Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors

FULL PAPER

DOI: 10.1002/chem.200501128

Synthesis and Evaluation of Acyl Protein Thioesterase 1 (APT1) Inhibitors

Markus Biel,^[b]Patrick Deck,^[a]Athanassios Giannis,*^[b]and Herbert Waldmann*^[a]

Abstract: Lipid-modified proteins play

decisive roles in important biological

processes such as signal transduction,

organisation of the cytoskeleton and

vesicular transport. Lipidation of these

proteins is essential for correct biologi-

cal function. Among the modifications

with lipids, prenylation and myristoyla-

tion are well understood. However, the

machinery of palmitoylation is still

under investigation. Recently, an

enzyme, acyl protein thioesterase 1

(APT1), that may play a regulatory

role in the palmitoylation cycle of H-

Ras and G-protein a subunits, was pu-

rified. Motivated by this work, several

inhibitors of APT1 were designed, syn-

thesized and biologically evaluated

leading to highly active compounds.

Keywords:

palmitoylation

·

peptidomimetics

signal transduction

·

proteins

Introduction

genic Ras

line PC12, nearly 80% of injected cells developed neurites.^[6]

When truncated Ras(G12V)DC181 protein lacking both the

isoprenylation motif and one of two possible palmitoylation

sites was microinjected, no neurites were formed. These

findings strongly indicate that palmitoylation has regulatory

functions in living cells.

Unlike myristoylation or farnesylation, palmitoylation is a

dynamic process due to the reversibility of the thioester

modification. Palmitoylated proteins cycle between acylated

and deacylated states many times during their existence

within a cell.^[7]The regulation of reversible protein palmi-

toylation has been formally demonstrated for endothelial

nitric oxide synthase, the b-adrenergic receptor, the m2 mus-

carinic receptor and the a subunit of the heterotrimeric G-

protein G_s.^[8–11]

Although protein-acyltransferase (PAT) activity was de-

tected in membrane fractions of different cell types already

20 years ago, the identification of a first bona fide PAT has

been difficult. The inherent instability of PAT activity during

purification employing detergents like Triton-X-100 did not

A

Numerous lipid-modified proteins are involved in important

biological events, such as signal transduction, organisation

of the cytoskeleton and vesicular transport.^[1–3]Lipidation of

these proteins is a prerequisite for a correct biological func-

tion, and the lipid groups are believed to participate in pro-

tein–protein and protein–membrane interactions, which, for

instance, may determine the selective intracellular localiza-

tion of lipid-modified proteins.

The most common lipid modifications are prenylation

with farnesyl or geranylgeranyl moieties and acylation with

myristoyl or palmitoyl residues. It was shown that peptides

that resemble the dual lipidation motifs of Ras or G-protein

a subunits are efficiently palmitoylated and localized at the

plasma membrane.^[4,5]The effect of palmitoylation on differ-

entiation of cells was investigated by microinjection experi-

ments. After microinjection of full-length recombinant onco-

[a] Dr. P. Deck, Prof. Dr. H. Waldmann

Max-Planck-Institute of Molecular Physiology

Department of Chemical Biology, Otto-Hahn-Strasse 11

44227 Dortmund (Germany)

allow

a final identification and isolation of the PAT

enzyme.^[12–14]Furthermore, enzymes of the lipid metabolism

are able to mimic PAT activity. The amino acid sequencing

of a protein, described as a H-Ras palmitoylating enzyme

matched the sequence of a previously isolated peroxysomal

thiolase A, an enzyme required for fatty acid b-oxidation.^[15]

In addition, several proteins such as G-protein a subunits

are palmitoylated in vitro at the appropriate cysteine resi-

dues in a non enzymatic manner by incubation with palmito-

yl-CoA.^[16–18]This observation led to the concept of an auto-

catalytic acylation process in vivo.

and:

University of Dortmund

Department of Chemical Biology, Otto-Hahn-Strasse 6

44227 Dortmund (Germany)

Fax : (+49)231-133-2499

E-mail: herbert.waldmann@mpi-dortmund.mpg.de

[b] Dipl.-Chem. M. Biel, Prof. Dr. A. Giannis

University of Leipzig, Institute of Organic Chemistry

Johannisallee 29, 04103 Leipzig (Germany)

Fax : (+49)341-97365-99

E-mail: giannis@chemie.uni-leipzig.de

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4121

In a genetic approach using Saccharomyces cerevisiae, the

protein complex Erf2/Erf4 could be identified as the Ras2p

palmitoylating enzyme.^[19]Although the ERF2-gene has or-

thologues in various eukaryotes, proteins with the corre-

sponding S-palmitoylating activity have not been identified

to date and the Erf2/Erf4 complex does not accept mamma-

lian H-Ras as substrate. Furthermore homologues of the

ERF4-gene exist only in closely related yeast. In contrast to

the principle of biocatalysis, Erf2 mediated protein acylation

in vitro is only performed at equimolar concentrations of

substrate and enzyme, a finding which remains to be clari-

fied.^[20]Thus, palmitoylation in eukaryontes is still not well

understood.

The enzymatic process which antagonizes acylation is pro-

tein deacylation. A palmitoyl protein thioesterase (PPT1)

that is able to deacylate Ras and G_a-proteins was purified

and the cDNA was cloned.^[21]Subsequent studies revealed

that the enzyme is a lysosomal resident, and mutations in

the PPT1 gene cause a fatal lysosomal storage disease, in-

fantile neuronal ceroid lipofuscinosis.^[22,23]Recently, a novel

candidate for a regulatory enzyme of palmitoylated signal-

ling proteins has been identified.^[24]A cytosolic acyl protein

thioesterase (APT1) was purified from rat liver which cata-

lyzes the depalmitoylation of G-protein a subunits and H-

Ras. Amino acid sequencing showed that APT1 was previ-

ously identified as a rat lysophospholipase,^[25]but its affinity

to acyl protein substrates is 250-fold higher compared with

lysophosphatidylcholine, the substrate for lysophospholi-

pase. In mammalian cells continuously expressing APT1, the

rate of palmitate turnover is significantly faster compared

with control cells.^[24]Therefore, APT1 is proposed to be the

first bona fide player in the regulated palmitoylation of in-

tracellular signalling proteins in mammalian cells. Genetic

or pharmacological inactivation of APT1 might serve to illu-

minate the role of the enzymes in reversible protein palmi-

toylation of G_asubunits or other palmitoylated proteins like

H-Ras.

A strain of Saccharomyces cerevisiae, which lacks the

APT1 gene, appears normal with regard to growth and spor-

ulation when compared with the wild-type strain.^[26]Al-

though extracts, prepared from this strain have a significant

reduction in biochemically detectable palmitoyl-G_ia1thioes-

terase activity, the yeast shows no obvious phenotypic alter-

ations. Due to the significant differences in amino acid se-

quence and substrate selectivity between APT1, isolated

from yeast and rat liver the development of potent inhibi-

tors is important for the study of the physiological impor-

tance of the mammalian enzyme. In this paper we describe

the design, synthesis and chemical evaluation of several dif-

ferent inhibitors of acyl protein thioesterase 1 (APT1).^[27]

Scheme 1. Analysis for the possible inhibitors.

In a first step, several peptide analogues with intact peptide

backbone consisting of three or five amino acids were plan-

ned to be synthesized. Therefore, the labile thioester group

was transformed into either a sulfonamide moiety or a

simple secondary alcohol function. In a second step, confor-

mational rigidity should be introduced into the desired com-

pounds. Therefore, we thought of bridging the peptide back-

bone and thus to synthesize proline and lactam derivatives.

Finally, a peptide-imitating benzodiazepinedione core^[28]was

chosen as the underlying scaffold and equipped with a hy-

drolysis-stable sulfonamide to mimic the transition state of

the hydrolysis of the thioester moiety. With this methodolo-

gy we were able to generate different classes of possible in-

hibitors for APT1, and with those in hand it was possible to

study structure–activity relationships.

Synthesis of tri- and pentapeptide analogues: For the syn-

thesis of the tri- and pentapeptide analogues, the two diami-

nopropionic acid derivatives 4 and 6 had to be generated

(Scheme 2). Starting from (2S,3)-diaminopropionic acid hy-

drochloride (1), allyl ester 2 was prepared following a

known procedure.^[29]Then, the b-amino functionality was

converted into a hexadecyl sulfonamide; subsequently the

allyl group was removed leading to free acid 4. Additionally,

the known bishydrotosylate 5 could be easily transformed

into amine 6.

The tripeptide synthesis commenced with the coupling of

Results and Discussion

Ac-Dap(SO₂C₁₆H₃₃)-OH (4) to H-Lys

(Scheme 3). After cleavage of the tert-butyl ester, H-Cys-

(Far)-OMe (10) was condensed with the resulting free acid

to yield the protected tripeptide 11. Finally, the Aloc group

was removed leading to analogue 12.

A

The human H-Ras protein is a substrate for APT1 in vitro.

Based on this fact, the possible inhibitors we designed to

mimic the C-terminal end of the natural protein (Scheme 1).

ACHTREUNG

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Scheme 3. Synthesis of the tripeptide analogue 12. a) H-Lys

(7), EDC, HOBt, CH₂Cl₂, 08C ! RT, 82%; b) TFA/CH₂Cl₂1:1, quant.

(9); c) H-Cys

(Far)-OMe (10), EDC, HOBt, CH₂Cl₂, 08C ! RT, 90 %;

d) [Pd(PPh₃)₄], DMB, THF, 73%. Aloc=allyloxycarbonyl, Dap=diami-

nopropionic acid, EDC=N-ethyl-N’-(3-dimethylaminopropyl)carbodi-

imide hydrochloride, Far=farnesyl, HOBt=1-hydroxybenzotriazole,

TFA=trifluoroacetic acid.

G

AHCTREUNG

Scheme 2. Synthesis of the diaminopropionic acids

4

and 6. a)

(PPh₃)₄], DMB,

RT, 72%. All=allyl,

AHCTREUNG

C₁₆H₃₃SO₂Cl, NEtiPr₂, DMF, 08C ! RT; 75 % (3); b) [Pd

ACHTREUNG

THF, 99%; c) C₁₆H₃₃SO₂Cl, NEt₃, ꢀ708C

!

DMB=N,N’-dimethylbarbituric acid, TsOH = para-toluenesulfonic acid.

The synthesis of the first pentapeptide 21 started with the

preparation of dipeptide 16 (Scheme 4). To this end, Ac-

Met-OH (13) and HCl·H-Ser-OBzl (14) were coupled and

the benzyl ester group was cleaved by hydrogenation. Con-

densation of H-Dap(SO₂C₁₆H₃₃)-OAll (6) with free dipep-

tide 16 and subsequent deprotection of the allyl ester yield-

ed unprotected tripeptide 18. This intermediate was coupled

elevated temperatures,^[30]converted diallyl amine 26 into the

free amino acid 27.

The synthesis of the second pentapeptide started with the

condensation of Ac-Met-Ser-OH (16) with the C₂₁amino

acid 27 (Scheme 6). Attempts to deprotect the tert-butyl

ester under acidic conditions resulted in lactone formation

between the secondary alcohol of the C₂₁acid and the C-ter-

minal carboxylic acid in 28. Therefore, basic saponification

conditions were tried, and fortunately it was possible to

cleave the tert-butyl group without any side reactions. Subse-

with freshly prepared H-Lys

N

ing to protected pentapeptide 20. Final Pd⁰-catalyzed allyl

transfer to N,N’-dimethylbarbituric acid gave the first penta-

peptide analogue 21 in high yields.

quently, H-Lys

N

In order to prepare pentapeptide 30, it was first necessary

to synthesize the central C₂₁

the resulting acid and the protected pentapeptide 29 could

amino acid 27 (Scheme 5).

Therefore, H-l-Glu(OMe)-OH

G

(22) was fully protected using

the tert-butyl ester group for

the carboxylic acid and a diallyl

protection for the amino group.

The two allyl groups were used

to prevent the molecule from

undesired deprotonation in a

subsequent Grignard reaction.

Glutamic acid 24 was then re-

duced at the methyl ester func-

tion with lithiumborohydride as

reducing agent. Next, alcohol

25 was oxidized with Dess–

Martin periodinane and the re-

sulting aldehyde was subse-

quently treated with hexadecyl

magnesium bromide at ꢀ788C

to yield the secondary alcohol

26. Final deprotection of the

Scheme 4. Synthesis of the pentapeptide analogue 21. a) EDC, HOBt, NEt₃, CH₂Cl₂, 08C ! RT, 86 % (15);

b) H₂, Pd/C (10%), MeOH, 99%; c) H-Dap(SO₂C₁₆H₃₃)-OAll (6), EDC, HOBt, DMF, 08C ! RT, 73 % (17);

d) [Pd

(PPh₃)₄], DMB, THF, 99%; e) H-Lys

U

(Far)-OMe (19), EDC, HOBt, CH₂Cl₂, 08C ! RT,

amine using Pd⁰chemistry at 50%; f) [Pd

(PPh₃)₄], DMB, THF, 82%. Bzl=benzyl.

G

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H. Waldmann, A. Giannis et al.

Scheme 7. Synthesis of the central proline building block 38. a) Boc₂O,

NaOH, dioxane/H₂O 2:1, 08C ! RT, 95 % (32); b) Cs₂CO₃, MeOH;

c) MeI, DMF, 88%, two steps; d) DMP, CH₂Cl₂, 81% (34);

e) (EtO)₂OPCH₂CN (35), NaH, THF, 85%; f) H₂, PtO₂·H₂O, EtOH,

CHCl₃; g) AlocCl, NEtiPr₂, CH₂Cl₂, 95% (37), two steps; h) TFA/CH₂Cl₂

1:1, quant. Boc=tert-butyloxycarbonyl.

