Structural modification of Fas C-terminal tripeptide and its effects on the inhibitory activity of Fas/FAP-1 binding

Article abstract of DOI:10.1021/jm980617f

We report the structural requirements of the C-terminal tripeptide derivative of Fas (Ac-Ser-Leu-Val-OH, 1) for the inhibitory activity of Fas/FAP-1 binding. The presence of a carboxyl group and a L-Val residue at the C-terminus is essential for the inhib

Full text of DOI:10.1021/jm980617f

J . Med. Chem. 1999, 42, 3289-3299
3289
Str u ctu r a l Mod ifica tion of F a s C-Ter m in a l Tr ip ep tid e a n d Its Effects on th e
In h ibitor y Activity of F a s/F AP -1 Bin d in g
Eiji Sawa,* Motoo Takahashi, Masaru Kamishohara, Tetsushi Tazunoki, Kaname Kimura, Midori Arai,
Tetsuko Miyazaki, Shiro Kataoka, and Tsuyoshi Nishitoba
Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., 3 Miyahara-cho, Takasaki, Gumma 370-1295, J apan
Received November 3, 1998
We report the structural requirements of the C-terminal tripeptide derivative of Fas (Ac-Ser-
Leu-Val-OH, 1) for the inhibitory activity of Fas/FAP-1 binding. The presence of a carboxyl
group and a L-Val residue at the C-terminus is essential for the inhibitory activity, and the
hydroxyl group of Ser plays an important role as the donor of a hydrogen bond. The introduction
of hydrophobic groups to the N-terminal region of 1, especially the phenylaminocarbonyl group
(41), showed a remarkable increase in potency. Further improvement was observed by the
attachment of the Glu residue to the meta-position of the phenyl ring of 41 (51). The ester
derivative of 41 (56) had the ability to induce apoptosis which was dependent on the
concentration of anti-Fas antibody in the colon cancer cell line, DLD-1, which expresses both
Fas and FAP-1 and is resistant to Fas-induced apoptosis. We are now investigating whether
FAP-1 is a main target of 56 and whether the inhibition of Fas/FAP-1 binding by 56 retrieves
the apoptotic signal.
Ta ble 1. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Replaced at Each Residue with D- or N-Methylated Amino
Acids at a Concentration of 1 mM
In tr od u ction
Fas (APO-1/CD95) is a cell surface receptor, which is
a member of the tumor necrosis factor receptor (TNFR)
superfamily, including p55-TNFR, the p75-nerve growth
factor receptor (NGFR), and CD40.1 Binding of the anti-
Fas antibody or ligand for Fas induces apoptosis of cells
expressing Fas.2-4 Genetic mutations of Fas or its ligand
have been related to lymphoproliferative and auto-
immune diseases in mice.5,6 Furthermore, changes in
the Fas expression level have been associated with the
induction of apoptosis of unnecessary cells such as
human immunodeficiency virus (HIV)-infected T-cells.7
Recently, Fas is reported to interact with several signal-
transducing molecules, such as Fas-associated phos-
phatase-1 (FAP-1),8 FADD,9-11 and RIP12 which were
identified using the yeast two-hybrid system and bio-
chemical method. FAP-1 binds to the negative regula-
tory region (C-terminal 15 amino acids) of Fas with its
third PDZ (GLGF) domain and suppresses Fas-mediated
apoptosis. It is reported that FAP-1 is involved in the
acquisition of resistance to anti-Fas antibody-mediated
apoptosis in human pancreatic adenocarcinoma cells13
and AIDS-associated Kaposi’s sacroma cells.14
a
Inhibitory activity was calculated by the methods described
under In Vitro Binding Assay (see Experimental Section).
other amino acids retained the potency with the excep-
tion of Gly. On the basis of these results, we previously
proposed the consensus motif of tS/T-X-V/L/I.16 In this
report, we detail our continuing efforts to identify the
structural requirements for inhibition against Fas/
FAP-1 binding and to develop more potent inhibitors.
Resu lts a n d Discu ssion
We think that an inhibitor of Fas/FAP-1 binding will
be a good candidate as an anticancer agent. We have
already reported that the C-terminal tripeptide (Ac-
SLV-OH, 1) of Fas was necessary and sufficient both
for binding to the third PDZ domain of FAP-1 and for
inhibiting Fas/FAP-1 binding in vitro.8,16 Furthermore,
the inhibitory activity of Fas/FAP-1 binding was studied
using the scanned tripeptides to determine the essential
amino acids for inhibition. Replacement of the first
position from the C-terminus15 with other amino acids
except for Ile or Leu considerably reduced the potency.
Additionally, the potency was reduced by replacement
of the third position with other amino acids except for
Thr. However, replacement of the second position with
To determine the structural requirements for the
inhibitory activity of Fas/FAP-1 binding, our initial
studies focused on the replacement of each amino acid
of the C-terminal tripeptide derivative of Fas (Ac-Ser-
Leu-Val-OH, 1) with several unusual amino acids. These
peptides were synthesized by the ordinary solid-phase
method.
First of all, we replaced the amino acid residues of 1
with D- and N-methylated analogues, and the results
of the in vitro inhibition assay of Fas/FAP-1 binding are
shown in Table 1. Each substitution of the first and
second residues with corresponding D-amino acids dis-
played a detrimental effect on the inhibitory potency
(compounds 2 and 3), suggesting that the L-stereochem-
10.1021/jm980617f CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/05/1999

3290 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Sawa et al.
Ta ble 2. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides Replaced at the 2nd Position Leu with Unusual Amino Acids at a
Concentration of 30 µM
a
b
See Table 1. Concentration is 1 mM.
istry at both positions is essential for the activity.
However, the D-configuration at the third position
(compound 4) did not reduce the potency as much as
those in the first and second positions, indicating that
both configurations at the third position were probably
acceptable in the case of the tripeptide derivatives. On
the other hand, N-methylated analogues (compounds 5
and 6) significantly reduced the potency, which indicates
that the amide nitrogens play an important role in
interacting with FAP-1 to possess the inhibitory activity.
The inhibitory potency was also studied by replace-
ment of the first Val position. As mentioned above, Val,
Ile, or Leu is essential to retain the potency at the first
position, so the unusual amino acids having a hydro-
phobic side chain were tried as replacements. However,
as shown in Table 3, all trials to improve the activity
by replacing Val with Abu (18), NorVal (19), tBuGly
(20), tBuAla (21), or Chg (22) ended in failure. This
result suggests that the isopropyl group is very suitable
for fitting into the hydrophobic pocket of FAP-1.
Final replacement with unusual amino acids was
carried out at the third position. These results are
shown in Table 4. As previously described, the hydroxyl
group at the third residue is essential for the inhibitory
activity. Therefore, the unusual amino acids having a
hydroxyl group were used for replacement. All attempts
by replacement of Ser with Hse (23), cisHyp (24),
transHyp (25), or statine (26) failed to improve the
potency, suggesting that the spatial position of the
hydroxyl group is important to maintain the inhibitory
activity.
Next, we proved the importance of the hydroxyl group
at the third position of Ser by modifying it. Any
modification, such as acetylation (28), benzoylation (30),
and benzylation (31), resulted in considerable losses of
potency, suggesting that this hydroxyl group plays an
important role as a donor of a hydrogen bond (Table 5).
Furthermore, it was confirmed by the following
several modifications that the presence of a carboxyl
group at the C-terminus was essential for the inhibitory
activity. Conversion of the carboxylic group to the
corresponding methyl ester (33) had a harmful effect
Previously, we described that the consensus motif of
tS/T-X-V/L/I is essential for inhibition of Fas/FAP-1
binding; therefore, we next investigated the effect of
replacement of the most tolerable second position with
unusual amino acids. At a concentration of 1 mM for
each peptide, the inhibitory potency was retained in
most of the cases. Further investigation at a lower
concentration (30 µM) of each peptide was carried out,
and the results are shown in Table 2. The potency was
retained by replacement with the hindered residues Chg
(7), tBuGly (8), and Thy(OAc) (9), but it was reduced
by replacement with the less hindered residues Abu
(10), NorVal (11), and Phg (12). On the other hand,
replacement with â-Ala (13) or statine (14) reduced the
potency. These results suggest the existence of a large
hydrophobic pocket around the side chain of the second
position. Furthermore, replacement of Leu with the R,R-
disubstituted residue Aib (15) or the conformationally
restricted residue aminobenzoic acid (16 and 17) caused
a significant loss in potency. This result suggests that
these rigid analogues cannot have the active conforma-
tion of the initial tripeptide 1.

Structural Modification of Fas C-Terminal Tripeptide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3291
Ta ble 3. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Replaced at the 1st Position Val with Unusual Amino Acids at
a Concentration of 50 µM
Ta ble 4. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Replaced at the 3rd Position Ser with Unusual Amino Acids at
a Concentration of 50 µM
a
See Table 1.
Ta ble 5. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Modified in the Hydroxyl Group at the 3rd Position Ser at a
Concentration of 100 µM
a
See Table 1.
on the potency, indicating that negative charge is
necessary at the C-terminus (Table 6). However, con-
version into a tetrazole derivative (38), which is re-
garded as an isoster of a carboxylic acid and was
prepared as shown in Scheme 1, showed a remarkable
loss in potency. This suggested that the spatial volume
of the negative charge is probably a crucial factor for
the inhibitor to fit into the FAP-1 pocket.
