.59±2.78 (m, 2H), 3.08±3.32
(m, 3H), 3.45±3.59 (m, 1H), 4.53±4.62 (m, 1H), 5.40 (d,
1H, J=8.6 Hz) 7.16±7.32 (m. 5H); IR (KBr) 2976, 2934,
2875, 1706, 1639, 1497, 1455, 1382, 1172, 1048, 1024,
are tabulated as m/z. Column chromatography was
carried out using SK-34 (Kishida, 70±230 mesh). Pre-
parative thin-layer chromatography (PTLC) was per-
formed using 60 F254 plates (Merck art. 5744). Optical
rotations ([a]d) were measured on a Jasco DIP-360
polarimeter.
5-Fluoro-3-hydroxy-2-methylbenzoic acid (1). To a stir-
red solution of 5-¯uoro-2-methylbenzoic acid (1.01 g,
6.55 mmol) in H2SO4 (98%, 8 mL) was added a mixture
of HNO3 (fuming, 0.33 mL, 7.9 mmol) and H2SO4
(98%, 1.5 mL) at 15ꢀC. The reaction mixture was
warmed to 0ꢀC and stirred for 30 min at this tempera-
ture. The mixture was poured into ice-cold water, stir-
red for another 10 min and extracted with EtOAc. The
combined organic layer was washed (brine), dried
(Na2SO4) and concentrated in vacuo. The residue was
used in the next step.
1
.
701 cm . Anal. calcd for C19H30N2O3 1/2H2O: C,
66.44; H, 9.10; N, 8.16. Found: C, 66.26; H, 9.23; N,
8.09; MS m/z 335 (M+H)+; [a]d25 4.8ꢀ (c 1.1, CHCl3).
(2-Oxo-1(S)-phenethyl-but-3-enyl)-carbamic acid tert-
butyl ester (3).
To a stirred solution of 2 (2.41 g, 7.21 mmol) in tetra-
hydrofuran (THF) (45 mL), was added a solution of
vinyl magnesium bromide (1M in THF, 35 mL) at 0ꢀC
and the reaction mixture was gradually warmed to room
temperature. After stirring the mixture for 4 h, the reac-
tion was quenched with 5% aq KHSO4 and extracted
The solution of the above crude product, K2CO3 (1.0 g,
7.3 mmol) and benzylbromide (0.87 mL, 7.7 mmol) was
stirred for 1 h at 50ꢀC. The reaction was stopped by
adding H2O and extracted with EtOAc. The organic
extracts were washed with brine, dried (Na2SO4) and

2068
E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
with EtOAc. The combined organic extracts were
washed with successively satd aq NaHCO3 and brine.
The organic extracts were dried (Na2SO4) and con-
centrated in vacuo. The residue was puri®ed by ¯ash
column chromatography (hexane:EtOAc=8:2) to
a€ord 3 (1.20 g, 58%) as a white crystalline solid: mp
3.73 (s, 3H), 3.99±4.07 (m, 1H), 4.26±4.34 (m, 1H), 4.84
(d, 1H, J=9.3 Hz) 7.14±7.31 (m. 5H); [a]2d5 +0.7ꢀ (c
ꢀ
1
0.76, CHCl3). epi-5 mp 90 C; H NMR (CDCl3) d 1.42
(s, 9H), 1.86±2.07 (m, 2H), 2.61±2.80 (m, 2H), 3.14 (d,
1H, J=5.0 Hz), 3.79 (s, 3H), 4.00±4.14 (m, 1H), 4.18 (d,
1H, J=3.5 Hz), 4.68 (s, 1H), 7.10±7.31 (m. 5H); [a]d27
40.4ꢀ (c 0.84, CHCl3).
46±48ꢀC; H NMR (CDCl3) d 1.50 (s, 9H), 1.75±1.90
1
(m, 1H), 2.08±2.19 (m, 1H). 2.55±2.74 (m, 2H), 4.68±
4.73 (m, 1H), 5.35 (d, 1H, J=7.2 Hz), 5.85 (d, 1H,
J=10.2 Hz), 6.30 (d, 1H, J=17.5 Hz), 6.45 (dd, 1H,
J1=17.5 Hz, J2=10.2 Hz), 7.15±7.31 (m, 5H); IR (KBr)
3344, 2981, 2932, 1706, 1680, 1617, 1522, 1369, 1319,
1278, 1165, 876, 700 cm 1. Anal. calcd for C17H23
(tert-Butoxycarbonyl)-2(S)-hydroxy-3(S)-amino-5-phenyl-
A suspension of 5 (155 mg,
pentanoic acid (6).
.
0.48 mmol) and LiOH H2O (361 mg, 8.5 mmol) in
MeOH (5 mL) was stirred for 1 day at room tempera-
ture. The reaction was quenched by adding 5% aq citric
acid and extracted with EtOAc. The extracts were
washed with brine, dried (Na2SO4) and concentrated in
vacuo. The residue (152 mg) was used in the next step
without further puri®cation.
.
NO3 1/4H2O: C, 69.48; H, 8.06; N, 4.77. Found: C,
69.36; H, 7.94; N, 4.65; MS m/z 290 (M+H)+.
{2(R,S)-Hydroxy-1(S)-phenethyl-but-3-enyl}-carbamic
.
acid tert-butyl ester (4). To a suspension of CeCl3 7H2O
(745 mg, 2.0 mmol) and NaBH4 (168 mg, 4.4 mmol) in
EtOH (8 mL) was added 3 (1.20 g, 4.16 mmol) at 0ꢀC
and the reaction mixture was stirred for 4 h at this tem-
perature. The reaction was quenched by adding
CH3CO2H and stirred for 5 min and then the mixture
was extracted with EtOAc. The combined organic layer
was successively washed with saturated aqueous
NaHCO3 and brine. The organic extracts were dried
(Na2SO4) and concentrated in vacuo. The residue was
puri®ed by ¯ash column chromatography (hexane:
Et2O=7:3) to a€ord 4 (1.12 g, 90%) as a diastereo-
mixture (colourless oil): MS m/z 291 (M)+.