Scheme 5. Synthesis of the C₂₁amino acid 27. a) Isobutene, H₂SO₄, diox-

ane, 55% (23); b) AllBr, NEt₃, TBAI, THF, 80%; c) LiBH₄, THF, 71%;

d) DMP, CH₂Cl₂; e) C₁₆H₃₃MgBr, THF, ꢀ788C, 63%, two steps; f) [Pd-

A

DMP=Dess–Martin periodinane.

Coupling

of

Ac-Dap-

(SO₂C₁₆H₃₃)-OH (4) to com-

pound 38 and again saponifica-

tion and condensation of H-

Cys(Far)-OMe (10) yielded tri-

U

peptide 44. The Aloc group was

then cleaved off leading to pro-

line analogue 45.

Synthesis of the lactam deriva-

tives: For the synthesis of the

lactam derivatives, (2S)-amino-

butyrolactone

hydrobromide

(46) was doubly protected at

the amino function with Boc

groups (Scheme 9). The result-

Scheme 6. Synthesis of the pentapeptide analogue 30. a) H-Hhe-OtBu (27), EDC, HOBt, CH₂Cl₂, 08C ! RT,

69%; b) 1m NaOH, MeOH; c) H-Lys

(Aloc)-Cys

U

steps; d) [Pd(PPh₃)₄], DMB, THF, 80%. Hhe=(2S)-amino-5-hydroxyheneicosanic acid.

G

ing lactone 47 was then opened

by saponification by using

be obtained in acceptable yield. Finally, Aloc deprotection

led to the second pentapeptide analogue 30.

CsOH and subsequently the obtained cesium salt of the car-

boxylic acid was esterified with methyl iodide to give homo-

serine 48. After Swern oxidation^[32]of the alcohol, TMSCN

was used to generate a cyanohydrin from which the remain-

ing TMS group was cleaved off by using ammonium fluo-

ride. The free hydroxyl group of cyanohydrin 49 was then

reprotected with the tert-butyldiphenylsilyl group to yield

compound 50 in order to prevent the alcohol function from

side reactions during ring closure. For this purpose, the

cyano function was hydrogenated with 10% Pd on charcoal

and the resulting amine was heated under reflux in toluene

to give the lactam core structure 51 in good yield. The silyl

group was cleaved, the resulting alcohol was oxidized with

Synthesis of the proline derivatives: The synthesis of proline

derivatives 42 and 45 commenced with the protection of

(4R)-hydroxy-l-proline (31) (Scheme 7). First, the amino

function was blocked by a Boc group, and then the carbox-

ylic acid was protected as a methyl ester. The resulting alco-

hol 33 was oxidized to the ketone by using Dess–Martin pe-

riodinane. A subsequent Horner–Wadsworth–Emmons reac-

tion^[31]yielded acrylic nitrile 36. This intermediate was re-

duced via hydrogenation by using Adamꢁs catalyst, and,

without further purification, the resulting amino group was

protected by an Aloc moiety to obtain proline derivative 37.

Cleavage of the Boc group yielded trifluoroacetate salt 38,

which is the central building block for the following synthe-

ses of the two proline derivatives 42 and 45.

Dess–Martin periodinane, and

a

Horner–Wadsworth–

Emmons reaction led to the Z-configured acrylic nitrile 53

as the single product. Structure determination was carried

out by means of NOE spectroscopy. The coupling of the vi-

nylic hydrogen with the b-hydrogens but not with the d-hy-

drogens of the cyclic system is a strong argument for Z con-

figuration of the double bond.

Compound 38 was coupled to C₁₆H₃₃SO₂-b-Ala-OH (39),

and, after cleavage of the methyl ester with sodium hydrox-

ide, H-Cys(Far)-OMe (10) was condensed to the C-terminus

G

(Scheme 8). The resulting tripeptide 41 was finally depro-

tected to yield proline derivative 42.

For the forthcoming alkylation of the lactam amide, the

corresponding electrophile was needed. To this end, thioest-

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Thioesterase Inhibitors

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Scheme 9. Synthesis of the lactam building block 53. a) Boc₂O, NEt₃,

CH₂Cl₂, 08C ! RT; b) Boc₂O, DMAP, CH₃CN, 94% (47), two steps; c)

CsOH, MeOH; d) MeI, DMF, 79%, two steps; e) (COCl)₂, DMSO,

ꢀ608C, NEtiPr₂, ꢀ608C ! RT; f) TMSCN, CH₂Cl₂; g) NH₄F, 90 % (49),

three steps; h) TBDPSCl, imidazole, DMF, 90%; i) H₂, Pd/C (10%),

MeOH, 0.1% HOAc; j) toluene, reflux, 67%, two steps; k) TBAF, THF,

quant. (52); l) DMP, CH₂Cl₂; m) (EtO)₂OPCH₂CN (35), NaH, THF,

90%, two steps. DMAP=N,N-dimethylaminopyridine, TBAF=tetrabu-

tylammonium fluoride, TBDPSCl=tert-butyldiphenylsilyl chloride,

TMSCN=trimethylsilyl cyanide.

Scheme 8. Synthesis of the proline analogues 42 and 45. a) C₁₆H₃₃SO₂-b-

Ala-OH (39), EDC, HOBt, NEtiPr₂, CH₂Cl₂, 08C ! RT, 81 % (40); b)

1m NaOH, MeOH; c) H-Cys(Far)-OMe (10), EDC, HOBt, CH₂Cl₂, 08C

G

! RT, 87%, two steps; d) [Pd

O₂C₁₆H₃₃)-OH (4), EDC, HOBt, NEtiPr₂, CH₂Cl₂, 08C ! RT, 76 % (43);

f) 1m NaOH, MeOH; g) H-Cys(Far)-OMe (10), EDC, HOBt, CH₂Cl₂,

08C ! RT, 87%, two steps; h) [Pd(PPh₃)₄], DMB, THF, 64%.

A

Scheme 10. Synthesis of the mesylate 58. a) NaOMe, MeOH; b) 56,

DMF, 88%, two steps; c) TsOH, MeOH; d) MsCl, pyridine, 08C ! RT,

quant., two steps.

er 55, which was accessible following a literature proce-

dure,^[33]was treated with sodium methylate, and subsequent-

ly the free thiol group was protected as a tert-butyl disulfide

using the methodology of Wünsch et al. (Scheme 10).^[34]The

methoxy tetrahydropyran group in 57 was cleaved and the

resulting alcohol was then transformed into the desired me-

sylate 58.

Several alkylation procedures were tried and the follow-

ing proved to be the best: Mesylate 58 and lactam 53 were

dissolved in THF and sodium hydride was added to deproto-

nate the lactam NH. After stirring for several hours the al-

kylated lactam 59 could be isolated in acceptable yield

(Scheme 11). Deprotection of the Boc groups yielded the

central structure 60. Coupling of C₁₆H₃₃SO₂-b-Ala-OH (39)

and Ac-Dap(SO₂C₁₆H₃₃)-OH (4), respectively, gave the two

lactam derivatives 61 and 62. Unfortunately, the acrylic ni-

trile functionality in both compounds could not be reduced

without complete loss of the disulfide moiety. However, the

structures obtained had very interesting substitution pat-

terns, so that a biological evaluation seemed worthwhile in

any case. Therefore, further alternative syntheses were not

investigated.

Synthesis of the benzodiazepine derivatives: The synthesis

of the benzodiazepines started with the condensation of

(4R)-hydroxy-l-proline (31) and 5-bromoisatoic acid anhy-

dride (63) yielding the benzodiazepine core 64 (Scheme 12).

A subsequent Heck reaction^[35]with tert-butyl acrylate gave

compound 65 in high yield. Then, the alcohol function was

converted into a mesylate which was subsequently treated

with NaN₃to obtain azide 66. Deprotonation of the amide

NH and alkylation with bromoacetonitrile led to compound

67, in which the acrylic double bond was cleaved by using

RuCl₃/NaIO₄.^[36]The resulting free carboxylic acid was pro-

tected with the tert-butyl group yielding benzodiazepine 68

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H. Waldmann, A. Giannis et al.

Scheme 11. Synthesis of the two lactams 61 and 62. a) 58, NaH, 08C !

RT, 57 % (59); b) TFA/CH₂Cl₂1:1, 99%; c) C₁₆H₃₃SO₂-b-Ala-OH (39),

EDC, HOBt, NEtiPr₂, 08C ! RT, 67%; d) Ac-Dap(SO₂C₁₆H₃₃)-OH (4),

EDC, HOBt, NEtiPr₂, 08C ! RT, 62 %.

in very good yield. Selective hydrogenation of the azide

using Pd on BaSO₄gave amine 69. The sulfonamide moiety

was introduced by reaction with hexadecylsulfonic acid

chloride leading to 70; subsequent hydrogenation with PtO₂

catalysis generated amine 71. From here, simple acidic

cleavage of the tert-butyl ester gave the first benzodiazepine

derivative 72.

Scheme 12. Synthesis of the benzodiazepines 72 and 76. a) 1408C,

Alternatively, protection of amine 71 with the Aloc group,

deprotection of the resulting compound 73 with trifluoroace-

DMSO, 93%; b) tert-butyl acrylate, [Pd

N

1008C, sealed tube, 97%; c) MsCl, pyridine, 08C ! RT; d) NaN₃, DMF,

458C, 89% (66), two steps; e) NaH, THF, ꢀ408C; f) BrCH₂CN, ꢀ408C

! RT, 95 % (67), two steps; g) RuCl₃, NaIO₄, H₂O/CH₃CN/CCl₄2:1:1; h)

tic acid and subsequent coupling of H-Cys(Far)-OMe (10)

G

to the free acid 74 gave protected benzodiazepine 75 in very

good yield. Finally, the Aloc group was removed leading to

the dually lipidated benzodiazepine 76.

For the synthesis of monolipidated benzodiazepines, com-

pound 69 was mesylated at the amino group to give 77, and

subsequently the nitrile function was hydrogenated by using

Adamꢁs catalyst (Scheme 13). The free amine in 78 was then

blocked with an Aloc group yielding 79; after the tert-butyl

Me₃CBr, K₂CO₃, Et₃(PhCH₂)NCl, DMA, 558C, 90% (68), two steps; i)

N

H₂, Pd/BaSO₄, MeOH, CHCl₃, quant.; j) C₁₆H₃₃SO₂Cl, NEtiPr₂, DMF,

08C ! RT, 82 % (70); k) H₂, PtO₂·H₂O, EtOH, CHCl₃, 81%; l) HCl/

Et₂O, quant.; m) AlocCl, NEt₃, CH₂Cl₂, 84% (73); n) TFA/CH₂Cl₂1:1,

quant. (74); o) H-Cys

A

89%; p) [Pd(PPh₃)₄], DMB, THF, 80%. DMA=dimethylacetamide.

AHCTREUNG

IC₅₀value is always in the low nanomolar range (see com-

pounds 12, 21, 30, 42, 45, 71, 72, 75 and 76). This high activi-

ty of the compounds is important for their application in bi-

ological systems. The long fatty acid chains of the inhibitors

are crucial for their inhibitory activity but they also decrease

the polarity of the molecules and limit their solubility in

aqueous solutions at high concentrations. Nevertheless, the

high inhibitory activity of the compounds enables their ap-

plication in several biological experiments.

The fact, that the tripeptide analogues are slightly more

active than the corresponding pentapeptides, may result

from a higher conformational flexibility of the pentapeptide

backbone compared with the tripeptides. This may also ex-

plain the observation that the conformationally fixed proline

ester was cleaved off under acidic conditions, H-Cys(Far)-

U

OMe (10) was condensed with the resulting free acid. Final

deprotection of 80 using again Pd⁰chemistry led to the mon-

olipidated benzodiazepine 81.

Results of the biological evaluation: The syntheses detailed

above had given access to 16 different compounds for bio-

chemical evaluation. The results of the assay for inhibition

of APT1 are shown in Table 1. A detailed procedure for the

assay accompanied with the synthesis of a substrate peptide

for APT1 is provided in the Experimental Section.

Comparison of the data given in Table 1 reveals that if a

hexadecylsulfonamide moiety is present in the molecule, the

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Thioesterase Inhibitors

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Experimental Section

General procedures: ¹H and ¹³C NMR spectra were recorded on

a

Bruker AC 250, AM 400 or DRX 500 and a Varian Mercury 400 spec-

trometer at room temperature. Mass spectra and high-resolution mass

spectra (HRMS) were measured on a Finnigan MAT MS70 spectrometer.

The optical rotation was determined with a Perkin–Elmer polarimeter

241.

Materials: When not otherwise indicated, all reactions were performed

under argon atmosphere with freshly distilled and dried solvents. The sol-

vents were dried following standard methods. Silica gel (40–60 mm) was

used for column chromatography. Commercial reagents were used with-

out further purification.

(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionic acid allyl

ester(3) : At 08C, NEtiPr₂(1.04 mL, 6.06 mmol) and a solution of hexa-

decanesulfonic acid chloride (965 mg, 2.91 mmol) in DMF (10 mL) were

subsequently added to a solution of hydrochloride 2 (540 mg, 2.43 mmol)

in DMF (5 mL). The solution was warmed to room temperature and stir-

red for 20 h. Then, the reaction mixture was diluted with ethyl acetate

(150 mL) and extracted with 1n HCl (330 mL). The organic layer was

dried over Na₂SO₄, and, after removal of the solvent under reduced pres-

sure, the residue was purified by chromatography (n-hexane/ethyl acetate

Scheme 13. Synthesis of the benzodiazepine 81. a) MsCl, NEtiPr₂, DMF,

08C ! RT, 92 % (77); b) H₂, PtO₂·H₂O, EtOH, CHCl₃, 85%; c) AlocCl,

NEt₃, CH₂Cl₂, 83% (79); d) TFA/CH₂Cl₂1:1; e) H-Cys(Far)-OMe (10),

A

EDC, HOBt, CH₂Cl₂, 08C ! RT, 89 % (80), two steps; f) [Pd

A

DMB, THF, 65%.

