Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array

Article abstract of DOI:10.1016/S0968-0896(00)00069-9

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylamino butyric acid (or SC-ααδ9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B₂. Two compounds, FY3-αα09 and FY21-αα09, were partial competitive inhibitors of Cdc25B₂ with K(i) values of 7.6±0.5 and 1.6±0.2 μM, respectively. FY21-αα09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-αα09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-αα09 also inhibited the G₂/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-αα09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00069-9

Bioorganic & Medicinal Chemistry 8 (2000) 1451±1466
Identi®cation of New Cdc25 Dual Speci®city Phosphatase
Inhibitors in a Targeted Small Molecule Array
Alexander P. Ducruet,a Robert L. Rice,a Kenji Tamura,a Fumiaki Yokokawa,b
Shiho Yokokawa,b Peter Wipf b and John S. Lazoa,*
aDepartment of Pharmacology, the Combinatorial Chemistry Center and the Molecular Therapeutic/Drug Discovery Program
of the University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
bDepartment of Chemistry and the Combinatorial Chemistry Center, University of Pittsburgh, Pittsburgh, PA 15260, USA
Received 23 December 1999; accepted 18 February 2000
AbstractÐDual speci®city protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle.
Few potent or speci®c inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a
combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-
diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylamino butyric acid (or SC-aad9), which is the most active ele-
ment in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.;
Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identi®ed as e€ective
inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-aa09
and FY21-aa09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6Æ0.5 and 1.6Æ0.2 mM, respectively. FY21-aa09
possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-aa09 inhibited growth
in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-aa09 also inhibited the G2/M transition in tsFT210 cells,
consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modi®cation of
the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure
of FY21-aa09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be
generated. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
and substrate speci®city. The protein phosphatases
(PPases) hydrolyze phosphomonoester bonds found
speci®cally on serine or threonine amino acid residues,
while the protein tyrosine phosphatases (PTPases) are
phosphotyrosine speci®c, removing the phosphomono-
ester from the phosphorylated tyrosine residue, return-
ing it to the analogous initial alcohol.4,5 A PTPase
sub-class is formed by the dual speci®city protein phos-
phatases (DSPases), which are capable of hydrolyzing
the phosphomonoester bonds on both phosphotyrosine
and phosphoserine/threonine residues found on the
same protein substrate.5 While PTPases have extremely
limited sequence similarity, they share the active site
signature motif (I/V) HCXAGXGR(S/T)G, harboring
the catalytic cysteine residue that plays an essential role
in the phosphatase-mediated phosphotyrosine phos-
phomonoester bond cleavage.6,7
Phosphorylation and dephosphorylation by the con-
comitant actions of kinases and phosphatases are cri-
tical control mechanisms for numerous physiological
functions.2 More speci®cally, protein phosphorylation is
the major component of signal transduction pathways,
highly regulated processes by which cells convey infor-
mation from the cell surface to their nucleus or other
remote subcellular sites.3,4 This information then con-
trols such processes as cell growth and di€erentiation,
metabolism, cell cycle regulation, cytoskeletal functions
and even transformation.2,5
Phosphatases are generally divided into two main
groups based on their enzymatic mechanism, structure
*Corresponding author. Department of Pharmacology, Biomedical
Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA
15261, USA. Tel.: +1-412-648-9319; fax: +1-412-648-2229; e-mail:
lazo+@pitt.edu
The PTPases have diverse biochemical and cellular
roles. For example, PTP1B regulates both epidermal
growth factor and insulin signaling pathways.6,8 10
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00069-9

1452
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
PTP1B can also reverse the transformation of cells by
v-src, v-crk and v-ras when over-expressed in these cells.11
The DSPase VHR (vaccina human-related) regulates
mitogenic signaling by speci®cally dephosphorylating
members of the MAPK (mitogen-activated protein
kinase) family, namely the extracellular regulated kina-
ses ERK1 and ERK2.5,12,13 The Cdc25 DSPases coor-
dinate the cell cycle by dephosphorylating, and thereby
activating, cell cycle dependent kinases. For example,
Cdc25B and Cdc25C dephosphorylate p34cdc2 (also
known as cdkl) and permit entry into mitosis.14 17
Since members of the PTPase family appear to play an
important role in cell signaling, cell cycle control and
oncogenesis, speci®c inhibitors of these proteins would
be highly desirable. Unfortunately, selective and potent
inhibitors of PTPases are not currently available.
Present crystallographic data, however, suggest that
selective inhibitors may be obtainable as signi®cant
di€erences have been observed in the active site of
Cdc25A, VHR and PTP1B phosphatases.13,14,18 The
shallow 6 A pocket of VHR and the even shallower
pocket of Cdc25A can accommodate both phosphotyr-
osine as well as phosphoserine/threonine residues.13,14
In contrast, PTP1B possesses a deeper 9 A catalytic
pocket where only the more extended phosphotyrosine
side chain, unencumbered by any adjacent phosphoser-
ine/threonine, can reach the nucleophilic cysteine at the
base of the catalytic pocket.5,18 The structural di€erences
present in these phosphatases should be exploitable in
the selection of targeted inhibitors. Furthermore, inhi-
bitors may be helpful in biochemically de®ning other
unidenti®ed intracellular functions.
Figure 1. SC pharmacophore and variable core region substituents.
General chemical structure for the founding SC pharmacophore series.
Rn indicate the combinatorial sites on the pharmacophore and are
sequentially listed after the core pre®x: (A) the variable core region;
(B) chemical structure of SC-aad9; (C) structure of variable core
diamine linker substitutions.
Table 1. Inhibition of Cdc25B2 phosphatase activity and lipophilicity
of SC-aad9 analoguesa
Cdc25B2 (% Inhibition)
clog P valueb
We have used a previously described non-electrophilic
phosphatase inhibitor, namely SC-aad9,1 as a pharma-
cophore to synthesize a new targeted library of small
molecules and examined each element for selective
DSPase inhibition. Minimal structural changes in the
pharmacophore resulted in signi®cant di€erences in
inhibitory properties. In addition, the hydrophobic
domain of the core pharmacophore was essential for
anti-phosphatase activity. We have de®ned a structure
with enhanced selectivity for the dual speci®city phos-
phatase Cdc25B2. This compound, FY21-aa09, also
blocked the proliferation of human breast cancer cells in
vitro.
Compound
3 mM
100 mM
Carboxylate Free acid
(R)-SC-aad9
(S)-SC-aad9
SC-aadA
3Æ3
2Æ2
85Æ2
83Æ2
78Æ2
76Æ3
78Æ3
79Æ4
13Æ2
23Æ2
1.2
1.2
NDc
3.6
4.9
4.9
NDc
8.4
6Æ6
SC-aad15
11Æ9
13Æ10
7Æ7
SC-aad17A
SC-aad17B
SC-aad6III
SC-aad4II
4.3
4.4
8.9
9.0
7Æ7
2.0
1.8
1.4
1.9
6Æ4
aEach value is the percent inhibition from untreated control and the
mean and SEM from three or more independent determinations.
bclog P values represent logarithms of the octanol±water partition
coecient and were calculated based on energy-minimized extended
conformations by the method of Alkorta and Villar.26
cND, not determined.
Results
Phosphatase inhibition by SC-ꢀꢀꢁ9 enantiomers and R4
analogues
Previous observations lead us to speculate that the
hydrophobic nonyl substituent of our SC pharmaco-
phore was critical for phosphatase inhibition.1 To
investigate this hypothesis, we replaced the nonyl moi-
ety (R4) of the SC-aad9 core pharmacophore with
hydrophobic substituents of variable length (Fig. 2).
Compounds such as SC-aad15, which contains a penta-
decyl side chain, had a greater clog P value than SC-
aad9 but had similar Cdc25B2 inhibitory activity as
compared to that seen with SC-aad9 (Table 1). Simi-
larly, SC-aad17A and SC-aadl7B, which had greater
Because we previously found that racemic SC-aad9
(Fig. 1B) was an inhibitor of dual speci®city phospha-
tases,1 we further examined the structural basis for
inhibition by synthesizing and testing both the (R)-SC-
aad9 and (S)-SC-aad9 enantiomers. Both enantiomers
inhibited Cdc25B2 phosphatase activity by approxi-
mately 85% at 100 mM (Table 1). At 3 mM, neither
enantiomers had signi®cant inhibitory activity.

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1453
on the C5 oxazole (R2) position and the R3 amine. The
importance of the R2 aromatic moiety was further
established with the full FY series, where -aa09 com-
pounds were generally more inhibitory than the -a109
compounds (Table 2). When we examined the inhibition
of Cdc25B2 with 100 mM of the -aa09 series, we found
that enzyme inhibition was moderately resistant to
modi®cation of the core pharmacophore structure
(Table 2), although several cores, such as FY9 and
FY10, exhibited decreased Cdc25 inhibitory activity.
FY21-aa09 was the most e€ective inhibitor of Cdc25B2
at 3 mM (Table 2), causing a 36% decrease in enzyme
activity. In the -a109 series, no compound was as
e€ective as FY21-aa09. Many congeners were similar
to SC-a109, although notably, FY3-a109, FY5-a109,
FY7-a109 and FY8-a109 exhibited 2- to 5-fold higher
inhibitory activity against Cdc25B2 than SC-a109. None
of the compounds in the rigidi®ed FY series, however,
caused marked inhibition of VHR or PTP1B at 3 mM
(Table 2). FY5-aa09 was the only compound with a
distinct preference for VHR over Cdc25B2 or PTP1B.
FY3-aa09, FY8-aa09 and FY21-aa09 showed no sig-
ni®cant in vitro preference for Cdc25B2, VHR or
PTP1B in these preliminary studies at 100 mM. A more
comprehensive study of FY21-aa09, however, revealed
that PTP1B (IC50=41.4 mM) was approximately 4-fold
less sensitive to the compound than Cdc25B2 or VHR
(IC50s of 7.0 mM and 12.1 mM, respectively) (Fig. 4).
Inhibition kinetics of compounds
We next determined the kinetic characteristics of
Cdc25B2 inhibition with two of our most potent com-
pounds, FY3-aa09 and FY21-aa09. We found that
FY3-aa09 and FY21-aa09 were partial competitive
inhibitors of Cdc25B2, with Ki values of 7.6Æ0.5 mM
and 1.6Æ0.2 mM, respectively (Fig. 5). Kinetic studies
using 1 to 60 mM FY3-aa09 with VHR and rhPTP1B
were most consistent with an S-parabolic noncompetitive
inhibition model (data not shown), although higher
FY3-aa09 concentrations presented a more mixed pro-
®le. The di€erences in the FY3-aa09 kinetic pro®les
seen with VHR, PTP1B and Cdc25B2 may re¯ect the
structural dissimilarities of these phosphatases.
Figure 2. Chemical structures of SC-aad9 R4 analogues.
clog P values than SC-aad15, maintained anti-Cdc25B2
activity, while the ether-containing SC-aad6III and
SC-aad4II, with the lowest clog P values of these
compounds, had no inhibitory activity against Cdc25B2
up to a concentration of 100 mM. These results demon-
strated a critical role for hydrophobicity rather than just
the length of the R4 substituent for Cdc25B2 phospha-
tase inhibition.
Phosphatase inhibition by modi®ed core pharmacophore
library members
Cell proliferation assays
Previous studies with the founding core library members
indicated that only SC-aad9 caused marked, concentra-
tion-dependent inhibition of tumor cell growth in cul-
ture.19 We found with the current FY series that only
FY21-aa09 caused greater than 90% inhibition of
MDA-MB-231 cell growth at 100 mM, while 100 mM of
either FY8-aa09 or FY21-aa09 inhibited MCF-7 cell
growth by greater than 90% (Fig. 6). Studies with
tsFT210 cells indicated that a 6 h exposure to 100 mM
FY21-aa09 partially blocked cell cycle progression at
the G2/M checkpoint while the biochemically inactive
congener SC-aa09 did not cause any block (Fig. 7).
FY21-aa09 at 100 mM was as e€ective inhibiting the
G2/M transition as the tubulin binder nocodazole at
1 mM. The G2/M block observed with FY21-aa09 is
consistent with intracellular inhibition of Cdc25B2.
Conformational rigidity can be achieved by replacing
the ¯exible ethyldiamine portion of the original SC-
aad9 pharmacophore with a series of cyclic diamines
(Figs. 1 and 3).
Speci®cally, cyclohexyldiamine, piperazine and phe-
nyldiamine substructures were used to enhance rigidity
of the variable core region and alter overall size. We
also examined an extended alkyl linker, a propyldiamine
substructure, to determine if the length of the linker was
important. Because of the absence of stereospeci®city in
the parental SC structure, all congeners were synthe-
sized as racemic mixtures. We found SC-aad9> SC-
aa09> SC-a109 as inhibitors of Cdc25B2 (Tables 1
and 2) revealing the importance of an aromatic moiety

