Structure - Activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

Article abstract of DOI:10.1016/S0968-0896(00)00346-1

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1′, Val as P2 and P2′ residues, and a variety of amino acids at the P3 and P3′ positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3′ residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.

Full text of DOI:10.1016/S0968-0896(00)00346-1

Bioorganic & Medicinal Chemistry 9 (2001) 1185±1195
Structure±Activity Studies of FIV and HIV Protease
Inhibitors Containing Allophenylnorstatine
Van-Duc Le,a Chi Ching Mak,a Ying-Chuan Lin,b John H. Elderb and Chi-Huey Wonga,*
aDepartment of Chemistry and the Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bDepartment of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 10 October 2000; accepted 7 December 2000
AbstractÐThe interaction of P1and P3 side chains with the combining S1and S3 hydrophobic subsites of HIV and FIV proteases
has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-
phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as
P10, Val as P2 and P20 residues, and a variety of amino acids at the P3 and P30 positions. All inhibitors showed competitive inhi-
bition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most e€ective
inhibitor against FIV protease, and it contains Phe at P3, but no P30 residue. VLE776 also exhibited potent antiviral activities
against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition
activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the
protease inhibitor and another enzyme inhibitor in one molecule. # 2001Elsevier Science Ltd. All rights reserved.
Introduction
therapy.3g,h However, clinically isolated drug-resistant
HIV variants often contain multiple mutations in their
protease.2,3c,e The higher number of substitutions in the
enzyme also increases the chance of developing cross-
resistance to a wide range of structurally diverse inhibi-
tors.3c,e This suggests that combination therapy with
multiple HIV PR inhibitors may not be a solution to
combat resistance selection. Therefore, it is necessary to
develop new protease inhibitors which are ecacious
against both the wild-type and drug-resistant HIV pro-
teases and less prone to resistance development.
Human immunode®ciency virus protease (HIV PR) has
been identi®ed as an important target enzyme for inhi-
bition in order to suppress HIV replication. In the last
several years, an extensive research e€ort has been
devoted to the search for therapeutically useful inhibitors
of the enzyme to stop the progression of human
acquired immunode®ciency syndrome (AIDS). Five
competitive inhibitors of the enzyme have already been
approved and several others are in clinical trials.1
However, recent reports2 indicate that 45 distinct drug-
resistant single mutations in HIV PR have been identi-
®ed, and the number of mutations has increased by
250% over a 3-year period. The development of drug-
resistance is the consequence of incomplete suppression
of HIV replication. The rapid replication rate of HIV
and its inherent genetic variation result in the generation
of numerous viral variants.2,3 This genetic ¯exibility of
HIV creates a new challenge for the development of the
next generation of HIV inhibitors. Previous attempts to
tackle the drug-resistance problems have been focused
on the modi®cation of inhibitors to overcome the e€ects
of single mutations,3a or have relied on combination
As a ®rst step toward this goal, we have developed
potent inhibitors against feline immunode®ciency virus
protease (FIV PR), which has been shown to be a useful
model for drug-resistant HIV PRs.4 FIV is a retrovirus
which causes an immunode®ciency syndrome in cats
comparable to AIDS in humans.5 FIV PR is also a C2-
symmetric homodimeric aspartic protease and is
responsible for processing both the structural proteins
of gag and the enzymes encoded by pol from poly-
protein during FIV replication.6 This enzyme has a
superimposable active site structure and identical
mechanism of catalysis to HIV PR.4c,7 Furthermore, six
mutated residues in HIV PR causing drug resistance
(K20I, V32I, I50V, N88D, L90M, Q92K)2,8 are found
in the structurally aligned native residues of FIV PR.7
In addition, FIV PR also contains ®ve other amino acid
*Corresponding author. Tel.: +1-858-784-2487; fax: + 1-858-784-2409;
e-mail: wong@scripps.edu
0968-0896/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00346-1

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V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
residues (L17, N39, I57, I85, I98)7 closely related to
mutations observed in drug-resistant HIV PRs, i.e.,
T12I, E34Q, G48V, A71V, P81T, respectively.2 Many
HIV protease mutants have a shrunken P3 binding site,
which is a structural characteristic of FIV protease.4,7
Kinetic studies have also demonstrated that various
structurally diverse potent HIV PR inhibitors lost their
eciency as inhibitors against FIV PR by a factor of
100 or more.4 These observations suggest that FIV PR
may serve as a model for drug-resistant HIV PRs con-
taining multiple mutations and may contribute to the
understanding of HIV resistance to inhibitors. Further-
more, the cat may be used as an animal model to test
the e€ectiveness of potential therapeutic drugs in vivo to
accelerate the drug development process.
Results and Discussion
Chemistry
Scheme 1illustrates the procedures to synthesize the
tripeptide derivatives 18a, b and 19. BOC-protected 1,3-
thiazolidine-4-carboxylic acid derivatives 16a, b were
prepared according to known procedures.9,10 Compounds
17a, b were prepared in good yields from the acids 16a,
b by using valine methyl ester and HBTU as a con-
densation reagent. Removal of the BOC group in 17a, b
followed by coupling of the acid11 gave the tripeptides
18a,b. Hydrolysis of the ester 18a with LiOH followed
by activation of the acid with N-hydroxysuccinimide
gave the activated ester. This activated ester was con-
densed with aqueous methylamine to give the tripeptide
19 in good yields.
Our ®rst investigation4a,b on the development of broad-
based protease inhibitors ecacious against both HIV
and FIV was focused on the systematic analysis of the
S3 and S30 subsite speci®cities of the enzymes. A series
of C2-symmetric inhibitors containing (1S, 2R, 3R, 4S)-
1,4-diamino-1,4-dibenzyl-2,3-butan-diol as P1 and P10
core were studied and these e€orts led to the develop-
ment of the potent FIV PR inhibitor TL3, which has a
remarkable versatility to control FIV, HIV, and SIV
infections in tissue culture with virtually the same degree
of e€ectiveness.4b Although TL3 showed highly potent
anti-HIV activity, it displays low oral bioavailability in
cat and mouse. In the present study, we described our
continuing e€ort to generate novel small-sized HIV and
FIV protease inhibitors.
Scheme 2 illustrates the synthesis of compound 23,
which began with the removal of the BOC group in 17a
followed by condensation with an epoxide4a to give
compound 20. Deprotection of the BOC group in 20
followed by peptide coupling with Cbz-AlaVal-OH gave
compound 23 in 80% yield.
The inhibitors 22, 27±33 were prepared from the corre-
sponding tripeptides 18a, b and 19 and are shown in
Schemes 3 and 4.
Scheme 2. (a) 4 N HCl, dioxane; (b) pH7; (c) MeOH, Et3N, 80 ꢀC,
90%; (d) 4 N HCl, dioxane; (e) Cbz-Ala-Val-OH, HBTU, DMF,
Et3N, 68%.
Scheme 3. (a) 4 N HCl, dioxane; (b) dipeptide, HBTU, DMF, Et3N,
70±90%.
Scheme 1. (a) HCHO, py, H2O, 85%; (b) BOC2O, THF/H2O,
NaHCO3, 77%; (c) Val-OMe, HBTU, DMF, Et3N, 90%; (d) 4 N HCl,
dioxane; (e) pH7; (f) HBTU, DMF, Et3N, 89%; (g) LiOH, 20%
MeOH/THF, H2O, 100%; (h) NHS, HOBt, DCC, DCE then MeNH2
78%.
Scheme 4. (a) 4 N HCl, dioxane; (b) Ac-Trp-Val-OH, HBTU, DMF,
Et3N, 82%.

V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
1187
Structure±activity relationship
produced the potent HIV PR inhibitor JE-2147 now in
clinical trials.12 Compounds 21±24 contain a thiazoli-
dine ring, a bio-isostere of proline, at the P10 portion;
they were tested against both the wild-type FIV and
HIV PRs and the results are summaries in Table 2.