1:1) to yield 3 as a colourless solid (865 mg, 75%). M.p. 708C; [a]_D²⁰

=

+17.5 (c=1.0 in CHCl₃); R_f=0.20 (cyclohexane/ethylacetate 1:1);

¹H NMR (CDCl₃, 400 MHz): d=0.84 (t, J=6.6 Hz, 3H, w-CH₃hexadec-

yl), 1.24 (m, 24H, 12CH₂hexadecyl), 1.38 (m, 2H, g-CH₂hexadecyl),

1.72 (m, 2H, b-CH₂hexadecyl), 2.08 (s, 3H, CH₃acetyl), 2.95 (m, 2H,

a-CH₂hexadecyl), 3.54 (m, 2H, b-CH₂Dap), 4.62 (m, 1H, -OCH_2aCH=

CH₂), 4.73 (m, 2H, a-CH Dap, -OCH_2bCH=CH₂), 5.23–5.36 (m, 2H,

-OCH₂CH=CH₂), 5.66 (t, J=6.8 Hz, 1H, NH), 5.91 (m, 1H, -OCH₂CH=

CH₂), 7.11 (d, J=7.6 Hz, 1H, NH); ¹³C NMR (CDCl₃, 100.6 MHz): d=

14.3 (w-CH₃hexadecyl), 22.8 (CH₃acetyl), 23.1 (CH₂), 23.8 (CH₂), 28.4

(CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.8

(CH₂), 29.8 (CH₂), 29.9 (CH₂), 30.0 (CH₂), 30.2 (CH₂), 32.1 (CH₂), 44.6

(b-CH₂Dap), 53.2 (a-CH₂hexadecyl), 66.9 (OCH₂CH=CH₂), 119.5

(OCH₂CH=CH₂), 131.5 (OCH₂CH=CH₂), 170.2 (C=O), 171.0 (C=O);

HRMS (FAB, 3-NBA): m/z: calcd for C₂₄H₄₇N₂O₅S: 475.3206, found:

475.3230 [M+H]⁺.

and benzodiazepine derivatives are the most active inhibi-

tors. If analogues with an Aloc group on the central amine

are compared to those with a free amino group, the conclu-

sion can be drawn, that the inhibitory potency of the pro-

tected compounds is about one order of magnitude lower

(cf. 11/12, 20/21, 44/45 and 75/76). The findings for lactam

derivatives 61 and 62, which have only a nitrile function,

agree with this conclusion. Comparison of the benzodiaze-

pines reveals that compounds 78 and 81, which lack a hexa-

decyl group, are nearly inactive. The presence of a farnesyl

group in 81 leads to an IC₅₀value that is about one order of

magnitude better than the corresponding tert-butyl ester 78.

Thus, at least one lipid group has to be present in the mole-

cule for inhibition. In summary, a long chain sulfonamide

moiety may be essential for nanomolar activity. If addition-

ally a central free amine group is present, the activity is

about one order of magnitude higher. Compounds with dual

lipidation patterns are better inhibitors than monolipidated

analogues, but the presence of a farnesyl group seems to be

not essential for high activity.

(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionic acid (4):

[Pd(PPh₃)₄] (77 mg, 0.07 mmol) was added to a solution of allyl ester 3

A

(530 mg, 1.12 mmol) and N,N’-dimethylbarbituric acid (104 mg,

0.67 mmol) in THF (40 mL). The mixture was stirred at room tempera-

ture for 1.5 h. After removal of the solvent under reduced pressure, the

residue was purified by chromatography (CH₂Cl₂/EtOH 10:1

+ 1%

HOAc) to yield 4 as a colorless solid (480 mg, 99%). M.p. 918C; [a]_D²⁰

=

+15.7 (c=1.0 in MeOH); R_f=0.22 (CH₂Cl₂/EtOH 10:1 + 1% HOAc);

¹H NMR (CD₃OD, 400 MHz): d=0.87 (t, J=6.6 Hz, 3H, w-CH₃hexa-

decyl), 1.30 (m, 24H, 12CH₂hexadecyl), 1.41 (m, 2H, g-CH₂hexadec-

yl), 1.79 (m, 2H, b-CH₂hexadecyl), 2.03 (s, 3H, CH₃acetyl), 3.04 (m,

2H, a-CH₂hexadecyl), 3.52 (m, 2H, b-CH₂Dap), 4.51 (m, 1H, a-CH

Dap); ¹³C NMR (CD₃OD, 100.6 MHz): d=13.2 (w-CH₃hexadecyl), 21.6

(CH₃acetyl), 23.1 (CH₂), 23.8 (CH₂), 28.4 (CH₂), 29.5 (CH₂), 29.6 (CH₂),

29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂),

30.0 (CH₂), 30.2 (CH₂), 32.1 (CH₂), 43.3 (b-CH₂Dap), 52.0 (a-CH₂hexa-

decyl), 53.3 (a-CH Dap), 170.2 (C=O), 171.3 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₂₁H₄₂N₂NaO₅S: 457.2714, found: 457.2682

[M+Na]⁺.

Conclusion

In conclusion, we have designed, synthesized, and evaluated

different inhibitors of acyl protein thioesterase 1. Several in-

hibitors with an activity in the low nanomolar range could

be found. A combinatorial approach starting from the

highly active benzodiazepines may lead to even better inhib-

itors of APT1 with higher diversity. Following this approach,

more precise structure–activity relationships could be ob-

tained. Additionally, the application of the highly potent

compounds in biophysical and biological experiments can

serve to elucidate the physiological significance of APT1 in

the reversible palmitoylation of signalling proteins.^[27]

(2S)-Amino-3-(hexadecane-1-sulfonylamino)propionic acid allyl ester (6):

At ꢀ708C,

a solution of hexadecanesulfonic acid chloride (136 mg,

0.41 mmol) in CH₂Cl₂(2 mL) was added to a solution of hydrotosylate 5

(200 mg, 0.41 mmol) and NEt₃(174 mL, 2.37 mmol) in CH₂Cl₂(2 mL).

The solution was warmed to room temperature and stirred for 15 h.

Then, the reaction mixture was diluted with CH₂Cl₂(20 mL) and washed

with Na₂CO₃solution (10%, 10 mL). The organic layer was dried over

Na₂SO₄, and, after removal of the solvent under reduced pressure, the

residue was purified by chromatography (CHCl₃/MeOH 5:1) to yield 6 as

a colorless solid (129 mg, 72%). M.p. 738C; [a]²_D⁰= +29.5 (c=1.0 in

CHCl₃); R_f=0.58 (CHCl₃/MeOH 5:1); ¹H NMR (CDCl₃, 400 MHz): d=

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4127

H. Waldmann, A. Giannis et al.

Table 1. Inhibition of APT1 by the synthesized compounds.^[a]

0.84 (t, J=6.5 Hz, 3H, w-CH₃hexa-

decyl), 1.22 (m, 24H, 12CH₂hexa-

decyl), 1.36 (m, 2H, g-CH₂hexadecyl),

1.76 (m, 2H, b-CH₂hexadecyl), 2.98

(m, 2H, a-CH₂hexadecyl), 3.20 (dd,

J=6.8, 13.1 Hz, 1H, b-CH_2aDap), 3.40

(dd, J=4.5, 13.1 Hz, 1H, b-CH_2bDap),

3.63 (dd, J=4.5, 6.8 Hz, 1H, a-CH

Dap), 4.61 (m, 2H, -OCH₂CH=CH₂),

5.21–5.36 (m, 2H, -OCH₂CH=CH₂),

5.91 (m, 1H, -OCH₂CH=CH₂);

¹³C NMR (CDCl₃, 100.6 MHz): d=

14.3 (w-CH₃hexadecyl), 22.8 (CH₂),

23.7 (CH₂), 28.5 (CH₂), 28.6 (CH₂),

28.7 (CH₂), 28.9 (CH₂), 29.1 (CH₂),

29.2 (CH₂), 29.3 (CH₂), 29.3 (CH₂),

29.3 (CH₂), 29.5 (CH₂), 29.8 (CH₂),

32.1 (CH₂), 46.2 (b-CH₂Dap), 53.0 (a-

CH Dap), 54.5 (a-CH₂hexadecyl),

Entry

Compound

Structure

IC₅₀[nm]

1

11

153Æ21

2

3

4

12

20

21

65Æ3

1583Æ240

291Æ11

66.3

(OCH₂CH=CH₂),

119.2

(OCH₂CH=CH₂), 131.7 (OCH₂CH=

CH₂), 173.1 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₂₂H₄₅N₂O₄S:

433.3101, found: 433.3120 [M+H]⁺.

N^a-[(2S)-Acetylamino-3-(hexadecane-

1-sulfonylamino-propionyl]-N^e-(allyl-

oxycarbonyl)-l-lysine tert-butyl ester

(8): At 08C, EDC (54 mg, 0.28 mmol)

was added to a solution of diamino-

propionic

0.23 mmol),

0.23 mmol)

acid

lysine

and

(4)

(100 mg,

(66 mg,

(72 mg,

7

HOBt

0.46 mmol) in CH₂Cl₂(8 mL). The sol-

ution was warmed to room tempera-

ture and stirred for 18 h. Then, the re-

action mixture was diluted with ethyl

acetate (50 mL) and subsequently

washed with 1n HCl (20 mL) and

Na₂CO₃solution (10%, 20 mL). The

organic layer was dried over Na₂SO₄,

and, after removal of the solvent

under reduced pressure, the residue

was purified by chromatography (cy-

clohexane/ethyl acetate 1:3) to yield 8

5

6

30

42

147Æ20

as

M.p. 808C; [a]²_D⁰

CHCl₃); R_f=0.24 (cyclohexane/ethyl

acetate 1:3); (CDCl₃,

¹H NMR

a

colorless solid (132 mg, 82%).

=

ꢀ12.1 (c=1.0 in

400 MHz): d=0.81 (t, J=6.5 Hz, 3H,

w-CH₃hexadecyl), 1.48 (s, 9H, C-

(CH₃)₃), 1.12–1.52 (m, 30H, 13CH₂

61Æ6

hexadecyl, 2CH₂Lys), 1.54–1.85 (m,

4H, g-CH₂hexadecyl, CH₂Lys), 2.02

(s, 3H, CH₃acetyl), 2.96 (m, 2H, a-

CH₂hexadecyl), 3.02–3.29 (m, 3H, b-

CH_2aDap, e-CH₂Lys), 3.56 (m, 1H, b-

CH_2bDap), 4.30 (m, 1H, a-CH Lys),

4.52 (m, 3H, a-CH Dap, -OCH₂CH=

CH₂), 4.95 (m, 1H, NH), 5.10–5.25 (m,

2H, -OCH₂CH=CH₂), 5.76 (m, 1H,

NH), 5.88 (m, 1H, -OCH₂CH=CH₂),

6.70 (m, 1H, NH), 7.19 (m, 1H, NH);

¹³C NMR (CDCl₃, 100.6 MHz): d=

14.3 (w-CH₃hexadecyl), 22.9 (CH₃

acetyl), 23.1 (CH₂), 23.8 (CH₂), 28.3

7

44

1223Æ258

(C(CH₃)₃), 28.4 (CH₂), 28.5 (CH₂),

A

29.2 (CH₂), 29.3 (CH₂), 29.5 (CH₂),

29.6 (CH₂), 29.7 (CH₂), 29.7 (CH₂),

29.7 (CH₂), 29.8 (CH₂), 29.9 (CH₂),

4128

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

Table 1. (Continued)

FULL PAPER

29.9 (CH₂), 30.0 (CH₂), 30.2 (CH₂),

32.1 (CH₂), 43.3 (b-CH₂Dap), 44.6 (e-

CH₂Lys), 53.5 (a-CH), 54.2 (a-CH₂

Entry

Compound

Structure

IC₅₀[nm]

hexadecyl),

(OCH₂CH=CH₂), 82.4 (C

115.1 (OCH₂CH=CH₂),

56.0

(a-CH),

67.3

ACHTREUNG

(OCH₂CH=CH₂), 157.5 (C=O), 172.1

(C=O), 174.7 (C=O), 174.8 (C=O);

HRMS (FAB, 3-NBA): m/z: calcd for

8

45

140Æ18

C₃₅H₆₆N₄NaO₈S:

725.4501,

found:

725.4512 [M+Na]⁺.