To improve the inhibitory potency, we finally inves-
tigated the introduction of more hydrophobic groups to
the N-terminal region of Ac-SLV-OH (1), instead of the
acetyl group (Table 7). Several acyl groups were intro-
duced by coupling the free amino group of the growing
peptide on the resin with appropriate acids: as a result,
benzoyl (39) and cyclohexylcarbonyl (40) groups in-
creased the potency. Further improvements were ob-
served when modified with appropriate isocyanates, and
the introduction of the phenylaminocarbonyl group (41)
gave a remarkable increase in activity. Compound 41
was more potent than the derivative possessing 6-amino
acids of the Fas C-terminus (43). These hydrophobic
groups probably fit the hydrophobic region which is
occupied by the side chains of Glu and Ile at the sixth
and fifth positions of the Fas C-terminal peptides.
a
See Table 1.
meta-position is most efficient for the activity (Table 8).
Ortho-substitution has almost no influence on the
activity, while para-substitution slightly improved it.
Next, we studied whether these peptide derivatives
had the ability to induce apoptosis in the colon cancer
cell line, DLD-1, which expresses both Fas and FAP-1
and is resistant to Fas-induced apoptosis. We prepared
an ester derivative of 41 to improve its permeability into
cells. As shown in Figure 1, DLD-1 cells survived when
they are incubated with only the anti-Fas antibody. On
the other hand, Fas-induced apoptosis, which was
dependent on the concentration of the anti-Fas antibody,
was observed during the existence of the tripeptide ester
We also examined the effect of the introduction of the
Glu residue at the phenyl ring of 41, because our
preliminary data showed that the presence of Glu at
the sixth position improved the inhibitory potency.
Three derivatives (47, 51, and 55) were prepared as
shown in Scheme 2, and the introduction of Glu at the

3292 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Sawa et al.
Sch em e 1
Ta ble 6. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Modified in the C-Terminal Region at a Concentration of 1 mM
These obtained structure-activity relationships are
almost in good agreement with the following informa-
tion which was obtained from the X-ray structure of the
third PDZ domain of the synaptic protein PSD-95 in
complex with its peptide ligand.17 (1) The C-terminal
carboxyl group of the peptide makes tight hydrogen
bonds with amide nitrogens in the carboxylate-binding
loop of the PDZ domain. (2) The side chain of the
C-terminal Val dips deeply into a prominent cavity on
the surface of the PDZ domain and makes tight contacts
with it. (3) The side chain of the second residue is away
from the interactive surface. (4) The hydroxyl oxygen
of the third residue Thr makes hydrogen bonds with the
N-3 nitrogen of His-372 of PSD-95. (5) Only the C-
terminal four residues of the peptide are clearly defined
in the crystal structure of the PDZ-peptide complex.
The first and second results agree with our results
that the presence of a carboxyl group and a Val residue
at the C-terminus is essential for the inhibitory activity.
The third one is in agreement with our result that the
side chain of the second position is tolerable for the
inhibitory potency. The fourth result supports our study
that the hydroxyl group at the third position of Ser plays
an important role as a donor of a hydrogen bond.
Finally, the fifth result suggests that there is a pos-
sibility for further improvement in the inhibitory activ-
ity and specificity by introducing an appropriate group
to the N-terminal region for interaction with the PDZ
domain. Indeed, the introduction of hydrophobic groups
into the N-terminal region of the tripeptide showed a
significant increase in the inhibitory activity against
Fas/FAP-1 binding.
a
See Table 1.
Ta ble 7. Fas/FAP-1 Binding Inhibitory Potency of Tripeptides
Modified in the N-Terminal Region at a Concentration of 10 µM
As already mentioned, the active conformation of the
C-terminal tripeptide of Fas binds to the third PDZ
domain of FAP-1 in a way similar to that observed in
the case of PSD-95 and its binding peptide. So it would
be effective to introduce several new groups into the
N-terminal region of the peptide for developing a specific
inhibitor for Fas/FAP-1 binding. Furthermore, it is
necessary to restrict the conformation of the inhibitor
to fit the PDZ cavity of FAP-1 and increase the inhibi-
tory potency. We are now investigating these subjects
in detail.
a
See Table 1.
Ph-NHCO-SLV-OEt (56). In a previous report,16 we
confirmed that microinjection of 1 induced apoptosis in
DLD-1 cells and the methyl ester 33 did not show any
Fas/FAP-1 binding inhibition in vitro; therefore, 56 was
probably hydrolyzed and converted into the active form
of 41 in the DLD-1 cells.
On the other hand, many PDZ domain-containing
proteins were recently found to bind target proteins
through interaction with the consensus C-terminal
sequence motif (tS/T-X-V).18,19 Therefore, 56 may bind
to several PDZ domain-containing proteins related to
the apoptotic signal transduction through Fas in addi-

Structural Modification of Fas C-Terminal Tripeptide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3293
Sch em e 2
Ta ble 8. Fas/FAP-1 Binding Inhibitory Potency of Peptides
Introduced by a Glu Residue in the N-Terminal Region at a
Concentration of 10 µM
a
See Table 1.
F igu r e 2. Fas-induced apoptosis that was dependent on the
concentration of anti-Fas antibody (CH-11) during the exist-
ence of the tripeptide ester PhNHCO-SLV-OEt (57).
peptide synthesizer on a 0.045-0.2-mmol scale except for
compounds 27-38 and 44-57. N-Fmoc protection was used
for all amino acids during the solid-phase synthesis. Peptides
with a C-terminal carboxylate were synthesized by employing
Wang resin (ACT or Watanabe Chemical Industries), and a
peptide with a C-terminal amide was synthesized using Rink
amide resin (ACT). Each amino acid was sequentially coupled
to the peptide chain from the C- to N-terminus employing the
N-hydroxybenzotriazole (HOBt) active ester coupling via di-
isopropylcarbodiimide (DIC) and subsequent 2-(1H-benzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophos-
phate (HBTU) as the coupling agents. Removal of the N-Fmoc
protective group was effected with 20% piperidine in DMF.
After the last amino acid was coupled, the growing peptide on
the resin was capped by acetic anhydride, appropriate acids,
or isocyanates. Peptides were cleaved from the resin with
simultaneous deprotection using TFA at room temperature for
2-3 h in the presence of 5% water or 2.5% water + 2.5%
triisopropylsilane as the scavenger. The resin was filtered and
washed twice with TFA. The filtrate was evaporated in vacuo,
and the residue was triturated with ether. The crude peptide
was collected by centrifuging, washed twice with ether, and
dried in vacuo.
F igu r e 1. Fas-induced apoptosis that was dependent on the
concentration of anti-Fas antibody (CH-11) during the exist-
ence of the tripeptide ester PhNHCO-SLV-OEt (56).
tion to FAP-1, and so further work needs to investigate
whether FAP-1 is a main target of 56 and whether the
inhibition of Fas/FAP-1 binding by 56 retrieves the
apoptotic signal.
Exp er im en ta l Section
P u r ifica tion a n d Ch a r a cter iza tion . The crude peptides
were purified by preparative reverse-phase HPLC (Nomura
Chemical Develosil ODS-HG-5, 20 × 250 mm) using various
P ep tid e Syn th esis. Peptides were synthesized by the solid-
phase method using an Advanced ChemTech (ACT) 357

3294 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Ta ble 9. HPLC Analysis Dataa
Sawa et al.
a
b
HPLC on a column of Develosil ODS-HG-5 (4.6 × 15 mm). 2: B, 5% f 53% in 32 min. 3: B, 30% f 73% in 32 min. A: 0.1% TFA.
B: 0.1% TFA in CH3CN. c 1: B, 0% f 32% in 32 min. 2: B, 5% f 53% in 32 min. 3: B, 30% f 73% in 32 min. A: 0.1% NH4HCO3. B:
CH3CN.
linear gradients of water containing 0.1% TFA and acetonitrile
containing 0.1% TFA at a flow rate of 10 mL/min for elution.
The fractions containing the product were pooled and lyo-
philized. The peptides were characterized by NMR and MS.
The purity was further checked by analytical reverse-phase
HPLC (Nomura Chemical Develosil ODS-HG-5, 4.6 × 150 mm)
using various linear gradients of water containing 0.1% TFA
and acetonitrile containing 0.1% TFA and also 0.1% aqueous
ammonium hydrocarbonate-acetonitrile solvent systems at a
flow rate of 1 mL/min with UV detection at 220 nm. All
compounds were found to be homogeneous in both HPLC
systems (Table 9). Total yield of peptides were different from
one another; for example, compound 41 was obtained in 15%
yield after HPLC purification (from Fmoc-Val-Wang resin).

Structural Modification of Fas C-Terminal Tripeptide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3295
Ac-Ser -Leu -Va l-OH (1): FAB-MS (M+ + H) 360; 1H NMR
(500 MHz, CD3OD) δ 4.48 (dd, J ) 10.4, 4.9 Hz, 1H), 4.44 (t,
J ) 6.1 Hz, 1H), 4.29 (d, J ) 6.1 Hz, 1H), 3.76 (m, 2H), 2.15
(m, 1H), 1.99 (s, 3H), 1.73-1.55 (m, 3H), 0.95 (m, 12H).
