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

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,^aRobert L. Rice,^aKenji Tamura,^aFumiaki Yokokawa,^b

Shiho Yokokawa,^bPeter Wipf ^band John S. Lazo^a,*

^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 eective

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 Cdc25B₂with K_ivalues 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

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.^4,5A 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.⁵While 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.²More 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.^3,4This information then con-

trols such processes as cell growth and dierentiation,

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}

PII: S0968-0896(00)00069-9

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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.^5,12,13The Cdc25 DSPases coor-

dinate the cell cycle by dephosphorylating, and thereby

activating, cell cycle dependent kinases. For example,

Cdc25B and Cdc25C dephosphorylate p34^cdc2(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

dierences have been observed in the active site of

Cdc25A, VHR and PTP1B phosphatases.^13,14,18The

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.^5,18The structural dierences

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.

R_nindicate 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 Cdc25B₂phosphatase activity and lipophilicity

of SC-aad9 analogues^a

Cdc25B₂(% Inhibition)

clog P value^b

We have used a previously described non-electrophilic

phosphatase inhibitor, namely SC-aad9,¹as 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 dierences 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

ND^c

3.6

4.9

ND^c

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

coecient 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.¹To

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 Cdc25B₂inhibitory 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,¹we further examined the structural basis for

inhibition by synthesizing and testing both the (R)-SC-

aad9 and (S)-SC-aad9 enantiomers. Both enantiomers

inhibited Cdc25B₂phosphatase 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 C₅oxazole (R₂) position and the R₃amine. The

importance of the R₂aromatic 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 Cdc25B₂with 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 eective inhibitor of Cdc25B₂

at 3 mM (Table 2), causing a 36% decrease in enzyme

activity. In the -a109 series, no compound was as

eective 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 Cdc25B₂than 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 Cdc25B₂or 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 Cdc25B₂or VHR

(IC₅₀s of 7.0 mM and 12.1 mM, respectively) (Fig. 4).

Inhibition kinetics of compounds

We next determined the kinetic characteristics of

Cdc25B₂inhibition 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 K_ivalues 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 dierences in the FY3-aa09 kinetic pro®les

seen with VHR, PTP1B and Cdc25B₂may re¯ect the

structural dissimilarities of these phosphatases.

Figure 2. Chemical structures of SC-aad9 R₄analogues.

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 R₄substituent for Cdc25B₂phospha-

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.¹⁹We 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 eective 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

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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 activity^a

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.¹⁴To 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 Cdc25B₂phosphatase activity.

Compounds with R₄substituents of a more hydro-

phobic nature than the nonyl group found in SC-aad9

displayed an inhibitory pro®le against Cdc25B₂similar

to that of SC-aad9. On the other hand, compounds with

R₄substituents 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 Cdc25B₂inhibitory activity at concentrations up

to 100 mM. Because kinetic studies with SC-aad9 sug-

gest that its mode of inhibition of Cdc25B₂is most

consistent with a competitive model, the importance of

a hydrophobic moiety in the R₄position of the phar-

macophore suggested that the Cdc2SB₂active site, with

the exception of the phosphate binding site, is hydro-

phobic in nature.^1,20

Figure 5. Kinetic analysis of Cdc25B₂inhibition by FY3-aa09 and

FY21-aa09. Panel A: Lineweaver±Burk plot of Cdc25B₂inhibition 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

Cdc25B₂inhibition 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 scaold, such as in

FY3-aa09, FY4-aa09 and FY21-aa09, we did not sig-

ni®cantly alter the anti-Cdc25B₂activity; these three

compounds also eciently 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

Cdc25B₂depends on the previously reported shallow

nature of the enzyme's active site relative to that of

PTP1B.¹⁴In addition to rigidifying the pharmacophore,

we also varied the substituents at the R₁and R₂posi-

tions. All the new compounds with anti-phosphatase

activity displayed a strong preference for an aromatic

moiety in the R₂position. 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-Fit^TM

.

In examining a subset of compounds, we found that the

better in vitro inhibitors of DSPases were eective 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.²²Nonetheless, 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 eects of selected compounds on MDA-MB-231 and MCF-7 human breast cancer cells. Antiproliferative eect 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

eect 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.²⁴A 3-a-

azido-B-homo-6-oxa-4-cholesten-7-one derivative had

an IC₅₀of 2 mM for Cdc25A, although there is no

information concerning its selectivity.²⁵Although

FY21-aa09 eciently inhibited both DSPases and

PTPases in vitro, unlike SC-aad9,¹it exhibited some

preference for the DSPases. As a partial competitive

inhibitor, FY21-aa09 interacted with Cdc25B₂in the

vicinity of the active site though was unable to com-

pletely impede substrate binding. R₄substituents in the

original SC pharmacophore revealed the importance of

a hydrophobic moiety for Cdc25B₂inhibition 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 Cdc25B₂inhibitors reported to date. Dysidiolide,

which was reported to have an IC₅₀of 9.4 mM against

Cdc25A,²³has more recently been found to lack potent

system used for our phosphatase library elements.¹The

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.¹Alkyl 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 R₃site 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.¹⁹A 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 KHCO₃and 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 NaHCO₃and 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

NaHCO₃and 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 CH₂Cl₂and 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 NaHCO₃and

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 NaHCO₃and

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 NaHCO₃and 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 KHCO₃and 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 NaHCO₃and 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 CH₂Cl₂were 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 NaHCO₃and 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 NaHCO₃and 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 NaHCO₃and 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 CH₂CI₂and 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 NaHCO₃and brine. The organic layer

was dried (Na₂SO₄), 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

NaHCO₃and 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 NaHCO₃and

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 NaHCO₃and 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

NaHCO₃and 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 NaHCO₃and 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 NaHCO₃and 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

NaHCO₃and 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 Cdc25B₂were 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

K_mwere 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.¹⁹All results were nor-

malized to vehicle treated control values.

For cell cycle studies, we used the previously described

tsFT210 cells.²⁷Cells were plated at 2Â10⁵cells/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 G₂blockage 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 buered 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

Initial enzyme rates with ®ve to eight inhibitor con-

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The V₀for each substrate concentration was determined

and then ®t to the Michaelis±Menten equation as pre-

viously described¹using GraphPad Prism 3.0 (Graph-

Pad Software, Inc., San Diego, CA). The best ®t kinetic

model and inhibition constants were obtained using EZ-

Fit^TM(Perrella Scienti®c, Amherst, NH).

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

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

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