N^a-[(2S)-Acetylamino-3-(hexadecane-

1-sulfonylamino)propionyl]-N^e-allyl-

oxycarbonyl-l-lysine (9): TFA (10 mL)

was added to a solution of tert-butyl

ester 8 (117 mg, 0.17 mmol) in CH₂Cl₂

(10 mL) at room temperature. The sol-

ution was stirred for 4 h. Then, the sol-

vent was removed under reduced pres-

sure. Coevaporation of the residue

with toluene yielded 9 as a colorless

solid (107 mg, 100%). M.p. 838C;

[a]²_D⁰= ꢀ14.0 (c=1.0 in MeOH); R_f=

0.01 (cyclohexane/ethyl acetate 1:3);

¹H NMR (CD₃OD, 400 MHz): d=0.85

(t, J=6.5 Hz, 3H, w-CH₃hexadecyl),

1.12–1.52 (m, 30H, 13CH₂hexadec-

yl, 2CH₂Lys), 1.54–1.85 (m, 4H, g-

CH₂hexadecyl, CH₂Lys), 2.02 (s, 3H,

CH₃acetyl), 2.96 (m, 2H, a-CH₂hexa-

decyl), 3.02–3.29 (m, 3H, b-CH_2aDap,

e-CH₂Lys), 3.56 (m, 1H, b-CH_2b

Dap), 4.30 (m, 1H, a-CH Lys), 4.52

(m, 3H, a-CH Dap, -OCH₂CH=CH₂),

5.10–5.25 (m, 2H, -OCH₂CH=CH₂),

5.88 (m, 1H, -OCH₂CH=CH₂);

¹³C NMR (CD₃OD, 100.6 MHz): d=

14.2 (w-CH₃hexadecyl), 22.9 (CH₃

acetyl), 23.1 (CH₂), 23.8 (CH₂), 28.3

(CH₂), 28.4 (CH₂), 28.5 (CH₂), 29.4

(CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.7

(CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8

(CH₂), 29.9 (CH₂), 29.9 (CH₂), 30.0

(CH₂), 30.2 (CH₂), 32.1 (CH₂), 43.3 (b-

CH₂Dap), 44.6 (e-CH₂Lys), 53.5 (a-

CH), 54.2 (a-CH₂hexadecyl), 56.0 (a-

CH), 67.3 (OCH₂CH=CH₂), 115.1

(OCH₂CH=CH₂), 137.4 (OCH₂CH=

CH₂), 157.5 (C=O), 172.1 (C=O),

174.7 (C=O), 175.8 (C=O); HRMS

(FAB, 3-NBA): m/z: calcd for

9

61

62

725Æ19

10

975Æ7

11

12

71

72

148Æ6

97Æ8

13

75

149Æ30

C₃₁H₅₈N₄NaO₈S:

669.3875,

found:

669.3887 [M+Na]⁺.

N^a-[(2S)-Acetylamino-3-(hexadecane-

1-sulfonylamino)propionyl]-N^e-allyl-

oxycarbonyl-l-lysyl-S-farnesyl-l-cys-

teine methyl ester(11) : At 08C, EDC

(36 mg, 0.19 mmol) was added to a sol-

14

15

76

78

27Æ5

ution of dipeptide

0.16 mmol), cysteine 10 (53 mg,

0.16 mmol) and HOBt (72 mg,

9

(100 mg,

0.46 mmol) in CH₂Cl₂(10 mL). The

solution was warmed to room temper-

ature and stirred for 18 h. Then, the

reaction mixture was diluted with

ethyl acetate (50 mL) and subsequent-

ly washed with 1n HCl (20 mL) and

Na₂CO₃solution (10%, 20 mL). The

organic layer was dried over Na₂SO₄,

252208Æ159000

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4129

H. Waldmann, A. Giannis et al.

N-Acetyl-l-methionyl-l-serine benzyl

Table 1. (Continued)

ester(15)

20.63 mmol) was added to a solution

of Ac-Met-OH 13 (3.288 g,

17.19 mmol), HCl·H-Ser-OBzl 14

: At 08C, EDC (4.036 g,

Entry

Compound

Structure

IC₅₀[nm]

(3.983 g, 17.19 mmol), HOBt (5.372 g,

34.38 mmol) and NEt₃(3.6 mL,

25.79 mmol) in CH₂Cl₂(100 mL). The

solution was warmed to room temper-

ature and stirred for 20 h. Then, the

reaction mixture was subsequently

washed with 1n HCl (50 mL) and

Na₂CO₃solution (10%, 50 mL). The

organic layer was dried over Na₂SO₄,

and, after removal of the solvent

under reduced pressure, the residue

was purified by chromatography

16

81

26975Æ17000

[a] For the determination of IC₅₀values, the described compounds were incubated with enzyme (APT1) and

substrate (a palmitoylated C-terminal fragment of the H-Ras protein) at six different concentrations. The re-

leased palmitate, which directly correlates with the enzyme activity was determined according to the ADIFAB

assay protocol in two independent measurements.

and, after removal of the solvent under reduced pressure, the residue was

(CHCl₃/MeOH 50:1) to yield 15 as a colorless solid (5.447 g, 86%). M.p.

578C; [a]²_D⁰= ꢀ21.4 (c=1.0 in CHCl₃); R_f=0.17 (cyclohexane/ethyl ace-

tate 1:3); ¹H NMR (CD₃OD, 400 MHz): d=1.92 (m, 1H, b-CH_2aMet),

2.01 (s, 3H, CH₃acetyl), 2.06 (m, 1H, b-CH_2bMet), 2.08 (s, 3H, CH₃

Met), 2.55 (m, 2H, g-CH₂Met), 3.85 (dd, J=3.7, 11.3 Hz, 1H, b-CH_2a

Ser), 3.99 (dd, J=3.7, 11.3 Hz, 1H, b-CH_2bSer), 4.54 (dd, J=6.1, 8.0 Hz,

1H, a-CH Met), 4.61 (t, J=3.9 Hz, 1H, a-CH Ser), 5.21 (s, 2H, -OCH₂-),

7.31–7.38 (m, 5H, arom. CH); ¹³C NMR (CD₃OD, 100.6 MHz): d=18.7

(CH₃acetyl), 25.9 (CH₃Met), 33.9 (g-CH₂Met), 35.5 (b-CH₂Met), 56.5

(a-CH Met), 59.0 (a-CH Ser), 65.7 (b-CH₂Ser), 71.2 (OCH₂), 132.15

(arom. CH), 132.3 (arom.CH), 132.5 (arom. CH), 139.4 (arom. C), 174.1

(C=O), 176.0 (C=O), 176.3 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₁₇H₂₅N₂O₅S: 369.1485, found: 369.1493 [M+H]⁺.

purified by chromatography (cyclohexane/ethyl acetate 1:5) to yield 11 as

a colorless oil (135 mg, 90%). [a]²_D⁰= +5.8 (c=0.5 in CHCl₃); R_f=0.22

(cyclohexane/ethyl acetate 1:5); H NMR (CDCl₃/CD₃OD 1:1, 400 MHz):

1

d=0.81 (t, J=6.5 Hz, 3H, w-CH₃hexadecyl), 1.24 (m, 24H, 12CH₂

hexadecyl), 1.58 (s, 6H, 2CH₃Far), 1.67 (s, 6H, 2CH₃Far), 1.35–1.84

(m, 8H, 2CH₂Lys, 2 CH₂hexadecyl), 2.04 (s, 3H, CH₃acetyl), 1.85–

2.14 (m, 10H, CH₂Lys, 4 CH₂Far), 2.68–3.28 (m, 10H, a-CH₂hexadec-

yl, b-CH₂Cys, -S-CH₂, b-CH₂Dap, e-CH₂Lys), 3.71 (s, 3H, OCH₃), 4.40

(m, 1H, a-CH Lys), 4.52 (m, 3H, a-CH Dap, -OCH₂CH=CH₂), 4.72 (m,

1H, a-CH Cys), 5.12–5.30 (m, 5H, 3CH Far, -OCH₂CH=CH₂), 5.91 (m,

1H, -OCH₂CH=CH₂), 6.10 (m, 1H, NH), 6.99 (m, 2H, NH), 7.21 (m,

1H, NH); ¹³C NMR (CDCl₃/CD₃OD 1:1, 100.6 MHz): d=14.3 (w-CH₃

hexadecyl), 16.5 (CH₃acetyl), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.3 (CH₃

Far), 23.1 (CH₂), 23.8 (CH₂), 25.3 (CH₃Far), 28.3 (CH₂), 28.4 (CH₂), 28.5

(CH₂), 29.4 (CH₂), 29.5 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8

(CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 30.0 (CH₂), 30.2

(CH₂), 32.1 (CH₂), 33.0 (CH₂), 33.3 (CH₂), 34.4 (CH₂), 39.8 (CH₂Far),

40.5 (CH₂Far), 42.0 (b-CH₂Dap), 44.6 (e-CH₂Lys), 50.4 (OCH₃), 53.4

(a-CH), 54.2 (a-CH), 54.5 (a-CH₂hexadecyl), 56.0 (a-CH), 67.3

(OCH₂CH=CH₂), 115.1 (OCH₂CH=CH₂), 117.1 (CH Far), 121.7 (CH

Far), 122.5 (CH Far), 133.0 (C Far), 137.5 (OCH₂CH=CH₂), 138.8 (C

Far), 139.3 (C Far), 157.5 (C=O), 170.9 (C=O), 172.0 (C=O), 174.7 (C=

O), 174.8 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₅₀H₈₉N₅NaO₉S₂:

990.6002, found: 990.6030 [M+Na]⁺.

N-Acetyl-l-methionyl-l-serine (16): Pd (10% on charcoal, 354 mg) was

added to a degassed solution of dipeptide 15 (2 g, 5.43 mmol) in MeOH

(125 mL) and acetic acid (12.5 mL). The reaction mixture was hydrogen-

ated under hydrogen atmosphere at room temperature for 3 d. Then, the

suspension was filtered through a pad of Celite. After removal of the sol-

vents under reduced pressure, the residue was purified by chromatogra-

phy (CHCl₃/MeOH 10:1) to yield 16 as a colorless solid (1.496 g, 99%).

M.p. 938C; [a]²_D⁰= ꢀ17.0 (c=1.0 in MeOH); R_f=0.19 (CHCl₃/MeOH

5:1); ¹H NMR (CD₃OD, 400 MHz): d=1.98 (m, 1H, b-CH_2aMet), 2.05

(s, 3H, CH₃acetyl), 2.13 (m, 1H, b-CH_2bMet), 2.14 (s, 3H, CH₃Met),

2.60 (m, 2H, g-CH₂Met), 3.85 (dd, J=3.7, 11.3 Hz, 1H, b-CH_2aSer), 3.99

(dd, J=3.7, 11.3 Hz, 1H, b-CH_2bSer), 4.39 (dd, J=6.1, 8.0 Hz, 1H, a-CH

Met), 4.54 (t, J=3.9 Hz, 1H, a-CH Ser); ¹³C NMR (CD₃OD,

100.6 MHz): d=18.7 (CH₃acetyl), 25.9 (CH₃Met), 33.9 (g-CH₂Met),

35.5 (b-CH₂Met), 56.5 (a-CH Met), 60.9 (a-CH Ser), 64.7 (b-CH₂Ser),

174.1 (C=O), 176.0 (C=O), 176.3 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₁₀H₁₉N₂O₅S: 279.1015, found: 279.1028 [M+H]⁺.

N^a-[(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionyl]-l-

lysyl-S-farnesyl-l-cysteine methyl ester(12)

:

[Pd(PPh₃)₄] (7 mg,

A

0.01 mmol) was added to a solution of tripeptide 11 (61 mg, 0.06 mmol)

and N,N’-dimethylbarbituric acid (10 mg, 0.06 mmol) in THF (6 mL). The

mixture was stirred at room temperature for 8 h. After removal of the

solvent under reduced pressure, the residue was purified by chromatogra-

phy (CH₂Cl₂/EtOH 5:1 + 1% NEt₃) to yield 12 as a colorless oil (41 mg,

73%). [a]²_D⁰= +8.1 (c=0.5 in MeOH); R_f=0.04 (CH₂Cl₂/EtOH 1:1);

¹H NMR (CD₃OD, 400 MHz): d=0.81 (t, J=6.5 Hz, 3H, w-CH₃hexa-

decyl), 1.24 (m, 24H, 12CH₂hexadecyl), 1.58 (s, 6H, 2CH₃Far), 1.67

(s, 6H, 2CH₃Far), 1.35–1.84 (m, 8H, 2CH₂Lys, 2 CH₂hexadecyl),

2.04 (s, 3H, CH₃acetyl), 1.85–2.14 (m, 10H, CH₂Lys, 4 CH₂Far), 2.68–

3.28 (m, 10H, a-CH₂hexadecyl, b-CH₂Cys, -S-CH₂, b-CH₂Dap, e-CH₂

Lys), 3.71 (s, 3H, OCH₃), 4.20 (m, 1H, a-CH Lys), 4.25 (m, 1H, a-CH

Dap), 4.45 (m, 1H, a-CH Cys), 4.90 (m, 2H, 2CH Far), 5.02 (m, 1H,

CH Far); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.3 (w-CH₃hexadecyl),

16.5 (CH₃acetyl), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.3 (CH₃Far), 23.1

(CH₂), 23.8 (CH₂), 25.3 (CH₃Far), 28.3 (CH₂), 28.4 (CH₂), 28.5 (CH₂),

29.4 (CH₂), 29.5 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂),

29.8 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 30.0 (CH₂), 30.2 (CH₂),

32.1 (CH₂), 33.0 (CH₂), 33.3 (CH₂), 34.4 (CH₂), 39.8 (CH₂Far), 40.5 (CH₂

Far), 42.0 (b-CH₂Dap), 44.6 (e-CH₂Lys), 50.4 (OCH₃), 53.4 (a-CH), 54.2

(a-CH), 54.5 (a-CH₂hexadecyl), 56.0 (a-CH), 117.1 (CH Far), 121.7 (CH

Far), 122.5 (CH Far), 133.0 (C Far), 138.8 (C Far), 139.3 (C Far), 170.9

(C=O), 172.0 (C=O), 174.7 (C=O), 174.8 (C=O); HRMS (FAB, 3-NBA):

m/z: calcd for C₄₆H₈₆N₅O₇S₂: 884.5969, found: 884.5936 [M+H]⁺.