(tert-Butoxycarbonyl)-2(S)-hydroxy-3(S)-amino-5-phenyl-
pentanoyl-4(S)-Cl-Pro-NH-tert-Bu (7). A solution of 6
.
(152 mg, 0.48 mmol), EDCI HCl (293 mg, 1.53 mmol),
HOBt (111 mg, 0.82 mmol) and 4(S)-Cl-Pro-tert-butyl-
amide (311 mg, 1.50 mmol) in DMF (3 mL) was stirred
for 1 day. The reaction mixture was diluted with EtOAc
and successively washed with H2O and brine. The
organic extracts were dried (Na2SO4) and concentrated
in vacuo. The residue was puri®ed by PTLC (CH2Cl2:
MeOH=9:1)ꢀto a€ord 7 (175 mg, 78%) as a colourless
1
form. Mp 61 C; H NMR (CDCl3) d 1.32 (s, 9H), 1.48
(s, 9H), 1.68±1.77 (m, 2H), 2.10±2.81 (m, 4H), 3.37±4.10
(m, 4H), 4.12±4.39 (m, 2H), 4.44 (d, 1H, J=9.0 Hz),
4.93 (d, 1H, J=9.0 Hz), 6.31 (s, 1H), 7.10±7.66 (m, 5H);
(tert-Butoxycarbonyl)-2(S)-hydroxy-3(S)-amino-5-phenyl-
pentanoic acid methy ester (5). O3 (ca. 3% in O2) was
bubbled (25 mL/h) into a solution of 4 (213 mg,
0.73 mmol) in MeOH (10 mL) at 78ꢀC for 4 h. Excess
O3 was purged by bubbling N2. To this was added Me2S
(0.50 mL, 6.8 mmol) at 78ꢀC and the reaction mixture
was gradually warmed to room temperature and stirred
for 30 min. After removal of the solvent in vacuo, the
residue was used in the next step.
IR (KBr) 3333, 2975, 1692, 1650, 1575, 1454, 17, 1170,
1
.
1027, 701 cm . Anal. calcd for C25H38N3O5Cl 3/4H2O:
C, 58.92; H, 7.81; N, 8.25; Cl, 6.92. Found: C, 58.86; H,
7.32; N, 8.01; Cl, 6.82; MS m/z 496 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-amino-
5-phenylpentanoyl)-4(S)-Cl-Pro-NH-t-Bu (8). A solution
of 7 (24.5 mg, 49.4 mmol) in 1,4-dioxane (2 mL, con-
tained 4N-HCl) was stirred for 4 h. After removal of the
solvent in vacuo, the residue was used in the next step
without further puri®cation.
To the solution of this residue and 2-methyl-2-buten
(0.40 mL, 3.8 mmol) in tert-butylalcohol (2 mL) was
added an aq solution (2 mL) of NaClO2 (230 mg,
2.54 mmol) and NaH2PO4 (340 mg, 2.83 mmol) at room
temperature, and stirred continuously for 5 h. The reac-
tion was stopped by adding brine and the mixture was
extracted with EtOAc. The combined extracts were
washed successively with satd aq NaS2O3 and brine. The
organic extracts were dried (Na2SO4) and concentrated
in vacuo. The residue (291 mg) was used without further
puri®cation in the next step.
The solution of 3-hydroxy-2-methylbenzoic acid (19.1 mg,
.
0.126 mmol), EDCI HCl (45.5 mg, 0.237 mmol), HOBt
(16.6 mg, 0.123 mmol), NEt3 (16.6 mg, 0.164 mmol) and
the above crude compound in THF (1 mL) was stirred
for 1 day at room temperature. The reaction was quen-
ched with brine and this reaction mixture was extracted
with EtOAc. The organic layer was washed with brine
and dried over Na2SO4. After removal of the solvent in
vacuo, the residue was puri®ed by PTLC (CH2Cl2:
MeOH=9:1) to a€ord 8 (15.6 mg) in 60% yield. Mp
To the stirred solution of the residue (291mg) in benzene:
MeOH (2:1, 3 mL) was added a solution of tetra-
methyldiazometane in n-hexane (10%, 1.5 mL) and stir-
red for 20 min. After removal of the solvent in vacuo,
the residue was puri®ed by PTLC (hexane:EtOAc=5:5)
to a€ord 5 (123 mg, 52%) and epi-5 {(tert-butoxy-
carbonyl)-2(R)-hydroxy-3(S)-amino-5-phenylpentanoic
111±112ꢀC; H NMR (CDCl3) d 1.26 (s, 9H), 1.80±1.90
1
(m, 2H), 2.35 (s, 3H), 2.26±2.81 (m, 3H), 3.51±3.83 (m,
4H), 4.18±4.59 (m, 4H), 6.13±6.50 (m, 2H), 6.85±7.65
(m, 9H); IR (KBr) 3325, 1646, 1529, 1455, 1367, 1283,
1119, 749, 700 cm 1; MS m/z 530 (M+H)+.
acid methyl ester} (36.5 mg, 16%). 5 mp 78±79ꢀC; H
(3-Hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-amino-
phenylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (9). Mp 117ꢀC;
1H NMR (CDCl3) d 1.30 (s, 9H), 1.96 (s, 3H), 2.21±2.77
1
NMR (CDCl3) d 1.50 (s, 9H), 1.50±1.64 (m, 1H), 1.70±
1.86 (m, 1H), 2.54±2.80 (m, 2H), 3.15 (d, 1H, J=5.6 Hz),

E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
2069
(m, 4H), 3.48±4.18 (m, 2H), 4.30±4.73 (m, 4H), 5.94 (s,
1H), 6.17±6.84 (m, 3H), 6.93±7.03 (m, 1H), 7.14±7.33
(m, 5H); IR (KBr) 3325, 2969, 2931, 1648, 1586, 1526,
(m, 1H), 6.77±6.89 (m, 1H), 6.94±7.34 (m, 6H). Anal.