Compound 21 bearing the same residues as TL3, except
for the P10 residue, exhibited similar FIV PR inhibitory
activity (IC50=95 nM), but lost its potency against HIV
PR by 5-fold. Removal of the P30 residue from com-
pound 21 resulted in a 2-fold decrease in FIV PR inhi-
bitory activity, but still retained its potency against HIV
PR (IC50=18 nM). Deletion of the 1-oxo group in 22
led to compound 23, which showed a signi®cant loss of
FIV PR activity (IC50=5.2 mM), but still retained its
binding anity to HIV PR. The con®guration of the
hydroxyl, which forms hydrogen bonds with the cataly-
tic aspartic acids, plays an important role in binding: the
(2R)-alcohol 24 displays a 96- and 26-fold reduction in
potency against FIV and HIV PRs respectively as com-
pared to the (2S)-alcohol 22. This is in line with the
results reported by Mimoto et al.13
Our ®rst e€ort to improve the activity of TL3 involves
structural modi®cation of P4 and P40 and the results are
shown in Table 1. All the C2-symmetric diols tested in
this study showed competitive inhibition of both the
feline and human lentivirus PRs, but with higher potency
against HIV PR by at least an order of magnitude. The
sulfonamides 3±7 and the amide 8 derivatives displayed
approximately 1±3-fold less active against FIV PR and
about 1±20-fold less active against HIV PR. Two notable
exceptions are compounds 2 and 9, which displayed the
highest potency with IC50 values of 17 and 62 nM
against FIV PR respectively. However, the urea deriva-
tives 10±13 showed only 37±61% inhibition at 200 mM
against FIV PR. Compounds 2 and 9 were tested
against drug-resistant mutant HIV PRs, G48V and
V82F, and were found to be quite e€ective.
In order to ascertain the optimal length of the C2-sym-
metrical inhibitors derived from TL3, we next examined
asymmetrically substituted derivatives 14 and 15 (Fig. 1).
The chain length signi®cantly a€ected the inhibitory
activity. We found that removal of the P30 residue from
TL3 reduces the potency against HIV and FIV PRs by
4-fold and 22-fold respectively. Compound 15, which
contains no P30 and P20 residues, exhibited marginal
activity with IC50 value of 200 mM against FIV PR and
100 nM against HIV PR. Such reduction in potency in
14 and 15 may also be attributed to Cbz group not
properly bound in S2 and S3 pockets and so contribute
to the poor activity.
In the course of our study on C2 symmetrical diol inhi-
bitors, we have observed that large hydrophobic P3
groups reduced the potency of inhibitors against FIV
PR considerably,4a as in the case of compound 25.
However, compound 26, which contains a tryptophan
as P3 residue and N-acetyl as the N-terminal group, has
a reasonable binding anity with FIV PR (212 nM).
This observation was in agreement with an independent
study reported by Dunn laboratory,14 indicating that in
this case the P3 residue has moved away from the P3
binding site and oriented toward outside the cleft.
In an e€ort to increase the antiviral activity and pharmaco-
kinetic pro®les of TL3 derivatives, we used allophenyl-
In the allophenylnorstatine series, compound 27, a
tryptophan as a P3 residue and N-Cbz as the terminal
group, shows an IC50 value of 1 mM against FIV PR
(Table 3). Interestingly, compound 28, with an N-acetyl
norstatine
((2S,3S)-3-amino-2hydroxy-4-phenylbutyric
acid) as a transition-state isostere.10 Using hydro-
xymethyl-carboxamide as a transition-state mimic has
Table 1. Structure and activity of TL3 derivatives against FIV, HIV and drug-resistant HIV proteasesa
Inhibitor
R
FIV PRb
IC50 (nM)
HIV PRc
IC50 (nM)
HIV (G48V)c
IC50 (nM)
HIV (V82F)c
IC50 (nM)
TL3
1
2
CBz
CBz-Ser
4-MePhSO2
PhSO2
100
128
17
138
4
17
5
46
21 (X5)
202 (X6)
28 (X6)
15 (X4)
58 (X3)
6 (X1)
3
4
5
6
7
8
9
4-BrPhSO2
4-O2NPhSO2
4-MeOPhSO2
PhCH2SO2
PhCO
141
228
310
118
156
62
70
79
20
23
17
11
3-Pyr-CO
53 (X5)
43 (X4)
10
11
12
13
4-F3COPhNHCO
4-F3CPhNHCO
4-MePhNHCO
4-MeOPhNHCO
119,000
55% (200 mM)
37% (200 mM)
61% (200 mM)
1800
2000
160
14
aIC50 values were determined in duplicate.
bData obtained at pH 5.25 at 37 ꢀC in 0.1M NaH 2PO4, 0.1M sodium citrate, 0.2 M NaCl, 0.1mM DTT, 5% glycerol, and 5% DMSO in volume.
cData obtained at pH 5.25 at 37 ꢀC in 0.1M MES, 5% glycerol, and 5% DMSO in volume.

1188
V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
Table 3. Structure and activity of unsymmetrically substituted inhi-
bitors of HIV and FIV proteases
Figure 1. Inhibition of TL3 and its derivatives against the wild-type
HIV and FIV proteases.
Table 2. Structure and activity of unsymmetrically substituted inhi-
bitors of HIV and FIV proteases
Inhibitors
26 27 28 29 30 31 32 33
Wild-type proteases
25
FIV PR (IC50, nM) 92,000 212 1000 256 1200 80 48 117 182
HIV PR (IC50, nM) 25
1
0
1
2
1
8
1
2
1
5
8
1
Therefore with phenylalanine in both P1and P3 is the
appropriate combination for good binding of 31.
Based on the X-ray crystallography studies of LP130
complex with FIV PR14 and JE2147 complex with HIV
PR,15 a chemical structure of VLE776 with binding
pockets were constructed and is shown in Figure 2. The
orientation of the phenylalanine side-chain in HIV PR
is completely di€erent from that observed in FIV PR,
due to the presence of the Phe53 side chain in the ¯ap
region of HIV PR. The phenyl ring at P3 is tightly
packed between Pro81and Phe53. In FIV PR, the
phenyl ring at P3 rotates away from the ¯ap and is in
closed contact with the three residues, Q99, I98 and I57.
Inhibitors
Wild-type proteases
TL3
21
22
23
24
FIV PR (IC50, nM)
HIV PR (IC50, nM)
100
4
95
20
200
18
5200
15
19,200
475
A resistance surmountable inhibitors, 31/VLE776
HIV PR also develops in vitro a high level of resistance
to TL3 by acquiring mutations in the protease-encoding
gene, though it takes a relatively long time.16 Mutations
conferring TL3-resistance were L24I, M46I, F53L,
L63P, V77I and V82A. The IC50 values of TL3 against
wild-type HIV PR and TL3-resistant HIV PR were 4 and
144 nM respectively. In this study, compound 31
(VLE776) was found to be active against both wild-type
HIV PR (IC50=8 nM) and the TL3-resistant protease
(IC50=40 nM). Other FDA approved drugs were also
tested against the TL3-resistant HIV PR and they showed
about 12±22-fold decrease in potency as compared to the
wild-type HIV PR. (Table 4).
group, displays a 4-fold higher activity. Removal of the
dimethyl group (29) from the thiazolidine ring reduces
the potency of compound 28 against FIV PR by 5-fold,
whereas no e€ect was observed against HIV PR. Con-
version of the methyl ester 28 into methyl amide 30 was
found to increase the inhibitory activity against FIV PR
by a factor of 3. Replacing the tryptophan group in 28
with a phenylalanine group in 31 (VLE776) as the P3
site resulted in a 2- and 5-fold increase in the binding
anity to HIV and FIV PRs respectively. However,
compound 33, with the alanine group at the P3 site
shows only a 1.4-fold increase in IC50 against FIV PR
compared to that of 28. Therefore, current observation
suggests that the steric interaction between neighboring
P1and P3 side chains is still a crucial factor to facilitate
proper binding in the active site of FIV PR. With phe-
nylalanine as the P3 binding site, it is not too big or too
small compared to tryptophan and alanine, respectively.
Preliminary study on the synthesis of bifunctional
inhibitors
Our initial e€ort in this regard is to incorporate a gp-
120 glycosidase inhibitor as the N-terminal protecting

V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
1189
Conclusion
In summary, we have designed and synthesized a series
of novel TL3 derivatives containing allophenyl-
norstatine with a hydroxymethylcarboxamide isostere
as transition-state analogue inhibitors of HIV and FIV
proteases. From our SAR study on HIV and FIV pro-
tease inhibition, we have identi®ed VLE776 as the best
FIV PR inhibitor to date. Also VLE776 showed a
strong inhibition against the drug-resistant mutant PRs
(G48V and V82F) and the TL3-resistant HIV PR. Studies
to determine the pharmacokinetic pro®les of VLE776
are currently in progress.