1454
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
Figure 3. Chemical structures of representative analogues of the -aa09 series.
Table 2. Inhibition of dual speci®city and tyrosine-speci®c phosphatase activitya
Cdc25B2 (% inhibition)
VHR (% inhibition)
rhPTP1B (% inhibition)
Compound
3 mM
100 mM
57Æ8.0
3 mM
100 mM
3 mM
100 mM
SC-aa09
SC-a109
2.1Æ1.3
2.8Æ1.7
4.0Æ2.4
3.3Æ2.3
4.1Æ2.4
1.4Æ1.0
0.9Æ0.9
3.3Æ3.0
17Æ4.4
3.1Æ1.8
4.4Æ2.8
1.1Æ0.6
0.1Æ0.1
2.5Æ4.1
2.3Æ1.3
3.2Æ3.2
36Æ1.3
3.0Æ0.7
4.5Æ1.4
8.4Æ1.1
5.5Æ0.3
5.2Æ0.2
1.3Æ0.3
6.4Æ1.1
1.1Æ2.1
10Æ0.5
6.4Æ0.1
6.5Æ1.0
1.7Æ0.6
2.2Æ0.9
3.4Æ0.7
4.4Æ0.2
2.6Æ2.1
11Æ2.7
6.4Æ3.6
12Æ1.4
99Æ 0.4
31Æ1.6
61Æ2.4
42Æ0.8
95Æ0.9
65Æ1.2
73Æ3.3
35Æ1.2
95Æ1.9
87Æ2.4
9.0Æ1.0
4.5Æ2.2
11Æ2.7
8.3Æ3.0
95Æ2.6
0.2Æ0.6
0.6Æ0.7
4.5Æ1.2
4.3Æ0.7
4.3Æ1.3
0.3Æ0.2
0.7Æ0.4
0.8Æ0.4
1.9Æ0.8
3.1Æ0.6
5.9Æ0.9
3.8Æ0.9
0.3Æ0.9
0.3Æ0.5
0.4Æ0.3
0.5Æ0.3
3.4Æ1.6
1.3Æ2.5
3.5Æ1.3
89Æ2.1
16Æ1.4
33Æ3.3
6.7Æ0.8
40Æ6.3
23Æ5.5
17Æ1.0
12Æ1.3
90Æ2.3
69Æ2.4
3.2Æ1.4
6.3Æ1.3
3.7Æ1.0
3.8Æ1.0
78Æ1.7
15Æ7.7
72Æ4.4
25Æ9.8
73Æ1.0
19Æ7.1
63Æ4.3
48Æ3.3
80Æ3.5
68Æ2.7
85Æ2.6
44Æ8.8
51Æ5.4
4.1Æ2.4
28Æ8.3
16Æ6.9
83Æ0.6
FY3-aa09
FY3-a109
FY4-aa09
FY4-a109
FY5-aa09
FY5-a109
FY7-aa09
FY7-a109
FY8-aa09
FY8-a109
FY9-aa09
FY9-a109
FY10-aa09
FY10-a109
FY21-aa09
aEach value is the percent inhibition from untreated control and the mean and SEM from three or more independent determinations.
Discussion
small molecules of a more hydrophobic nature may
possess enhanced anti-phosphatase activity.14 To test
the hypothesis that the hydrophobic nonyl substituent
in our founding pharmacophore was essential for phos-
phatase inhibition, we replaced the SC-aad9 nonyl
moiety, which has an extended length of 22 A, with
various more hydrophobic substituents (Fig. 2). There
seemed to be a parallel relationship between clog P
values and inhibition of Cdc25B2 phosphatase activity.
Compounds with R4 substituents of a more hydro-
phobic nature than the nonyl group found in SC-aad9
displayed an inhibitory pro®le against Cdc25B2 similar
to that of SC-aad9. On the other hand, compounds with
R4 substituents with more hydrophilic character and
The dual speci®city and tyrosine speci®c phosphatases
share low sequence homology except for conserved
residues involved in phosphate binding and catalysis.
Based on their crystal structures, Cdc25 and VHR
appear to be more accommodating to substrates and
less sterically hindered than PTP1B. Our structure±
activity relationship results support this conclusion:
several of the rigid compounds show marked preference
for Cdc25 and VHR rather than PTP1B.
The crystal structure of Cdc25 reveals a hydrophobic
region within 20±22 A of the active site, suggesting that

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1455
Figure 4. Inhibition of dual speci®city and tyrosine-speci®c phospha-
tases by FY21-aa09. Concentration-dependent phosphatase inhibition
by FY21-aa09 (representative results from one experiment). -&-,
Cdc25B2; -^-, VHR; -~-, PTP1B. Enzyme activities were determined
as described in Experimental. Non-linear regressions were performed
using GraphPad Prism 3.0.
therefore lower clog P values, such as the ether-con-
taining SC-aad6III and SC-aad4II, demonstrated negli-
gible Cdc25B2 inhibitory activity at concentrations up
to 100 mM. Because kinetic studies with SC-aad9 sug-
gest that its mode of inhibition of Cdc25B2 is most
consistent with a competitive model, the importance of
a hydrophobic moiety in the R4 position of the phar-
macophore suggested that the Cdc2SB2 active site, with
the exception of the phosphate binding site, is hydro-
phobic in nature.1,20
Figure 5. Kinetic analysis of Cdc25B2 inhibition by FY3-aa09 and
FY21-aa09. Panel A: Lineweaver±Burk plot of Cdc25B2 inhibition by
FY3-aa09. -&-, 0 mM; -!-, 0.1 mM; -*-, 1 mM; -&-, 10 mM -~-, 50
mM; and -!-, 100 mM FY3-aa09. Panel B: Lineweaver±Burk plot of
Cdc25B2 inhibition by FY21-aa09. -&-, 0 mM -!-, 5 mM; -^-, 15
mM; -*-, 30 mM; and -&-, 60 mM FY21-aa09. Enzyme activities were
determined as described in Experimental procedures and the data ®t to
the Michaelis±Menten equation. The best ®t kinetic model was deter-
Rigidi®cation of our basic SC pharmacophore model in
our founding targeted array improved the inhibitory
pro®le against DSPases. By replacing the ethyldiamine
linker with a cyclohexyldiamine sca€old, such as in
FY3-aa09, FY4-aa09 and FY21-aa09, we did not sig-
ni®cantly alter the anti-Cdc25B2 activity; these three
compounds also eciently inhibited VHR. FY4-aa09,
however, demonstrated signi®cant preference for the
DSPases and did not inhibit PTP1B. Perhaps this is due
to the less extended nature of FY4-aa09 as compared
with the other cyclohexyldiamine congeners. FY21-
aa09 was even more potent against DSPases at 100 mM
compared with FY4-aa09, but it also inhibited PTP1B;
at 3 mM, however, FY21-aa09 displayed a signi®cant
preference for Cdc25B2. Perhaps this speci®city for
Cdc25B2 depends on the previously reported shallow
nature of the enzyme's active site relative to that of
PTP1B.14 In addition to rigidifying the pharmacophore,
we also varied the substituents at the R1 and R2 posi-
tions. All the new compounds with anti-phosphatase
activity displayed a strong preference for an aromatic
moiety in the R2 position. This may be due to the more
hydrophobic nature of DSPase active sites and the
documented second aryl phosphate binding site adja-
cent to the active site of PTP1B.1,20,21
mined using EZ-FitTM
.
In examining a subset of compounds, we found that the
better in vitro inhibitors of DSPases were e€ective inhi-
bitors of breast cancer proliferation. MDA-MB-231
cells were more selective and intrinsically less sensitive
to the compounds tested than MCF-7 cells. Cell
proliferation was inhibited in a clear concentration
response manner, with FY21-aa09 being the most
potent agent tested. The growth inhibition seen with
several of the compounds, such as FY3-a109, which
lacked signi®cant anti-DSPase activity in vitro, seems
most likely due to other activities. Cdc25 is the major
regulator of the G2/M checkpoint and blockage seen
with FY21-aa09 is similar to that observed with other
Cdc25 inhibitors.22 Nonetheless, we recognize that the
G2/M block seen with FY21-aa09 could be the result of
mechanisms other than inhibition of Cdc25. Additional
studies are required to con®rm our hypothesis regarding
the mechanism of G2/M arrest.