.
calcd for C27H33N3O5ClF 2/3H2O: C, 59.39; H, 6.34; N,
7.70; Cl, 6.49; F, 3.48. Found: C, 59.10; H, 6.48; N,
7.03; Cl, 6.06; F, 2.91; MS m/z 534 (M+H)+.
1455, 1367, 1282, 1225, 1208, 1176, 1112, 1093, 700
1
.
cm . Anal. calcd for C27H34N3O5Cl 1/2H2O: C, 61.77;
H, 6.72; N, 8.00; Cl, 6.75. Found: C, 61.46; H, 6.77; N,
7.85; Cl, 6.64; MS m/z 515 (M+)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(3,4-di¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu
(16). H NMR (CD3OD) d 1.34 (s, 9H), 2.01 (s, 3H),
1
(3-Hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-amino-
1
2.67±2.81 (m, 2H), 3.03±3.09 (m, 1H), 3.84±3.91 (m,
1H), 4.06±4.17 (m, 1H), 4.32±4.58 (m, 5H), 6.95±7.01
(m, 1H), 7.14±7.19 (m, 3H), 7.26±7.36 (m, 2H); MS m/z
552 (M+H)+.
4-p-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (10). H NMR
(CDCl3) d 1.30 (s, 9H), 2.02 (s, 3H), 2.32 (s, 3H), 2.63±
2.84 (m, 4H), 3.81 (d, 1H, J=6.4 Hz), 3.99±4.08 (m,
2H), 4.30±4.39 (m, 2H), 4.41±4.60 (m, 1H), 4.67±4.73
(m, 1H), 6.06 (d, 1H, J=8.1 Hz), 6.28 (s 1H), 6.69±6.84
(m, 5H), 7.00±7.17 (m, 5H); MS m/z 530 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(3,5-di¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu
1
(17). H NMR (CDCl3) d 1.34 (s, 9H), 2.09 (s, 3H),
(3-Hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-amino-
1
2.50±2.90 (m, 4H), 3.89±4.03 (m, 2H), 4.27±4.48 (m,
4H), 4.60±4.69 (m, 2H), 5.97 (s, 1H), 6.16 (d, 1H,
J=8.9 Hz), 6.67±6.91 (m, 5H), 7.06 (t, 1H, J=7.7 Hz);
MS m/z 552 (M+H)+.
4-m-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (11). H NMR
(CDCl3) d 1.31 (s, 9H), 2.02 (s, 3H), 2.27 (s, 3H), 2.62±
2.84 (m, 4H), 3.66±3.77 (m, 2H), 4.00±4.10 (m, 2H),
4.31±4.41 (m, 2H), 4.50±4.62 (m, 1H), 4.69±4.72 (m,
1H), 6.08 (d, 1H, J=8.6 Hz), 6.29 (s, 1H), 6.68 (d, 1H,
J=8.6 Hz), 6.74 (s, 1H), 6.64±7.06 (m, 4H), 7.15±7.23
(m, 1H); MS m/z 530 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(2,4-di¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu
1
(18). H NMR (CDCl3) d 1.31 (s, 9H), 2.06 (s, 3H),
2.47±2.66 (m, 2H), 2.84±2.97 (m, 2H), 3.96±4.06 (m, 2H),
4.28±4.47 (m, 3H), 4.59±4.67 (m, 2H), 5.83 (s, 1H), 6.15
(s, 1H), 6.33 (d, 1H, J=8.5Hz), 6.76±6.89 (m, 3H), 6.98±
7.11 (s, 2H), 7.29±7.38 (m, 1H); MS m/z 552 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-amino-
1
4-o-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (12). H NMR
(CDCl3) d 1.29 (s, 9H), 2.08 (s, 3H), 2.36 (s, 3H), 2.59±
2.72 (m, 3H), 3.95±4.00 (m, 1H), 4.06±4.15 (m, 1H),
4.32±4.40 (m, 2H), 4.47±4.52 (m, 1H), 4.69±4.75 (m,
1H), 4.76±4.82 (m, 1H), 5.36 (s, 1H), 6.12 (d, 1H,
J=8.5 Hz), 6.33 (s, 1H), 6.62 (d, 1H, J=7.1 Hz), 6.76 (t,
1H, J=8.3 Hz), 6.97 (t, 1H, J=7.8 Hz), 7.04±7.26 (m,
4H); MS m/z 530 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(4-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-
1
Bu (19). H NMR (DMSO-d6) d 1.24 (s, 9H), 1.72 (s,
3H), 1.91±2.00 (m, 1H), 2.69±2.92 (m,3H), 3.07±3.50 (m,
1H), 4.35±4.44 (m, 4H), 4.52±4.55 (m, 1H), 5.25 (d, 1H,
J=6.9 Hz), 6.56 (d, 1H, J=7.4 Hz), 6.77 (d, 1H,
J=8.1 Hz), 6.95 (t, 1H, 7.7 Hz), 7.55±7.62 (m, 5H), 8.27
(d, 1H, 8.4 Hz), 9.28 (s, 1H); MS m/z 584 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(4-¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (13).