Ac-Ser -Leu -D-Va l-OH (2): FD-MS (M+ + H) 360; 1H NMR
(400 MHz, CDCl3) δ 8.12 (br, 1H), 7.61 (br, 1H), 4.67 (m, 2H),
4.37 (m, 1H), 3.84 (br, 1H), 3.76 (m, 1H), 3.61 (m, 1H), 2.18
(m, 1H), 2.04 (s, 3H), 1.72-1.56 (m, 3H), 1.00-0.90 (m, 12H)
Ac-Ser -D-Leu -Va l-OH (3): FD-MS (M+ + H) 360; 1H NMR
(400 MHz, CDCl3/CD3OD) δ 4.52 (dd, J ) 8.8, 5.9 Hz, 1H),
4.47 (m, 1H), 4.36 (d, J ) 5.1 Hz, 1H), 4.00 (dd, J ) 11.5, 3.7
Hz, 1H), 3.66 (dd, J ) 11.5, 4.6 Hz, 1H), 2.22 (m, 1H), 2.06 (s,
3H), 1.73-1.54 (m, 3H), 0.99-0.91 (m, 12H).
J ) 7.9 Hz, 1H), 7.41 (t, J ) 7.9 Hz, 1H), 4.50 (m, 2H), 3.83
(m, 2H), 2.28 (m, 1H), 2.14 (s, 3H), 1.05 (d, J ) 6.7 Hz, 6H).
Ac-Ser -4-Abz-Va l-OH (17): FAB-MS (M+ + H) 366; 1H
NMR (500 MHz, CD3OD) δ 7.82 (d, J ) 8.5 Hz, 2H), 7.67 (d,
J ) 8.5 Hz, 2H), 4.49 (m, 2H), 3.87 (m, 1H), 3.83 (m, 1H), 2.27
(m, 1H), 2.14 (s, 3H), 1.04 (d, J ) 6.7 Hz, 6H).
Ac-Ser -Leu -Abu -OH (18): FAB-MS (M+ + H) 346; 1H
NMR (500 MHz, CD3OD) δ 4.48 (dd, J ) 10.4, 4.9 Hz, 1H),
4.43 (t, J ) 6.1 Hz, 1H), 4.26 (dd, J ) 8.5, 4.9 Hz, 1H), 3.78
(dd, J ) 10.4, 6.1 Hz, 1H), 3.73 (dd, J ) 10.4, 6.1 Hz, 1H),
2.00 (s, 3H), 1.91-1.84 (m, 1H), 1.77-1.56 (m, 4H), 0.99-0.92
(m, 9H).
Ac-Ser -Leu -Nor Va l-OH (19): FAB-MS (M+ + H) 360; 1H
NMR (500 MHz, CD3OD) δ 4.48 (dd, J ) 10.4, 4.9 Hz, 1H),
4.43 (t, J ) 6.1 Hz, 1H), 4.33 (dd, J ) 9.2, 4.9 Hz, 1H), 3.78
(dd, J ) 11.0, 5.5 Hz, 1H), 3.73 (dd, J ) 11.0, 6.1 Hz, 1H),
2.00 (S, 3H), 1.84-1.56 (m, 5H), 1,45-1.36 (m, 2H), 0.97-0.92
(m, 9H).
Ac-Ser -Leu -tBu Gly-OH (20): FAB-MS (M+ + H) 374; 1H
NMR (500 MHz, CD3OD) δ 4.50 (m, 1H), 4.45 (t, J ) 6.1 Hz,
1H), 4.29 (m, 1H), 3.75 (m, 2H), 2.00 (s, 3H), 1.75-1.57 (m,
3H), 1.01 (s, 9H), 0.96 (d, J ) 6.7 Hz, 3H), 0.92 (d, J ) 6.1 Hz,
3H).
Ac-D-Ser -Leu -Va l-OH (4): FAB-MS (M+ + H) 360; 1H
NMR (500 MHz, CD3OD) δ 4.47 (dd, J ) 9.2, 5.5 Hz, 1H), 4.40
(t, J ) 5.5 Hz, 1H), 4.29 (d, J ) 5.5 Hz, 1H), 3.76 (m, 2H),
2.16 (m, 1H), 2.00 (s, 3H), 1.68 (m, 2H), 1.62 (m, 2H), 0.95 (m,
12H).
Ac-Ser -NMe-Leu -Va l-OH (5): FAB-MS (M+ + Na) 396;
1H NMR (500 MHz, CD3OD) δ 5.25 (dd, J ) 9.8, 5.5 Hz, 1H),
4.97 (m, 1H), 4.28 (m, 1H), 3.75 (d, J ) 7.3 Hz, 2H), 3.11 (s,
3H), 2.15 (m, 1H), 1.97 (s, 3H), 1.77-1.66 (m, 2H), 1.53 (m,
1H), 0.96-0.89 (m, 12H).
Ac-Ser -Leu -tBu Ala -OH (21): FAB-MS (M+ + H) 388; 1H
NMR (500 MHz, CD3OD) δ 4.48-4.42 (m, 3H), 3.74 (m, 2H),
2.00 (s, 3H), 1.80-1.54 (m, 5H), 0.95 (s, 9H), 0.94 (m, 6H).
Ac-Ser -Leu -Ch g-OH (22): FAB-MS (M+ + H) 400; 1H
NMR (500 MHz, CD3OD) δ 4.49 (dd, J ) 10.4, 4.9 Hz, 1H),
4.44 (t, J ) 6.1 Hz, 1H), 4.28 (d, J ) 6.1 Hz, 1H), 3.75 (m,
2H), 2.00 (s, 3H), 1.82-1.56 (m, 9H), 1.30-1.09 (m, 5H), 0.96
(d, J ) 6.7 Hz, 3H), 0.92 (d, J ) 6.1 Hz, 3H).
Ac-NMe-Ser -Leu -Va l-OH (6): FAB-MS (M+ + H) 374; 1H
NMR (500 MHz, CD3OD) δ 4.60 (m, 1H), 4.45 (m, 1H), 4.27
(m, 1H), 3.97 (m, 1H), 3.88 (m, 1H), 2.65 (s, 3H), 2.18 (m, 1H),
2.15 (s, 3H), 1.78-1.60 (m, 3H), 0.95 (m, 12H).
Ac-Ser -Ch g-Va l-OH (7): FAB-MS (M+ + H) 386; 1H NMR
(500 MHz, CD3OD) δ 4.48 (m, 1H), 4.31 (d, J ) 7.3 Hz, 1H),
4.28 (d, J ) 5.5 Hz, 1H), 3.74 (m, 2H), 2.15 (m, 1H), 2.00 (s,
3H), 1.81-1.65 (m, 6H), 1.26-1.07 (m, 5H), 0.96 (d, J ) 6.7
Hz, 6H).
Ac-Hse-Leu -Va l-OH (23): FAB-MS (M+ + H) 374; 1H NMR
(500 MHz, CD3OD) δ 4.46 (m, 2H), 4.30 (d, J ) 5.5 Hz, 1H),
3.62 (m, 2H), 2.17 (m, 1H), 2.00 (m, 1H), 1.97 (s, 3H), 1.72 (m,
1H), 1.60 (m, 2H), 0.96 (d, J ) 6.7 Hz, 9H), 0.92 (d, J ) 6.7
Hz, 3H).
Ac-Ser -tBu Gly-Va l-OH (8): FAB-MS (M+ + H) 360; 1H
NMR (500 MHz, CD3OD) δ 4.48 (t, J ) 5.5 Hz, 1H), 4.37 (m,
1H), 4.29 (m, 1H), 3.75 (d, J ) 5.5 Hz, 2H), 2.14 (m, 1H), 2.01
(s, 3H), 1.02 (s, 9H), 0.96 (d, J ) 6.7 Hz, 6H).
1
Ac-cisHyp -Leu -Va l-OH (24): FAB-MS (M+ + H) 386; H
Ac-Ser -Th y(OAc)-Va l-OH (9): FAB-MS (M+ + H) 544; 1H
NMR (500 MHz, CD3OD) δ 7.24 (d, J ) 8.6 Hz, 2H), 7.06 (d,
J ) 9.2 Hz, 2H), 6.97 (d, J ) 9.2 Hz, 2H), 6.89 (d, J ) 8.6 Hz,
2H), 4.70 (m, 1H), 4.40 (m, 1H), 4.29 (m, 1H), 3.70 (m, 2H),
3.17 (dd, J ) 14.0, 4.9 Hz, 1H), 2.93 (dd, J ) 14.0, 8.5 Hz,
1H), 2.26 (s, 3H), 2.15 (m, 1H), 1.96 (s, 3H), 0.96 (m, 6H).