1456
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
Figure 6. Antiproliferative e€ects of selected compounds on MDA-MB-231 and MCF-7 human breast cancer cells. Antiproliferative e€ect of com-
pounds. MDA-MB-231 cells: 10 mM (&) 30 mM ( ) and 100 mM (&), MCF-7 cells: 10 mM ( ), 30 mM ( ) and 100 mM ( ). The antiproliferative
e€ect of compounds was determined as described in Experimental procedures and results were normalized to vehicle treated controls. Error bars
represent standard deviations (n=6).
inhibitory activity against this phosphatase.24 A 3-a-
azido-B-homo-6-oxa-4-cholesten-7-one derivative had
an IC50 of 2 mM for Cdc25A, although there is no
information concerning its selectivity.25 Although
FY21-aa09 eciently inhibited both DSPases and
PTPases in vitro, unlike SC-aad9,1 it exhibited some
preference for the DSPases. As a partial competitive
inhibitor, FY21-aa09 interacted with Cdc25B2 in the
vicinity of the active site though was unable to com-
pletely impede substrate binding. R4 substituents in the
original SC pharmacophore revealed the importance of
a hydrophobic moiety for Cdc25B2 inhibition and
rigidi®cation of the variable core region, and modi®ca-
tion of the side chain moieties of the pharmacophore
suggests several structural requirements for DSPase
inhibition. The structures of FY5-aa09, FY7-aa09 and
FY21-aa09 should provide excellent platforms for
future analogue development. Our results indicate that
selective inhibition of DSPases and PTPases is indeed
Figure 7. Inhibition of tsFT210 cell cycle progression at G2/M by
possible.
FY21-aa09. (A) tsFT210 cells were cultured at the permissive tem-
perature of 32.0 ꢀC and then (B) incubated for 17 h at the non-per-
missive temperature of 39.4 ꢀC. Cells were released from cycle arrest
by shifting to the 32.0 ꢀC medium. The cells were then incubated for 6
Experimental
h in the presence of (C) DMSO vehicle, (D) 1 mM nocodazole, (E) 100
mM FY21-aa09 or (F) 100 mM SC-aa09. Fluorescence corresponding
to 2C or 4C DNA content is represented by vertical bars.
Chemical compounds
We have previously described the general nomenclature
With FY21-aa09, we have identi®ed one of the most
potent Cdc25B2 inhibitors reported to date. Dysidiolide,
which was reported to have an IC50 of 9.4 mM against
Cdc25A,23 has more recently been found to lack potent
system used for our phosphatase library elements.1 The
alphanumeric pre®x provides a unique indicator of
the core pharmacophore. Thus, the `SC' pre®x was used
to designate the pharmacophore used in the founding

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1457
phosphatase inhibitor library (Fig. 1A). Replacement of
the ethylenediamine in the founding pharmacophore
with a trans cyclohexyl-1,2-diamine produced the FY3
series, cis cyclohexyl-1,2-diamine produced the FY4
series, para-phenyldiamine produced the FY5 series,
meta-phenyldiamine produced the FY7 series, ortho-
phenyldiamine produced the FY8 series, propyldiamine
produced the FY9 series, piperazine produced the FY10
series and a trans cyclohexyl-1,4-diamine produced the
FY21 series. The FY6 series utilizes ethylenediamine as
the core structure. The four descriptors following the
pre®x indicate the chemical nature of the substituents
on the four variable domains of the pharmacophore.
The ®rst descriptor always described the substituent
(R1) furthest from the assumed surrogate phosphate, a
carbonyl moiety in both the SC and FY families. Greek
letters indicate an aromatic moiety so that the addition
of a phenyl was termed a, a phenethyl was b, benzyl was
d and a styryl was g.1 Alkyl chains were designated on
the basis of carbon length with Roman numerals and
capital letters added if there was chain branching or the
inclusion of non-carbon-based linkers. Thus, each com-
pound has a unique name. For most compounds in this
study the R3 site was protonated and this was desig-
nated as 0. Compounds were resuspended in DMSO
prior to use and stocks (powder and resuspended) were
stored at 20 ꢀC.
The general synthesis of compounds SC-aad9 and SC-
aa09 has been previously described.19 A slightly mod-
i®ed strategy was used for the synthesis of the new
compounds SC-aad4II and SC-aad6III. dl-Glutamate
(1) was selectively side-chain esteri®ed with trimethyl-
silyl chloride in allyl alcohol, N-protected with b,b,b-
trichloroethoxycarbonyl chloride, and methylated to
give Troc-Glu(allyl)-OMe (2b) in 49% overall yield
(Scheme 1). After Pd(0)-catalyzed deallylation, the side-
chain carboxyl terminus was extended by PyBroP-
mediated coupling with BnNHCH2CH2NHAlloc,
renewed Pd(0)-catalyzed deallylation and coupling with
the oxazole segment to give 4. After deprotection of the
N-terminus with zinc in acetic acid, EDCI-mediated
coupling provided SC-aad4II and SC-aad6II in 5±10%
overall yield from dl-glutamate.
Scheme 1.

1458
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
For the synthesis of FY21-aa09, protected glutamate
2a was acylated at the N-terminus after removal
of the Troc group, deallylated and coupled to diamine
8 to give the segment 9 in 52% yield from 2a (Scheme
2). Acidolytic cleavage of the Boc group and PyBroP-
mediated coupling with oxazole 10 provided ester
11, which was readily converted to the desired FY21-
aa09.
extracted with EtOAc (3Â). The resulting organic layer
was dried (Na2SO4) and concentrated in vacuo to give
8.76 g of carboxylic acid as a yellow oil. To a solution of
the resulting oil in 32 mL of DMF was added 3.87 g
(38.7 mmol) of KHCO3 and 3.4 mL (29 mmol) of benzyl
bromide. The reaction mixture was stirred at 22 ꢀC for
14 h, diluted with EtOAc, and washed with 10% HCl,
H2O, saturated aqueous NaHCO3 and brine. The
organic layer was dried (Na2SO4), concentrated in
vacuo, and chromatographed on SiO2 (hexanes:EtOAc,
6:1!5:1!4:1!3:1) to give 5.70 g (19% over three
steps) of 2a as a pale yellow oil: IR (neat) 3344, 2953,
1745, 1519, 1327, 1269, 1203 cm 1; 1H NMR (CDCl3) d
7.31 (m, 5H), 5.94±5.83 (m, 1H), 5.69 (d, 1H, J=8.1
Hz), 5.34±5.21 (m, 2H), 5.19 (s, 2H), 4.71 (AB, 2H,
J=11.9 Hz), 4.57 (d, 2H, J=4.7 Hz), 4.51±4.44 (m, 1H),
2.50±2.33 (m, 2H), 2.32±2.24 (m, 1H), 2.10±2.01 (m,
1H); 13C NMR (CDCl3) d 172.2, 171.3, 154.2, 135.0,
131.9, 128.6, 128.3, 118.4, 95.3, 74.5, 67.4, 65.4, 53.5,
29.9, 27.2.
Detailed experimental protocols for FY21-aa09, FY3-
aa09, FY4-aa09, FY5-aa09, FY7-aa09, FY8-aa09,
FY9-aa09, FY10-aa09, SC-aad17A, SC-aad17B, SC-
aadA, SC-aad4II, and SC-aad6III are listed below.
FY21-ꢀꢀ09
2-(2,2,2-Trichloroethoxycarbonylamino)-pentanoic acid
5-allyl ester 1-benzyl ester (2a). To a stirred suspension
of 10 g (68 mmol) of d,l-glutamic acid (1) in 170 mL of
allyl alcohol was added dropwise 21.6 mL (170 mmol)
of chlorotrimethylsilane. The suspension was stirred at
22 ꢀC for 22 h and concentrated in vacuo. The resulting
oil was dissolved in 180 mL of dioxane. The resulting
solution was treated with 237.9 mmol of aqueous
sodium carbonate (25.21 g in 180 mL of H2O) at 0 ꢀC,
stirred for 5 min, and treated with 9.5 mL (69.3 mmol)
of TrocCl. The reaction mixture was stirred at 0 ꢀC for
10 min and warmed to 22 ꢀC, stirred for 17 h, con-
centrated in vacuo, poured into 300 mL of H2O and
washed with Et2O. The aqueous layer was acidi®ed to
pH 1 with concentrated HCl, salted out with NaCl, and
2-Decanoylamino-pentanedioic acid 5-allyl ester 1-benzyl
ester (7). To a solution of 5.698 g (12.59 mmol) of 2a
in 16.5 mL of acetic acid and 24.7 mL of THF was
added 16.455 g (251.73 mmol) of zinc dust. The sus-
pension was stirred at 22 ꢀC for 1 h. The reaction mix-
ture was ®ltered through a Celite pad and the ®ltrate
was concentrated in vacuo. The resulting residue was
diluted with EtOAc and washed with saturated aqueous
NaHCO3 and brine. The organic layer was dried
(Na2SO4) and concentrated in vacuo to give 2.68 g
Scheme 2.