Experimental
Analytical TLC was performed on pre-coated plates
(Merck, silica gel 60F-254). Silica gel used for ¯ash col-
umn chromatography was Mallinckrodt Type 60 (230±
400 mesh). NMR (1H, 13C) spectra were recorded either
on a Bruker AMX-400, AMX-500 or AMX-600 MHz
fourier transform spectrometer. Coupling constants (J)
are reported in hertz, and chemical shifts are reported in
parts per million (d) relative to tetramethylsilane (TMS,
Figure 2. Comparison of subsites of HIV and FIV proteases.
1
0.0 ppm) or DMSO (2.49 ppm for H and 39.5 ppm for
13C) or CD3OD (3.30 ppm for 1H and 49.0 ppm for 13C)
as internal reference. All new compounds were homo-
geneous by TLC and were characterized by satisfactory
1H, 13C NMR, and mass spectra.
group of an HIV protease inhibitor e€ective against the
wild-type and some resistant mutants. Previous studies
indicate that the N-terminal protecting group of inhibitors
with a small P3 group has little e€ect on the activity.4 We
therefore decided to attach deoxynojirimycin,17 a gp-
120 glycosidase inhibitor, to the N-terminus of the pro-
tease inhibitor 354a via a linker (Scheme 5). Compound
36 was shown to be active against HIV protease
(IC50=39 nM) and work is in progress to investigate its
activity against di€erent glycosidases.
Compound 1. To a solution of compound TL34a,4b (1g,
1.10 mmol) in 2,2-dimethoxypropane (20 mL) was
added catalytic amounts of p-TsOH. The reaction mix-
ture was heated at room temperature for 24 h then
Table 4. Comparation of inhibitory activity of FDA approved drugs and 31 against HIV-WT, drug-resistant and TL3-resistant proteases
IC50, nM
Inhibitors
WT
G48V
V82F
V82A
V82A, F53L M46I
V82A, V77I, L63P F53L, M46I, L24I
TL3
SQV
NFV
RTV
31
4
2
3
2
8
21(5X)
ND
ND
ND
68(8X)
15(4X)
ND
ND
ND
28(4X)
13(3X)
7(4X)
6(2X)
7((4X)
16(2X)
51(3X)
20(10X)
19(6X)
25(13X)
ND
144(36X)
37(19X)
35(12X)
43(22X)
40(5X)
Scheme 5. (a) HBTU, DMF, Et3N, 60% (b) H2, Pd/C, AcOH, 56%.

1190
V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
diluted with EtOAc. The organic solution was washed
with satd aq NaHCO3 and satd aq NaCl, dried over
MgSO4, ®ltered and concentrated in vacuo to give acet-
onide as a white solid. The above acetonide in MeOH
(30 mL) containing 10% Pd/C (170 mg) was stirred
under H2 (1atm) at 20 ꢀC for 6 h. The reaction mixture
was ®ltered through Celite and then concentrated in
vacuo to give diamine in nearly quantitative yields. To
the above diamine (20 mg, 0.029 mmol) in DMF (2 mL)
were added Cbz-Ser-OH (14 mg, 0.058 mmol) and
HBTU (22.4 mg, 0.058 mmol). The reaction mixture was
stirred at room temperature overnight then diluted with
EtOAc. The organic solution was washed with satd aq
NaHCO3 and satd aq NaCl, dried over MgSO4, ®ltered
and concentrated in vacuo. The residue was dissolved in
MeOH (2 mL) and pTsOH (4 mg) was added. The reac-
tion mixture was stirred for 24 h and then water (2 mL)
was added. Compound 1 which precipitated out was
collected and dried (78%): 1H NMR (600 MHz,
DMSO-d6) 8.08 (d, 1H, J=7.4), 7.62 (d, 1H, J=9.0),
7.29±7.37 (m, 7H), 7.14±7.16 (m, 3H), 7.07±7.10 (m,
1H), 5.02 (d, 1H, J=12.7), 5.00 (d, 1H, J=12.7), 4.41±
4.45 (m, 1H), 4.28 (dt, 1H, J=14.1, 7.0), 4.09 (dd, 1H,
J=13.8, 6.4), 4.03 (dd, 1H, J=8.6, 6.9), 3.58 (dd, 1H,
J=10.9, 5.8), 3.52 (dd, 1H, J=10.8, 6.7), 3.22 (s, 1H),
2.74 (dd, 1H, J=13.9, 10.3), 2.59 (dd, 1H, J=13.7, 3.8),
1.75±1.81 (m, 1H), 1.15 (d, 3H, J=7.0), 0.69 (d, 3H,
J=6.7), 0.62 (d, 3H, J=6.7); 13C NMR (150 MHz,
DMSO-d6) 171.8, 170.3, 169.9, 155.9, 138.9, 136.9,
129.1, 128.3, 127.8, 127.7, 125.7, 73.2, 65.5, 61.8, 58.0,
57.0, 50.4, 48.3, 38.5, 30.4, 19.2, 18.1, 18.0; HRMS
(FAB+), calcd for C56H74N8O14Na m/e 1105.5222,
found m/e 1105.5181.
2H), 7.46±7.64 (m, 4H), 7.33 (d, 1H, J=9.5), 7.15±7.16
(m, 5H), 7.07±7.10 (m, 1H), 4.65 (s, 1H), 4.46 (m, 1H),
3.93 (dd, 1H, J=9.0, 6.5), 3.87 (m, 1H), 3.20 (s, 1H),
2.74 (dd, 1H, J=13.5, 10.0), 2.60 (dd, 1H, J=14.0,
10.0), 1.73 (m, 1H), 0.97 (d, 3H, J=7.0); HRMS
(FAB+), calcd for C46H60N6O10S2Cs m/e 1053.2867,
found m/e 1053.2904.
Compound 4. In the same manner, diamine was reacted
with 4-bromophenylsulfonyl chloride followed by
deprotection to give compound 4 (70%) as a white
1
solid; H NMR (500 MHz, DMSO-d6), 8.09 (bs, 1H),
7.65±7.75 (m, 5H), 7.32 (d, 1H, J=9.5), 7.14±7.15 (m,
1H), 7.07±7.10 (m, 1H), 4.65 (s, 2H), 4.44 (m, 1H), 3.84±
3.92 (m, 2H), 3.19 (s, 1H), 2.73 (dd, 1H, J=14.0, 10.0),
2.56 (m, 1H), 1.65±1.70 (m, 1H), 1.00 (d, 3H, J=7.0),
0.58 (d, 3H, J=7.0), 0.54 (d, 3H, J=7.0); HRMS
(FAB+), calcd for C46H58N6O10S2Br2Cs m/e
1211.1056, found m/e 1211.1021.
Compound 5. In the same manner, diamine was reacted
with 4-nitrophenylsulfonyl chloride followed by depro-
1
tection to give compound 5 (74%) as a white solid: H
NMR (500 MHz, DMSO-d6), 8.35 (d, 2H, J=8.5), 8.02
(d, 2H, J=8.5), 7.70 (d, 1H, J=8.5), 7.34 (d, 1H,
J=9.0), 7.13±7.14 (m, 5H), 7.08 (m, 1H), 4.64 (s, 2H),
4.42 (m,1H), 4.00 (m, 1H), 3.89 (dd, 1H, J=8.5, 6.5),
3.18 (s, 1H), 2.72 (m, 1H), 2.54 (m, 1H), 1.65 (m, 1H),
1.01 (d, 1H, J=.0), 0.56 (d, 1H, J=6.5), 0.5 (d, 1H,
J=6.5).