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-3-(hexadecane-1-sulfonylami-

no)]-propionic acid allyl ester (17): At 08C, EDC (43 mg, 0.22 mmol) was

added to a solution of H-Dap(SO₂C₁₆H₃₃)-OAll 6 (80 mg, 0.19 mmol),

Ac-Met-Ser-OH 16 (62 mg, 0.22 mmol) and HOBt (58 mg, 0.37 mmol) in

DMF (5 mL). The solution was warmed to room temperature and stirred

for 18 h. After removal of the solvent under reduced pressure, the resi-

due was dissolved in ethyl acetate and subsequently washed with 1n HCl

(15 mL) and Na₂CO₃solution (10%, 15 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (CH₂Cl₂/EtOH 15:1, then

10:1) to yield 17 as a colorless solid (93 mg, 73%). M.p. 788C; [a]_D²⁰

=

ꢀ9.4 (c=1.0 in MeOH); R_f=0.28 (CH₂Cl₂/EtOH 10:1); ¹H NMR

(CD₃OD, 400 MHz): d=0.87 (t, J=6.5 Hz, 3H, w-CH₃hexadecyl), 1.25

(m, 24H, 12CH₂hexadecyl), 1.40 (m, 2H, g-CH₂hexadecyl), 1.75 (m,

2H, b-CH₂hexadecyl), 1.95 (m, 1H, b-CH_2aMet), 2.02 (s, 3H, CH₃

acetyl), 2.11 (s, 3H, CH₃Met), 2.12 (m, 1H, b-CH_2bMet), 2.56 (m, 2H,

g-CH₂Met), 2.98 (m, 2H, a-CH₂hexadecyl), 3.53 (m, 2H, b-CH₂Dap),

3.78 (dd, J=3.7, 11.3 Hz, 1H, b-CH_2aSer), 3.92 (dd, J=3.8, 11.3 Hz, 1H,

b-CH_2bSer), 4.40 (t, J=4.3 Hz, 1H, a-CH Ser), 4.45 (dd, J=6.0, 8.1 Hz,

1H, a-CH Met), 4.59 (t, J=5.4 Hz, 1H, a-CH Dap), 4.66 (m, 2H,

-OCH₂CH=CH₂), 5.24–5.36 (m, 2H, -OCH₂CH=CH₂), 5.93 (m, 1H,

-OCH₂CH=CH₂); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.2 (w-CH₃hexa-

4130

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

decyl), 22.6 (CH₃acetyl), 22.9 (CH₃Met), 23.4 (CH₂), 23.7 (CH₂), 28.5

(CH₂), 28.6 (CH₂), 28.7 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.2 (CH₂), 29.3

(CH₂), 29.3 (CH₂), 29.3 (CH₂), 29.5 (CH₂), 29.8 (CH₂), 30.3 (CH₂), 31.2

(g-CH₂Met), 32.1 (b-CH₂Met), 43.5 (b-CH₂Dap), 53.2 (a-CH Dap),

53.3 (a-CH Met), 55.6 (a-CH₂hexadecyl), 60.9 (a-CH Ser), 61.9 (b-CH₂

Ser), 66.8 (OCH₂CH=CH₂), 119.2 (OCH₂CH=CH₂), 131.6 (OCH₂CH=

CH₂), 169.5 (C=O), 170.8 (C=O), 172.6 (C=O), 172.8 (C=O); HRMS

(FAB, 3-NBA): m/z: calcd for C₃₂H₆₁N₄O₈S₂: 693.3932, found: 693.3944

[M+H]⁺.

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-3-(hexadecane-1-sulfonylami-

no)propionyl]-l-lysyl-S-farnesyl-l-cysteine methyl ester(21) : [Pd(PPh₃)₄]

R

(7 mg, 0.01 mmol) was added to a desgassed solution of pentapeptide 20

(21 mg, 0.02 mmol) and N,N’-dimethylbarbituric acid (3 mg, 0.02 mmol)

in THF (3 mL). The mixture was stirred at room temperature for 4 h.

After removal of the solvent under reduced pressure, the residue was pu-

rified by chromatography (CH₂Cl₂/MeOH 1:1 + 1% NEt₃) to yield 21 as

slightly yellow solid (16 mg, 82%). M.p. 868C; [a]²_D⁰= +10.3 (c=0.5 in

MeOH); R_f=0.01 (CH₂Cl₂/EtOH 10:1); ¹H NMR (CD₃OD, 400 MHz):

d=0.78 (t, J=6.6 Hz, 3H, w-CH₃hexadecyl), 1.15 (m, 24H, 12CH₂

hexadecyl), 1.49 (s, 6H, 2CH₃Far), 1.57 (s, 6H, 2CH₃Far), 1.26–1.68

(m, 8H, 2CH₂Lys, 2 CH₂hexadecyl), 1.93 (s, 3H, CH₃acetyl), 2.00 (s,

3H, CH₃Met), 1.76–2.05 (m, 12H, b-CH₂Met, CH₂Lys, 4 CH₂Far),

2.46 (m, 2H, g-CH₂Met), 2.68–3.17 (m, 6H, a-CH₂hexadecyl, b-CH₂

Cys, -SCH₂-), 3.26–3.46 (m, 4H, b-CH₂Dap, e-CH₂Lys), 3.64 (s, 3H,

OCH₃), 3.68 (m, 1H, b-CH_2aSer), 3.87 (m, 1H, b-CH_2bSer), 4.25–4.54

(m, 5H, 5a-CH), 5.00 (m, 2H, 2CH Far), 5.14 (m, 1H, CH Far);

¹³C NMR (CD₃OD, 100.6 MHz): d=14.0 (w-CH₃hexadecyl), 16.5 (CH₃

acetyl), 16.8 (CH₃Met), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.3 (CH₃Far),

19.3 (CH₂), 21.3 (CH₂), 24.0 (CH₂), 24.3 (CH₂), 25.3 (CH₃Far), 28.6

(CH₂), 29.2 (CH₂), 29.3 (CH₂), 30.0 (CH₂), 30.0 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.9 (CH₂), 31.8 (CH₂), 32.5 (CH₂), 33.8 (CH₂), 34.4 (CH₂), 39.8

(CH₂Far), 40.5 (CH₂Far), 42.0 (b-CH₂Dap), 44.6 (e-CH₂Lys), 50.4

(OCH₃), 53.5 (a-CH), 54.2 (a-CH), 54.5 (a-CH₂hexadecyl), 56.0 (a-CH),

56.1 (a-CH), 57.1 (a-CH), 64.8 (b-CH₂Ser), 117.1 (CH Far), 121.7 (CH

Far), 122.5 (CH Far), 133.9 (C Far), 138.8 (C Far), 139.3 (C Far), 170.9

(C=O), 172.0 (C=O), 174.7 (C=O), 175.4 (C=O); HRMS (FAB, 3-NBA):

m/z: calcd for C₅₄H₁₀₀N₇O₁₀S₃: 1102.6695, found: 1102.6685 [M+H]⁺.

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-3-(hexadecane-1-sulfonylami-

no)]propionic acid (18): [Pd(PPh₃)₄] (15 mg, 0.01 mmol) was added to a

U

degassed solution of allyl ester 17 (90 mg, 0.13 mmol) and N,N’-dimethyl-

barbituric acid (12 mg, 0.08 mmol) in THF (5 mL). The mixture was stir-

red at room temperature for 1.5 h. After removal of the solvent under re-

duced pressure, the residue was purified by chromatography (CH₂Cl₂/

EtOH 5:1 + 1% HOAc) to yield 18 as a colorless solid (85 mg, 99%).

M.p. 828C; [a]²_D⁰= ꢀ8.6 (c=1.0 in CHCl₃); R_f=0.09 (CHCl₃/MeOH 5:1

1

+ 1% HOAc); H NMR (CD₃OD, 400 MHz): d=0.86 (t, J=6.5 Hz, 3H,

w-CH₃hexadecyl), 1.27 (m, 24H, 12CH₂hexadecyl), 1.43 (m, 2H, g-

CH₂hexadecyl), 1.77 (m, 2H, b-CH₂hexadecyl), 1.98 (m, 1H, b-CH_2a

Met), 2.09 (s, 3H, CH₃acetyl), 2.12 (s, 3H, CH₃Met), 2.13 (m, 1H, b-

CH_2bMet), 2.60 (m, 2H, g-CH₂Met), 3.01 (m, 2H, a-CH₂hexadecyl),

3.51 (m, 2H, b-CH₂Dap), 3.80 (dd, J=3.7, 11.3 Hz, 1H, b-CH_2aSer),

3.96 (dd, J=3.8, 11.3 Hz, 1H, b-CH_2bSer), 4.27 (m, 1H, a-CH Dap), 4.43

(t, J=4.3 Hz, 1H, a-CH Ser), 4.48 (dd, J=6.0, 8.1 Hz, 1H, a-CH Met);

¹³C NMR (CD₃OD, 100.6 MHz): d=14.2 (w-CH₃hexadecyl), 22.6 (CH₃

acetyl), 22.9 (CH₃Met), 23.4 (CH₂), 23.7 (CH₂), 28.5 (CH₂), 28.6 (CH₂),

28.7 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.2 (CH₂), 29.3 (CH₂), 29.3 (CH₂),

29.3 (CH₂), 29.5 (CH₂), 29.8 (CH₂), 30.3 (CH₂), 31.2 (g-CH₂Met), 32.1

(b-CH₂Met), 43.5 (b-CH₂Dap), 53.2 (a-CH Dap), 53.3 (a-CH Met), 55.6

(a-CH₂hexadecyl), 60.9 (a-CH Ser), 61.9 (b-CH₂Ser), 169.5 (C=O),

170.8 (C=O), 172.6 (C=O), 175.8 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₂₉H₅₆N₄NaO₈S₂: 675.3440, found: 675.3469 [M+Na]⁺.

l-Glutamic acid 1-tert-butyl ester5-methyl ester(23) : Isobutene (75 mL)

was added to a solution of H-l-Glu(OMe)-OH 22 (5 g, 31.03 mmol) in di-

U

oxane (45 mL) and conc. H₂SO₄(4.5 mL) in a sealed tube. After stirring

at room temperature for 3 d the reaction mixture was poured into a mix-

ture of ethyl acetate (250 mL) and saturated NaHCO₃solution (250 mL).

The organic layer was separated and washed again with saturated

NaHCO₃solution (50 mL). The organic layer was dried over Na₂SO₄and

the solvent was removed under reduced pressure to yield 23 as a color-

less oil (3.728 g, 55%). [a]_D²⁰= +25.7 (c=1.0 in CHCl₃); R_f=0.17 (cyclo-

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-3-(hexadecane-1-sulfonylami-

no)propionyl]-N^e-allyloxycarbonyl-l-lysyl-S-farnesyl-l-cysteine

methyl

ester(20) : At 08C, EDC (19 mg, 0.10 mmol) was added to a solution of

tripeptide 18 (52 mg, 0.08 mmol), freshly prepared H-Lys(Aloc)-Cys-

(Far)-OMe 19 (44 mg, 0.08 mmol) and HOBt (25 mg, 0.16 mmol) in

AHCTREUNG

ACHTREUNG

1

CH₂Cl₂(6 mL). The solution was warmed to room temperature and stir-

red for 16 h. Then, the reaction mixture was diluted with ethyl acetate

(30 mL) and subsequently washed with 1n HCl (10 mL) and Na₂CO₃sol-

ution (10%, 10 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent under reduced pressure, the residue was purified

by chromatography (CH₂Cl₂/EtOH 20:1, then 10:1) to yield 20 as color-

less solid (47 mg, 50%). M.p. 1018C; [a]²_D⁰= +6.1 (c=0.5 in CHCl₃);

R_f=0.30 (CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃/CD₃OD, 400 MHz): d=

0.78 (t, J=6.6 Hz, 3H, w-CH₃hexadecyl), 1.15 (m, 24H, 12CH₂hexa-

decyl), 1.49 (s, 6H, 2CH₃Far), 1.57 (s, 6H, 2CH₃Far), 1.26–1.68 (m,

8H, 2CH₂Lys, 2 CH₂hexadecyl), 1.93 (s, 3H, CH₃acetyl), 2.00 (s,

3H, CH₃Met), 1.76–2.05 (m, 12H, b-CH₂Met, CH₂Lys, 4 CH₂Far),

2.46 (m, 2H, g-CH₂Met), 2.68–3.17 (m, 6H, a-CH₂hexadecyl, b-CH₂

Cys, -SCH₂-), 3.26–3.46 (m, 4H, b-CH₂Dap, e-CH₂Lys), 3.64 (s, 3H,

OCH₃), 3.68 (m, 1H, b-CH_2aSer), 3.87 (m, 1H, b-CH_2bSer), 4.25–4.54

(m, 7H, 5a-CH, -OCH₂CH=CH₂), 5.00 (m, 2H, 2CH Far), 5.08–5.22

(m, 3H, CH Far, -OCH₂CH=CH₂), 5.74 (m, 1H, -OCH₂CH=CH₂);

¹³C NMR (CDCl₃/CD₃OD, 100.6 MHz): d=14.0 (w-CH₃hexadecyl), 16.5

(CH₃acetyl), 16.8 (CH₃Met), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.3 (CH₃

Far), 19.3 (CH₂), 21.3 (CH₂), 24.0 (CH₂), 24.3 (CH₂), 25.3 (CH₃Far), 28.6

(CH₂), 29.2 (CH₂), 29.3 (CH₂), 30.0 (CH₂), 30.0 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.9 (CH₂), 31.8 (CH₂), 32.5 (CH₂), 33.8 (CH₂), 34.4 (CH₂), 39.8

(CH₂Far), 40.5 (CH₂Far), 42.0 (b-CH₂Dap), 44.6 (e-CH₂Lys), 50.4

(OCH₃), 53.5 (a-CH), 54.2 (a-CH), 54.5 (a-CH₂hexadecyl), 56.0 (a-CH),

56.1 (a-CH), 57.1 (a-CH), 64.8 (b-CH₂Ser), 67.3 (OCH₂CH=CH₂), 115.1

(OCH₂CH=CH₂), 117.1 (CH Far), 121.7 (CH Far), 122.5 (CH Far), 133.9

(C Far), 137.5 (OCH₂CH=CH₂), 138.8 (C Far), 139.3 (C Far), 157.5 (C=

O), 170.9 (C=O), 172.0 (C=O), 174.7 (C=O), 175.4 (C=O); HRMS (FAB,

3-NBA): m/z: calcd for C₅₈H₁₀₃N₇NaO₁₂S₃: 1208.6727, found: 1208.6708

[M+Na]⁺.

hexane/ethyl acetate 1:1); H NMR (CDCl₃, 400 MHz): d=1.44 (brs, 2H,

NH₂), 1.46 (s, 9H, C(CH₃)₃), 1.78 (m, 1H, b-CH_2aGlu), 1.99 (m, 1H, b-

CH_2bGlu), 2.42 (t, J=7.8 Hz, 2H, g-CH₂Glu), 3.32 (dd, J=5.3, 8.2 Hz,

1H, a-CH Glu), 3.69 (d, 3H, OCH₃); ¹³C NMR (CDCl₃, 100.6 MHz): d=

G

28.1 (C

A

54.5 (OCH₃), 81.4 (C(CH₃)₃), 173.8 (C=O), 175.0 (C=O); HRMS (FAB,

H

3-NBA): m/z: calcd for C₁₀H₂₀NO₄: 218.1393, found: 218.1372 [M+H]⁺.