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1459
of free amine. The resulting residue was dissolved in
42 mL of CH2Cl2 and treated with 5.3 mL (38 mmol) of
triethylamine and 3.9 mL (19 mmol) of decanoyl chlo-
ride at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC for
15 min and warmed to 22 ꢀC, stirred for 12 h, con-
centrated in vacuo, diluted with EtOAc, and washed
with 10% HCl, H2O, saturated aqueous NaHCO3 and
brine. The organic layer was dried (Na2SO4), con-
centrated in vacuo, and chromatographed on SiO2
(hexane:EtOAc, 5:1!3:1) to give 3.69 g (68% over two
1H), 5.22±5.12 (m, 2H), 4.56 (m, 1H), 4.37 (m, 1H), 3.70
(m, 1H), 3.41 (m, 1H), 2.24±2.17 (m, 5H), 1.19±1.15 (m,
5H), 1.59±1.53 (br, 4H), 1.44 (s, 9H), 1.26 (br, 12H),
0.90±0.86 (m, 5H).
2-Decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
trans-1,4-cyclohexyl}-carbamoyl)-butyric acid benzyl ester
(11). A mixture of 167 mg (0.284 mmol) of 9 in a HCl/
dioxane solution was stirred at 0 ꢀC for 5 min, warmed
to 22 ꢀC, stirred for 4 h, and concentrated in vacuo. The
resulting residue and 75 mg (0.29 mmol) of oxazole
segment 10 were dissolved in 0.95 mL of THF. To this
solution was added 99 ml (0.71 mmol) of triethylamine
followed by 238 mg (0.511 mmol) of PyBroP at 0 ꢀC.
After stirring at 0 ꢀC for 1 h and at 22 ꢀC for 12 h, the
reaction mixture was diluted with EtOAc and washed
with 10% HCl, H2O, saturated aqueous NaHCO3 and
brine. The organic layer was dried (Na2SO4), con-
centrated in vacuo, and chromatographed on SiO2
(hexanes:EtOAc, 1:1!1:2!1:3) to give 70 mg (33%
over two steps) of 11 as a white solid: 1H NMR (CDCl3)
d 8.38±8.36 (m, 2H), 7.70±7.22 (m, 13H), 5.21±5.10 (m,
2H), 4.54±4.53 (m, 1H), 3.93±3.92 (m, 1H), 3.76 (m,
1H), 2.29±1.82 (m, 8H), 1.61 (br, 2H), 1.47±1.38 (m,
4H), 1.26 (br, 12H), 0.89±0.85 (m, 5H).
steps) of 9 as a yellow oil: IR (neat) 3293, 3063, 2924,
1
2855, 1740, 1649, 1534, 1453, 1379, 1175, 986, 930 cm
;
1H NMR d 7.26 (s, 5H), 6.68 (d, 1H, J=7.8 Hz), 5.85±
5.75 (m, 1H), 5.22 (d, 1H, J=17.3 Hz), 5.14 (d, 1H,
J=10.4 Hz), 5.08 (s, 2H), 4.63±4.57 (m, 1H), 4.48 (d,
2H, J=5.6 Hz), 2.38±2.28 (m, 2H), 2.2±2.1 (m, 3H),
2.0±1.9 (m, 1H), 1.55 (t, 2H, J=6.9 Hz), 1.20 (bs, 12H),
0.82 (t, 3H, J=5.9 Hz); 13C NMR d 173.0, 172.1, 171.6,
135.0, 131.7, 128.2, 128.1, 127.8, 117.9, 66.8, 64.9,
51.3, 36.0, 31.6, 29.9, 29.1, 29.0, 26.8, 25.3, 22.3,
13.8; MS (EI) m/z (relative intensity) 431 (M+, 12),
319 (21), 296 (51), 142 (100), 124 (31), 91(91); HRMS
(EI) m/z calcd for C25H37NO5: 431.2672; found:
431.2673.
trans-1-Amino-4-t-butoxycarbonylamino-cyclohexane (8).
To a solution of 1.0 g (8.8 mmol) of trans-1,4-diamino-
cyclohexane in 30 mL of MeCN was added 1.93 g (8.85
mmol) of Boc2O. The reaction was stirred at 22 ꢀC for
12 h, concentrated in vacuo, poured into 50 mL of H2O,
and washed with Et2O. The aqueous layer was acidi®ed
to pH 4 with 10% citric acid, salted out with NaCl, and
extracted with EtOAc (3Â). The resulting organic layer
was dried (Na2SO4) and concentrated in vacuo to give
401 mg (21%) of 8 as a colorless solid: 1H NMR
(CDCI3) d 4.35 (br, 1H), 3.38 (br, 1H), 2.63±2.62 (m,
1H), 2.01±1.97 (m, 2H), 1.87±1.84 (m, 2H), 1.44 (s, 9H),
1.40 (m, 2H), 1.26±1.11 (m, 4H); HRMS (EI) m/z calcd
for C7H13N2O2 (M-t-Bu): 157.0977; found: 157.0980.
2-Decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
trans-1,4-cyclohexyl}-carbamoyl)-butyric acid (FY21-
ꢀꢀ09). To a solution of 23.6 mg (0.0321 mmol) of 11
in 0.24 mL of dioxane was added 0.385 mmol of aqu-
eous lithium hydroxide (16.2 mg in 0.08 mL of H2O) at
0 ꢀC. The reaction mixture was stirred at 0 ꢀC for 10 min
and at 22 ꢀC for 3 h, poured into 20 mL of H2O and
washed with Et2O. The aqueous layer was acidi®ed
to pH 1 with 10% HCl, salted out with NaCl, and
extracted with EtOAc (3Â). The resulting organic layer
was dried (Na2SO4) and concentrated in vacuo to give
1
12.1 mg (58%) of FY21-aa09 as a pale yellow solid: H
NMR (CDCl3) d 7.62±7.19 (m, 10H), 4.26±4.23 (m, 1H),
3.88 (m, 1H), 3.63±3.61 (m, 1H), 2.19±1.76 (m, 19H),
1.55 (m, 2H), 1.41±1.34 (m, 4H), 1.18 (br, 12H), 0.82±
0.78 (m, 5H); MS (EI) m/z 626 ([M±H2O]+).
4-[(trans-4-t-Butoxycarbonylamino-1-cyclohexyl)-carb-
amoyl]-2-decanoylamino-butyric acid benzyl ester (9). To
a solution of 518.1 mg (1.201 mmol) of 9 in 8.6 mL of
THF was added 1.0 mL (12 mmol) of morpholine fol-
lowed by 83 mg (0.072 mmol) of tetrakistriphenyl-
phosphine Pd(0) under a nitrogen atmosphere at 22 ꢀC.
After stirring for 30 min, the reaction mixture was dilu-
ted with EtOAc and washed with 10% HCI and brine.
The organic layer was dried (Na2SO4) and concentrated
in vacuo to give 557.2 mg of carboxylic acid as a pale
yellow solid. The carboxylic acid (50 mg, 0.13 mmol)
and 27 mg (0.13 mmol) of diamine fragment 8 were
dissolved in 0.85 mL of THF. To this solution was
added 28 mL (0.20 mmol) of triethylamine followed by
25 mL (0.17 mmol) of diethylphosphoryl cyanide
(DEPC) at 0 ꢀC. After stirring at 0 ꢀC for 1 h and at
22 ꢀC for 14 h, the reaction mixture was diluted with
EtOAc, washed with 10% citric acid, H2O, saturated
aqueous NaHCO3 and brine. The organic layer was
dried (Na2SO4), concentrated in vacuo, and chromato-
graphed on SiO2 (hexanes:EtOAc, 1:4!EtOAc) to give
FY3-ꢀꢀ09
4-[(trans-2-t-Butoxycarbonylamino-1-cyclohexyl)-carb-
amoyl]-2-decanoylamino-butyric acid benzyl ester.
According to the procedure described for 9, 7 (100 mg,
0.255 mmol) was converted to 4-[(trans-2-t-butoxy-
carbonylamino-1-cyclohexyl)-carbamoyl]-2-decanoyl-
amino-butyric acid benzyl ester (162 mg, 30%) as an
amorphous solid: 1H NMR (CDCl3) d 7.38±7.28 (m,
5H), 6.91±6.82 (m, 1H), 6.47±6.45 (m, 1H), 5.16±5.11
(m, 2H), 4.74±4.68 (m, 1H), 4.55±4.51 (m, IH), 3.51±
3.50 (m, 1H), 3.34±3.31 (m, 1H), 2.24±1.97 (m, 8H),
1.73±1.61 (m, 4H), 1.39 (m, 9H), 1.25 (m, 16H), 0.88 (m,
3H).
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
trans-1,2-cyclohexyl}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(trans-
2-t-butoxycarbonylamino-1-cyclohexyl)-carbamoyl]-2-
decanoylamino-butyric acid benzyl ester was converted
1
58.2 mg (77%) of 9 as an amorphous solid: H NMR
(CDCl3) d 7.35 (br, 5H), 6.54±6.52 (br, 1H), 5.93 (br,