Compound 8. In the same manner, diamine was reacted
with benzoyl chloride followed by deprotection to give
compound 8 (88%) as a white solid: 1H NMR
(500 MHz, DMSO-d6), 8.52 (d, 1H, J=7.4), 7.88 (d, 2H,
J=7.0), 7.40±7.61(m, 6H), 7.06±7.81 (m, 6H), 4.50±
4.55 (m, 1H), 4.43±4.49 (m, 1H), 4.07 (dd, 1H, J=8.4,
6.6), 3.27 (s, 1H), 2.77 (dd, 1H, J=13.6, 10.3), 1.81±1.88
(m, 1H), 1.30 (d, 3H, J=7.4), 0.70 (d, 3H, J=7.0), 0.66
(d, 3H, J=7.0); 13C NMR (125 MHz, DMSO-d6), 171.9,
170.2, 166.2, 138.9, 134.1, 131.3, 129.0, 128.2, 127.7,
127.4, 125.6, 73.2, 57.8, 50.4, 48.9, 38.5, 30.4, 19.3, 17.8,
17.6; HRMS (FAB+), calcd for C48H60N6O8Na m/e
871.4370, found m/e 871.4373.
Compound 2. To the above diamine (31mg, 0.046 mmol)
in dry pyridine (2 mL) at 0 ꢀC was added a solution of p-
toluenesulfonyl chloride (17.4 mg, 0.091 mmol) in
CH2Cl2 (0.5 mL). The reaction mixture was stirred at
room temperature for 24 h then diluted with EtOAc.
The organic solution was washed with 1N HCl, satd aq
NaHCO3 and satd aq NaCl, dried over MgSO4, ®ltered
and concentrated in vacuo. The residue was dissolved in
MeOH (2 mL) and pTsOH (4 mg) was added. The reac-
tion mixture was stirred for 24 h and then water (2 mL)
was added. Compound 2 which precipitated out was
collected and dried (88%): 1H NMR (500 MHz,
DMSO-d6) 7.85 (d, 1H, J=8.5), 7.64 (d, 2H, J=8.0),
7.58 (d, 1H, J=8.5), 7.32 (m, 4H), 7.14 (m, 7H), 4.64
(s, 2H), 4.45 (m, 1H), 3.91 (dd, 1H, J=9.0, 6.5), 3.82
(m, 1H), 3.18 (m, 1H), 2.75 (m, 1H), 2.57 (m, 1H), 2.34
(s, 3H), 1.70 (m, 1H), 0.95 (d, 3H, J=7.0), 0.58 (d, 3H,
J=7.0), 0.54 (d, 3H, J=7.0); HRMS (FAB+), calcd
for C48H64N6O10S2Cs m/e 1081.3180, found m/e
1081.3219.
Compound 9. In the same manner, diamine was coupled
with 6-methylnicotinic acid followed by deprotection to
give compound 9 (55%) as a white solid: 1H NMR
(500 MHz, DMSO-d6), 8.91 (s, 1H), 8.67 (d, 1H,
J=7.4), 8.11 (d, 1H, J=8.0), 7.64 (d, 1H, J=8.8), 7.42
(d, 1H, J=9.6), 7.35 (d, 1H, J=8.1), 7.08±7.28 (m, 7H),
4.68 (s, 1H), 4.47±4.53 (m, 2H), 4.06 (m, 1H), 3.26 (s,
1H), 2.76 (m, 1H), 2.61 (m, 1H), 2.55 (s, 3H),1.82-1.86
(m, 1H), 1.29 (d, 3H, J=7.0), 0.70 (d, 3H, J=6.6),
0.66(d, 3H, J=6.6); 13C NMR (125 MHz, DMSO-d6),
179.8, 174.2, 170.4, 157.6, 148.5, 144.9, 138.5, 137.6,
137.3, 136.4, 135.1, 132.1, 82.9, 67.3, 59.9, 58.4, 51.2,
40.0, 33.6, 28.8, 27.4, 27.1; HRMS (FAB+), calcd for
C48H62N8O8Na m/e 901.4583, found m/e 901.4608.
The preparations of compounds 3±13 were carried out
using the general procedures for coupling and depro-
tection.
Compound 3. In the same manner, diamine was reacted
with phenylsulfonyl chloride followed by deprotection
to give compound 3 (58%) as a white solid: H NMR
(500 MHz, DMSO-d6) 8.00 (d, 1H, J=8.5), 7.78 (m,
Compound 10. In the same manner, diamine was reacted
with 4-trifuoromethylphenyl isocyanate followed by
deprotection to give compound 10 (67%) as a white
1

V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
1191
solid: 1H NMR (400 MHz, DMSO-d6), 8.87 (s, 1H),
7.84 (d, 1H, J=8.9), 7.44 (d, 2H, J=8.9), 7.13±7.21 (m,
8H), 7.08±7.10 (m, 1H), 6.43 (d, 1H, J=7.6), 4.67 (s,
1H), 4.46±4.51 (m, 1H), 4.26±4.33 (m, 1H), 4.06 (dd,
1H, J=8.6, 7.0), 3.24 (s, 1H), 2.76 (dd, 1H, J=13.8,
10.5), 2.58±2.63 (m, 1H), 1.77±1.86 (m, 1H), 1.14 (d, 3H,
J=6.8), 0.70 (d, 3H, J=6.8), 0.65 (d, 3H, J=6.5);
HRMS (FAB+), calcd for C50H60N8O10F6Cs m/e
1179.3391, found m/e 1179.3350.
Compound 15. In the same manner, amine was coupled
with Cbz-Cl to give compound 15 (70%) as a white
solid: 1H NMR (400 MHz, CD3OD) 7.13±7.35 (m,
15H), 6.39±6.47 (m, 1H), 5.09 (s, 2H), 5.04 (s, 2H),
4.05±4.30 (m, 4H), 3.76 (m, 2H), 2.75±2.87 (m, 4H),
1.82±1.90 (m, 1H), 1.20 (d, 3H, J=7.2), 0.86 (m, 3H),
0.75 (m, 3H); HRMS (FAB+), calcd for
C42H50N4O8Na m/e 761.3629, found m/e 761.3640.
Compound 17a. To a solution of acid 16a9 (261mg,
1mmol) in DMF (4 mL) was added Val-OMe.HCl
(168 mg, 1mmol), HBTU (379 mg, 1mmol) and Et 3N
(0.28 mL). The reaction mixture was stirred overnight
and then diluted with EtOAc. The organic solution was
washed with satd aq NaHCO3 and satd aq NaCl, dried
over MgSO4, ®ltered and concentrated in vacuo. The
residue was puri®ed by ¯ash chromatography in 10%
EtOAc/Hexane to give compound 17a (85%): 1H NMR
(500 MHz, CD3OD) 4.65 (d, 1H, J=9.1), 4.60 (d,
J=9.1), 4.21±4.37 (m, 2H), 3.70 (s, 3H), 2.12±2.19 (m,
1H), 1.56 (s, 3H), 1.43 (s, 9H), 1.38 (s, 3H), 0.98 (m,
6H); HRMS (FAB+), calcd for C17H30N2O5SNa m/e
397.1768, found m/e 397.1787.
Compound 12. In the same manner, diamine was reacted
with 4-methylylphenyl isocyanate followed by depro-
1
tection to give compound 12 (70%) as a white solid: H
NMR (400 MHz, DMSO-d6), 8.46 (d, 1H, J=7.3), 7.79
(d, 2H, J=7.9), 7.59 (d, 1H, J=9.1), 7.42 (d, 1H,
J=9.4), 7.26 (d, 2H, J=7.9), 7.08±7.17 (m, 5H), 4.38±
4.51 (m, 4H), 4.06 (dd, 1H, J=8.8, 6.4), 3.25 (s, 1H),
2.76 (dd, 1H, J=13.8, 10.3), 2.61 (dd, 1H, J=13.8, 3.8),
2.35 (s, 3H), 1.80±1.88 (m, 1H), 1.28 (d, 3H, J=7.04),
0.69 (d, 3H, J=7.0), 0.65 (d, 3H, J=6.8); 13C NMR
(100 MHz, DMSO-d6), 172.1, 170.3, 166.1, 141.2, 139.0,
131.3, 129.1, 128.8, 128.1, 127.8, 127.5, 125.7, 73.3, 57.8,
50.4, 48.9, 38.6, 30.5, 21.0, 20.8, 19.3, 17.8, 17.6; HRMS
(FAB+), calcd for C50H64N6O8Na m/e 899.4683, found
m/e 899.4690.