Ac-Ser -Abu -Va l-OH (10): FAB-MS (M+ + H) 322; 1H NMR
(500 MHz, CD3OD) δ 4.45 (m, 1H), 4.35 (m, 1H), 4.30 (m, 1H),
3.78 (dd, J ) 11.0, 5.5 Hz, 1H), 3.73 (dd, J ) 11.0, 6.1 Hz,
1H), 2.16 (m, 1H), 2.00 (s, 3H), 1.89 (m, 1H), 1.68 (m, 1H),
0.97 (m, 9H).
NMR (500 MHz, CD3OD) δ 4.45 (m, 2H), 4.37 (m, 1H), 4.28
(d, J ) 6.1 Hz, 1H), 3.75 (dd, J ) 11.0, 4.9 Hz, 1H), 3.57 (m,
1H), 2.38 (m, 1H), 2.17 (m, 1H), 2.08 (s, 3H), 2.00 (m, 1H),
1.77 (m, 1H), 1.70-1.59 (m, 2H), 0.98-0.91 (m, 12H).
Ac-tr a n sHyp -Leu -Va l-OH (25): FAB-MS (M+ + H) 386;
1H NMR (500 MHz, CD3OD) δ 4.58-4.43 (m, 3H), 4.30 (d, J )
5.5 Hz, 1H), 3.75 (dd, J ) 11.0, 4.3 Hz, 1H), 3.51 (m, 1H), 2.38-
2.00 (m, 3H), 2.07 (s, 3H), 1.78 (m, 1H), 1.62 (m, 2H), 0.99-
0.93 (m, 12H).
Ac-Sta -Leu -Va l-OH (26): FAB-MS (M+ + H) 430; 1H NMR
(500 MHz, CD3OD) δ 4.46 (dd, J ) 9.2, 6.1 Hz, 1H), 4.30 (d, J
) 6.1 Hz, 1H), 3.97 (m, 2H), 2.32 (m, 2H), 2.17 (m, 1H), 2.03
(s, 3H), 1.72 (m, 1H), 1.62-1.49 (m, 4H), 1.33 (m, 1H), 0.97-
0.90 (m, 18H).
1
Ac-Ser -Nor Va l-Va l-OH (11): FAB-MS (M+ + H) 346; H
NMR (500 MHz, CD3OD) δ 4.44 (m, 2H), 4.29 (m, 1H), 3.77
(dd, J ) 11.0, 5.5 Hz, 1H), 3.73 (dd, J ) 11.0, 6.1 Hz, 1H),
2.16 (m, 1H), 2.00 (s, 3H), 1.82 (m, 1H), 1.64 (m, 1H), 1.41 (m,
2H), 0.95 (m, 9H).
Ac-Ser (OAc)-Leu -Va l-OtBu (27). To a solution of Ac-Ser-
Leu-Val-OtBu (50 mg, 0.12 mmol) in CH2Cl2 (1 mL) were
added Ac2O (15 mg, 0.15 mmol) in CH2Cl2 (0.5 mL) and Et3N
(0.03 mL, 0.22 mmol). The mixture was stirred for 6 h.
Additional Ac2O (7.5 mg, 0.074 mmol) in CH2Cl2 (1 mL), Et3N
(0.03 mL, 0.22 mmol), and CH2Cl2 (1 mL) were added. The
mixture was stirred for an additional 16 h and partitioned
between water and ethyl acetate. The organic layer was
washed with brine, dried (Na2SO4), and concentrated. The
residue was adsorbed on a plate of silica gel, and the plate
was developed with 10% methanol/chloroform to afford 35 mg
(64%) of 27: FD-MS (M+ + H) 458; 1H NMR (500 MHz, CDCl3)
δ 6.76 (br, 1H), 6.66 (br, 1H), 6.60 (br, 1H), 4.75 (m, 1H), 4.53
(m, 1H), 4.41 (dd, J ) 9.2, 4.9 Hz, 1H), 4.31 (dd, J ) 11.0, 5.5
Hz, 1H), 4.18 (dd, J ) 11.0, 6.1 Hz, 1H), 2.15 (m, 1H), 2.07 (s,
3H), 2.05 (s, 3H), 1.70-1.53 (m, 3H), 1.47 (s, 9H), 0.94 (d, J )
6.7 Hz, 3H), 0.92 (d, J ) 6.7 Hz, 3H), 0.90 (d, J ) 6.7 Hz, 3H),
0.88 (d, J ) 6.7 Hz, 3H).
Ac-Ser -P h g-Va l-OH (12): FAB-MS (M+ + H) 380; 1H NMR
(500 MHz, CD3OD) δ 7.45 (d, J ) 7.3 Hz, 2H), 7.33 (m, 3H),
5.60 (s, 1H), 4.50 (m, 1H), 4.33 (m, 1H), 3.78 (m, 2H), 2.17 (m,
1H), 2.00 (s, 3H), 0.98 (d, J ) 6.7 Hz, 6H).
Ac-Ser -â-Ala -Va l-OH (13): FAB-MS (M+ + H) 318; 1H
NMR (500 MHz, CD3OD) δ 4.36 (dd, J ) 5.5, 4.9 Hz, 1H), 4.32
(d, J ) 6.1 Hz, 1H), 3.78 (dd, J ) 11.0, 5.5 Hz, 1H), 3.73 (dd,
J ) 11.0, 4.9 Hz, 1H), 3.50 (m, 1H), 3.45 (m, 1H), 2.49 (m,
2H), 2.18 (m, 2H), 2.02 (s, 3H), 0.98 (m, 6H).
Ac-Ser -Sta -Va l-OH (14): FAB-MS (M+ + H) 404; 1H NMR
(500 MHz, CD3OD) δ 4.40 (t, J ) 5.5 Hz, 1H), 4.31 (d, J ) 5.5
Hz, 1H), 4.02-3.94 (m, 2H), 3.79 (d, J ) 5.5 Hz, 2H), 2.38 (m,
2H), 2.18 (m, 1H), 2.02, (s, 3H), 1.67-1.55 (m, 2H), 1.32 (m,
1H), 0.98 (d, J ) 7.3 Hz, 6H), 0.93 (d, J ) 6.1 Hz, 3H), 0.90 (d,
J ) 6.7 Hz, 3H).
Ac-Ser -Aib-Va l-OH (15): FAB-MS (M+ + H) 322; 1H NMR
(500 MHz, CD3OD) δ 4.36 (m, 1H), 4.28 (d, J ) 5.5 Hz, 1H),
3.75 (m, 2H), 2.14 (m, 1H), 1.99 (s, 3H), 1.49 (d, J ) 6.1 Hz,
6H), 0.95 (d, J ) 6.7 Hz, 3H), 0.91 (d, J ) 6.7 Hz, 3H).
Ac-Ser -3-Abz-Va l-OH (16): FAB-MS (M+ + H) 366; 1H
NMR (500 MHz, CD3OD) δ 7.97 (s, 1H), 7.72 (m, 1H), 7.75 (d,
Ac-Ser (OAc)-Leu -Va l-OH (28). A solution of 27 (33 mg,
0.072 mmol) in trifluoroacetic acid (TFA; 1 mL) and water
(0.025 mL) was stirred for 1 h and concentrated. The residue
was dissolved in ethyl acetate, and the solution was washed
with water and brine. The organic layer was dried (Na2SO4)

3296 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Sawa et al.
and concentrated. The residue was washed with Et2O to
completely remove TFA and dried under vacuum to afford 19
temperature and concentrated. The residue was washed with
Et2O and partitioned between CHCl3 and saturated aqueous
NaHCO3. The organic layer was dried (Na2SO4) and concen-
trated to afford 181 mg (25%) of 34: FD-MS (M+) 250; 1H NMR
(500 MHz, CDCl3) δ 7.40-7.30 (m, 5H), 5.78 (br, 1H), 5.39 (br,
1H), 5.30 (br, 1H), 5.12 (s, 2H), 4.03 (t, J ) 7.3 Hz, 1H), 2.16
(m, 1H), 1.00 (d, J ) 6.7 Hz, 3H), 0.95 (d, J ) 6.7 Hz, 3H).
1
mg (66%) of 28: FAB-MS (M+ + H) 402; H NMR (500 MHz,
CDCl3) δ 7.58 (br, 1H), 7.11 (br, 1H), 6.95 (br, 1H), 4.74 (m,
1H), 4.50 (m, 1H), 4.30 (dd, J ) 11.0, 5.5 Hz, 1H), 4.25 (dd, J
) 11.0, 5.5 Hz, 1H), 2.20 (m, 1H), 2.05 (s, 3H), 2.04 (s, 3H),
1.66-1.55 (m, 3H), 0.94 (m, 12H).