1460
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
to 2-decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carb-
onyl]-trans-1,2-cyclohexyl}-carbamoyl)-butyric acid
benzyl ester (53.8 mg, 78%): H NMR (CDCl3) d 8.34±
8.32 (m, 2H), 8.11±8.10 (m, 2H), 7.51±7.14 (m, 11H),
6.68±6.57 (m, 1H), 6.41±6.38 (m, 1H), 5.05±4.86 (m,
2H), 4.43±4.36 (m, 1H), 3.81±3.74 (m, 2H), 2.17±1.18 (m,
8H), 1.65±1.32 (m, 8H), 1.24 (br, 12H), 0.87 (m, 3H).
carbamoyl]-2-decanoyl-amino-butyric acid benzyl ester
1
(295 mg, 80%): H NMR (CDCl3) d 7.70±7.63 (m, 2H),
1
7.58±7.30 (m, 7H), 6.48 (m, 2H), 5.21±5.11 (m, 2H), 4.65
(m, 1H), 2.37±2.18 (m, 4H), 1.97 (m, 1H), 1.72 (m, 1H),
1.61 (m, 2H), 1.51 (s, 9H), 1.25 (br. 12H), 0.87 (m, 3H).
2-Decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
1,4-phenyl}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(4-t-
butoxycarbonylamino-1-phenyl)-carbamoyl}-2-decanoyl-
amino-butyric acid benzyl ester was converted to 2-
decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
1,4-phenyl}-carbamoyl)-butyric acid benzyl ester (22.4
2-Decanoylamino-44{4-[2,5-diphenyl-oxazole-4-carbonyl]-
trans-1,2-cyclohexyl}-carbamoyl)-butyric acid (FY3-
ꢀꢀ09). According to the procedure described for
FY21-aa09, 2-decanoylamino-4-({4-[2,5-diphenyl-oxa-
zole-4-carbonyl]-trans-1,2-cyclohexyl}-carbamoyl)-but-
yric acid benzyl ester was converted to FY3-aa09 (40.8
1
mg, 12%) as a pale yellow oil: H NMR (CDCl3) d
7.70±7.13 (m, 19H), 5.16 (m, 2H), 4.66 (m, 1H), 2.40±
1.81 (m, 6H), 1.61 (m, 2H), 1.25 (br, 12H), 0.87 (m, 3H).
1
mg, 86%): H NMR (CDCl3) d 8.30±8.28 (m, 2H), 8.09
(br, 2H), 7.60±7.36 (m, 6H), 4.31 (m, 1H), 3.93 (m, 1H),
3.80 (m, 1H), 2.50±1.83 (m, 8H), 1.52±1.43 (m, 8H), 1.23
(br, 12H), 0.87 (br, 3H); HRMS (EI) m/z calcd for
C37H48N4O6: 644.3574; found: 644.3595.
2-Decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
1,4-phenyl}-carbamoyl)-butyric acid (FY5-ꢀꢀ09).
According to the procedure described for FY21-aa09,
2-decanoylamino-4-({5-[2,5-diphenyl-oxazole-4-carbonyl]-
1,4-phenyl}-carbamoyl)-butyric acid benzyl ester was
converted to FY5-aa09 (13.9 mg, 71%): 1H NMR
(CDCl3) d 7.62±7.24 (m, 14H), 4.64 (m, 1H), 2.50 (m,
1H), 2.19±1.89 (m, 5H), 1.55 (m, 2H), 1.19 (br, 12H), 0.83
(m, 3H); MS (EI) m/z 638 (M+), 620 ([M H2O]+).
FY4-ꢀꢀ09
4-[(cis-2-t-Butoxycarbonylamino-1-cyclohexyl)-carbamoyl]-
2-decanoylamino-butyric acid benzyl ester. According to
the procedure described for 9, 7 (250 mg, 0.637 mmol)
was converted to 4-[(cis-2-t-butoxycarbonylamino-1-
cyclohexyl)-carbamoyl]-2-decanoylamino-butyric acid
1
benzyl ester (159 mg, 42%): H NMR (CDCl3) d 7.35
FY7-ꢀꢀ09
(br, 5H), 6.72 (m, 1H), 6.60 (m, 1H), 5.23±5.12 (m, 2H),
4.56 (m, 1H), 4.05 (m, 1H), 3.82 (m, 1H), 2.29±2.18 (m,
4H), 2.02 (m, 2H), 1.70±1.62 (m, 4H), 1.44 (br, 9H),
1.32±1.26 (m, 16H), 0.88 (m, 3H).
4-[(3-t-Butoxycarbonylamino-1-phenyl)-carbamoyl]-2-de-
canoyl-amino-butyric acid benzyl ester. According to the
procedure described for 9, 7 (300 mg, 0.766 mmol) was
converted to 4-[(3-t-butoxycarbonylamino-1-phenyl)-
carbamoyl]-2-decanoyl-amino-butyric acid benzyl ester
(194 mg, 43%) as a pale yellow oil: 1H NMR (CDCl3) d
7.64 (br, 1H), 7.34 (br, 5H), 7.20 (m, 3H), 6.70 (m, 1H),
6.58 (m, 1H), 5.20±5.15 (m, 2H), 4.65 (m, 1H), 2.39±2.17
(m, 5H), 2.03 (m, 1H), 1.60 (br, 2H), 1.50 (br 9H), 1.24
(br, 12H), 0.89 (m, 3H).
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
cis-1,2-cyclohexyl}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(cis-
2-t-butoxycarbonylamino-1-cyclohexyl)-carbamoyl]-2-
decanoylamino-butyric acid benzyl ester was converted
to 2-decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carb-
onyl]-cis-1,2-cyclohexyl}-carbamoyl)-butyric acid ben-
zyl ester (41.2 mg, 41%): 1H NMR (CDCl3) d 8.39±8.34
(m, 2H), 8.11±8.02 (m, 2H), 7.52±7.16 (m, 11H), 5.17±
4.79 (m, 2H), 4.65±4.56 (m, 1H), 4.36 (1H), 4.16±4.09
(m, 1H), 2.24±1.54 (m, 16H), 1.19 (br, 12H), 0.86 (m,
3H).
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
1,3-phenyl}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(3-t-
butoxycarbonylamino-1-phenyl)-carbamoyl]-2-decanoyl-
amino-butyric acid benzyl ester was converted to 2-
decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
1,3-phenyl}-carbamoyl)-butyric acid benzyl ester (92.7
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
cis-1,2-cyclohexyl}-carbamoyl)-butyric acid (FY4-ꢀꢀ09).
According to the procedure described for FY21-aa09,
2-decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carb-
onyl]-cis-1,2-cyclohexyl}-carbamoyl)-butyric acid ben-
zyl ester was converted to FY4-aa09 (15.4 mg, quant.)
1
mg, 79%): H NMR (CDCl3) d 8.35 (m, 1H), 8.14±8.02
(m, 2H), 7.52±7.24 (m, 16H), 6.70 (m, 1H), 5.13 (m,
2H), 4.66 (m, 1H), 2.41±1.97 (m, 6H), 1.58 (m, 2H), 1.21
(br, 12H), 0.84 (m, 3H); HRMS (EI) m/z calcd for
C44H48N4O6: 728.3574; found: 728.3552.
1
as a pale yellow oil: H NMR (CDCl3) d 8.67±8.21 (m,
2H), 8.04±7.98 (m, 2H), 7.45±7.19 (m, 6H), 4.33 (m,
1H), 4.01±3.97 (m, 2H), 2.11±1.47 (m, 16H), 1.16 (m,
12H), 0.78 (m, 3H); MS (EI) m/z 644 (M+).
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl)-
1,3-phenyl}-carbamoyl)-butyric acid (FY7-ꢀꢀ09).
According to the procedure described for FY21-aa09,
2-decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
1,3-phenyl}-carbamoyl)-butyric acid benzyl ester was
converted to FY7-aa09 (31.9 mg, quant.): 1H NMR
(CDCl3) d 8.24 (m, 1H), 8.04±7.95 (m, 2H), 7.44±7.19
(m, 11H), 4.50 (m, 1H), 2.51±2.44 (m, 2H), 2.20±1.98
(m, 3H), 1.76 (m 1H), 1.50 (m, 2H), 1.12 (br, 12H), 0.76
(m, 3H); MS (EI) m/z 620 ([M H2O]+).
FY5-ꢀꢀ09
4-[(4-t-Butoxycarbonylamino-1-phenyl)-carbamoyl]-2-de-
canoyl-amino-butyric acid benzyl ester. According to the
procedure described for 9, 7 (247 mg, 0.631 mmol) was
converted to 4-[(4-t-butoxycarbonylamino-1-phenyl)-

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1461
FY8-ꢀꢀ09
carbamoyl)butyric acid benzyl ester was converted to
FY9-aa09 (169 mg, quant.) as a pale yellow oil: 1H
NMR (CDCl3) d 8.30±7.24 (m, 10H), 4.49 (m, 1H),
3.49±3.32 (m, 4H), 2.40 (m, 2H), 2.21 (m, 2H), 1.98 (m,
2H), 1.79 (m, 2H), 1.57 (m, 2H), 1.20 (br, 12H), 0.84 (m,
3H); MS (EI) m/z 604 (M+), 586 ([M±H2O]+).
4-[(2-t-Butoxycarbonylamino-1-phenyl)-carbamoyl]-2-de-
canoyl-amino-butyric acid benzyl ester. According to the
procedure described for 9, 7 (301 mg, 0.769 mmol)
was converted to 4-[(2-t-butoxycarbonylamino-1-phenyl)-
carbamoyl]-2-decanoyl-amino-butyric acid benzyl ester
(239 mg, 53%) as a brown oil: 1H NMR (CDCl3) d 7.57
(m, 1H), 7.48 (m, 1H), 7.34 (br, 5H), 7.19±7.06 (m, 2H),
6.54 (m, 1H), 5.16 (m, 2H), 4.63 (m, 1H), 2.43±2.19 (m,
4H), 2.04±1.82 (m, 2H), 1.60 (m, 2H), 1.49 (br, 9H), 1.25
(br, 12H), 0.88 (m, 3H).
FY10-ꢀꢀ09
4-[(2-t-Butoxycarbonylamino-piperazine)-carbamoyl]-2-
decanoyl-amino-butyric acid benzyl ester. According to
the procedure described for 9, 7 (300 mg, 0.766 mmol)
and tert-butyl 1-piperazinecarboxylate (143 mg, 0.768
mmol) were converted to 4-[(2-t-butoxycarbonylamino-
piperazine)-carbamoyl]-2-decanoyl-amino-butyric acid
2-Decanoylamino-4-({3-[2,5-diphenyl-oxazole-4-carbonyl]-
1,2-phenyl}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(2-t-
butoxycarbonylamino-1-phenyl)-carbamoyl]-2-decanoyl-
amino-butyric acid benzyl ester was converted to 2-
decanoylamino-4-({3-[2,5-diphenyl-oxazole-4-carbonyl]-
1,2-phenyl}-carbamoyl)-butyric acid benzyl ester (113
1
benzyl ester (366 mg, 85%) as a yellow oil: H NMR
(CDCl3) d 7.36 (br, 5H), 6.65 (m, 1H), 5.17 (m, 2H),
4.59 (m, 1H), 3.57 (m, 2H), 3.43 (m, 4H), 3.29 (m, 2H),
2.35±2.05 (m, 5H), 1.70 (m, 1H), 1.61 (m, 2H), 1.47 (br,
9H), 1.26 (br, 12H), 0.88 (m, 3H).
1
mg, 64%) as a pale yellow solid: H NMR (CDCl3) d
8.36 (m, 1H), 7.95 (br, 2H), 7.42±7.15 (m, 16H), 6.67 (m,
1H), 5.03 (m, 2H), 4.58 (m, 1H), 2.47±1.89 (m, 6H), 1.50
(m, 2H), 1.09 (br, 12H), 0.86 (m, 3H).
2-Decanoylamino-4-{{3-[2,5-diphenyl-oxazole-4-carbonyl]-
piperazine}-carbamoyl)-butyric acid benzyl ester.
According to the procedure described for 11, 4-[(2-t-
butoxycarbonylamino-piperazine)-carbamoyl]-2-decan-
oyl-amino-butyric acid benzyl ester was converted to
2-decanoylamino-4-({3-[2,5-diphenyl-oxazole-4-carbonyl]-
piperazine}-carbamoyl)-butyric acid benzyl ester (160
mg, 68%): 1H NMR (CDCl3) d 8.10±7.29 (m, 15H),
6.65 (m, 1H), 5.14 (m, 2H), 4.60 (m, 1H), 3.78±3.34 (m,
8H), 2.38±2.08 (m, 5H), 1.86 (m, 1H), 1.59 (m, 2H), 1.24
(br, 12H), 0.86 (m, 3H).
2-Decanoylamino-4-{{3-[2,5-diphenyl-oxazole-4-carbonyl)-
1,2-phenyl}-carbamoyl)-butyric acid benzyl ester (FY8-
ꢀꢀ09). According to the procedure described for
FY21-aa09, 2-decanoylamino-4-({3-[2,5-diphenyl-oxa-
zole-4-carbonyl]-1,2-phenyl}-carbamoyl)-butyric acid
benzyl ester was converted to FY8-aa09 (93.3 mg,
1
94%): H NMR (CDCl3) d 8.23 (m, 1H), 7.99 (m, 2H),
7.40±7.05 (m, 11H), 4.37 (m, 1H), 2.52±1.69 (m, 6H),
1.38 (m, 2H), 1.10 (br, 12H), 0.77 (m, 3H); MS (EI) m/z
638 (M+), 620 ([M H2O]+).
2-Decanoylamino-4-({3-[2,5-dlphenyl-oxazole-4-carbonyl-
piperazine-carbamoyl)-butyric acid (FY10-ꢀꢀ09).
According to the procedure described for FY21-aa09,
2-decanoylamino-4-({3-[2,5-diphenyl-oxazole-4-carbonyl]-
piperazine}-carbamoyl)-butyric acid benzyl ester was
converted to FY10-aa09 (146 mg, quant.): 1H NMR
(CDCl3) d 8.05±7.24 (m, 10H), 4.42 (m, 1H), 3.62±3.43
(m, 8H), 2.53 (m, 2H), 2.20 (m, 2H), 2.02 (m, 2H), 1.53
(m, 2H), 1.20 (br, 12H), 0.84 (m, 3H); MS (EI) m/z 616
(M+), 598 ([M H2O]+).
FY9-ꢀꢀ09
4-[(3-t-Butoxycarbonylamino-propyl)-carbamoyl]-2-de-
canoylamino-butyric acid benzyl ester. According to the
procedure described for 9, 7 (304 mg, 0.776 mmol) was
converted to 4-[(3-t-butoxycarbonylamino-propyl)-car-
bamoyl]-2-decanoylamino-butyric acid benzyl ester (279
1
mg, 66%): H NMR (CDCl3) d 7.36 (m, 5H), 6.72 (m,
1H), 5.16 (m, 2H), 4.56 (m, 1H), 3.29±3.11 (m, 4H),
2.27±2.15 (m, 4H), 1.99 (m, 1H), 1.73 (m, 2H), 1.63±1.57
(m, 3H), 1.44 (br, 9H), 1.26 (br, 12H), 0.88 (m, 3H).
SC-ꢀꢀꢁ17A
DL-2-(2,2,2-Trichloroethoxycarbonylamino)-pentanoic
acid 5-allyl ester 1-methyl ester (2b). To a stirred sus-
pension of 5.0 g (34 mmol) of dl-glutamic acid (1) in 85
mL of allyl alcohol was added dropwise 10.8 mL (85
mmol) of chlorotrimethylsilane. The suspension was
stirred at 22 ꢀC for 13 h and concentrated in vacuo. The
resulting oil was dissolved in 90 mL of dioxane. This
solution was treated with 118.9 mmol of aqueous
sodium carbonate (12.61 g in 90 mL of H2O) at 0 ꢀC,
stirred for 5 min, and treated with 4.80 mL (34.7 mmol)
of TrocCl. The reaction mixture was stirred at 0 ꢀC for
10 min and warmed to 22 ꢀC, stirred for 16 h, con-
centrated in vacuo, quenched with 150 mL of H2O and
washed with Et2O. The aqueous layer was acidi®ed to
pH 1 with concentrated HCl, salted out with NaCl, and
extracted with EtOAc (3Â). The resulting organic layer
was dried (Na2SO4) and concentrated in vacuo to give
2-Decanoylamino-4-{{4-[2,5-diphenyl-oxazole-4-carbonyl]-
propyl}-carbamoyl)-butyric acid benzyl ester. According
to the procedure described for 11, 4-[(3-t-butoxy-
carbonylamino-propyl)-carbamoyl]-2-decanoylamino-
butyric acid benzyl ester was converted to 2-decanoyl-
amino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-propyl}-
carbamoyl)-butyric acid benzyl ester (131 mg, 63%): 1H
NMR (CDCl3) d 8.37±7.33 (m, 15H), 6.79 (m, 2H), 5.16
(m, 2H), 4.58 (m, 1H), 3.53±3.32 (m, 4H), 2.32±2.17 (m,
6H), 1.87±1.73 (m, 4H), 1.23 (br, 12H), 0.86 (m, 3H).
2-Decanoylamino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-
propyl}-carbamoyl)-butyric acid (FY9-ꢀꢀ09). According
to the procedure described for FY21-aa09, 2-decanoyl-
amino-4-({4-[2,5-diphenyl-oxazole-4-carbonyl]-propyl}-