1H NMR (CDCl3) d 1.31 (s, 9H), 2.03 (s, 3H), 2.53±
2.71 (m, 2H), 2.74±2.86 (m, 2H), 3.96±4.01 (m, 1H),
4.08±4.19 (m, 1H), 4.30±4.45 (m, 3H), 4.55±4.64 (m,
2H), 6.20±6.29 (m, 2H), 6.65±6.81 (m, 3H), 6.94±7.03
(m, 3H), 7.11±7.25 (m,2H). Anal. calcd for C27H33
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(4-methoxy)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu
1
(20). H NMR (CD3OD) d 1.30 (s, 9H), 2.03 (s, 3H),
2.59±2.64 (m, 2H), 2.73±2.85 (m, 2H), 3.76 (s, 3H),
3.96±4.13 (m, 2H), 4.25±4.35 (m, 2H), 4.40±4.46 (m,
2H), 4.57±4.66 (m, 2H), 6.28 (t, 2H, J=7.8 Hz), 6.62±
6.85 (m, 5H), 7.14 (d, 1H, J=5.1 Hz); MS m/z 546
(M+H)+.
.
N3O5ClF 3/2H2O: C, 57.80; H, 6.47; N, 7.49; Cl, 6.32;
F, 3.39. Found: C, 58.03; H, 6.02; N, 7.14; Cl, 6.03; F,
3.08; MS m/z 534 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(3-¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (14).
1H NMR (CDCl3) d 1.31 (s, 9H), 2.06 (s, 3H), 2.61±
2.70 (m, 2H), 2.74±2.90 (m, 2H), 3.91±4.14 (m, 2H),
4.29±4.36 (m, 1H), 4.41±4.49 (m, 1H), 4.60±4.70 (m,
2H), 5.56 (s, 1H), 6.11±6.16 (m, 2H), 6.69±7.06 (m, 6H),
7.24±7.30 (m, 2H). Anal. calcd for C27H33N3O5
ClF H2O: C, 58.74; H, 6.39; N, 7.61; Cl, 6.42; F, 3.44.
Found: C, 58.61; H, 6.33; N, 7.45; Cl, 6.35; F, 3.27; MS
m/z 534 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(3-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-
Bu (21). H NMR (DMSO-d6) d 1.26 (s, 9H), 1.81 (s,
3H), 1.81±2.03 (m, 1H), 2.70±2.92 (m, 3H), 3.71±3.77
(m, 1H), 4.28±4.59 (m, 5H), 5.11 (d, 1H, J=6.1 Hz),
6.57 (d, 1H, J=7.5 Hz), 6.79 (d, 1H, J=7.5 Hz), 6.95 (t,
1H, J=7.5 Hz), 7.52±7.61 (m, 3H), 7.75 (t, 1H, J=
5.7 Hz), 7.87 (s, 1H), 8.32 (d, 1H, J=9.5 Hz), 9.40 (s,
1H); MS m/z 584 (M+H)+.
1
.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(2-¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (15).
1H NMR (CDCl3) d 1.31 (s, 9H), 2.04 (s, 3H), 2.60±
2.72 (m, 2H), 2.82±3.02 (m, 2H), 3.98±4.16 (m, 2H),
4.27±4.36 (m, 1H), 4.40±4.49 (m, m, 2H), 4.63±4.70 (m,
1H), 6.27 (s, 1H), 6.36 (s, 1H), 6.47 (s, 1H), 6.67±6.72
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(4-hydroxy)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (22).
1H NMR (CD3OD) d 1.33 (s, 9H), 1.89 (s, 3H), 2.60±
3.03 (m, 4H), 3.84±3.90 (m, 1H), 4.40±5.51 (m, 5H),
6.61±6.77 (m, 5H), 6.95±7.17 (m, 2H); MS m/z 532
(M+H)+.

2070
E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(4-phenyl)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (23).
1H NMR (CDCl3) d 1.31 (s, 9H), 2.05 (s, 3H), 2.61±
2.66 (m, 2H), 2.83±2.97 (m, 2H), 3.95±4.11 (m, 2H),
4.28±4.36 (m, 2H), 4.42±4.48 (m, 1H), 4.67±4.75 (m,
2H), 6.22±6.29 (m, 1H), 6.60±6.99 (m, 2H), 7.13±7.57
(m, 12H); MS m/z 592 (M+H)+.
(5-Fluoro-3-hydroxy-2-methylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-phenylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (30). 1H
NMR (CDCl3) d 1.31 (s, 9H), 1.90 (s, 3H), 2.41±2.96
(m, 4H), 3.98 (d, 1H, J=4.0 Hz), 4.00±4.14 (m, 1H),
4.30±4.42 (m, 1H), 4.48±4.52 (m, 1H), 4.58±4.71 (m,
1H), 5.65 (br, 1H), 6.06 (d, 1H, J=8.4 Hz), 6.18 (br,
1H), 6.35±6.47 (m, 1H), 6.52±6.61 (m, 1H), 7.15±7.34
(m, 5H); MS m/z 534 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-2(S)-hydroxy-3(S)-amino-
4-(3,4-dimethyl)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu
(24). H NMR (CDCl3) d 1.31 (s, 9H), 2.05 (s, 3H),
2.22 (s, 3H), 2.23 (s, 3H), 2.57±2.89 (m, 4H), 3.83±4.07
(m, 3H), 4.25±4.60 (m, 4H), 4.67±4.72 (m, 1H), 6.10 (d,
1H, J=8.2 Hz), 6.33 (s, 1H), 6.89±7.07 (m 6H); MS m/z
544 (M+H)+.