Compound 17b. In the similar manner, acid 16b9 was
coupled with ValOMe.HCl to give compound 17b
1
Compound 13. In the same manner, diamine was reacted
with 4-methoxyphenyl isocyanate followed by depro-
(80%) as a white solid: H NMR (500 MHz, CDCl3)
4.73 (bs, 1H), 4.66 (m, 1H), 4.53 (m, 1H), 4.38 (bs, 1H),
3.73 (s, 3H), 2.14±2.20 (m, 1H), 1.50 (s, 9H), 0.93 (d,
3H, J=6.6), 0.89 (d, 3H, J=6.6); 13C NMR (125 MHz,
CDCl3) 171.6, 169.8, 153.7, 81.7, 56.9, 51.8, 49.2, 31.1,
27.9, 18.7, 17.3; HRMS (FAB+), calcd for
C15H26N2O5SNa m/e 369.1455, found m/e 369.1490.
1
tection to give compound 13 (70%) as a white solid: H
NMR (500 MHz, DMSO-d6), 8.45 (s, 1H), 7.80 (d, 1H,
J=9.0), 7.33 (d, 1H, J=8.9) 7.25 (d, 1H, J=8.6), 7.14±
7.17 (m, 4H), 7.08±7.11 (m, 1H), 6.74 (d, 1H, J=0.3),
6.25 (d, 1H, J=7.7), 4.65 (s, 1H), 4.48 (m, 1H), 4.27 (dt,
1H, J=13.8, 6.7), 4.05 (t, 1H, J=7.5), 3.70 (s, 3H), 3.25
(s, 1H), 2.76 (dd, 1H, J=13.8, 10.7), 2.60 (dd, 1H,
J=13.7, 3.5), 1.79±1.85 (m, 1H), 1.14 (d, 3H, J=6.74),
0.70 (d, 3H, J=6.7), 0.65 (d, 3H, J=6.6); HRMS
(FAB+), calcd for C50H66N8O10Cs m/e 1071.3956,
found m/e 1071.3929.
Compound 18a. To a solution of compound 17a
(374 mg, 1mmol) in CH 2Cl2 (4 mL) was added 4 N HCl
in dioxane (4 mL). The reaction mixture was stirred for
4 h and the solvent was removed to dryness. To a solu-
tion of this residue in DMF (8 mL) was added the acid11
(295 mg, 1mmol) HBTU (379 mg, 1mmol) and Et N
3
Compound 14. To a solution of amine 14a18 (50 mg,
0.13 mmol) in DMF (2 mL) was added Cbz-AlaVal-OH
(40.3 mg, 0.13 mmol) and HBTU (47.4 mg, 0.13 mmol).
The reaction mixture was stirred for 2 h then diluted
with EtOAc. The organic solution was washed with satd
aq NaHCO3 and satd aq NaCl, dried over MgSO4, ®l-
tered and concentrated in vacuo. The residue was dis-
solved in 4 N HCl in dioxane (2 mL) and stirred for 2 h.
After removal of the solvents to dryness, the residue was
dissolved in DMF and Cbz-Val-OH (31mg, 0.13 mmol)
and HBTU (47 mg) and Et3N (52 mL, 0.38 mmol) was
added. The reaction mixture was stirred for 6 h then
diluted with EtOAc. The organic solution was washed
with satd aq NaHCO3 and satd aq NaCl, dried over
MgSO4, ®ltered and concentrated in vacuo to give
(0.14 mL). The reaction mixture was stirred overnight
and then diluted with EtOAc. The organic solution was
washed with satd aq NaHCO3 and satd aq NaCl, dried
over MgSO4, ®ltered and concentrated in vacuo. The
residue was puri®ed by ¯ash chromatography in 20%
EtOAc/Hexane to give compound 18a (72%): 1H NMR
(600 MHz, CD3OD) 7.14±7.26 (m, 5H), 5.05 (d, 1H,
J=9.2), 4.93 (d, 1H, J=9.2), 4.62 (s, 1H), 4.50 (d, 1H,
J=3,9), 4.44 (d, 1H, J=5.7), 4.01(m,1H), 3.70 (s, 3H),
2.81(dd, 1H, J=14.0, 3.5), 2.62 (dd, 1H, J=13.6, 11.0),
2.17 (m, 1H), 1.60 (s, 3H), 1.44 (s, 3H), 1.31 (s, 9H), 0.96
(t, 6H, J=7.0); 13C NMR (150 MHz, CD3OD) 173.4,
172.3, 170.9, 157.8, 140.2, 130.6, 129.2, 127.1, 80.1, 73.3,
73.1, 59.0, 56.0, 52.4, 35.7, 32.0, 30.1, 28.7, 25.3, 19.5,
18.4; HRMS (FAB+), calcd for C27H41N3O7SNa m/e
574.2665, found m/e 574.2693.
1
compound 14 (65%): H NMR (500 MHz, DMSO-d6),
7.09±7.35 (m, 20H), 5.04 (d, 2H, J=12.5), 5.02 (s, 2H),
4.71±4.77 (m, 2H), 4.47±4.50 (m, 2H), 4.14 (m, 1H), 4.08
(t, 2H, J=8.1), 3.76 (t, 1H, J=8.8), 3.25 (m, 3H), 2.76
(m, 2H), 2.60 (m, 2H), 1.80 (m, 2H), 1.18 (d, 3H,
J=7.0), 1.15 (d, 3H, J=7.0), 0.61±0.72 (m, 12H);
HRMS (FAB+), calcd for C47H59N5O9Na m/e
860.4313, found m/e 860.4390.
Compound 18b. To a solution of compound 17b
(346 mg, 1mmol) in CH 2Cl2 (4 mL) was added 4 N HCl
in dioxane (4 mL). The reaction mixture was stirred for
4 h and the solvent was removed to dryness. To a solu-
tion of this residue in DMF (8 mL) was added the acid
(295 mg, 1mmol), HBTU (379 mg, 1mmol) and Et N
3

1192
V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
(0.14 mL). The reaction mixture was stirred overnight
and then diluted with EtOAc. The organic solution was
washed with satd aq NaHCO3 and satd aq NaCl, dried
over MgSO4, ®ltered and concentrated in vacuo. The
residue was puri®ed by ¯ash chromatography in 20%
EtOAc/Hexane to give compound 18b (68%); 1H NMR
(500 MHz, CD3OD) 7.13±7.29 (m, 5H), 5.00 (d, 1H,
J=9.5), 4.96 (t, 1H, J=7.0), 4.77 (d, 1H, J=9.9), 4.59
(d, 1H, J=3.3), 4.41(d, 1H, J=5.5), 4.02±4.05 (m, 2H),
3.70 (s, 3H), 3.40 (dd, 1H, J=11.7, 7.7), 3.14 (dd, 1H,
J=11.7, 6.6), 2.89 (dd, 1H, J=14.0, 3.0), 2.66 (m, 1H),
2.16 (m, 1H), 1.32 (s, 9H), 0.95 (d, 6H, J=6.6); 13C
NMR (125 MHz, CD3OD) 173.4, 172.2, 171.8, 157.7,
140.0, 130.5, 129.1, 127.1, 80.0, 73.3, 65.3, 64.0, 61.4,
59.1, 56.0, 52.5, 51.0, 34.0, 32.0, 28.7, 19.5, 18.4; HRMS
(FAB+), calcd for C25H37N3O7SNa m/e 546.2244,
found m/e 546.2239.
1H, J=4.7), 3.95 (d, 1H, J=10.0), 3.80 (m, 1H), 3.75 (s,
3H), 3.59 (m, 1H), 3.16 (s, 1H), 2.98 (d, 2H, J=12.3),
2.86 (dd, 1H, J=12.7, 9.6), 2.76 (m, 1H), 2.21 (m, 1H),
1.63 (s, 3H), 1.41 (s, 3H), 1.31 (s, 9H), 0.98 (d, 3H,
J=6.9), 0.93 (d, 3H, J=6.8); 13C NMR (125 MHz,
CDCl3) 174.4, 171.4, 155.5, 138.0, 129.4, 128.2, 126.2,
80.7, 79.2, 72.0, 62.0, 60.3, 58.6, 56.8, 55.6, 54.6, 52.5,
35.9, 30.4, 29.6, 28.2, 25.3, 19.4, 17.7, 14.1; HRMS
(FAB+), calcd for C27H43N3O6SNa m/e 560.2765,
found m/e 560.2742.