Ac-Ser (OBz)-Leu -Va l-OtBu (29). To a solution of Ac-Ser-
Leu-Val-OtBu (50 mg, 0.12 mmol) in THF (6 mL) were added
benzoyl chloride (0.017 mL, 0.15 mmol) and Et3N (0.03 mL,
0.22 mmol). The mixture was then stirred for 18 h. Additional
benzoyl chloride (0.009 mL, 0.078 mmol) and Et3N (0.02 mL,
0.14 mmol) were added, and the mixture was stirred for an
additional 3 h. To the mixture was added 4-(dimethylamino)-
pyridine (7 mg, 0.06 mmol). The mixture was stirred for 1.5 h
and partitioned between water and ethyl acetate. The organic
layer was washed with brine, dried (Na2SO4), and concen-
trated. The residue was adsorbed on a plate of silica gel, and
the plate was developed with 10% methanol/chloroform to
afford 36 mg (60%) of 29: FD-MS (M+ + H) 520; 1H NMR (500
MHz, CDCl3) δ 8.05 (d, J ) 7.3 Hz, 2H), 7.57 (t, J ) 7.3 Hz,
1H), 7.44 (t, J ) 7.3 Hz, 2H), 6.83 (d, J ) 7.9 Hz, 1H), 6.65 (d,
J ) 7.3 Hz, 1H), 6.56 (d, J ) 8.5 Hz, 1H), 4.87 (m, 1H), 4.63
(dd, J ) 11.6, 6.1 Hz, 1H), 4.51 (m, 1H), 4.50 (dd, J ) 11.6,
5.5 Hz, 1H), 4.35 (dd, J ) 8.5, 4.3 Hz, 1H), 2.07 (m, 1H), 2.05
(s, 3H), 1.72-1.55 (m, 3H), 1.46 (s, 9H), 0.90 (d, J ) 6.1 Hz,
6H), 0.83 (d, J ) 6.1 Hz, 3H), 0.81 (d, J ) 6.7 Hz, 3H).
Ac-Ser (OBz)-Leu -Va l-OH (30). The titled compound was
prepared in 70% yield according to the procedure as described
for 28 using 29 instead of 27: FAB-MS (M+ + H) 464; 1H NMR
(500 MHz, CDCl3) δ 8.00 (d, J ) 7.9 Hz, 2H), 7.75 (br, 1H),
7.57 (t, J ) 7.3 Hz, 1H), 7.44 (t, J ) 7.3 Hz, 1H), 7.13 (br, 1H),
4.87 (t, J ) 5.5 Hz, 1H), 4.54 (t, J ) 4.9 Hz, 1H), 4.50 (m, 2H),
4.37 (d, J ) 4.9 Hz, 1H), 2.12 (m, 1H), 2.04 (s, 3H), 1.66-1.53
(m, 3H), 0.90 (d, J ) 6.7 Hz, 3H), 0.89 (d, J ) 6.7 Hz, 3H),
0.85 (d, J ) 6.7 Hz, 6H).
Z-Va l-CN (35). To a suspension of 34 (170 mg, 0.68 mmol)
in pyridine (3 mL) was added POCl3 (0.175 mL, 1.88 mmol) at
-15 °C. The mixture was stirred for 1.5 h at the same
temperature. After water was added, the solution was ex-
tracted with ethyl acetate. The extract was washed with water,
1 N HCl, saturated aqueous NaHCO3, and brine and was dried
(Na2SO4) and concentrated to afford 35 (158 mg, quantitative)
which was used without further purification: FD-MS (M+) 232;
1H NMR (500 MHz, CDCl3) δ 7.40-7.30 (m, 5H), 5.14 (s, 2H),
4.51 (dd, J ) 7.3, 7.3 Hz, 1H), 2.04 (m, 1H), 1.09 (d, J ) 6.7
Hz, 3H), 1.07 (d, J ) 7.3 Hz, 3H).
Z-Va l-Tet (36). A solution of 35 (140 mg, 0.60 mmol), NaN3
(41 mg, 0.63 mmol), and NH4Cl (36 mg, 0.67 mmol) in DMF
(2 mL) was stirred for 6 h at 105 °C. Additional NaN3 (20 mg,
0.30 mmol) and NH4Cl (20 mg, 0.37 mmol) were added, and
the mixture was stirred for 16 h at 105 °C. Additional NaN3
(41 mg, 0.63 mmol) and NH4Cl (36 mg, 0.67 mmol) were added,
and the mixture was stirred for 10 h at 120 °C. After ice chips
and 1 N HCl were added, the solution was extracted with ethyl
acetate. The organic layer was washed with brine, dried (Na2-
SO4), and concentrated. The residue was washed with Et2O/
hexane to afford 36 (78 mg, 47%) which was used without
further purification: FD-MS (M+) 275; 1H NMR (500 MHz,
CDCl3) δ 13.68 (br, 1H), 7.40-7.30 (m, 5H), 5.60 (d, J ) 7.9
Hz, 1H), 5.14 (d, J ) 12.2 Hz, 1H), 5.11 (d, J ) 12.2 Hz, 1H),
4.78 (br, 1H), 2.48 (m, 1H), 1.07 (d, J ) 6.1 Hz, 3H), 0.92 (d,
J ) 7.3 Hz, 3H).
Ac-Ser (OtBu )-Leu -Va l-Tet (37). Palladium on carbon
(10%, 20 mg) was added to a solution of 36 (200 mg, 0.72 mmol)
in MeOH (15 mL). A balloon containing hydrogen gas was
attached to the flask, and the mixture was stirred for 2 h. The
mixture was filtered through Celite, and the filtrate was
concentrated. The residue was dissolved in DMF (15 mL). To
the solution were added Ac-Ser(OtBu)-Leu-OH (230 mg, 0.72
mmol), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hy-
drochloride (EDC; 150 mg, 0.78 mmol), and HOBt (110 mg,
0.81 mmol), and the mixture was stirred for 8 h. After water
was added, the solution was extracted with ethyl acetate. The
extract was washed with brine (3×), dried (Na2SO4), and
concentrated. The residue was washed with Et2O to afford 37
(243 mg, 77%) which was used without further purification:
FD-MS (M+ + H) 440; 1H NMR (500 MHz, DMSO-d6) δ 16.18
(br, 1H), 8.33 (d, J ) 8.5 Hz, 1H), 7.95 (d, J ) 7.9 Hz, 1H),
7.85 (d, J ) 7.9 Hz, 1H), 4.93 (t, J ) 7.9 Hz, 1H), 4.40 (m,
1H), 4.29 (m, 1H), 3.45 (dd, J ) 9.2, 5.5 Hz, 1H), 3.40 (dd, J )
9.2, 5.5 Hz, 1H), 2.20 (m, 1H), 1.89 (s, 3H), 1.60-1.36 (m, 3H),
1.09 (s, 9H), 0.91 (d, J ) 6.7 Hz, 3H), 0.83 (d, J ) 6.1 Hz, 3H),
0.81 (d, J ) 6.1 Hz, 3H), 0.75 (d, J ) 6.7 Hz, 3H).
Ac-Ser (OBn )-Leu -Va l-OH (31). A solution of Ac-Ser(OBn)-
Leu-Val-OtBu (100 mg, 0.198 mmol) in TFA (2 mL) and water
(0.05 mL) was stirred for 2.5 h and concentrated. The residue
was washed with Et2O and dried under vacuum to afford 83
1
mg (93%) of 31: FD-MS (M+) 449; H NMR (500 MHz, CD3-
OD) δ 7.35-7.25 (m, 5H), 4.59-4.50 (overlapped, 4H), 4.27
(m, 1H), 3.74 (dd, J ) 9.8, 5.5 Hz, 1H), 3.70 (dd, J ) 9.8, 5.5
Hz, 1H), 2.13 (m, 1H), 2.00 (s, 3H), 1.68 (m, 1H), 1.59 (m, 2H),
0.93-0.90 (m, 12H).
H-Ser (OtBu )-Leu -Va l-OMe (32). A solution of Fmoc-Ser-
(OtBu)-Leu-Val-OMe (200 mg, 0.33 mmol) in 20% piperidine/
DMF (1 mL) was stirred for 25 min. After water was added,
the mixture was extracted with ethyl acetate. The extract was
washed with brine (3×), dried (Na2SO4), and concentrated. The
residue was chromatographed (silica gel, hexane/ethyl acetate
) 9/1 f CHCl3/MeOH ) 100/1 f 100/2 f 100/5) to afford 128
mg (quantitative) of 32: FAB-MS (M+ + H) 388; 1H NMR (90
MHz, CDCl3) δ 6.64 (m, 1H), 4.50 (m, 2H), 3.73 (s, 3H), 3.55
(m, 3H), 2.04 (m, 1H), 1.69-1.54 (m, 3H), 1.18 (s, 9H), 0.93
(m, 12H).
Ac-Ser -Leu -Va l-Tet (38). A solution of 37 (100 mg, 0.228
mmol) in TFA (1.90 mL) and water (0.1 mL) was stirred for
1.5 h. After the solvent was removed under vacuum, the
residue was subjected to preparative reverse-phase HPLC
(Nomura Chemical Develosil ODS-HG-5, 20 × 250 mm) using
a linear gradient of (A) water containing 0.1% TFA and (B)
acetonitrile containing 0.1% TFA (5-70% B, in 40 min) at a
flow rate of 10 mL/min. Fractions containing the major peak
were pooled and lyophilized to yield 19 mg (22%) of 38: FD-
MS (M+ + H) 384; 1H NMR (500 MHz, DMSO-d6) δ 16.15 (br,
1H), 8.32 (d, J ) 8.6 Hz, 1H), 8.02 (d, J ) 7.9 Hz, 1H), 7.98 (d,
J ) 7.9 Hz, 1H), 4.92 (t, J ) 7.9 Hz, 1H), 4.30 (m, 2H), 3.53
(m, 2H), 2.20 (m, 1H), 1.86 (s, 3H), 1.57 (m, 1H), 1.46 (m, 2H),
0.91 (d, J ) 6.7 Hz, 3H), 0.85 (d, J ) 6.7 Hz, 3H), 0.81 (d, J )
6.7 Hz, 3H), 0.75 (d, J ) 6.7 Hz, 3H).
Ac-Ser -Leu -Va l-OMe (33). A solution of 32 (135 mg, 0.35
mmol), Ac2O (0.04 mL, 0.42 mmol), and Et3N (0.06 mL, 0.43
mmol) in CH2Cl2 (2 mL) was stirred for 15 h at room
temperature under argon. The mixture was partitioned be-
tween CHCl3 and water. The organic layer was dried (Na2-
SO4) and concentrated. The residue was dissolved in TFA (2
mL) and water (0.1 mL). The solution was stirred for 35 min,
diluted with ice-cooled water, and extracted with ethyl acetate.