(3-Hydroxy-2,6-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (31). Mp
134±136ꢀC; H-NMR (CDCl3) d 1.30 (s, 9H), 1.91 (s,
3H), 1.97 (s, 3H), 2.36±2.90 (m, 3H), 3.71±4.19 (m, 4H),
4.31±4.85 (m, 3H), 5.04±7.00 (m, 3H), 7.11±7.52 (m,
7H); IR (KBr) 3335, 2968, 1645, 1532, 1455, 1276, 1223,
1118, 872, 749, 700 cm 1. Anal. calcd for C28H36
N3O5Cl 1.78H2O: C, 59.84; H, 7.09; N, 7.47; Cl, 6.31.
Found: C, 60.07; H, 6.75; N, 7.09; Cl, 6.57; MS m/z
529 (M+)+.
1
.
(3-Hydroxy-2-methylbenzoyl)-2(S)-hydroxy-3(S)-amino-
4-(benzo[b]thiophene-5-yl)-butanoyl}-4(S)-Cl-Pro-NH-t-
1
Bu (25). H NMR (CDCl3) d 1.29 (s, 9H), 1.85±1.98
(m, 3H), 2.59±3.31 (m, 6H), 3.93±4.03 (m, 1H), 4.27±
4.38 (m, 2H), 4.41±4.46 (m, 1H), 4.58±4.71 (m, 2H),
6.24±6.41 (m, 2H), 7.06±7.29 (m, 5H), 7.41±7.44 (m,
1H), 7.59±7.68 (m, 1H), 7.73±7.81 (m, 1H); MS m/z 572
(M+H)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(3-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-
Pro-NH-t-Bu (32). H NMR (CDCl3) d 1.31 (s, 9H),
2.05 (s, 3H), 2.22 (s, 3H), 2.56±2.96 (m, 4H), 3.83 (d,
1H, J=6.1 Hz), 3.97±4.17 (m, 2H), 4.30±4.36 (m, 2H),
4.40±4.48 (m, 1H), 4.61±4.79 (m, 2H), 6.04 (s, 1H), 6.11
(d, 1H, 8.7 Hz), 6.63 (d, 1H, 7.6 Hz), 6.91 (d, 1H,
7.6 Hz), 7.28±7.53 (m, 4H); MS m/z 598 (M+H)+.
1
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(ꢀ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-Bu (26). 1H
NMR (CD3OD) d 1.33 (s, 9H), 1.72 (s, 3H), 2.11±2.18
(m, 1H), 2.69±2.70 (m, 1H), 3.11±3.17 (m, 1H), 3.57±
3.66 (m, 1H), 3.71±3.76 (m, 1H), 3.91 (dd, 1H, J1=
11.1, J2=7.6 Hz), 4.39±4.54 (m, 4H), 6.52 (d, 1H,
J=7.0 Hz), 6.72 (d, 1H, J=7.9 Hz), 6.89±6.93 (m, 1H),
7.38±7.59 (m, 4H), 7.77 (d, 1H, J=7.9 Hz), 7.87 (d, 1H,
J=7.8 Hz), 8.34 (d, 1H, J=8.4 Hz); MS m/z 566
(M+H)+.
(3-Hydroxy-2,5-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(3-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-
1
Pro-NH-t-Bu (33). H NMR (CDCl3) d 1.31 (s, 9H),
1.97 (s, 3H), 2.20 (s, 3H), 2.43±2.96 (m, 4H), 3.86 (d,
1H, J=6.40 Hz), 3.94±4.22 (m, 2H), 4.29±4.37 (m, 2H),
4.42±4.49 (m, 1H), 4.60±4.76 (m, 2H), 6.03 (br, 1H),
6.07 (d, 1H, J=8.8 Hz), 6.48 (s, 1H), 6.62 (s, 1H), 7.35±
7.55 (m, 4H); MS m/z 598 (M+H)+.
(3-Hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-3(S)-amino-
4-(ꢁ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-Bu (27). 1H
NMR (CD3OD) d 1.34 (s, 9H), 1.66 (s, 3H), 2.10±2.17
(m, 1H), 2.71±2.78 (m, 1H), 2.91 (dd, 1H, J1=14.0,
J2=11.5 Hz), 3.25±3.31 (m, 2H), 3.91 (dd, 1H, J1=10.8,
J2=7.8 Hz), 4.36±4.55 (m, 4H), 4.66±4.88 (m, 1H), 6.55
(d, 1H, J=7.2 Hz), 6.71 (d, 1H, J=7.9 Hz), 6.89 (t, 1H,
J=7.9 Hz), 7.40±7.46 (m, 2H), 7.53 (dd, 1H, J1=8.2,
J2=1.4 Hz), 7.71±7.83 (m, 4H); MS m/z 566 (M+H)+.
(5-Fluoro-3-hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-
3(S)-amino-4-(3-tri¯uoromethyl)-phenylbutanoyl}-4(S)-
1
Cl-Pro-NH-t-Bu (34). H NMR (CDCl3) d 1.31 (s, 9H),
1.91 (s, 3H), 2.45±2.96 (m, 4H), 3.87 (d, 1H, J=6.2 Hz),
3.94±4.06 (m, 1H), 4.07±4.37 (m, 2H), 4.44±4.48 (m,
1H), 4.62±4.71 (m, 2H), 5.48 (br, 1H), 5.97 (br, 1H),
6.11 (d, 1H, J=8.8 Hz), 6.41±6.46 (m, 1H), 6.54±6.62
(m, 1H), 7.35±7.54 (m, 4H); MS m/z 602 (M+H)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
1
amino-4-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-Bu (28). H
(3-Hydroxy-2,6-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(3-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-
Pro-NH-t-Bu (35). H NMR (CDCl3) d 1.31 (s, 9H),
1.85 (s, 3H), 1.92 (s, 3H), 2.42±3.09 (m, 4H), 3.77±3.80
(m, 1H), 4.00±4.20 (m, 2H), 4.29±4.44 (m, 2H), 4.46 (t,
1H, J=7.8 Hz), 4.54±4.88 (m, 3H), 6.01 (s, 1H), 6.65 (d,
1H, J=8.2 Hz), 6.78±6.87 (m, 1H), 7.30±7.54 (m, 4H);
MS m/z 598 (M+H)+.