Compound 23. To a solution of compound 20 (54 mg,
0.1mmol) in CH 2Cl2 (2 mL) was added 4 N HCl in
dioxane (2 mL). The reaction mixture was stirred for 4 h
and the solvent was removed to dryness. To a solution
of crude amine in DMF (2 mL) was added Cbz-AlaVal-OH
(32 mg, 0.1mmol), HBTU (38 mg, 0.1mmol) and Et N
3
(28 mL, 0.2 mmol). The reaction mixture was stirred for
12 h and then diluted with EtOAc. The organic solution
was washed with satd aq NaHCO3 and satd aq NaCl,
dried over MgSO4, ®ltered and concentrated in vacuo.
The residue was puri®ed by ¯ash chromatography in
15% EtOAc/Hexane to give compound 23 (80%) as a
white solid: 1H NMR (600 MHz, CD3OD) 7.12±7.35
(m, 10H), 5.09 (s, 2H), 5.05 (s, 1H), 4.53 (d, 1H, J=9.5),
4.34 (dd, 1H, J=10.6, 5.4), 3.95±4.18 (m, 4H), 3.73 (s,
3H), 2.94±2.98 (m, 1H), 2.72±2.76 (m, 2H), 2.16±2.21
(m, 1H), 1.87±1.92 (m, 1H), 1.57 (s, 3H), 1.37 (s, 3H),
1.32 (d, 3H, J=7.1), 0.94±0.96 (m, 6H), 0.77 (t, 3H,
J=7.0), 0.60 (t, 3H, J=7.0); 13C NMR (150 MHz,
CD3OD), 175.5, 174.3, 173.6, 173.1, 158.4, 140.0, 138.1,
130.6, 129.5, 129.3, 129.0, 127.2, 82.6, 74.1, 67.7, 61.5,
60.4, 58.7, 58.5, 55.5, 53.1, 54.9, 52.8, 52.1, 37.0, 32.0,
31.4, 30.3, 26.7, 19.9, 19.7, 18.4, 18.0; HRMS (FAB+),
calcd for C38H55N5O8SNa m/e 764.3664, found m/e
764.3689.
Compound 19. To a solution of compound 18a (551mg,
1mmol) in 20% MeOH in THF (8 mL) was added a
solution of LiOH (72 mg, 3 mmol) in water (0.5 mL).
The reaction mixture was stirred for 4 h and then dilu-
ted with EtOAc. The organic solution was washed with
10% citric acid and water, dried over MgSO4, ®ltered
and concentrated in vacuo. To a solution of the residue
in 1,2-dichloroethane (10 mL) was added N-hydroxy-
succinimide (115 mg, 1 mmol), HOBt (153 mg, 1 mmol)
and DCC (206 mg, 1mmol). The reaction mixture was
stirred for 24 h and excess of solvents was removed to
dryness. The residue was dissolved in THF (10 mL) and
methylamine (0.7 mL, 2 M in THF, 1.4 mmol) was
added. The reaction mixture was stirred for 1h and the
precipitate which forms was removed by ®ltration.
Excess of solvents were removed and the crude product
was puri®ed by ¯ash chromatography to give com-
1
pound 19 (90%) as a white solid: H NMR (500 MHz,
CD3OD) 7.13±7.18 (m, 5H), 5.09 (d, 1H, J=9.6), 4.95
(d, 1H, J=8.8), 4.57 (s, 1H), 4.54 (d, 1H, J=4.4), 4.18
(m, 1H), 3.94 (m, 1H), 2.90 (m, 1H), 2.70 (s, 3H), 2.03
(m, 1H), 1.57 (s, 3H), 1.40 (s, 3H), 1.31 (s, 9H), 0.96 (d,
3H, J=6.6), 0.91(d, 3H, J=7.0); 13C NMR (125 MHz,
CD3OD) 173.9, 172.5, 170.5, 157.7, 140.0, 130.5, 129.1,
127.1, 80.0, 73.5, 61.4, 56.1, 52.3, 35.9, 32.0, 30.4, 28.7,
26.3, 26.1, 25.1, 20.9, 19.7, 19.1 14.5; HRMS (FAB+),
calcd for C27H42N4O6SNa m/e 573.2717, found m/e
573.2726.
1
Compound 24. H NMR (600 MHz, CD3OD) 8.27 (d,
1H, J=8.76), 7.86 (d, 1H, J=9.24), 7.26±7.34 (m, 10H),
7.18±7.20 (m, 1H), 5.10 (d, 1H, J=12.7), 5.06 (d, 1H,
J=12.2), 4.65 (d, 1H, J=8.3), 4.53 (m, 1H), 4.47 (d, 1H,
J=7.86), 4.35 (s, 1H), 4.30±4.32 (m, 2H), 4.18 (dd, 1H,
J=14.0, 7.0), 4.13 (t, 1H, J=6.6), 3.67 (s, 3H), 2.99 (dd,
1H, J=12.6, 8.8), 2.87 (dd, 1H, J=12.3, 6.6), 2.11 (dt,
1H, J=21.1, 14.5, 7.5), 1.91±1.93 (m, 1H), 1.53 (s, 3H),
1.35 (s, 3H), 1.32 (d, 3H, J=7.0), 0.94 (t, 3H, J=5.7),
0.80 (dd, 3H, J=9.7, 7.0); 13C NMR (150 MHz,
CD3OD,) 173.3, 173.1, 172.1, 171.7, 171.6, 158.5, 139.3,
138.1, 130.5, 129.6, 129.5, 129.0, 128.7, 127.7, 73.4, 70.6,
67.7, 60.1, 59.3, 52.9, 52.6, 52.4, 51.9, 38.8, 32.0, 31.9,
31.3, 24.3, 20.0, 19.5, 18.7, 18.5, 18.0; HRMS (FAB+),
calcd for C38H53N5O9SNa m/e 778.3564, found m/e
778.3590.
Compound 20. To a solution of compound 17a (374 mg,
1mmol) in CH 2Cl2 (4 mL) was added 4 N HCl in diox-
ane (4 mL). The reaction mixture was stirred for 4 h and
the solvent was removed to dryness. The residue was
dissolved in water and neutralized with 2 MNaOH. The
aqueous solution was extracted with EtOAc and the
organic layer was dried over MgSO4, ®ltered and con-
centrated in vacuo. To a solution of this residue in dry
MeOH (10 mL) was added epoxide4a (263 mg, 1mmol)
and Et3N (0.28 mL). The reaction mixture was stirred at
80 ꢀC for 24 h. After allowing to cool to room tempera-
ture, excess of solvent was removed and the crude pro-
duct was puri®ed by ¯ash chromatography to give
Compound 25. See ref 4a.
Compound 26. 1H NMR (600 MHz, DMSO-d6), 8.10 (d,
1H, J=8.3), 7.68 (d, 1H, J=9.2), 7.60 (d, 1H, J=7.9),
7.46 (d, 1H, J=7.9), 7.31(d, 1H, J=7.9), 6.95±7.21(m,
15H), 4.52±4.55 (m, 1H), 4.44±4.48 (m, 1H), 4.08 (dd,
1H, J=8.8, 6.5), 3.29 (s, 1H), 3.01 (dd, 1H, J=12.6,
1.6), 2.85 (dd, 1H, J=14.9, 10.5), 2.76±2.81 (m, 1H),
2.64 (dd, 1H, J=14.0, 4.0), 1.83±1.86 (m, 1H), 1.74 (s,
1
compound 20 (62%): H NMR (500 MHz, CDCl3) 7.97
(d, 1H, J=9.0), 7.17±7.30 (m, 5H), 4.71 (d, 1H, J=8.7),
4.52 (d, 1H, J=10.0), 4.48 (dd, 1H, J=9.4, 4.9), 4.42 (d,

V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
1193
3H), 0.68 (d, 3H, J=7.0), 0.65 (d, 3H, J=7.0); 13C
NMR (150 MHz, DMSO-d6), 171.6, 170.3, 169.2, 139.0,
136.0, 129.1, 128.8, 128.0, 127.8, 127.3, 125.7, 125.5,
123.3, 120.8, 118.5, 118.1, 111.2, 110.5, 73.3, 57.9, 53.2,
50.6, 38.6, 30.5, 27.4, 22.5, 19.3, 17.9; HRMS (FAB+),
calcd for C54H66N8O8Na m/e 977.4896, found m/e
977.4882.