The extract was washed with saturated aqueous NaHCO3 and
brine. Concentrating under vacuum gave a syrup, which was
washed with Et2O to afford 31 mg (24%) of 33: FAB-MS (M+
+ H) 374; 1H NMR (500 MHz, CDCl3) δ 6.99 (br, 1H), 6.84
(br, 1H), 6.59 (br, 1H), 4.56 (m, 2H), 4.46 (m, 1H), 4.02 (m,
1H), 3.74 (s, 3H), 3.61 (dd, J ) 11.0, 7.3 Hz, 1H), 2.15 (m, 1H),
2.04 (s, 3H), 1.75-1.57 (m, 3H), 0.92 (m, 12H).
Bz-Ser -Leu -Va l-OH (39): FAB-MS (M+ + H) 422; 1H NMR
(500 MHz, CD3OD) δ 7.87 (d, J ) 7.3 Hz, 2H), 7.53 (m, 1H),
7.46 (m, 2H), 4.68 (t, J ) 6.1 Hz, 1H), 4.52 (dd, J ) 9.8, 4.9
Z-Va l-NH2 (34). Ammonia gas was bubbled into a solution
of Z-Val-OSu (1.0 g, 2.87 mmol) in CHCl3 (10 mL) at 0 °C for
15 min with stirring. The mixture was stirred for 1 h at room

Structural Modification of Fas C-Terminal Tripeptide
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3297
Hz, 1H), 4.29 (d, J ) 5.5 Hz, 1H), 3.90 (m, 2H), 2.16 (m, 1H),
1.75 (m, 1H), 1.65 (m, 2H), 0.95 (m, 12H).
toluene (5 mL), and the suspension was concentrated. The
residue was dissolved in a 50/50 mixture of 50% AcOH and
DMSO and subjected to preparative reverse-phase HPLC
(Nomura Chemical Develosil ODS-HG-5, 20 × 250 mm) using
a linear gradient of (A) water containing 0.1% TFA and (B)
acetonitrile containing 0.1% TFA (5-60% B, in 35 min) at a
flow rate of 10 mL/min. Fractions containing the major peak
were pooled and lyophilized to yield 48 mg (70%) of 47: FAB-
MS (M+ + H) 581; 1H NMR (500 MHz, CD3OD) δ 7.60 (m, 1H),
7.38 (m, 1H), 7.22 (m, 2H), 4.49 (m, 1H), 4.38 (m, 1H), 4.27 (d,
J ) 5.5 Hz, 1H), 4.18 (m, 1H), 3.88 (m, 1H), 3.79 (m, 1H), 2.57
(m, 2H), 2.25 (m, 2H), 2.13 (m, 1H), 1.75-1.60 (m, 3H), 0.93
(m, 12H).
m -NO2-P h -NHCO-Ser (OtBu )-Leu -Va l-OtBu (48). The
titled compound was prepared in quantitative yield according
to the procedure as described for 44 using 3-nitrophenyl
isocyanate instead of 2-nitrophenyl isocyanate: FD-MS (M+)
593; 1H NMR (500 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.49 (s, 1H),
7.93 (d, J ) 8.6 Hz, 1H), 7.89 (d, J ) 8.6 Hz, 1H), 7.73 (d, J )
7.9 Hz, 1H), 7.56 (d, J ) 7.9 Hz, 1H), 7.48 (t, J ) 7.9 Hz, 1H),
6.47 (m, 1H), 4.48 (m, 1H), 4.28 (m, 1H), 3.97 (m, 1H), 3.59
(m, 1H), 3.43 (m, 1H), 1.99 (m, 1H), 1.60 (m, 1H), 1.45 (m,
2H), 1.37 (s, 9H), 1.09 (s, 9H), 0.84 (m, 12H).
Cyh -CO-Ser -Leu -Va l-OH (40): FAB-MS (M+ + H) 428;
1H NMR (500 MHz, CD3OD) δ 4.44 (m, 2H), 4.29 (d, J ) 5.5
Hz, 1H), 3.73 (m, 2H), 2.24 (m, 1H), 2.15 (m, 1H), 1.78 (m,
4H), 1.66 (m, 4H), 1.41 (m, 2H), 1.28 (m, 3H), 0.94 (m, 12H).
P h -NHCO-Ser -Leu -Va l-OH (41): FAB-MS (M+ + H) 437;
1H NMR (500 MHz, CD3OD) δ 7.35 (d, J ) 8.5 Hz, 2H), 7.24
(m, 2H), 6.97 (m, 1H), 4.50 (dd, J ) 9.8, 4.9 Hz, 1H), 4.40 (dd,
J ) 6.1, 4.9 Hz, 1H), 4.29 (d, J ) 5.5 Hz, 1H), 3.86 (dd, J )
10.4, 4.9 Hz, 1H), 3.74 (dd, J ) 10.4, 6.1 Hz, 1H), 2.17 (m,
1H), 1.77-1.60 (m, 3H), 0.95 (m, 12H).
Cyh -NHCO-Ser -Leu -Va l-OH (42): FAB-MS (M+ + H)
443; 1H NMR (500 MHz, CD3OD) δ 4.48 (m, 1H), 4.29 (m, 2H),
3.78 (dd, J ) 10.4, 5.5 Hz, 1H), 3.66 (m, 1H), 3.47 (m, 1H),
2.16 (m, 1H), 1.85 (m, 2H), 1.71 (m, 3H), 1.60 (m, 2H), 1.34
(m, 3H), 1.17 (m, 3H), 0.95 (m, 12H).
Ac-Glu -Ile-Gln -Ser -Leu -Va l-OH (43): FAB-MS (M+
+
Na) 753; 1H NMR (500 MHz, CD3OD) δ 4.48 (m, 1H), 4.40 (m,
2H), 4.29 (m, 2H), 4.13 (d, J ) 7.3 Hz, 1H), 3.86 (dd, J ) 11.0,
5.5 Hz, 1H), 3.78 (dd, J ) 11.0, 5.5 Hz, 1H), 2.41-2.33 (m,
4H), 2.18-2.09 (m, 3H), 1.99 (s, 3H), 1.92-1.86 (m, 2H), 1.72
(m, 1H), 1.66 (m, 3H), 1.56 (m, 1H), 1.22 (m, 1H), 0.97-0.90
(m, 18H).
m -NH2-P h -NHCO-Ser (OtBu )-Leu -Va l-OtBu (49). The
titled compound was prepared in 89% yield according to the
procedure as described for 45 using 48 instead of 44: FD-MS
o-NO2-P h -NHCO-Ser (OtBu )-Leu -Va l-OtBu (44). To a
solution of H-Ser(OtBu)-Leu-Val-OtBu (500 mg, 1.16 mmol)
in DMF (3 mL) was added 2-nitrophenyl isocyanate (210 mg,
1.28 mmol). The mixture was stirred for 10 min at room
temperature and cooled to 0 °C. The mixture was partitioned
between water and ethyl acetate. The organic layer was
washed with water and brine and dried (MgSO4). The solvent
was removed under vacuum to afford 44 (691 mg, 100%) which
1
(M+) 563; H NMR (500 MHz, DMSO-d6) δ 8.52 (s, 1H), 7.91
(d, J ) 8.5 Hz, 1H), 7.79 (d, J ) 7.9 Hz, 1H), 6.79 (t, J ) 7.9
Hz, 1H), 6.65 (s, 1H), 6.49 (d, J ) 7.3 Hz, 1H), 6.26 (d, J ) 7.3
Hz, 1H), 6.09 (d, J ) 7.9 Hz, 1H), 4.84 (s, 2H), 4.47 (m, 1H),
4.24 (m, 1H), 3.98 (m, 1H), 3.57 (m, 1H), 3.36 (dd, J ) 9.2, 4.9
Hz, 1H), 1.99 (m, 1H), 1.59 (m, 1H), 1.44 (m, 2H), 1.38 (s, 9H),
1.09 (s, 9H), 0.86 (m, 12H).