N-Diallyl-l-glutamic acid 1-tert-butyl ester5-methyl ester(24) : A solu-

tion of H-Glu(OMe)-OtBu 23 (8 g, 36.82 mmol), tetrabutylammoniumio-

H

dide (1.36 g, 3.68 mmol), NEt₃(12.8 mL, 92.05 mmol) and allyl bromide

(64.7 mL, 736.39 mmol) in THF (184 mL) was stirred under reflux for

2 d. The suspension was cooled down to room temperature and the sol-

vent was removed under reduced pressure. The residue was dissolved in

ethyl acetate (500 mL) and washed with Na₂CO₃solution (10%, 2

50 mL). The organic layer was dried over Na₂SO₄and the solvent was re-

moved under reduced pressure. The residue was purified by chromatog-

raphy (cyclohexane/ethyl acetate 8:1) to yield 24 as a colorless oil (8.76 g,

80%). [a]²_D⁰= +17.3 (c=1.0 in CHCl₃); R_f=0.49 (cyclohexane/ethyl ace-

tate 2:1); ¹H NMR (CDCl₃, 400 MHz): d=1.42 (s, 9H, C

(CH₃)₃), 1.84

E

(m, 2H, b-CH₂Glu), 2.32 (m, 2H, g-CH₂Glu), 2.97 (dd, J=8.0, 14.4 Hz,

2H, 2-NCH_2aCH=CH₂), 3.28 (m, 3H, a-CH Glu, 2-NCH_2bCH=CH₂),

3.60 (s, 3H, OCH₃), 4.98–5.12 (m, 4H, 2-NCH₂CH=CH₂), 5.64 (m, 2H,

2-NCH₂CH=CH₂); ¹³C NMR (CDCl₃, 100.6 MHz): d=24.7 (b-CH₂

Glu), 28.4 (C

ACHTREUNG

61.3 (NCH₂CH=CH₂), 80.9 (C

AHCTREUNG

(NCH₂CH=CH₂), 171.9 (C=O), 173.8 (C=O); HRMS (FAB, 3-NBA): m/

z: calcd for C₁₆H₂₈NO₄: 298.2019, found: 209.2027 [M+H]⁺.

(2S)-Diallylamino-5-hydroxypentanoic acid tert-butyl ester(25) : A 2m

solution of LiBH₄in THF (12.2 mL, 24.48 mmol) was added to a solution

of diester 24 (5.6 g, 18.83 mmol) in THF (90 mL). The solution was stir-

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4131

H. Waldmann, A. Giannis et al.

red at room temperature for 36 h. Then, saturated NaCl solution (50 mL)

was added and the mixture was extracted with ethyl acetate (3100 mL).

The combined organic layers were dried over Na₂SO₄, and, after removal

of the solvents under reduced pressure, the residue was purified by chro-

matography (cyclohexane/ethyl acetate 3:1) to yield 25 as a colorless oil

(3.601 g, 71%). [a]²_D⁰= 24.0 (c=1.0 in CHCl₃); R_f=0.31 (cyclohexane/

ethyl acetate 2:1); ¹H NMR (CDCl₃, 400 MHz): d=1.48 (s, 9H, C-

action mixture was diluted with ethyl acetate (100 mL) and subsequently

washed with 1n HCl (50 mL) and Na₂CO₃solution (10%, 50 mL). The

organic layer was dried over Na₂SO₄, and, after removal of the solvent

under reduced pressure, the residue was purified by chromatography

(CH₂Cl₂/EtOH 15:1, then 10:1) to yield 28 as colorless oil (536 mg,

69%). R_f=0.23 (CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃, 400 MHz): d=

0.87 (t, J = 6.6 Hz, 3H, w-CH₃Hhe), 1.24 (m, 28H, 14CH₂Hhe), 1.40

A

(m, 4H, d-CH₂Hhe, b-CH₂Hhe), 1.48 (s, 9H, C(CH₃)₃), 1.70–2.14 (m,

N

2-NCH_2aCH=CH₂), 3.32 (m, 1H, a-CH), 3.45 (m, 2H, 2-NCH_2bCH=

CH₂), 3.53 (m, 1H, d-CH_2a), 3.62 (m, 1H, d-CH_2b), 5.10–5.24 (m, 4H, 2

-NCH₂CH=CH₂), 5.80 (m, 2H, 2-NCH₂CH=CH₂); ¹³C NMR (CDCl₃,

4H, g-CH₂Hhe, b-CH₂Met), 2.01 (s, 3H, CH₃acetyl), 2.10 (s, 3H, CH₃

Met), 2.53 (m, 2H, g-CH₂Met), 3.54 (m, 1H, -CH(OH)-), 3.72 (dd, J=

3.7, 11.3 Hz, 1H, b-CH_2aSer), 3.95 (dd, J=3.8, 11.3 Hz, 1H, b-CH_2bSer),

4.44 (m, 1H, a-CH Hhe), 4.60 (m, 1H, a-CH Ser), 4.71 (dd, J=6.1,

8.0 Hz, 1H, a-CH Met), 7.02 (m, 1H, NH), 7.62 (m, 2H, 2NH);

¹³C NMR (CDCl₃, 100.6 MHz): d=14.3, (w-CH₃Hhe), 22.9 (CH₃acetyl),

100.6 MHz): d=27.7 (b-CH₂), 28.5 (C

ACHTREUNG

62.8 (NCH₂CH=CH₂), 63.6 (d-CH₂), 81.2 (C

AHCTREUNG

CH₂), 136.0 (NCH₂CH=CH₂), 172.3 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₁₅H₂₈NO₃: 270.2070, found: 270.2063 [M+H]⁺.

23.2 (CH₃Met), 25.6–30.3 (17CH₂Hhe), 30.1 (C

Met), 53.0 (a-CH Hhe), 53.4 (a-CH Met), 54.9 (a-CH Ser), 62.9 (b-CH₂

Ser), 71.4 (CHOH), 82.5 (C(CH₃)₃), 170.3 (C=O), 171.0 (C=O), 171.5

A

(2S)-Diallylamino-5-hydroxyheneicosanic acid tert-butyl ester(26) : A sol-

ution of Dess–Martin periodinane in CH₂Cl₂(15%, 9.59 mL, 4.51 mmol)

was added to a solution of alcohol 25 (810 mg, 3.01 mmol) in CH₂Cl₂

(27 mL). The mixture was stirred at room temperature for 1 h. The solu-

tion was diluted with ethyl acetate (100 mL) and a mixture of saturated

Na₂S₂O₃solution (10 mL) and saturated NaHCO₃solution (30 mL) was

added. After the two phases had become clear, the separated organic

layer was washed with saturated NaHCO₃solution (30 mL). The organic

layer was dried over Na₂SO₄, and, after removal of the solvents under re-

duced pressure, the residue was purified by chromatography (cyclohex-

ane/ethyl acetate 4:1) to yield the aldehyde as a colorless oil (800 mg,

97%).

AHCTREUNG

(C=O), 172.1 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₃₅H₆₇N₃NaO₇S [M+Na]⁺: 696.4600, found: 696.4612.

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-5-hydroxy]heneicosanoyl-N^e-

(allyloxycarbonyl)-l-lysyl-S-farnesyl-l-cysteine methyl ester(29) : A 1m

NaOH solution (16 mL) was added to

a solution of tripeptide 28

(530 mg, 0.79 mmol) in MeOH (16 mL). The solution was stirred at room

temperature for 3 h. Then, the solvent was removed under reduced pres-

sure. The residue was dissolved in CHCl₃(100 mL) and then washed with

saturated NH₄Cl solution (50 mL). The organic layer was dried over

Na₂SO₄and the solvent was removed under reduced pressure to yield the

free acid as colorless solid (486 mg, 100%).

At ꢀ788C, a solution of hexadecyl magnesium bromide freshly prepared

from magnesium (109 mg, 4.48 mmol) and hexadecyl bromide (1.41 mL,

4.48 mmol) in THF (1.5 mL) was added to a solution of the above alde-

hyde (800 mg, 2.92 mmol) in THF (15 mL). After 15 min, water (5 mL)

was added to the reaction mixture. Then, the solution was diluted with

ethyl acetate (50 mL) and washed with water (10 mL). The organic layer

was dried over Na₂SO₄, and, after removal of the solvents under reduced

pressure, the residue was purified by chromatography (cyclohexane/ethyl

acetate 10:1) to yield 26 as a colorless oil (930 mg, 63%). R_f=0.44 (cyclo-

hexane/ethyl acetate 3:1); ¹H NMR (CDCl₃, 400 MHz): d=0.82 (t, J=

6.6 Hz, 3H, w-CH₃Hhe), 1.13–1.38 (m, 32H, b-CH₂Hhe, 15CH₂Hhe),

At 08C, EDC (32 mg, 0.16 mmol) was added to a solution of the above

acid (67 mg, 0.11 mmol), freshly prepared H-Lys

N

(59 mg, 0.11 mmol) and HOBt (34 mg, 0.22 mmol) in CH₂Cl₂(5 mL) and

DMF (3 mL). The solution was warmed to room temperature and stirred

for 15 h. Then, the reaction mixture was diluted with ethyl acetate

(50 mL) and subsequently washed with 1n HCl (15 mL) and Na₂CO₃sol-

ution (10%, 15 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent under reduced pressure, the residue was purified

by chromatography (cyclohexane/ethyl acetate 1:1, then CH₂Cl₂/EtOH

20:1, then 10:1) to yield 29 as colorless oil (55 mg, 44%). R_f=0.11

(CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃/CD₃OD, 400 MHz): d=0.80 (t,

J=6.6 Hz, 3H, w-CH₃Hhe), 1.52 (s, 6H, 2CH₃Far), 1.61 (s, 6H, 2

CH₃Far), 1.13–1.70 (m, 36H, 15CH₂Hhe, b-CH₂Hhe, 2CH₂Lys),

1.93 (s, 3H, CH₃acetyl), 2.00 (s, 3H, CH₃Met), 1.72–2.05 (m, 14H, g-

CH₂Hhe, b-CH₂Met, CH₂Lys, 4 CH₂Far), 2.49 (m, 2H, g-CH₂Met),

2.72 (m, 1H, b-CH_2aCys), 2.86 (m, 1H, b-CH_2bCys), 3.00–3.18 (m, 4H,

-SCH₂-, e-CH₂Lys), 3.50 (m, 1H, -CH(OH)-), 3.67 (s, 3H, OCH₃), 3.72

(m, 1H, b-CH_2aSer), 3.95 (m, 1H, b-CH_2bSer), 4.25–4.60 (m, 7H, 5a-

CH, -OCH₂CH=CH₂), 5.01 (m, 2H, 2CH Far), 5.08–5.22 (m, 3H, CH

Far, -OCH₂CH=CH₂), 5.80 (m, 1H, -OCH₂CH=CH₂); ¹³C NMR (CDCl₃/

CD₃OD, 100.6 MHz): d=14.3 (w-CH₃Hhe), 17.0 (CH₃Far), 17.2 (CH₃

Far), 19.3 (CH₃Far), 22.9 (CH₃acetyl), 23.2 (CH₃Met), 25.3 (CH₃Far),

23.6–34.2 (25CH₂), 37.9 (b-CH₂Met), 39.9 (CH₂Far), 40.5 (CH₂Far),

44.6 (e-CH₂Lys), 50.4 (OCH₃), 54.2 (a-CH), 55.9 (a-CH), 56.0 (a-CH),

57.1 (a-CH), 59.6 (a-CH), 64.9 (b-CH₂Ser), 67.3 (OCH₂CH=CH₂), 82.2

(CHOH), 115.1 (OCH₂CH=CH₂), 117.1 (CH Far), 121.7 (CH Far), 122.5

(CH Far), 133.9 (C Far), 137.5 (OCH₂CH=CH₂), 139.3 (C Far), 142.4 (C

Far), 157.5 (C=O), 170.9 (C=O), 172.1 (C=O), 174.7 (C=O), 175.1 (C=

O); HRMS (FAB, 3-NBA): m/z: calcd for C₆₀H₁₀₆N₆NaO₁₁S₂: 1173.7261,

found: 1173.7325 [M+Na]⁺.