NMR (CDCl3) d 1.30 (s, 9H), 2.11 (s, 3H), 2.22 (s, 3H),
2.59±2.89 (m, 4H), 3.81 (d, 1H, J=6.5 Hz), 3.99±4.12
(m, 2H), 4.31±4.39 (m, 2H), 4.39±4.53 (m, 1H), 4.59±
4.64 (m, 1H), 4.66±4.70 (m, 1H), 6.09 (d, 1H, J=
8.3 Hz), 6.26 (s, 1H), 6.61 (d, 1H, J=7.7 Hz), 6.90 (d,
1H, 7.7 Hz), 7.15±7.33 (m, 5H); MS m/z 530 (M+H)+.
1
(3-Hydroxy-2,5-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-phenylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (29). 1H
NMR (CDCl3) d 1.30 (s, 9H), 1.93 (s, 3H), 2.19 (s, 3H),
2.40±2.96 (m, 4H), 3.86 (d, 1H, J=6.5 Hz), 3.97±4.12
(m, 2H), 4.31±4.42 (m, 2H), 4.48±4.59 (m, 1H), 4.60±
4.74 (m, 2H), 6.03 (d, 1H, J=8.2 Hz), 6.25 (s, 1H), 6.46
(s, 1H), 6.64 (s, 1H), 7.16±7.52 (m, 5H); MS m/z 530
(M+H)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(4-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-
Pro-NH-t-Bu (36). H NMR (CDCl3) d 1.31 (s, 9H),
2.00 (s, 3H), 2.23 (s, 3H), 2.57±2.88 (m, 4H), 3.82 (d,
1H, 6.4 Hz), 3.92±4.18 (m, 2H), 4.30±4.36 (m, 2H),
4.41±4.51 (m, 1H), 4.64±4.79 (m, 2H), 6.03 (s, 1H), 6.06
(d, 1H, J=8.7 Hz), 6.64 (d, 1H, J=7.6 Hz), 6.92 (d, 1H,
1

E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
2071
J=7.6 Hz), 7.39 (d, 1H, 8.1 Hz), 7.56 (d, 1H, 8.1 Hz);
MS m/z 598 (M+H)+.
1H), 4.69±4.81 (m, 1H), 4.87 (t, 1H, J=7.7 Hz), 5.21 (s,
1H), 6.26±6.30 (m, 1H), 6.62 (dd, 1H, J1=27.9, J2=
7.7 Hz), 6.93 (dd, 1H, J1=26.0, J2=7.7 Hz) 7.01±7.07
(m, 2H), 7.08±7.15 (m, 2H); MS m/z 544 (M+H)+.
(3-Hydroxy-2,6-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(4-tri¯uoromethyl)-phenylbutanoyl}-4(S)-Cl-
1
Pro-NH-t-Bu (37). H NMR (CDCl3) d 1.31 (s, 9H),
(3-Hydroxy-2,6-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-o-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (44). 1H
NMR (CDCl3) d 1.26±1.33 (m, 9H), 1.89 (s, 3H), 2.11
(s, 3H), 2.35 (s, 3H), 2.53±3.02 (m, 5H), 3.79±3.89 (m,
1H), 3.95±4.05 (m, 2H), 4.20±4.47 (m, 3H), 4.49±4.60
(m, H), 4.75±4.81 (m, 1H), 4.90 (t, 1H, J=7.5 Hz), 6.84±
6.92 (m, 1H), 7.04±7.14 (m, 5H); MS m/z 544 (M+H)+.
1.82 (s, 3H), 1.90 (s, 3H), 2.44±2.96 (m, 4H), 3.79 (d,
1H, J=6.3 Hz), 4.00±4.19 (m, 1H), 4.31±4.39 (m, 2H),
4.44±4.52 (m, 1H), 4.66±4.75 (m, 2H), 4.82±4.85 (m,
1H), 5.94 (d, 1H, J=8.7 Hz), 6.01 (s, 1H), 6.64 (d, 1H,
8.2 Hz), 6.81 (d, 1H, 8.2 Hz), 7.39 (d, 1H, J=8.1 Hz),
7.56 (d, 2H, J=8.1 Hz); MS m/z 598 (M+H)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
1
(3-Hydroxy-2,5-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(4-phenyl)-phenylbutanoyl}-4(S)-Cl-Pro-NH-t-
Bu (45). H NMR (CDCl3) d 1.31(s, 9H), 1.90 (s, 3H),
2.12 (s, 3H), 2.60±2.66 (m, 2H), 2.77±2.97 (m, 2H),
3.94±4.07 (m, 2H), 4.28±4.36 (m, 2H), 4.43±4.53 (m,
1H), 4.63±4.76 (m, 2H), 6.01 (s, 1H), 6.23 (s, 1H), 6.46±
6.64 (m, 1H), 7.21±7.57 (m, 11H); MS m/z 606 (M+H)+.
amino-4-m-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (38). H
1
NMR (CDCl3) d 1.30 (s, 9H), 2.06 (s, 3H), 2.22 (s, 3H),
2.32 (s, 3H), 2.58±2.79 (m, 4H), 3.81 (d, 1H, J=7.4 Hz),
4.00±4.10 (m, 2H), 4.31±4.39 (m, 2H), 4.51±4.61 (m,
2H), 4.70±4.74 (m, 1H), 6.07 (d, 1H, J=8.2 Hz), 6.31 (s,
1H), 6.62 (d, 1H, J=7.8 Hz), 6.70±6.97 (m, 2H), 7.00±
7.05 (m, 2H), 7.13±7.21 (m, 1H); MS m/z 544 (M+H)+.