1
was used without further purification: FD-MS (M+) 593; H
m -(Boc-Glu (OtBu )-NH)-P h -NHCO-Ser (OtBu )-Leu -Va l-
OtBu (50). The titled compound was prepared in 90% yield
according to the procedure as described for 46 using 49 instead
of 45: FD-MS (M+) 848; 1H NMR (500 MHz, DMSO-d6) δ 9.88
(s, 1H), 8.93 (s, 1H), 7.98 (d, J ) 7.9 Hz, 1H), 7.90 (d, J ) 9.1
Hz, 1H), 7.70 (s, 1H), 7.13 (m, 3H), 7.03 (m, 1H), 6.37 (d, J )
7.9 Hz, 1H), 4.50 (m, 1H), 4.29 (m, 1H), 4.00 (m, 1H), 3.98 (m,
1H), 3.60 (m, 1H), 3.42 (m, 1H), 2.25 (m, 2H), 2.01 (m, 1H),
1.88 (m, 1H), 1.79 (m, 1H), 1.62 (m, 1H), 1.47 (m, 2H), 1.38 (s,
27H), 1.10 (s, 9H), 0.87 (m, 12H).
m -(Glu -NH)-P h -NHCO-Ser -Leu -Va l-OH (51). The titled
compound was prepared in 74% yield according to the proce-
dure as described for 47 using 50 instead of 46: FAB-MS (M+
+ H) 581; 1H NMR (500 MHz, CD3OD) δ 7.78 (s, 1H), 7.23 (m,
2H), 7.08 (d, J ) 8.0 Hz, 1H), 4.52 (m, 1H), 4.39 (m, 1H), 4.28
(m, 1H), 4.02 (m, 1H), 3.86 (m, 1H), 3.71 (m, 1H), 2.52 (m,
2H), 2.18 (m, 3H), 1.65 (m, 3H), 0.94 (m, 12H).
NMR (500 MHz, CD3OD) δ 8.19 (d, J ) 8.5 Hz, 1H), 8.02 (d,
J ) 6.7 Hz, 1H), 7.63 (t, J ) 7.3 Hz, 1H), 7.16 (t, J ) 7.3 Hz,
1H), 4.47 (t, J ) 7.3 Hz, 1H), 4.29 (t, J ) 4.9 Hz, 1H), 3.99 (d,
J ) 6.7 Hz, 1H), 3.56 (dd, J ) 9.2, 5.5 Hz, 1H), 3.45 (m, 1H),
2.00 (m, 1H), 1.63 (m, 1H), 1.48 (t, J ) 7.3 Hz, 2H), 1.39 (s,
9H), 1.12 (s, 9H), 0.89 (d, J ) 6.7 Hz, 3H), 0.86 (d, J ) 6.7 Hz,
3H), 0.85 (d, J ) 6.7 Hz, 6H).
o-NH2-P h -NHCO-Ser (OtBu )-Leu -Va l-OtBu (45). Palla-
dium hydroxide on carbon (18%, 120 mg) was added to a
solution of 44 (667 mg, 1.14 mmol) in EtOH (50 mL). A balloon
containing hydrogen was attached to the flask, and the
mixture was stirred for 16 h. The mixture was filtered through
Celite, and the filtrate was concentrated under vacuum. The
residue was chromatographed (silica gel, 25:1 CHCl3:MeOH).
Fractions containing the product were pooled and concentrated
to afford 600 mg (93%) of 45: FD-MS (M+) 563; 1H NMR (500
MHz, DMSO-d6) δ 7.99 (d, J ) 7.9 Hz, 1H), 7.96 (s, 1H), 7.85
(d, J ) 6.7 Hz, 1H), 7.29 (d, J ) 6.7 Hz, 1H), 6.79 (t, J ) 7.9
Hz, 1H), 6.68 (d, J ) 6.7 Hz, 1H), 6.51 (t, J ) 7.9 Hz, 1H),
6.41 (d, J ) 7.9 Hz, 1H), 4.74 (s, 2H), 4.51 (q, J ) 7.9 Hz, 1H),
4.24 (m, 1H), 3.99 (dd, J ) 7.9, 6.1 Hz, 1H), 3.60 (dd, J ) 9.2,
4.3 Hz, 1H), 3.42 (dd, J ) 9.2, 4.9 Hz, 1H), 2.00 (m, 1H), 1.63
(m, 1H), 1.47 (t, J ) 7.3 Hz, 2H), 1.39 (s, 9H), 1.10 (s, 9H),
0.87 (m, 12H).
o-(Boc-Glu (OtBu )-NH)-P h -NHCO-Ser (OtBu )-Leu -Va l-
OtBu (46). To a solution of 45 (200 mg, 0.355 mmol) in DMF
(2 mL) were added Boc-Glu(OtBu)-OH (130 mg, 0.426 mmol),
EDC (82 mg, 0.426 mmol), and HOBt (66 mg, 0.426 mmol).
The mixture was stirred for 20 h and partitioned between
water and ethyl acetate. The organic layer was washed with
brine, dried (MgSO4), and concentrated. The residue was
chromatographed (silica gel, 30:1 CHCl3:MeOH) to afford 46
(215 mg, 71%): FD-MS (M+) 848; 1H NMR (500 MHz, CD3-
OD) δ 7.55 (d, J ) 7.9 Hz, 1H), 7.42 (d, J ) 7.3 Hz, 1H), 7.12
(m, 1H), 7.04 (m, 1H), 4.52 (m, 1H), 4.30 (m, 1H), 4.08 (m,
1H), 3.98 (d, J ) 6.1 Hz, 1H), 3.62 (m, 1H), 3.44 (m, 1H), 2.28
(m, 2H), 1.99 (m, 2H), 1.81 (m, 1H), 1.63 (m, 1H), 1.48 (m,
2H), 1.40 (s, 18H), 1.39 (s, 9H), 1.10 (s, 9H), 0.87 (m, 12H).
o-(Glu -NH)-P h -NHCO-Ser -Leu -Va l-OH (47). A solution
of 46 (100 mg, 0.118 mmol) in 19:1 TFA:water (2 mL) was
stirred for 2.5 h and concentrated. To the residue was added
p-NO2-P h -NHCO-Ser (OtBu )-Leu -Val-OtBu (52). The titled
compound was prepared in 94% yield according to the proce-
dure as described for 44 using 4-nitrophenyl isocyanate instead
1
of 2-nitrophenyl isocyanate: FD-MS (M+) 593; H NMR (500
MHz, DMSO-d6) δ 9.73 (br, 1H), 8.23 (d, J ) 9.2 Hz, 2H), 8.04
(m, 2H), 7.70 (d, J ) 9.2 Hz, 2H), 6.69 (m, 1H), 4.60 (m, 1H),
4.41 (m, 1H), 4.09 (m, 1H), 3.71 (dd, J ) 9.2, 3.7 Hz, 1H), 3.54
(dd, J ) 9.2, 4.3 Hz, 1H), 2.11 (m, 1H), 1.71 (m, 1H), 1.56 (t,
J ) 7.3 Hz, 2H), 1.49 (s, 9H), 1.20 (s, 9H), 0.97 (m, 12H).
p -NH 2-P h -NH CO-Ser (Ot Bu )-Leu -Va l-Ot Bu (53). The
titled compound was prepared in 87% yield according to the
procedure as described for 45 using 52 instead of 44: FD-MS
1
(M+) 563; H NMR (500 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.96
(d, J ) 7.9 Hz, 1H), 7.79 (d, J ) 8.6 Hz, 1H), 7.01 (d, J ) 8.5
Hz, 2H), 6.46 (d, J ) 8.5 Hz, 2H), 6.12 (d, J ) 7.3 Hz, 1H),
4.65 (s, 2H), 4.49 (t, J ) 7.9 Hz, 1H), 4.22 (m, 1H), 3.99 (dd, J
) 8.5, 6.7 Hz, 1H), 3.58 (dd, J ) 9.2, 4.3 Hz, 1H), 3.38 (dd, J
) 9.2, 4.9 Hz, 1H), 2.02 (m, 1H), 1.62 (m, 1H), 1.46 (t, J ) 7.3
Hz, 2H), 1.39 (s, 9H), 1.10 (s, 9H), 0.87 (m, 12H).
p-(Boc-Glu (OtBu )-NH)-P h -NHCO-Ser (OtBu )-Leu -Va l-
OtBu (54). The titled compound was prepared in 84% yield
according to the procedure as described for 46 using 53 instead
of 45: FD-MS (M+ + H) 849; 1H NMR (500 MHz, DMSO-d6) δ
9.73 (s, 1H), 8.80 (s, 1H), 7.90 (d, J ) 7.9 Hz, 1H), 7.84 (d, J

3298 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Sawa et al.