1.40 (s, 9H, C(CH₃)₃), 1.52–1.80 (m, 2H, g-CH₂Hhe), 3.01 (m, 2H, 2-

G

NCH_2aCH=CH₂), 3.21 (m, 1H -CHOH), 3.24–3.46 (m, 3H, a-CH Hhe,

2-NCH_2bCH=CH₂), 5.02–5.14 (m, 4H, 2-NCH₂CH=CH₂), 5.86 (m,

2H, 2-NCH₂CH=CH₂); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.2 (w-CH₃

Hhe), 29.9 (C

ACHTREUNG

(NCH₂CH=CH₂), 71.3 (HCOH), 81.2 (C

AHCTREUNG

136.1 (NCH₂CH=CH₂), 172.3 (C=O); HRMS (FAB, 3-NBA): m/z: calcd

for C₃₁H₆₀NO₃: 494.4574, found: 494.4540 [M+H]⁺.

(2S)-Amino-5-hydroxyheneicosanic acid tert-butyl ester(27) : [Pd(PPh₃)₄]

U

(222 mg, 0.19 mmol) was added to a degassed solution of diallyl amine 26

(950 mg, 1.92 mmol) and N,N’-dimethylbarbituric acid (1.8 g,

11.59 mmolmmol) in CH₂Cl₂(100 mL). The mixture was stirred for 6 h at

358C. Then, the solution was diluted with ethyl acetate (250 mL) and

washed with Na₂CO₃solution (10%, 50 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (cyclohexane/ethyl acetate

3:1, then CH₂Cl₂/EtOH 10:1) to yield 27 as a yellowish solid (597 mg,

75%). M.p. 638C; R_f=0.29 (CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃,

400 MHz): d=0.88 (t, J=6.6 Hz, 3H, w-CH₃Hhe), 1.21–1.47 (m, 28H,

14CH₂Hhe), 1.48–1.52 (m, 4H, d-CH₂Hhe, b-CH₂Hhe), 1.49 (s, 9H,

C

ACHTREUNG

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-5-hydroxy]heneicosanoyl-l-

lysyl-S-farnesyl-l-cysteine methyl ester(30)

:

[Pd(PPh₃)₄] (1 mg,

A

ACHTREUNG

0.01 mmol) was added to a solution of pentapeptide 29 (7 mg, 0.01 mmol)

and N,N’-dimethylbarbituric acid (1 mg, 0.01 mmol) in THF (2 mL). The

mixture was stirred at room temperature for 6 h. After removal of the

solvent under reduced pressure, the residue was purified by chromatogra-

phy (CH₂Cl₂/EtOH 10:1, then CH₂Cl₂/MeOH 1:1 + 1% NEt₃) to yield

30 as a slightly yellow solid (5 mg, 80%). M.p. 718C; R_f=0.01 (CH₂Cl₂/

EtOH 10:1); ¹H NMR (CDCl₃/CD₃OD, 400 MHz): d=0.80 (t, J=6.6 Hz,

3H, w-CH₃Hhe), 1.52 (s, 6H, 2CH₃Far), 1.61 (s, 6H, 2CH₃Far),

(HCOH), 81.2 (C(CH₃)₃), 172.5 (C=O); HRMS (FAB, 3-NBA): m/z:

C

N-Acetyl-l-methionyl-l-seryl-[(2S)-amino-5-hydroxy]heneicosanic acid

tert-butyl ester(28) : At 08C, EDC (272 mg, 1.39 mmol) was added to a

solution of dipeptide 16 (388 mg, 1.39 mmol), H-Hhe-OtBu 27 (480 mg,

1.16 mmol) and HOBt (362 mg, 2.32 mmol) in CH₂Cl₂(25 mL). The solu-

tion was warmed to room temperature and stirred for 20 h. Then, the re-

4132

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

1.13–1.70 (m, 36H, 15CH₂Hhe, b-CH₂Hhe, 2CH₂Lys), 1.93 (s, 3H,

CH₃acetyl), 2.00 (s, 3H, CH₃Met), 1.72–2.05 (m, 14H, g-CH₂Hhe, b-

CH₂Met, CH₂Lys, 4 CH₂Far), 2.49 (m, 2H, g-CH₂Met), 2.72 (m, 1H,

b-CH_2aCys), 2.86 (m, 1H, b-CH_2bCys), 3.00–3.18 (m, 4H, -SCH₂-, e-CH₂

Lys), 3.50 (m, 1H, -CH(OH)-), 3.67 (s, 3H, OCH₃), 3.72 (m, 1H, b-CH_2a

Ser), 3.95 (m, 1H, b-CH_2bSer), 4.25–4.60 (m, 5H, 5a-CH), 5.01 (m,

2H, 2CH Far), 5.14 (m, 1H, CH Far); ¹³C NMR (CDCl₃/CD₃OD,

100.6 MHz): d=14.2 (w-CH₃Hhe), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.1

(CH₃Far), 22.7 (CH₃acetyl), 23.0 (CH₃Met), 25.3 (CH₃Far), 23.2–34.7

(25CH₂), 37.9 (b-CH₂Met), 39.9 (CH₂Far), 40.5 (CH₂Far), 44.6 (e-

CH₂Lys), 50.4 (OCH₃), 54.2 (a-CH), 55.9 (a-CH), 56.2 (a-CH), 57.1 (a-

CH), 59.6 (a-CH), 64.7 (b-CH₂Ser), 82.0 (CHOH), 117.3 (CH Far), 121.7

(CH Far), 122.5 (CH Far), 133.9 (C Far), 139.3 (C Far), 142.4 (C Far),

170.7 (C=O), 172.0 (C=O), 174.7 (C=O), 175.3 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₅₆H₁₀₃N₆O₉S₂: 1067.7229, found: 1067.7237

[M+H]⁺.

2.90 (m, 1H, b-CH_2bPro), 3.73 (s, 3H, OCH₃), 3.87 (m, 2H, d-CH₂Pro),

4.75 (m, 1H, a-CH Pro); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.4 (C-

AHCTREUNG

(FAB, 3-NBA): m/z: calcd for C₁₁H₁₈NO₅: 244.1186, found: 244.1191

[M+H]⁺.

N-tert-Butyloxycarbonyl-4-(cyanomethylen)-l-proline methyl ester (36):

Cyanomethyldiethylphosphonate 35 (4.4 mL, 28.01 mmol) was added to a

suspension of NaH (708 mg, 28.01 mmol) in THF (80 mL). After stirring

at room temperature for 10 min,

a solution of ketone 34 (1.974 g,

8 mmol) in THF (80 mL) was added. The reaction mixture was stirred

for 1.5 h and then saturated NH₄Cl solution (20 mL) was added. After re-

moval of the solvent under reduced pressure, the residue was dissolved in

ethyl acetate (200 mL) and washed subsequently with saturated NH₄Cl

solution (50 mL) and saturated NaCl solution (50 mL). The organic layer

was dried over Na₂SO₄, and, after removal of the solvent under reduced

pressure, the residue was purified by chromatography (cyclohexane/ethyl

acetate 6:1) to yield 36 as a yellowish oil (1.812 g, 85%). [a]²_D⁰= ꢀ23.3

(c=1.0 in CHCl₃); R_f=0.40 (cyclohexane/ethyl acetate 1:1); ¹H NMR

N-tert-Butyloxycarbonyl-(4R)-hydroxy-l-proline (32): At 08C,

a 1 m

NaOH solution (24 mL) and a solution of Boc₂O (4.118 g, 18.30 mmol) in

dioxane (6 mL) were subsequently added to a solution of (4R)-hydroxy-

l-proline 31 (2 g, 15.25 mmol) in dioxane/H₂O (2:1, 54 mL). The solution

was warmed to room temperature and stirred for 7 h. The solvents were

removed to 30 mL and the residue was dissolved in ethyl acetate

(200 mL). The solution was acidified with 1n HCl to pH 3 and extracted

with ethyl acetate (350 mL). The combined organic layers were dried

over Na₂SO₄and the solvent was removed under reduced pressure to

yield 32 as a colorless foam (3.36 g, 95%). M.p. 968C; [a]²_D⁰= ꢀ54.1 (c=

1.0 in CHCl₃); R_f=0.56 (CHCl₃/MeOH 1:1); ¹H NMR (CDCl₃,

(CDCl₃, 400 MHz): d=1.45 (s, 9H, C(CH₃)₃), 3.10–3.22 (m, 2H, b-CH₂

A

Pro), 3.72 (s, 3H, OCH₃), 4.12–4.40 (m, 2H, d-CH₂Pro), 4.97 (m, 1H, a-

CH Pro), 5.81 (m, 1H, -CHCN); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.5

(C

ACHTREUNG

Pro), 80.9 (C

AHCTREUNG

(C=CHCN), 171.2 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₁₃H₁₈N₂O₄: 266.1267, found: 266.1265 [M]⁺.

N-tert-Butyloxycarbonyl-(4S)-(N’-allyloxycarbonylaminoethyl)-l-proline

methyl ester(37) : PtO₂·H₂O (63 mg, 0.25 mmol) was added to a degassed

solution of nitrile 36 (670 mg, 2.52 mmol) in EtOH (114 mL) and CHCl₃

(2.2 mL). The reaction mixture was hydrogenated under hydrogen atmos-

phere at room temperature for 2.5 h. Then, the suspension was filtered

through a pad of Celite. The solvents were removed under reduced pres-

sure to yield the amine as a colorless oil (685 mg, 100%).

400 MHz): d=1.35 (s, 9H, C

3.36–3.58 (m, 2H, d-CH₂Pro), 4.30–4.43 (m, 2H, a-CH Pro, g-CH Pro);

¹³C NMR (CDCl₃, 100.6 MHz): d=20.9 (C

(CH₃)₃), 28.5 (b-CH₂Pro), 54.7

(d-CH₂Pro), 54.8 (a-CH Pro), 69.2 (g-CH Pro), 81.2 (C(CH₃)₃), 154.2

(CH₃)₃), 2.06–2.35 (m, 2H, b-CH₂Pro),

AHCTREUNG

(C=O), 178.4 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₀H₁₈NO₅:

232.1186, found: 232.1175 [M+H]⁺; elemental analysis calcd (%) for

C₁₀H₁₇NO₅: C 51.94, H 7.41, N 6.06; found: C 51.77, H 7.35, N 6.00.

AlocCl (413 mL, 3.77 mmol) was then added to a solution of the above

amine (685 mg, 2.52 mmol) and NEtiPr₂(861 mL, 5.03 mmol) in CH₂Cl₂

(27 mL). After stirring at room temperature for 18 h, the reaction mix-

ture was diluted with ethyl acetate (100 mL) and washed with 1n HCl

(30 mL). The organic layer was dried over Na₂SO₄, and, after removal of

the solvents under reduced pressure, the residue was purified by chroma-

tography (cyclohexane/ethyl acetate 1:1) to yield 37 as a colorless oil

(852 mg, 95%). [a]²_D⁰= ꢀ17.6 (c=0.5 in CHCl₃); R_f=0.31 (cyclohexane/

N-tert-Butyloxycarbonyl-(4R)-hydroxy-l-proline methyl ester (33):

Cs₂CO₃(2.56 g, 7.24 mmol) was added to a solution of acid 32 (3.35 g,

14.49 mmol) in MeOH (80 mL). After stirring at room temperature for

5 min, the solvent was removed under reduced pressure and the residue

was coevaporated with toluene. The resulting salt was dissolved in DMF

(100 mL). Methyl iodide (1.350 mL, 21.73 mmol) was added to this solu-

tion and the mixture was stirred at room temperature for 20 h. After re-

moval of the solvent under reduced pressure, the residue was dissolved in

ethyl acetate (200 mL) and washed subsequently with Na₂CO₃solution

(10%, 50 mL) and saturated NaCl solution (30 mL). The organic layer

was dried over Na₂SO₄, and, after removal of the solvent, the residue was

purified by chromatography (cyclohexane/ethyl acetate 1:1) to yield 33 as

a yellowish oil (3.138 g, 88%). [a]²_D⁰= ꢀ50.6 (c=1.0 in CHCl₃); R_f=0.22

(cyclohexane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.41 (s,

ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.38 (s, 9H, C

(CH₃)₃),

T

1.53 (m, 3H, g-CH Pro, -CH₂CH₂NH-), 2.10 (m, 1H, b-CH_2aPro), 2.41

(m, 1H, b-CH_2bPro), 2.98 (m, 1H, d-CH_2aPro), 3.14 (m, 2H,

-CH₂CH₂NH-), 3.62 (s, 3H, OCH₃), 3.62–3.74 (m, 1H, d-CH_2bPro), 4.08–

4.20 (m, 1H, a-CH Pro), 4.48 (m, 2H, -OCH₂CH=CH₂), 4.63 (m, 1H,

NH), 5.11–5.24 (m, 2H, OCH₂CH=CH₂), 5.84 (m, 1H, OCH₂CH=CH₂);

¹³C NMR (CDCl₃, 100.6 MHz): d=27.1 (b-CH₂Pro), 27.5 (g-CH Pro),

9H, C(CH₃)₃), 2.03 (m, 1H, b-CH_2aPro), 2.25 (m, 1H, b-CH_2bPro), 2.93

A

28.7 (C

ACHTREUNG

(brs, 1H, OH), 3.41–3.62 (m, 2H, d-CH₂Pro), 3.71 (s, 3H, OCH₃), 4.30–

4.48 (m, 2H, a-CH Pro, g-CH Pro); ¹³C NMR (CDCl₃, 100.6 MHz): d=

50.4 (OCH₃), 57.5 (a-CH Pro), 66.1 (OCH₂CH=CH₂), 80.9 (C

ACHTREUNG

118.06 (OCH₂CH=CH₂), 132.8 (OCH₂CH=CH₂), 156.6 (C=O), 159.4 (C=

O), 172.0 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₇H₂₉N₂O₆:

357.2026, found: 357.2022 [M+H]⁺.

28.6 (C

ACHTREUNG

(a-CH Pro), 69.4 (g-CH Pro), 80.6 (C

AHCTREUNG

HRMS (FAB, 3-NBA): m/z: calcd for C₁₁H₁₉NO₅: 246.1342, found:

246.1338 [M+H]⁺.