26.1, 25.2, 22.5, 19.8, 19.7, 19.6, 19.2, 18.5; HRMS
(FAB+), calcd for C40H55N7O7SNa m/e 800.3776,
found m/e 800.3793.
Compound 31. The above amine (49 mg, 0.1mmol) was
coupled with Ac-PheVal-OH (31mg, 0.1mmol) to give
compound 31 (72%) as a white solid: 1H NMR
(400 MHz, CD3OD) 7.15±7.31 (m, 10H), 5.08 (d, 1H,
J=9.4), 4.91(d, 1H, J=9.4), 4.63 (s, 1H), 4.57 (d, 1H,
J=10.0, 5.3), 4.50 (d, 1H, J=3.52), 4.43 (d, 1H,
J=5.88), 4.34±4.37 (m, 1H), 4.07 (d, 1H, J=7.3), 3.70
(s, 3H), 2.98 (dd, 1H, J=14.1, 5.0), 2.73±2.87 (m, 3H),
2.11±2.19 (m, 1H), 1.90±1.97 (m, 1H), 1.86 (s, 3H), 1.60
(s, 3H), 1.45 (s, 3H), 0.95 (dd, 3H, J=6.8, 3.2), 0.81(d,
3H, J=6.8); 13C NMR (100 MHz, CD3OD), 173.7,
173.5, 173.2, 173.1, 172.0, 170.9, 139.9, 138.5, 130.6,
130.2, 129.4, 129.3, 127.7, 127.3, 73.0, 72.7, 60.3, 59.1,
56.0, 54.8, 52.5, 52.4, 38.5, 34.7, 32.0, 30.2, 25.3, 22.3,
19.7, 19.5, 18.6, 18.5; HRMS (FAB+), calcd for
C38H53N5O8SNa m/e 762.3507, found m/e 762.3539.
General procedure for the synthesis of compounds 22, 27,
28, 29 and 31
To a solution of compound 18a (551mg, 1mmol) or 18b
(523 mg, 1mmol) or 19 (550 mg, 1mmol) in CH 2Cl2
(10 mL) was added 4 N HCl in dioxane (10 mL). The
reaction mixture was stirred for 4 h and the solvent was
removed to dryness. To a solution of the amine (49 mg,
0.1mmol) in DMF (2 mL) was added dipeptide
(0.1mmol), HBTU (38 mg, 0.1mmol) and Et N (28 mL,
3
0.2 mmol). The reaction mixture was stirred for 12 h and
then diluted with EtOAc. The organic solution was
washed with satd aq NaHCO3 and satd aq NaCl, dried
over MgSO4, ®ltered and concentrated in vacuo. The
residue was puri®ed by ¯ash chromatography in 15%
EtOAc/Hexane to give the title compound.
Compound 32. The above amine (49 mg, 0.1mmol) was
coupled with Ac-PheVal-OH (31mg, 0.1mmol) to give
compound 32 (79%) as a white solid: 1H NMR
(400 MHz, CD3OD) 8.03 (d, 1H, J=8.8), 7.30 (d, 1H,
J=7.4), 7.18±7.22 (m, 10H), 5.08 (d, 1H, J=9.4), 4.91
(m, 1H), 4.58 (dd, 1H, J=11.4, 5.0), 4.55 (s, 1H), 4.52
(d, 1H, J=4.1), 4.33±4.38 (m, 1H), 4.16 (d, 1H, J=8.2),
4.09 (d, 1H, J=7.3), 3.00 (dd, 1H, J=9.4, 5.0), 2.90 (dd,
1H, J=14.1, 3.2), 2.79 (dd, 1H, J=14.1, 10), 2.70 (s,
3H), 1.96±2.00 (m, 1H), 1.86 (s, 3H), 1.56 (s, 3H), 1.40
(s, 3H), 0.96 (m, 3H), 0.79 (t, 3H, J=6.8); 13C NMR
(100 MHz, CD3OD,) 173.9, 173.6, 173.4, 172.8, 172.2,
170.6, 139.9, 138.5, 130.5, 130.2, 129.4, 129.3, 127.7,
127.3, 73.4, 72.8, 60.4, 60.3, 55.9, 54.9, 52.5, 38.5, 34.9,
32.0, 31.9, 30.3, 26.1, 25.2, 22.3, 19.8, 19.6, 18.6, 17.7;
HRMS (FAB+), calcd for C38H54N6O7SNa m/e
761.3667, found m/e 761.3640.
Compound 22. The above amine (49 mg, 0.1mmol) was
coupled with Cbz-AlaVal-OH (32 mg, 0.1mmol) to give
compound 22 (75%) as a white solid: 1H NMR
(400 MHz, CD3OD) 7.11±7.34 (m, 10H), 5.07 (s, 2H),
5.04 (m, 1H), 4.62 (s, 1H), 4.35±4.48 (m, 3H), 4.05±4.13
(m, 2H), 3.70 (s, 3H), 2.79±2.86 (m, 2H), 2.13±2.16 (m,
1H), 1.60 (s, 3H), 1.45 (s, 3H), 0.93±0.97 (m, 9H), 0.77±
0.79 (m, 3H); HRMS (FAB+), calcd for
C38H53N5O9SNa m/e 778.3564, found m/e 778.3580.
Compound 29. The above amine (46 mg, 0.1mmol) was
coupled with Ac-TrpVal-OH (35 mg, 0.1mmol) to give
compound 29 (66%) as a white solid: 1H NMR
(500 MHz, CD3OD) 7.60 (m, 1H), 6.94±7.32 (m, 9H),
5.14 (d, 1H, J=9.2), 4.87 (d, 1H, J=9.2), 4.64 (s, 1H),
4.53 (m, 1H), 4.34±4.43 (m, 3H), 4.05 (d, 1H, J=6.3),
3.70 (s, 3H), 3.15±3.20 (m, 1H), 3.06 (m, 1H), 2.69 (m,
1H), 3.15±3.20 (m, 1H), 3.06 (m, 1H), 2.88 (m, 1H), 2.69
(m, 1H), 2.13 (m, 1H), 1.90 (s, 3H), 1.60 (s, 3H), 1.45 (s,
3H), 0.94 (t, 3H, J=7.0), 0.40 (dd, 3H, J=24.6, 7.0);
HRMS (FAB+), calcd for C38H50N6O8SNa m/e
773.3411, found m/e 773.3450.
Compound 33. The above amine (49 mg, 0.1mmol) was
coupled with Ac-AlaVal-OH (23 mg, 0.1mmol) to give
compound 33 (85%) as a white solid: 1H NMR
(600 MHz, CD3OD) 7.16±7.25 (m, 5H), 5.07 (d, 1H,
J=9.2), 4.92 (d, 1H, J=9.2), 4.62 (s, 1H), 4.48 (d, 1H,
J=3.5), 4.43 (d, 1H, J=5.7), 4.39 (m, 1H), 4.32 (dd,
1H, J=14.0, 7.0), 4.06 (d, 1H, J=7.4), 3.71(s, 3H), 2.83
(dd, 1H, J=14.0, 3.5), 2.73 (dd, 1H, J=14.0, 11.0), 2.16
(m, 1H), 1.95 (s, 3H), 1.60 (s, 3H), 1.45 (s, 3H), 0.96 (t,
3H, J=7.0), 0.80 (t, 3H, J=6.5); 13C NMR (150 MHz,
CD3OD) 175.0, 173.5, 173.3, 173.2, 172.0, 170.9, 139.8,
130.5, 129.3, 127.2, 73.0, 72.8, 60.4, 59.1, 54.6, 52.5,
52.4, 50.4, 34.9, 32.0, 31.9, 30.1, 25.3, 22.3, 19.7, 19.5,
18.5, 17.7; HRMS (FAB+), calcd for C32H49N5O8SNa
m/e 686.3302, found m/e 686.3320.