1462
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
7.92 g of carboxylic acid as a yellow oil. To a solution of
the resulting oil in 29 mL of DMF were added 3.50 g
(35.0 mmol) of KHCO3 and 1.60 mL (26.2 mmol) of
iodomethane. The reaction mixture was stirred at 22 ꢀC
for 16 h, diluted with EtOAc, and washed with 10%
HCl, H2O, saturated aqueous NaHCO3 and brine. The
organic layer was dried (Na2SO4), concentrated in
vacuo, and chromatographed on SiO2 (hexanes:EtOAc,
5:1!4:1!3:1!2:1) to give 6.26 g (49% over three
carbamoyl]-2-oleoyl-amino-butyric acid methyl ester:
1H NMR (CDCl3) d 7.69±7.13 (m, 5H), 6.65 (m, 1H),
5.91±5.85 (m, 2H), 5.35±5.17 (m, 4H), 4.61±4.45 (m,
5H), 3.73±3.36 (m, 7H), 2.45±2.00 (m, 10H), 1.60 (m,
2H), 1.27 (br, 20H), 0.86 (m, 3H); MS (EI) m/z 641
(M+).
2-Oleoylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-car-
bonyl]-ethyl}-carbamoyl)-butyric acid methyl ester. To a
solution of 169 mg (0.263 mmol) of 4-[(2-allyloxy-
carbonylamino-ethyl)-benzyl-carbamoyl]-2-oleoyl-amino-
butyric acid methyl ester in 2 mL of CH2Cl2 were added
0.031 mL (0.53 mmol) of acetic acid and 9 mg (0.008
mmol) of tetrakistriphenylphosphine Pd(0) followed by
0.092 mL (0.34 mmol) of tributyltin hydride under
nitrogen atmosphere at 22 ꢀC. After stirring for 30 min,
the reaction mixture was diluted with EtOAc and
washed with saturated aqueous NaHCO3 and brine.
The organic layer was dried (Na2SO4) and concentrated
in vacuo. The resulting residue and 70 mg (0.26 mmol)
of oxazole segment 10 were dissolved in 1 mL of THF.
To this solution was added 0.092 mL (0.66 mmol) of
triethylamine followed by 0.221 g (0.473 mmol) of
PyBroP at 0 ꢀC. After stirring at 0 ꢀC for 1 h and at
22 ꢀC for 12 h, the reaction mixture was diluted with
EtOAc and washed with 10% HCl, H2O, saturated
aqueous NaHCO3 and brine. The organic layer was
dried (Na2SO4), concentrated in vacuo, and chroma-
tographed on SiO2 (hexane:EtOAc, 2:1!1:1) to give
2-oleoylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-car-
bonyl]-ethyl}-carbamoyl)-butyric acid methyl ester (104
1
steps) of 2b as a pale yellow oil: H NMR (CDCl3) d
5.95±5.86 (m, 1H), 5.71 (d, 1H, J=7.2 Hz), 5.34±5.22
(m, 2H), 4.74, 4.70 (AB, 2H, J=13.9 Hz), 4.58 (d, 2H, J
=5.8 Hz), 4.47±4.40 (m, 1H), 3.77 (s, 3H), 2.50±2.42 (m,
2H), 2.32±2.23 (m, 1H), 2.07±2.00 (m, 1H); MS (CI) m/z
376 ([M+1]+).
2-Oleoylamino-pentanedioic acid 5-allyl ester 1-methyl
ester. To a solution of 947 mg (2.51 mmol) of 2b in 3.3
mL of acetic acid and 4.9 mL of THF was added 3.29 g
(50.3 mmol) of zinc dust. The reaction mixture was
stirred at 22 ꢀC for 1 h, ®ltered through a Celite pad,
and the ®ltrate was concentrated in vacuo. The resulting
residue was diluted with EtOAc and washed with satu-
rated aqueous NaHCO3 and brine. The organic layer
was dried (Na2SO4) and concentrated in vacuo to give
free amine. This amine (67.7 mg) was dissolved in 1.1
mL of CH2CI2 and treated with 0.14 mL (1.02 mmol) of
triethylamine and 0.17 mL (0.51 mmol) of oleoyl chlo-
ride at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC for
15 min and warmed to 22 ꢀC, stirred for 14 h, diluted
with EtOAc, and washed with 10% HCl, H2O, satu-
rated aqueous NaHCO3 and brine. The organic layer
was dried (Na2 SO4), concentrated in vacuo, and chro-
matographed on SiO2 (hexanes:EtOAc, 8:1!7:1!5:1)
to give 110 mg (70%) of 2-oleoylamino-pentanedioic
1
mg, 55%) as a pale yellow oil: H NMR (CDCl3) d
8.37±8.35 (m, 2H), 8.10 (m, 2H), 7.52±7.13 (m, 6H), 5.31
(m, 2H), 4.70±4.60 (m, 3H), 3.68±3.52 (m, 7H), 2.13±
1.95 (m, 10H), 1.64 (m, 2H), 1.26 (br, 20H), 0.90 (m,
3H); MS (EI) m/z 804 (M+).
1
acid 5-allyl ester 1-methyl ester as a pale yellow oil: H
NMR (CDCl3) d 6.12 (d, 1H, J=7.7 Hz), 5.98±5.85
(m, 1H), 5.37±5.23 (m, 4H), 4.68±4.58 (m, 3H), 3.76 (s,
3H), 2.47±2.17 (m, 6H), 2.07±1.95 (m, 4H), 1.60 (m,
2H), 1.30 (brd, 20H), 0.89 (m, 3H); MS (EI) m/z 465
(M+).
2-Oleoylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-car-
bonyl]-ethyl}-carbamoyl)-butyric acid (SC-ꢀꢀꢁl7A). To
a solution of 104 mg (0.129 mmol) of 2-oleoylamino-4-
(benzyl-{3-[2,5-diphenyl-oxazole-4-carbonyl]-ethyl}-car-
bamoyl)-butyric acid methyl ester in 0.5 mL of dioxane
was added 1.6 mmol of aqueous lithium hydroxide (65
mg in 0.2 mL of H2O) at 0 ꢀC. The reaction mixture was
stirred at 0 ꢀC for 10 min and at 22 ꢀC for 1 h, poured
into 50 mL of H2O and washed with Et2O. The aqueous
layer was acidi®ed to pH 1 with 10% HCl, salted out
with NaCl, and extracted with EtOAc (3Â). The result-
ing organic layer was dried (Na2SO4) and concentrated
in vacuo to give SC-aad17A (28.1 mg, 28%) as a pale
4-[(2-Allyloxycarbonylamino-ethyl)-benzyl-carbamoyl]-2-
oleoyl-amino-butyric acid methyl ester. To a solution of
110 mg (0.236 mmol) of 2-oleoylamino-pentanedioic
acid 5-allyl ester 1-methyl ester in 1 mL of THF was
added 0.20 mL (2.4 mmol) of morpholine followed by
27.3 mg (0.024 mmol) of tetrakistriphenylphosphine
Pd(0) under nitrogen atmosphere at 22 ꢀC. After stirring
for 30 min, the reaction mixture was diluted with
EtOAc, and washed with 10% HCl and brine. The
organic layer was dried (Na2SO4) and concentrated in
vacuo. The resulting residue and 27.0 mg (0.115 mmol)
of diamine segment were dissolved in 0.5 mL of THF.
The solution was treated with 0.03 mL (0.18 mmol) of
N,N-diisopropylethylamine and 0.070 g (0.15 mmol) of
PyBroP at 0 ꢀC, stirred for 1 h, warmed to 22 ꢀC and
stirred for 9 h. The reaction mixture was diluted with
EtOAc, washed with 10% HCl, H2O, saturated aqueous
NaHCO3 and brine. The organic layer was dried
(Na2SO4), concentrated in vacuo, and chromatographed
on SiO2 (hexanes:EtOAc, 1:1!1:2) to give 169 mg
(quant.) of 4-[(2-allyloxycarbonylamino-ethyl)-benzyl-
1
yellow oil: H NMR (CDCl3) d 8.26±8.23 (m, 2H), 8.01
(m, 2H), 7.43±7.07 (m, 11H), 5.25 (m, 2H), 4.69±4.49
(m, 2H), 4.34±4.26 (m, 1H), 3.82±3.45 (m, 6H), 2.19±
1.91 (m, 8H), 1.47 (m, 2H), 1.18 (br, 20H), 0.80 (m, 3H);
HRMS (EI) m/z calcd for C48H60N4O5 ([M H2O]+):
772.4564; found: 772.4540.
SC-ꢀꢀꢁ17B
4-[(2-Allyloxycarbonylamino-ethyl)-benzyl-carbamoyl]-2-
(2,2,2-trichloroethoxycarbonylamlno)-butyric acid methyl
ester (3). According to the procedure described for 4-
[(2-allyloxycarbonylamino-ethyl)-benzyl-carbamoyl]-2-