(3-Hydroxy-2,5-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(3,5-di¯uoro)-phenylbutanoyl}-4(S)-Cl-Pro-NH-
t-Bu (46). H NMR (CDCl3) d 1.34 (s, 9H), 1.87 (s,
(3-Hydroxy-2,5-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
1
1
amino-4-m-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (39). H
NMR (CDCl3) d 1.31 (s, 9H), 1.96 (s, 3H), 2.19 (s, 3H),
2.31 (s, 3H), 2.41±2.96 (m, 4H), 3.87 (d, 1H, J=6.5 Hz),
3.96±4.14 (m, 2H), 4.32±4.49 (m, 2H), 4.51±4.61 (m,
2H), 4.69±4.73 (m, 1H), 6.04 (d, 1H, J=8.2 Hz), 6.30 (s,
H), 6.44 (s, 1H), 6.60 (s, 1H), 6.94±7.06 (m, 3H), 7.15±
7.22 (m, 1H); MS m/z 544 (M+H)+.
3H), 2.05±2.16 (m, 1H), 2.20 (s, 3H), 2.70±2.81 (m, 2H),
3.08 (d, 1H, J=3.0 Hz), 3.88±3.89 (m, 1H), 4.40±4.58
(m, 5H), 6.46 (s, 1H), 6.61 (s, 1H), 6.74±6.83 (m, 1H),
7.00±7.07 (m, 2H); MS m/z 566(M+H)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(ꢁ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-Bu (47).
1H NMR (CDCl3) d 1.30 (s, 9H), 1.91 (s, 3H), 2.19 (s,
3H), 2.61±2.79 (m, 2H), 2.95±3.06 (m, 2H), 3.83 (d, 1H,
J=6.5 Hz), 3.92±4.07 (m, 2H), 4.33±4.45 (m, 2H), 4.54±
4.57 (m, 1H), 4.61±4.77 (m, 2H), 6.11 (d, 1H, J=
8.2 Hz), 6.29 (s, 1H), 6.59 (d, 1H, J=7.7 Hz), 6.84 (d,
1H, J=7.7 Hz), 7.29±7.49 (m, 3H), 7.60±8.02 (m, 4H);
MS m/z 580 (M+H)+.
(3-Hydroxy-2,6-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
1
amino-4-m-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (40). H
NMR (CDCl3) d 1.30 (s, 9H), 1.84 (s, 3H), 1.94 (s, 3H),
2.28 (s, 3H), 2.41±2.98 (m, 4H), 3.79 (d, 1H, J=6.3 Hz),
4.00±4.14 (m, 2H), 4.31±4.40 (m, 2H), 4.50±4.65 (m,
2H), 4.76±4.85 (m, 1H), 5.98 (d, 1H, J=8.1 Hz), 6.28 (s,
H), 6.63±6.73 (m, 1H), 6.79±6.85 (m, 1H), 6.90±7.04 (m,
3H), 7.12±7.19 (m, 1H); MS m/z 544 (M+H)+.
(3-Hydroxy-2,5-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(ꢁ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-Bu (48).
1H NMR (CDCl3) d 1.31 (s, 9H), 1.84 (s, 3H), 2.05 (s,
3H), 2.62±3.06 (m, 4H), 3.89 (d, 1H, J=6.5Hz), 3.92±
4.08 (m, 2H), 4.33±4.45 (m, 2H), 4.53±4.77 (m, 3H), 6.07
(d, 1H, J=8.2 Hz), 6.28 (s, 1H), 6.31 (s, 1H), 6.55 (s, 1H),
7.29±7.52 (m, 4H), 7.61±7.83 (m, 4H); MS m/z 579 (M)+.
(3-Hydroxy-2,4-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-p-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (41). 1H
NMR (CDCl3) d 1.29 (s, 9H), 2.06 (s, 3H), 2.23 (s, 3H),
2.31 (s, 3H), 2.60±2.83 (m, 4H), 3.77 (d, 1H, J=6.6 Hz),
3.97±4.10 (m, 2H), 4.31±4.39 (m, 2H), 4.51±4.67 (m,
2H), 4.69±4.72 (m, 1H), 6.03 (d, 1H, J=8.3 Hz), 6.30 (s,
1H), 6.64 (d, 1H, J=7.6 Hz), 6.91 (d, 1H, J=7.6 Hz),
6.95±7.15 (m, 4H); m/z 544 (M+H)+.
(3-Hydroxy-2,6-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(ꢁ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-Bu (49).
1H NMR (CDCl3) d 1.30 (s, 9H), 1.70 (s, 3H), 1.83 (s,
3H), 2.63±3.02 (m, 4H), 3.79 (d, 1H, J=6.1 Hz), 4.02±
4.10 (m, 2H), 4.35±4.43 (m, 2H), 4.53±4.65 (m, 1H),
4.74±4.82 (m, 2H), 6.00 (d, 1H, J=8.2 Hz), 6.28 (s, 1H),
6.60±6.67 (m, 1H), 6.74±6.83 (m, 1H), 7.29±7.52 (m,
3H), 7.59±7.82 (m, 4H); MS m/z 580 (M+H)+.