Compound 30. The above amine (49 mg, 0.1mmol) was
coupled with Ac-PheVal-OH (35 mg, 0.1mmol) to give
compound 30 (55%) as a white solid: 1H NMR
(500 MHz, CD3OD) 7.58 (d, 1H, J=7.7), 7.32 (d, 1H,
J=8.0), 7.17±7.24 (m, 6H), 7.03±7.14 (m, 2H), 6.97±
7.00 (m, 1H), 5.04 (d, 1H, J=9.2), 4.86 (d, 1H, J=9.2),
4.65 (m, 1H), 4.52 (dd, 1H, J=9.2, 3.7), 4.32 (m, 1H),
4.07±4.14 (m, 2H), 3.18 (dd, 1H, J=14.7, 5.9), 3.04 (dd,
1H, J=14.7, 8.1), 2.90 (m, 1H), 2.75±2.80 (m, 1H), 2.70
(s, 3H), 1.94±1.98 (m, 2H), 1.54 (s, 3H), 1.40 (s, 3H),
0.89±0.95 (m, 6H), 0.72 (d, 3H, J=8.0), 0.70 (d, 3H,
J=7.0); 13C NMR (125 MHz, CD3OD,) 174.1, 173.9,
173.4, 173.2, 172.3, 170.7, 139.9, 138.1, 130.6, 129.3,
127.5, 124.6, 122.4, 119.8, 119.4, 112.3, 110.9, 73.5, 72.6,
60.5, 60.4, 55.5, 55.0, 52.5, 34.8, 31.9, 31.6, 30.3, 28.7,
Compound 34. To a solution of deoxynojirimycin tetra-
benzyl ether19 (2.62 g, 5 mmol) in dry DMF (10 mL) was
added ethyl 4-bromobutyrate (0.98 g, 5 mmol) and tetra-
n-butylammonium iodide (1.85 g, 5 mmol). The reaction
mixture was stirred at 80 ꢀC for 12 h and then diluted
with EtOAc. The organic solution was washed with 1 N
HCl and satd aq NaCl, dried over MgSO4, ®ltered and
concentrated in vacuo. The residue was puri®ed by ¯ash

1194
V.-D. Le et al. / Bioorg. Med. Chem. 9 (2001) 1185±1195
chromatography in 10% EtOAc/Hexane to give ester
1
52.9, 51.9, 50.6, 37.4, 35.0, 34.7, 34.3, 32.0, 31.5, 28.9,
27.3, 27.0, 22.0, 21.3, 19.9, 18.5, 17.8; HRMS (FAB+),
calcd for C70H95N6O9S m/e 1163.7155, found m/e
1163.7139.
(3 g, 94%); H NMR (500 MHz, CDCl3) 7.26±7.33 (m,
18H), 7.13 (m, 2H), 4.96 (d, 1H, J=11.1), 4.86 (d, 1H,
J=10.9), 4.80 (d, 1H, J=11.1), 4.68 (d, 1H, J=11.6),
4.65 (d, 1H, J=11.6), 4.47 (d, 1H, J=12.1), 4.43 (d, 1H,
J=12.0), 4.41 (d, 1H, J=10.9), 4.11 (d, 1H, J=7.1),
4.08 (d, 1H, J=7.1), 3.60±3.68 (m, 3H), 3.55 (t, 1H,
J=9.2), 3.47 (t, 1H, J=9.0), 3.10 (dd, 1H, J=11.2, 4.9),
2.73 (m, 1H), 2.59 (m, 1H), 2.34 (m, 1H), 2.13±2.26 (m,
3H), 1.72 (m, 2H), 1.23 (t, 3H, J=7.2); 13C NMR
(125 MHz, CDCl3) 173.2, 138.9, 138.4, 138.3, 137.7,
128.2, 128.2, 127.7, 127.5, 127.4, 127.3, 87.2, 78.5, 78.4,
75.2, 75.0, 73.2, 72.6, 65.7, 63.8, 60.2, 54.3, 51.4, 31.9,
19.5, 14.2; HRMS (FAB+), calcd for C40H48NO6 m/e
638.3476, found m/e 638.3503.
To the above coupling product (1.16 g, 10 mmol) in
AcOH (20mL) containing 10% Pd/C (170mg) was stirred
under H2 (1atm) at 20ꢀC for 2 days. The reaction mixture
was ®ltered through Celite and then concentrated in vacuo
to give 36 (444 mg, 56%) as a white solid (lympholizer);
1H NMR (400 MHz, D2O) 7.29±7.32 (m, 5H), 4.99 (m,
4H), 4.75 (m, 1H), 4.67 (m, 1H), 4.22 (m, 2H), 4.02±4.11
(m, 3H), 3.75 (m, 2H), 3.65 (m, 2H), 3.02 (m, 1H), 2.91
(m, 3H), 2.53 (m, 2H), 1.82±2.01 (m, 9H), 1.63±1.70 (m,
6H), 1.47 (m, 4H), 1.35 (m, 4H), 1.22 (s, 9H), 1.15 (d,
3H, J=7.0), 0.70 (t, 6H, J=6.4); HRMS (FAB+),
calcd for C42H71N6O9 m/e 803.5204, found m/e
803.5309.
To a solution of the above ester (2.55 g, 4 mmol) in 20%
MeOH in THF (8 mL) was added a solution of LiOH
(192 mg, 8 mmol) in water (1 mL). The reaction mixture
was stirred for 4 h and then diluted with EtOAc. The
organic solution was washed with 10% citric acid and
water, dried over MgSO4, ®ltered and concentrated in
vacuo to give the acid 34 (2.3 g, 95%) as a white solid;
1H NMR (600 MHz, CDCl3) 7.26±7.31(m, 18H), 7.09
(m, 2H), 4.92 (d, 1H, J=11.1), 4.84 (d, 1H, J=10.8),
4.76 (d, 1H, J=11.1), 4.72 (d, 1H, J=11.3), 4.66 (d, 1H,
J=11.3), 4.44 (s, 2H), 4.42 (d, 1H, J=11.0), 4.10 (m,
1H), 3.90 (dd, 1H, J=11.6, 4.4), 3.83 (t, 1H, J=9.3),
3.75 (d, 1H, J=10.6), 3.56 (t, 1H, J=8.9), 3.50 (m, 1H),
3.22 (m, 1H), 3.02 (m, 1H), 2.90 (d, 1H, J=7.8), 2.59 (t,
1H, J=11.3), 2.30 (m, 2H), 1.82 (m, 2H); 13C NMR
(150 MHz, CDCl3) 175.7, 138.0, 137.5, 137.3, 136.8,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6,
107.8, 84.9, 75.6, 75.5, 75.0, 73.4, 73.1, 67.5, 64.9, 64.1,
52.5, 52.2, 31.5, 29.0, 23.8, 20.8, 18.1.
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(d, 1H, J=10.9), 4.74 (d, 1H, J=11.1), 4.68 (d, 1H,
J=11.6), 4.62 (d, 1H, J=11.6), 4.48 (d, 1H, J=11.9),
4.42 (d, 1H, J=11.0), 4.40 (d, 1H, J=11.9), 4.32 (m,
1H), 4.26 (dd, 1H, J=14.3, 7.1), 4.03 (d, 1H, J=7.0),
3.82 (m, 1H), 3.67 (s, 2H), 3.57 (dt, 1H, J=14.1, 9.9,
4.9), 3.48 (t, 1H, J=9.2), 3.40 (t, 1H, J=9.0), 3.14 (dd,
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1H, J=14.3, 3.1), 2.70±2.75 (m, 2H), 2.56±2.66 (m, 3H),
2.32 (d, 1H, J=9.5), 1.92±2.23 (m, 9H), 1.75 (m, 4H),
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1.25 (d, 3H, J=7.1), 0.78 (d, 3H, J=7.0), 0.77 (d, 3H,
J=6.9); 13C NMR (150 MHz, CDCl3) 176.0, 175.6,
174.9, 172.9, 140.3, 140.2, 140.0, 139.8, 139.3, 130.5,
129.6, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.8,
128.7, 128.6, 128.5, 128.4, 127.1, 88.2, 79.7, 79.4, 76.3,
76.2, 74.3, 73.5, 72.2, 66.8, 65.3, 60.4, 59.7, 55.2, 54.6,
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