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1463
oleoyl-amino-butyric acid methyl ester, 2b (217 mg,
0.577 mmol) was converted to 4-[(2-allyloxycarbonyl-
amino-ethyl)-benzyl-carbamoyl]-2-(2,2,2-trichloroeth-
oxycarbonyl-amino)-butyric acid methyl ester (3, 294
8.33±8.30 (m, 2H), 8.08 (m, 2H), 7.50±7.03 (m, 11H),
5.34 (m, 2H), 4.72±4.42 (m, 4H), 4.13±4.07 (m, 1H),
3.80±3.48 (m, 4H), 2.75 (m, 2H), 2.50±2.01 (m, 10H),
1.54 (m, 2H), 1.23 (br, 14H), 0.88 (m, 3H); HRMS (EI)
m/z calcd for C48H59N4O5 (M±OH): 771.4485; found:
771.4487.
1
mg, 92%) as an orange oil: H NMR (CDCl3) d 7.68±
7.11 (m, 5H), 6.08 (m, 1H), 5.91 (m, 1H), 5.32±5.19 (m,
2H), 4.8 1±4.40 (m, 5H), 3.80±3.65 (m, 5H), 3.40 (m,
2H), 2.61±2.31 (m, 6H); MS (EI) m/z 553 (M+).
SC-ꢀꢀꢁA
2-(2,2,2-Trichloroethoxycarbonylamino)-4-(benzyl-{3-
[2,5-diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-
butyric acid methyl ester (4). According to the proce-
dure described for 2-oleoylamino-4-(benzyl-{3-[2,5-di-
phenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
acid methyl ester, 4-[(2-allyloxycarbonylamino-ethyl)-
benzyl-carbamoyl]-2-(2,2,2-trichloroethoxycarbonyl-
amino)-butyric acid methyl ester (3, 887 mg, 1.604
mmol) was converted to 2-(2,2,2-trichloroethoxy-
carbonylamino)-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid methyl ester
(4, 819 mg, 71%) as a pale yellow oil: 1H NMR (CDCl3)
d 8.38±8.35 (m, 2H), 8.13±8.09 (m, 2H), 7.53±7.13 (m,
11H), 4.74±4.58 (m, 4H), 4.36 (m, 1H), 3.80±3.52 (m,
7H), 2.51±2.05 (m, 4H); MS (EI) m/z 716 (M+).
2-Citronellylamino-pentanedioic acid 5-allyl ester 1-
methyl ester. To a solution of 947 mg (2.51 mmol) of
2b in 3.3 mL of acetic acid and 4.9 mL of THF was
added 3.286 g (50.27 mmol) of zinc dust. The reaction
was stirred at 22 ꢀC for 1 h. The mixture was ®ltered
through a Celite pad and the ®ltrate was concentrated in
vacuo. The resulting residue was diluted with EtOAc
and washed with saturated aqueous NaHCO3 and
brine. The organic layer was dried (Na2SO4) and con-
centrated in vacuo to give free amine. The resulting
residue (68.7 mg) was dissolved in 1.1 mL of CH2Cl2
and treated with 4.2 mg (0.034 mmol) of DMAP, 69 mL
(0.38 mmol) of racemic citronellic acid and 79 mg (0.41
mmol) of EDCI at 0 ꢀC. The reaction mixture was
stirred at 0 ꢀC for 15 min and warmed to 22 ꢀC, stirred
for 14 h, diluted with EtOAc, and washed with 10%
HCl, H2O, saturated aqueous NaHCO3 and brine. The
organic layer was dried (Na2SO4), concentrated in
vacuo, and chromatographed on SiO2 (hexanes:EtOAc,
7:1!5:1) to give 83.9 mg (69%) of 2-citronellylamino-
pentanedioic acid 5-allyl ester 1-methyl ester as a color-
2-Linoleylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid methyl ester.
To a solution of 143 mg (0.199 mmol) of 2-(2,2,2-tri-
chloroethoxycarbonylamino)-4-(benzyl-{3-[2,5-diphenyl-
oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric acid
methyl ester in 0.3 mL of acetic acid and 0.5 mL of THF
was added 0.260 g (3.98 mmol) of zinc dust. The
reaction was stirred at 22 ꢀC for 1 h. The mixture was
®ltered through a Celite pad and the ®ltrate was con-
centrated in vacuo. The resulting residue was diluted
with EtOAc and washed with saturated aqueous
NaHCO3 and brine. The organic layer was dried
(Na2SO4) and concentrated in vacuo to give free amine.
The resulting residue was dissolved in 0.1 mL of CH2Cl2
and treated with 0.3 mg (0.003 mmol) of DMAP, 9.3 ml
(0.03 mmol) of linoleic acid and 6.2 mg (0.032 mmol) of
EDCI at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC
for 15 min and warmed to 22 ꢀC, stirred for 14 h, diluted
with EtOAc, and washed with 10% HCl, H2O, satu-
rated aqueous NaHCO3 and brine. The organic layer
was dried (Na2SO4), concentrated in vacuo, and chro-
matographed on SiO2 (hexanes:EtOAc, 7:1!5:1) to give
67.7 mg (42%) of 2-linoleylamino-4-(benzyl-{3-[2,5-
diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
acid methyl ester of as a colorless oil: 1H NMR (CDCl3)
d 8.37±8.35 (m, 2H), 8.13±8.07 (m, 2H), 7.52±7.13 (m,
11H), 5.33 (m, 4H), 4.70±4.51 (m, 3H), 3.75±3.52 (m,
7H), 2.76 (m, 2H), 2.45 (m, 2H), 2.23±1.99 (m, 8H), 1.55
(m, 2H), 1.26 (br, 14H), 0.88 (m, 3H); MS (EI) m/z 802
(M+).
1
less oil: H NMR (CDCl3) d 6.19 (d, 1H, J=7.4 Hz),
5.95±5.86 (m, 1H), 5.34±5.23 (m, 2H), 5.09 (m, 1H),
4.66±4.57 (m, 3H), 3.75 (s, 3H), 2.48±1.97 (m, 9H), 1.68
(s, 3H), 1.60 (s, 3H), 1.38±1.36 (m, 1H), 1.26±1.20 (m,
1H), 0.99±0.93 (m, 3H); MS (EI) m/z 353 (M+).
4-[(2-Allyloxycarbonylamino-ethyl)-benzyl-carbamoyl]-2-
citronellyl-amino-butyric acid methyl ester. According
to the procedure described for 4-[(2-allyloxycarbonyl-
amino-ethyl)-benzyl-carbamoyl]-2-oleoyl-amino-butyric
acid methyl ester, 2-citronellylamino-pentanedioic acid
5-allyl ester 1-methyl ester (83.9 mg, 0.237 mmol) was
converted to 4-[(2-allyloxycarbonylamino-ethyl)-benzyl-
carbamoyl]-2-citronellyl-amino-butyric acid methyl
ester (128 mg, quant.) as a brown oil: 1H NMR (CDCl3)
d 7.70±7.13 (m, 5H), 6.56 (m, 1H), 5.92±5.86 (m, 1H),
5.72 (br, 1H), 5.32±5.18 (m, 2H), 5.08 (m, 1H), 4.62±
4.45 (m, 5H), 3.74±3.71 (m, 5H), 3.42±3.23 (m, 4H),
2.50±1.93 (m, 9H), 1.67 (s, 3H), 1.59 (s, 3H), 1.37±1.32
(m, 1H), 1.26±1.18 (m, 1H), 0.92 (m, 3H); MS (FAB) m/
z 530 ([M+1]+).
2-Citronellylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid methyl ester.
According to the procedure described for 2-oleoyl-
amino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-carbonyl]-
ethyl}-carbamoyl)-butyric acid methyl ester, 4-[(2-allyl-
oxycarbonylamino - ethyl) - benzyl - carbamoyl]- 2- citro-
nellyl-amino-butyric acid methyl ester (128 mg, 0.241
mmol) was converted to 2-citronellylamino-4-(benzyl-
{3-[2,5-diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-
butyric acid methyl ester (152 mg, 92%) as a pale yellow
2-Linoleylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid (SC-ꢀꢀꢁ17B).
According to the procedure described for SCaad17A,
2-linoleylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid methyl ester
(67.7 mg, 0.0843 mmol) was converted to SC-aad17B
(66.4 mg, 99%) as a pale yellow oil: 1H NMR (CDCl3) d
1
oil: H NMR (CDCl3) d 8.37 (m, 2H), 8.10 (m, 2H),