) 8.5 Hz, 1H), 7.41 (d, J ) 9.2 Hz, 2H), 7.28 (d, J ) 8.5 Hz,
2H), 6.92 (br, 1H), 6.30 (br, 1H), 4.46 (m, 1H), 4.24 (m, 1H),
4.02 (m, 1H), 3.97 (m, 1H), 3.57 (m, 1H), 3.39 (m, 1H), 2.21
(m, 2H), 1.99 (m, 1H), 1.85 (m, 1H), 1.76 (m, 1H), 1.60 (m,
1H), 1.45 (m, 2H), 1.37 (s, 27H), 1.08 (s, 9H), 0.85 (m, 12H).
translation system (Promega, TNT lysate) and T7 RNA poly-
merase. The C-terminus regions of Fas (191-335) and Fas∆15
(191-320, Fas lacks FAP-1 binding region) were subcloned into
pGEX-4T-1 (Amersham Pharmacia Biotech). GST-fusion pro-
tein was purified with Glutathione-Sepharose (Amersham
Pharmacia Biotech) according to instructions of the manufac-
turer. The resulting 35S-labeled protein was incubated with
GST-Fas fusion proteins that had been immobilized on GST-
Sepharose 4B affinity beads (Amersham Pharmacia Biotech)
with a synthetic peptide in a buffer containing 150 mM NaCl,
50 mM Tris (pH 8.0), 5 mM dithiothreitol, 2 mM EDTA, 1.1%
Nonidet P-40, 1 mM phenylmethanesulfonyl fluoride, 50 µg/
mL leupeptin, 1 mM benzamidine, and 7 µg/mL pepstatin for
16 h at 4 °C. After four vigorous washings in the same buffer,
associated proteins were recovered with the Glutathione-
Sepharose beads by centrifugation, and radioactivity was
monitored by liquid scintillation counter. Specific binding was
calculated by subtraction of the binding to Fas∆15 from the
binding to Fas. The binding ratio (%) was calculated as
follows: [specific binding with peptides]/[specific binding
without peptides] × 100 (%). The “percent binding inhibition”
was calculated by subtraction of binding ratio (%) from 100
(%). Thus, 100% inhibition is defined as no binding between
Fas and FAP-1.
p-(Glu -NH)-P h -NHCO-Ser -Leu -Va l-OH (55). The titled
compound was prepared in 77% yield according to the proce-
dure as described for 47 using 54 instead of 46: FAB-MS (M+
1
+ H) 581; H NMR (500 MHz, CD3OD) δ 7.48 (d, J ) 8.5 Hz,
2H), 7.36 (d, J ) 8.5 Hz, 2H), 4.52 (dd, J ) 9.8, 4.9 Hz, 1H),
4.38 (t, J ) 5.5 Hz, 1H), 4.28 (d, J ) 6.1 Hz, 1H), 4.01 (t, J )
6.1 Hz, 1H), 3.86 (dd, J ) 10.4, 5.5 Hz, 1H), 3.73 (dd, J ) 10.4,
6.1 Hz, 1H), 2.53 (t, J ) 7.3 Hz, 2H), 2.20 (m, 3H), 1.80-1.55
(m, 3H), 0.95 (m, 12H).
P h -NHCO-Ser -Leu -Va l-OE t (56). A solution of 41 (192
mg, 0.442 mmol) in 10% HCl/EtOH (1.5 mL) was stirred for
12 h and concentrated. The residue was adsorbed on a plate
of silica gel, and the plate was developed with 10% methanol/
1
chloroform to afford 41 mg (20%) of 56: FD-MS (M+) 464; H
NMR (500 MHz, CDCl3/CD3OD) δ 7.37-7.26 (m, 5H), 4.44 (m,
2H), 4.39 (m, 1H), 4.18 (m, 2H), 3.90 (dd, J ) 11.0, 4.3 Hz,
1H), 3.65 (dd, J ) 11.0, 7.3 Hz, 1H), 2.15 (m, 1H), 1.73-1.56
(m, 3H), 1.29 (t, J ) 3.7 Hz, 3H), 0.92 (m, 12H).
P h -NHCO-Ser -Leu -Ala -OEt (57). A solution of Leu-Ala
(2.0 g, 9.1 mmol) in saturated HCl/EtOH (50 mL) was stirred
for 4 h at room temperature. Concentration and subsequent
separation between EtOAc and saturated aqueous NaHCO3
gave an organic layer, which was washed with brine, dried
over Na2SO4, and concentrated to afford Leu-Ala-OEt (0.51 g,
2.22 mmol) which was used without further purification. A
solution of Leu-Ala-OEt (0.50 g, 2.17 mmol), Fmoc-Ser(OtBu)-
OH (0.83 g, 2.22 mmol), and EDC (0.45 g, 2.35 mmol) in DMF
(10 mL) was stirred at room temperature for 3.5 h. After water
was added, the solution was extracted with ethyl acetate. The
extract was washed with brine (3×), dried over Na2SO4, and
concentrated. Obtained residue was dissolved in 20% piperi-
dine/DMF (10 mL) and stirred for 50 min. The solution was
partitioned between ethyl acetate and water. The organic layer
was washed with brine (3×), dried over Na2SO4, and concen-
trated. The crude products were washed with MeOH, and the
soluble part was subjected to a silica gel column (hexane f
hexane/ethyl acetate ) 9/1 f CHCl3/MeOH ) 100/1 f 100/5)
to afford 588 mg (5.8%, 3 steps) of Ser(OtBu)-Leu-Ala-OEt:
Cytotoxicity Assa y. After preincubation for 24 h, the
human colon cancer cell line, DLD-1 (2 × 104), was incubated
20 h at 37 °C with a graded concentration of Fas antibody CH-
11 (Medical & Biological Laboratories Co., Ltd., Aichi, J apan)
and synthetic peptide in 96-well plates. Each well was washed
twice by phosphate-buffered saline, and surviving cells were
determined using the MTT assay (Chemicon International Inc.,
El Segundo, CA).
Abbr evia tion s: FAP-1, Fas-associated phosphatase-1; Ac,
acetyl; Chg, cyclohexyl-L-glycine; tBu, tert-butyl; tBuGly, tert-
butyl-L-glycine; Thy(OAc), 4-acetoxyphenyl-L-tyrosine; Abu,
2-aminobutyric acid; NorVal, norvaline (2-aminovaleric acid);
Phg, phenyl-L-glycine; Sta, statine; Aib, aminoisobutyric acid;
Abz, aminobenzoic acid; tBuAla, tert-butyl-L-alanine; Hse,
homoserine; Hyp, 4-hydroxy-L-proline; Bz, benzoyl; Bn, benzyl;
Z, benzyloxycarbonyl; Su, succinimide; Tet, tetrazole; Cyh,
cyclohexyl; Ph, phenyl; Boc, tert-butoxycarbonyl; Fmoc, 9-fluo-
renylmethoxycarbonyl; HOBt, N-hydroxybenzotriazole; DIC,
diisopropylcarbodiimide; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; EDC, 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride; TFA, tri-
fluoroacetic acid; DMF, N,N-dimethylformamide; THF, tetra-
hydrofuran; DMSO, dimethyl sulfoxide; GST, glutathione
S-transferase; EDTA, ethylenediaminetetraacetic acid; SDS,
sodium dodecyl sulfate.
1
FD-MS (M+) 373; H NMR (500 MHz, CDCl3) δ 7.79 (d, J )
8.6 Hz, 1H), 6.74 (d, J ) 6.7 Hz, 1H), 4.51 (m, 1H), 4.43 (m,
1H), 4.19 (qd, J ) 7.3, 1.2 Hz, 2H), 3.56 (m, 2H), 3.50 (m, 1H),
1.78 (s, 2H), 1.69 (m, 2H), 1.58 (m, 1H), 1.39 (d, J ) 6.7 Hz,
3H), 1.28 (t, J ) 7.3 Hz, 3H), 1.18 (s, 9H), 0.95 (d, J ) 6.7 Hz,
3H), 0.92 (d, J ) 6.1 Hz, 3H).
A solution of Ser(OtBu)-Leu-Ala-OEt (400 mg, 1.07 mmol)
and phenyl isocyanate (150 µM, 1.38 mmol) in DMF (5 mL)
was stirred for 80 min at room temperature, and then it was
diluted with water and extracted with ethyl acetate. The
insoluble product was removed by filtration, and the filtrate
was washed with brine (3×), dried over Na2SO4, and concen-
trated to give a solid residue. The solid residue was suspended
in ethyl acetate and separated into soluble and insoluble parts.
Insoluble products were combined into afford 396 mg of Ph-
NHCO-Ser(OtBu)-Leu-Ala-OEt. A solution of Ph-NHCO-Ser-
(OtBu)-Leu-Ala-OEt (390 mg) in TFA (5 mL) and water (0.25
mL) was stirred for 1 h at room temperature and concentrated.
The oily residue were partitioned between water and ethyl
acetate. The organic layer was washed with saturated aqueous
NaHCO3 and brine, dried over Na2SO4, and then concentrated.
Crystalline solid was washed with Et2O/hexane to afford 318
mg (68%, 2 steps) of 57: FD-MS (M+) 464; 1H NMR (500 MHz,
CDCl3/CD3OD) δ 7.37-7.26 (m, 5H), 4.44 (m, 2H), 4.39 (m,
1H), 4.18 (m, 2H), 3.90 (dd, J ) 11.0, 4.3 Hz, 1H), 3.65 (dd, J
) 11.0, 7.3 Hz, 1H), 2.15 (m, 1H), 1.73-1.56 (m, 3H), 1.29 (t,
J ) 3.7 Hz, 3H), 0.92 (m, 12H).
Ack n ow led gm en t. We thank the staff in our labo-
ratory for the MS analyses. We also thank Ms. Yoshiko
Yanagisawa for the HPLC analyses. We are grateful to
Dr. Hiroshi Iijima for helpful discussions.
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