(3-Hydroxy-2,6-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-p-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (42). 1H
NMR (CDCl3) d 1.30 (s, 9H), 1.84 (s, 3H), 1.94 (s, 3H),
2.31 (s, 3H), 2.59±2.88 (m, 4H), 3.76 (d, 1H, J=6.4 Hz),
4.00±4.14 (m, 2H), 4.31±4.40 (m, 2H), 4.51±4.63 (m,
2H), 4.76±4.88 (m, 1H), 5.93 (d, 1H, J=8.2 Hz), 6.28 (s,
H), 6.64 (d, 1H, J=8.2 Hz), 6.80 (d, 1H, 8.2 Hz), 7.02±
7.13 (m, 4H); MS m/z 544 (M+H)+.
(5-Fluoro-3-hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-
3(S)-amino-4-(ꢁ-naphtyl)-butanoyl}-4(S)-Cl-Pro-NH-t-
1
(3-Hydroxy-2,4-dimethylbenzoyl)-(2(S)-hydroxy-3(S)-
amino-4-o-tolylbutanoyl)-4(S)-Cl-Pro-NH-t-Bu (43). 1H
NMR (CDCl3) d 1.27 (s, 9H), 2.02 (s, 3H), 2.17 (s, 3H),
2.28 (s, 3H), 2.38±2.61 (m, 1H), 2.68±3.06 (m, 4H),
3.83±4.05 (m, 2H), 4.24±4.45 (m, 2H), 4.57±4.59 (m,
Bu (50). H NMR (CDCl3) d 1.31 (s, 9H), 1.77 (s, 3H),
2.63±2.76 (m, 2H), 2.95±3.07 (m, 2H), 3.86±4.04 (m,
2H), 4.31±4.43 (m, 3H), 4.51±4.76 (m, 3H), 5.41 (s, 1H),
6.13±6.58 (m, 4H), 7.29±7.67 (m, 4H), 7.73±7.83 (m,
3H); MS m/z 584 (M+H)+.

2072
E. Takashiro et al. / Bioorg. Med. Chem. 7 (1999) 2063±2072
(3-Hydroxy-2,5-dimethylbenzoyl)-{2(S)-hydroxy-3(S)-
amino-4-(benzo[b]thiophene-5-yl)-butanoyl}-4(S)-Cl-Pro-
NH-t-Bu (51). H NMR (CDCl3) d 1.31 (s, 9H), 1.83±
1.96 (m, 3H), 2.06±2.18 (m, 3H), 2.57±3.33 (m, 6H),
3.92±4.06 (m, 2H), 4.28±4.36 (m, 2H), 4.43±4.58 (m,
1H), 4.66±4.71 (m, 2H), 6.01 (s, 1H), 7.06±7.28 (m, 4H),
7.42±7.45 (m, 2H); MS m/z 586 (M+H)+.
T. Tetrahedron. Lett. 1994, 35, 6123 and references cited
therein.
17. Sakurai, M.; Higashida, S.; Sugano, M.; Komai, T.; Yagi,
R.; Ozawa, Y.; Handa, H.; Nishigaki, T.; Yabe, Y. Bioorg.
Med. Chem. 1994, 2, 807.
1
18. Komai, T.; Higashida, S.; Sakurai, M.; Nitta, T.; Kasuya,
A.; Miyamoto, S.; Yagi, R.; Ozawa, Y.; Handa, H.; Mohri,
H.; Yasuoka, A.; Oka, S.; Nishigaki, T.; Kimura, S.; Shimada,
K.; Yabe, Y. Bioorg. Med. Chem. 1996, 4, 1365.
19. Takashiro, E.; Watanabe, T.; Nitta, T.; Kasuya, A.;
Miyamoto, S.; Ozawa, Y.; Yagi, R.; Nishigaki, T.; Shibayama,
T.; Nakagawa, A.; Iwatomo, A.; Yabe, Y. Bioorg. Med. Chem.
1998, 6, 595.
20. Kaldor, S. W.; Kalish, V. J.; Davies II, J. F.; Shetty, B. V.;
Fritz, J. E.; Appelt, K.; Burgess, J. A.; Campanale, K. M.;
Chirgadze, N. Y.; Clawson, D. K.; Dressman, B. A.; Hatch, S.
D.; Khalil, D. A.; Kosa, M. B.; Lubbehusen, P. P.; Muesing,
M. A.; Patick, A. M.; Reich, S. H.; Su, K. S.; Tatlock, J. H. J.
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(5-Fluoro-3-hydroxy-2-methylbenzoyl)-{2(S)-hydroxy-
3(S)-amino-4-(benzo[b]thiophene-5-yl)-butanoyl}-4(S)-Cl-
1
Pro-NH-t-Bu (52). H NMR (CDCl3) d 1.31 (s, 9H),
1.80 (s, 3H), 2.64±2.73 (m, 3H), 2.89±2.93 (m, 1H),
3.25±3.35 (m, 2H), 3.89±4.00 (m, 2H), 4.30±4.34 (m,
2H), 4.49±4.54 (m, 1H), 4.66±4.71 (m, 2H), 5.58±5.65
(m, 1H), 6.98±7.30 (m, 4H), 7.45 (d, 1H, J=5.5Hz), 7.69
(s, 1H), 7.83 (d, 1H, J=8.1 Hz); MS m/z 590 (M+H)+.
21. For Nel®navir, see: Jarvis, B.; Faulds, D. Drugs 1998, 56,
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