1464
A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
7.69±7.15 (m, 11H), 5.04 (m, 1H), 4.69±4.60 (m, 3H),
3.68±3.52 (m, 7H), 2.48±1.86 (m, 9H), 1.65 (s, 3H), 1.57
(s, 3H), 1.28±1.26 (m, 2H), 0.85 (m, 3H); MS (EI) m/z
692 (M+).
SC-ꢀꢀꢁ6III
2-2-[2-(2-Methoxyethoxy)ethoxy]acetyl}-amino-4-(benzyl-
{3-[2,5-diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-
butyric acid methyl ester (6). According to the proce-
dure described for 2-[2-(2-methoxyethoxy)acetyl]-
amino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-carbonyl]-
ethyl}-carbamoyl)-butyric acid methyl ester (5), 2-
(2,2,2-trichloroethoxycarbonylamino)-4-(benzyl-{3-[2,5-
diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
acid methyl ester (4) was converted to 2-{2-[2-(2-meth-
oxyethoxy)ethoxy]acetyl}-amino-4-(benzyl-{3-[2,5-di-
phenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
2-Citronellylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl)-ethyl}-carbamoyl)-butyric acid (SC-ꢀꢀꢁA).
According to the procedure described for SCaad17A,
2-citronellylamino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-
carbonyl]-ethyl}-carbamoyl)-butyric acid methyl ester
(152 mg, 0.219 mmol) was converted to SC-aadA (87.1
1
mg, 58%): H NMR (CDCl3) d 8.29 (m, 2H), 8.08 (m,
2H), 7.50±7.13 (m, 11H), 5.03 (m, 1H), 4.72±4.37 (m,
3H), 3.78±3.56 (m, 4H), 2.78±1.91 (m, 9H), 1.56 (s, 3H),
1.54 (s, 3H), 1.28±1.14 (m, 2H), 0.87 (m, 3H); HRMS
(EI) m/z calcd for C40H46N4O6: 678.3417; found:
678.3441.
1
acid methyl ester (6, 57.2 mg, 39%): H NMR (CDCl3)
d 8.37 (m, 2H), 8.11 (m, 2H), 7.51±7.13 (m, 11H), 4.73±
4.60 (m, 3H), 3.98 (m, 2H), 3.71±3.50 (m, 15H), 3.33 (s,
3H), 2.50±2.02 (m, 4H); MS (EI) m/z 700 (M+).
2-{2-[2-(2-Methoxyethoxy)ethoxy]acetyl}-amino-4-(benzyl-
{3-[2,5-diphenyl-oxazole-4-carbonyl-ethyl}-carbamoyl)-
butyric acid (SC-ꢀꢀꢁ6III). According to the procedure
described for SC-aad17A, 6 (57.2 mg, 0.0816 mmol) was
converted to SC-aad6III (52.9 mg, 94%) as a pale
SC-ꢀꢀꢁ4II
2-[2-(2-Methoxyethoxy)acetyl]-amino-4-(benzyl-{3-[2,5-
diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
acid methyl ester (5). To a solution of 290 mg (0.404
mmol) of 2-(2,2,2-trichloroethoxycarbonylamino)-4-
(benzyl-{3-[2,5-diphenyl-oxazole-4-carbonyl]-ethyl}-car-
bamoyl)-butyric acid methyl ester (4) in 0.53 mL of
acetic acid and 0.79 mL of THF was added 529 mg (8.09
mmol) of zinc dust. The reaction was stirred at 22 ꢀC for
1 h. The mixture was ®ltered through a Celite pad and
the ®ltrate was concentrated in vacuo. The resulting
residue was diluted with EtOAc and washed with satu-
rated aqueous NaHCO3 and brine. The organic layer
was dried (Na2SO4) and concentrated in vacuo to give
free amine. This amine (112 mg) was dissolved in 0.7
mL of THF and treated with 72 mL (0.52 mmol) of
triethylamine, 26 ml (0.23 mmol) of 2-(2-methoxyethoxy)-
acetic acid and 173 mg (0.372 mmol) of PyBroP at 0 ꢀC.
The reaction mixture was stirred at 0 ꢀC for 1 h and
warmed to 22 ꢀC, stirred for 11 h, diluted with EtOAc,
and washed with 10% HCl, H2O, saturated aqueous
NaHCO3 and brine. The organic layer was dried
(Na2SO4), concentrated in vacuo, and chromatographed
on SiO2 (hexanes:EtOAc, 1:1!1:4!1:5!EtOAc only)
1
yellow oil: H NMR (CDCl3) d 8.80 (brs, 1H), 8.24 (m,
2H), 8.01 (m, 2H), 7.42±7.06 (m, 11H), 4.65±4.45 (m,
3H), 3.90 (m, 2H), 3.63±3.43 (m, 12H), 3.26 (s, 3H),
2.90±2.20 (m, 4H); HRMS (EI) m/z calcd for C37H42
N4O9: 686.2952; found: 686.2958.
Plasmids and reagents
Plasmids pGEX2T-KG containing the GST-fusion of
full length human Cdc25B2 were previously described.1
Recombinant human PTP1B was a gift from Dr.
Zhong-Yin Zhang (Albert Einstein College of Medicine,
Bronx, NY). To generate recombinant human VHR, we
excised the coding sequence for VHR phosphatase from
pT7-7-VHR (a gift from Dr. Jack E. Dixon, University
of Michigan, Ann Arbor, MI) using Ned and EcoRI.
The resulting DNA was ligated into the pMTL 22 vec-
tor. The pMTL 22-VHR plasmid was used to transform
DH5a Escherichia coli, and the plasmid DNA was
puri®ed. The VHR phosphatase sequence from pMTL
22-VHR was excised with EcoRV and XhoI and ligated
into the pGEX-4T3 plasmid, which had been digested
with SmaI and XhoI. The pGEX-4T3 plasmids were
used to transform DH5a E. coli. This clone was then
used to produce the GST-VHR phosphatase protein as
were all other recombinant fusion proteins by our pre-
viously published methods.1
1
to give 68.2 mg (50%) of 5: H NMR (CDCl3) d 8.36
(m, 2H), 8.11 (m, 2H), 7.52±7.13 (m, 11H), 4.69±4.60
(m, 3H), 4.00 (m, 2H), 3.68±3.47 (m, 11H), 3.35 (s, 3H),
2.48±1.83 (m, 4H); HRMS (EI) m/z calcd for C36H40
N4O8: 656.2846; found: 656.2844.
2-[2-(2-Methoxyethoxy)acetyl]-amino-4-(benzyl-{3-[2,5-
diphenyl-oxazole-4-carbonyl]-ethyl}-carbamoyl)-butyric
acid (SC-ꢀꢀꢁ4II). According to the procedure descri-
bed for SC-aad17A, 2-[2-(2-methoxyethoxy)acetyl]-
amino-4-(benzyl-{3-[2,5-diphenyl-oxazole-4-carbonyl]-
ethyl}-carbamoyl)-butyric acid methyl ester (6, 68.2 mg,
0.104 mmol) was converted to SC-aad4II (70.3 mg,
Tyrosine speci®c phosphatase and dual speci®city
phosphatase assays
The activity of the GST-tagged dual speci®city phos-
phatases and recombinant tyrosine speci®c phosphatase
was measured in 96-well microtiter plates with O-
methyl¯uorescein monophosphate (OMFP) (Sigma, St.
Louis, MO) as the substrate in three to six independent
experiments. The phosphatases catalyzed the metabolism
of OMFP to the ¯uorescent product O-methyl¯uorescein
1
quant.) as a pale yellow oil: H NMR (CDCl3) d 9.28
(brs, 1H), 8.21 (m, 2H), 7.99 (m, 2H), 7.39±7.05 (m,
11H), 4.63±4.46 (m, 3H), 3.90 (m, 2H), 3.61±3.42 (m,
8H), 3.24 (s, 3H), 2.63±1.96 (m, 4H); HRMS (EI) m/z
calcd for C35H38N4O8: 642.2690; found: 642.2702.

A. P. Ducruet et al. / Bioorg. Med. Chem. 8 (2000) 1451±1466
1465
(OMF) (Sigma; St. Louis, MO). The reaction mixture
(150 mL) consisted of 30 mM Tris (pH 7.0), 75 mM
NaCl, 1 mM EDTA, 0.033% bovine serum albumin and
1 mM DTT for GST-VHR and rhPTP1B or 30 mM
Tris (pH 8.0), 75 mM NaCl, 1 mM EDTA, 0.033%
bovine serum albumin and 1 mM DTT for GST-
Cdc25B2. Substrate concentrations approximating the
Km were used: VHR, 10 mM; rhPTP1B, 200 mM;
Cdc25B2, 40 mM. Both the substrate and compounds
were resuspended in DMSO and diluted in water; the
®nal DMSO concentration remained at 7% for all
assays. The inhibitory potential for each compound was
monitored at 0, 3 and 100 mM reaction concentrations,
and the reactions were initiated by the addition of
ꢁ0.025 mg of VHR, ꢁ0.25 mg of rhPTP1B and ꢁ0.10 mg
Cdc25B2. Reaction length varied with each phospha-
tase: 21 min for GST-VHR, 14 min for rhPTP1B and
60 min for Cdc25B2. Fluorescent emissions from the
hydrolysis of OMFP to OMF were measured at 25 ꢀC
during the entire course of the reaction using a Persep-
tive Biosystems Cyto¯uor II (Framingham, MA) with
an excitation ®lter of 485 nm (20 nm band width) and
an emissions ®lter of 530 nm (30 nm band width). The
¯uorescent emissions were converted to units of product
formation using an OMF standard curve that was per-
formed with each experiment. Spontaneous hydrolysis
of OMFP to OMF as monitored in an enzyme free
reaction was normalized to the OMF standard curve.
Product formation with 0 mM inhibitor was also mon-
itored and determined to be linear for all enzymes over
the incubation period. Furthermore, the reaction was
directly proportional to both the enzyme and substrate
concentration.
well in RPMI-1640 culture medium containing 10%
fetal bovine serum. After a 24 h incubation, 100 mM of
selected compounds were added to the cells. The cells
were then incubated with the compounds for 72 h and
viable cell number was determined by our previously
described method using 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide.19 All results were nor-
malized to vehicle treated control values.
For cell cycle studies, we used the previously described
tsFT210 cells.27 Cells were plated at 2Â105 cells/mL and
maintained at 32.0 ꢀC until they were blocked at G2
phase by incubation at 39.4 ꢀC for 17 h. The synchro-
nized cells were then released from G2 blockage by re-
incubating at 32.0 ꢀC and treated immediately with
FY21-aa09, SC-aad9, nocodazole or the DMSO vehicle
to probe for G2/M arrest. After a 6 h incubation, cells
were harvested with phosphate bu€ered saline and
stained with a solution containing 50 mg/mL propidium
iodide and 250 mg/mL RNase A. Flow cytometry ana-
lyses were conducted with a Becton Dickinson FACS
Star (Becton Dickinson, Franklin Lakes, NJ).
Acknowledgements
We are grateful for the helpful molecular biology advice
of Dr. Iliya Lefterov, the cell culture results of Angela
Wang and the excellent technical assistance of Eileen
Southwick. This work was supported in part by Army
Breast Cancer Predoctoral Research Fellowship
DAMDI7-94-J4193 and Grant DAMD17-97-1-7229,
the Fiske Drug Discovery Fund, and USPHS NIH
Grants CA 78039 and GM 55433.
Steady-state kinetics
References and Notes
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The V0 for each substrate concentration was determined
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viously described1 using GraphPad Prism 3.0 (Graph-
Pad Software, Inc., San Diego, CA). The best ®t kinetic
model and inhibition constants were obtained using EZ-
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