(4S)-(N-Allyloxycarbonylaminoethyl)-l-proline methyl ester hydrotri-

fluoroacetate (38): TFA (15 mL) was added to a solution of proline 37

(698 mg, 1.96 mmol) in CH₂Cl₂(60 mL). After stirring at room tempera-

ture for 30 min the solvent was removed under reduced pressure and the

residue was coevaporated with toluene to yield 38 as a colorless oil

(725 mg, quant.). [a]_D²⁰= ꢀ39.8 (c=1.0 in MeOH); R_f=0.38 (CH₂Cl₂/

EtOH 5:1); ¹H NMR (CDCl₃, 400 MHz): d=1.56–1.74 (m, 3H, g-CH

Pro, -CH₂CH₂NH-), 2.46 (m, 1H, b-CH_2aPro), 2.59 (m, 1H, b-CH_2bPro),

2.96 (m, 1H, d-CH_2aPro), 3.14 (m, 2H, -CH₂CH₂NH-), 3.62 (s, 3H,

OCH₃), 3.54 (m, 1H, d-CH_2bPro), 4.36 (m, 1H, a-CH Pro), 4.48 (m, 2H,

-OCH₂CH=CH₂), 5.02 (brd, 1H, NH), 5.11–5.23 (m, 2H, -OCH₂CH=

CH₂), 5.83 (m, 1H, -OCH₂CH=CH₂); ¹³C NMR (CDCl₃, 100.6 MHz): d=

32.7 (b-CH₂Pro), 34.9 (g-CH Pro), 36.2 (CH₂CH₂NH), 39.4

(CH₂CH₂NH), 50.7 (d-CH₂Pro), 53.7 (OCH₃), 59.1 (a-CH Pro), 65.8

(OCH₂CH=CH₂), 117.8 (OCH₂CH=CH₂), 128.4 (CF₃COOH), 132.9

N-tert-Butyloxycarbonyl-4-oxo-l-proline methyl ester (34): A solution of

Dess–Martin periodinane in CH₂Cl₂(15%, 27.2 mL, 12.79 mmol) was

added to a solution of alcohol 33 (2.6 g, 10.6 mmol) in CH₂Cl₂(104 mL).

The mixture was stirred at room temperature for 1 h. The solution was

diluted with ethyl acetate (200 mL) and a mixture of saturated Na₂S₂O₃

solution (10 mL) and saturated NaHCO₃solution (30 mL) was added.

After the two phases had become clear, the separated organic layer was

washed with saturated NaHCO₃solution (30 mL). The organic layer was

dried over Na₂SO₄, and, after removal of the solvents under reduced

pressure, the residue was purified by chromatography (cyclohexane/ethyl

acetate 1:1) to yield 34 as a colorless oil (2.1 g, 81%). [a]²_D⁰= ꢀ36.7 (c=

0.5 in CHCl₃); R_f=0.42 (cyclohexane/ethyl acetate 1:1); ¹H NMR

(CDCl₃, 400 MHz): d=1.45 (s, 9H, C

A

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4133

H. Waldmann, A. Giannis et al.

(OCH₂CH=CH₂), 161.8 (C=O), 169.5 (C=O), 176.0 (CF₃COOH); HRMS

(FAB, 3-NBA): m/z: calcd for C₁₂H₂₁N₂O₄: 257.1502, found: 257.1512

[M+H]⁺.

sure, the residue was purified by chromatography (cyclohexane/ethyl ace-

tate 1:1, then 1:5) to yield 41 as colorless oil (701 mg, 87%). [a]²_D⁰= ꢀ8.4

(c=0.5 in CHCl₃); R_f=0.29 (cyclohexane/ethyl acetate); ¹H NMR

(CDCl₃, 400 MHz): d=0.86 (t, J=6.6 Hz, 3H, w-CH₃hexadecyl), 1.23

(m, 24H, 12CH₂hexadecyl), 1.37 (m, 2H, g-CH₂hexadecyl), 1.50 (s,

6H, 2CH₃Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃Far), 1.60 (m,

3H, g-CH Pro, -CH₂CH₂NH-), 1.76 (m, 2H, b-CH₂hexadecyl), 1.82–2.08

(m, 8H, 4CH₂Far), 2.29 (m, 1H, b-CH_2aPro), 2.45 (m, 1H, b-CH_2b

Pro), 2.58 (m, 2H, a-CH₂b-Ala), 2.72 (m, 2H, -SCH₂-), 2.96 (m, 3H, a-

CH₂hexadecyl, d-CH_2aPro), 3.20 (m, 4H, b-CH₂b-Ala, b-CH₂Cys), 3.41

(m, 3H, -CH₂CH₂NH-, d-CH_2bPro), 3.68 (s, 3H, OCH₃), 4.53 (m, 2H,

-OCH₂CH=CH₂), 4.76 (m, 1H, a-CH Cys), 4.92 (m, 1H, a-CH Pro),

5.04–5.29 (m, 6H, 3CH Far, NH, -OCH₂CH=CH₂), 5.54 (m, 1H, NH),

5.90 (m, 1H, -OCH₂CH=CH₂), 7.11 (m, 1H, NH); ¹³C NMR (CDCl₃,

100.6 MHz): d=14.3 (w-CH₃hexadecyl), 16.2 (CH₃Far), 16.4 (CH₃Far),

16.5 (CH₃Far), 17.9 (CH₃Far), 22.8 (CH₂), 22.9 (CH₂), 23.8 (CH₂), 24.7

(CH₂), 28.5 (CH₂), 28.6 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.5 (CH₂), 29.6

(CH₂), 29.8 (CH₂), 30.3 (CH₂), 30.5 (CH₂), 30.7 (CH₂), 31.5 (CH₂), 32.1

(CH₂), 32.3 (CH₂), 33.3 (CH₂), 33.4 (CH₂), 35.2 (g-CH Pro), 35.5 (CH₂),

36.7 (CH₂CH₂NH), 38.8 (b-CH₂b-Ala), 39.2 (CH₂Far), 39.4

(CH₂CH₂NH), 39.8 (CH₂Far), 42.7 (a-CH₂b-Ala), 52.5 (d-CH₂Pro),

52.9 (OCH₃), 57.4 (a-CH Cys), 59.1 (a-CH₂hexadecyl), 59.2 (a-CH Pro),

65.8 (OCH₂CH=CH₂), 117.9 (OCH₂CH=CH₂), 119.7 (CH Far), 123.9

(CH Far), 124.5 (CH Far), 132.3 (C Far), 133.0 (OCH₂CH=CH₂), 135.5

(C Far), 140.8 (C Far), 156.4 (C=O), 170.2 (C=O), 171.0 (C=O), 172.8

(C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₄₉H₈₇N₄O₈S₂: 923.5966,

found: 923.5955 [M+H]⁺.

3-(Hexadecane-1-sulfonylamino)propionic acid (39): TMSCl (651 mL,

5.13 mmol) was added to a suspension of b-alanine (229 mg, 2.56 mmol)

in CH₃CN (10 mL) and the reaction mixture was heated under reflux for

1 h. The solution was cooled down to room temperature and then NEt₃

(1.43 mL, 10.26 mmol) and a solution of hexadecanesulfonic acid chloride

(1 g, 3.08 mmol) in CH₃CN (10 mL) was subsequently added. After stir-

ring at room temperature for 16 h, the reaction mixture was diluted with

ethyl acetate (50 mL) and washed with 1n HCl (20 mL). The organic

layer was dried over Na₂SO₄, and, after removal of the solvents under re-

duced pressure, the residue was recrystallized from acetone to yield 39 as

a

colorless solid (900 mg, 93%). M.p. 1248C; ¹H NMR (CDCl₃,

400 MHz): d=0.72 (t, J=6.6 Hz, 3H, w-CH₃hexadecyl), 1.18 (m, 24H,

12CH₂hexadecyl), 1.24 (m, 2H, g-CH₂hexadecyl), 1.60 (m, 2H, b-CH₂

hexadecyl), 2.40 (t, J=6.4 Hz, 2H, a-CH₂b-Ala), 2.82 (m, 2H, a-CH₂

hexadecyl), 3.12 (t, J=6.4 Hz, 2H, b-CH₂b-Ala), 4.20 (brs, 1H, NH);

¹³C NMR (CDCl₃, 100.6 MHz): d=17.9 (w-CH₃hexadecyl), 26.7 (CH₂),

27.5 (CH₂), 32.3 (CH₂), 32.4 (CH₂), 32.6 (CH₂), 32.7 (CH₂), 33.0 (CH₂),

33.2 (CH₂), 33.7 (CH₂), 33.8 (CH₂), 34.6 (CH₂), 34.8 (CH₂), 35.2 (CH₂),

35.9 (CH₂), 38.8 (b-CH₂b-Ala), 42.7 (a-CH₂b-Ala), 56.5 (a-CH₂hexa-

decyl), 178.0 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₉H₄₀NO₄S:

378.2679, found: 378.2677 [M+H]⁺.

N-[3-(Hexadecane-1-sulfonylamino)propionyl]-(4S)-(N’-allyloxycarbonyl-

aminoethyl)-l-proline methyl ester (40): At 08C, EDC (279 mg,

1.43 mmol) was added to a solution of proline 38 (440 mg, 1.19 mmol), b-

alanine 39 (538 mg, 1.43 mmol), HOBt (371 mg, 2.38 mmol) and NEtiPr₂

(244 mL, 1.43 mmol) in CH₂Cl₂(45 mL). The solution was warmed to

room temperature and stirred for 18 h. Then, the reaction mixture was di-

luted with ethyl acetate (100 mL) and subsequently washed with 1n HCl

(30 mL) and Na₂CO₃solution (10%, 30 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (cyclohexane/ethyl acetate

1:2) to yield 40 as colorless oil (593 mg, 81%). [a]²_D⁰= ꢀ13.2 (c=1.0 in

CHCl₃); R_f=0.30 (cyclohexane/ethyl acetate 1:3); ¹H NMR (CDCl₃,

400 MHz): d=0.86 (t, J=6.6 Hz, 3H, w-CH₃hexadecyl), 1.23 (m, 24H,

12CH₂hexadecyl), 1.37 (m, 2H, g-CH₂hexadecyl), 1.60 (m, 3H, g-CH

Pro, -CH₂CH₂NH-), 1.76 (m, 2H, b-CH₂hexadecyl), 2.29 (m, 1H, b-CH_2a

Pro), 2.45 (m, 1H, b-CH_2bPro), 2.58 (m, 2H, a-CH₂b-Ala), 2.97 (m, 2H,

a-CH₂hexadecyl), 3.19 (m, 3H, b-CH₂b-Ala, d-CH_2aPro), 3.36 (m, 3H,

-CH₂CH₂NH-, d-CH_2bPro), 3.71 (s, 3H, OCH₃), 4.34 (m, 1H, NH), 4.53

(m, 2H, -OCH₂CH=CH₂), 4.92 (m, 1H, a-CH Pro), 5.16–5.34 (m, 3H,

NH, -OCH₂CH=CH₂), 5.88 (m, 1H, -OCH₂CH=CH₂); ¹³C NMR (CDCl₃,

100.6 MHz): d=14.3 (w-CH₃hexadecyl), 22.8 (CH₂), 24.7 (CH₂), 28.5

(CH₂), 28.6 (CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.8

(CH₂), 30.3 (CH₂), 30.5 (CH₂), 30.7 (CH₂), 31.5 (CH₂), 32.3 (CH₂), 33.3

(CH₂), 35.2 (g-CH Pro), 35.5 (CH₂), 36.7 (CH₂CH₂NH), 38.8 (b-CH₂b-

Ala), 39.4 (CH₂CH₂NH), 42.7 (a-CH₂b-Ala), 52.5 (d-CH₂Pro), 52.8

(OCH₃), 59.1 (a-CH₂hexadecyl), 59.2 (a-CH Pro), 65.8 (OCH₂CH=

CH₂), 117.9 (OCH₂CH=CH₂), 133.0 (OCH₂CH=CH₂), 156.4 (C=O),

170.2 (C=O), 172.8 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₃₁H₅₈N₃O₇S: 616.3996, found: 616.3976 [M+H]⁺.

N-[3-(Hexadecane-1-sulfonylamino)propionyl]-(4S)-(aminoethyl)-l-

prolyl-S-farnesyl-l-cysteine methyl ester(42)

:

[Pd(PPh₃)₄] (38 mg,

A

0.03 mmol) was added to a degassed solution of tripeptide 41 (300 mg,

0.33 mmol) and N,N’-dimethylbarbituric acid (51 mg, 0.33 mmol) in THF

(30 mL). The mixture was stirred at room temperature for 3 h. After re-

moval of the solvent under reduced pressure, the residue was purified by

chromatography (CH₂Cl₂/EtOH 20:1, then 10:1, then 5:1 + 1% NEt₃,

then 1:1 + 1% NEt₃) to yield 42 as a slightly yellow foam (216 mg,

79%). [a]²_D⁰= ꢀ14.5 (c=0.5 in CHCl₃); R_f=0.07 (CH₂Cl₂/EtOH 10:1);

¹H NMR (CD₃OD, 400 MHz): d=0.86 (t, J=6.6 Hz, 3H, w-CH₃hexa-

decyl), 1.23 (m, 24H, 12CH₂hexadecyl), 1.37 (m, 2H, g-CH₂hexadec-

yl), 1.50 (s, 6H, 2CH₃Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃

Far), 1.60 (m, 3H, g-CH Pro, -CH₂CH₂NH₂), 1.76 (m, 2H, b-CH₂hexa-

decyl), 1.82–2.08 (m, 8H, 4CH₂Far), 2.29 (m, 1H, b-CH_2aPro), 2.45

(m, 1H, b-CH_2bPro), 2.58 (m, 2H, a-CH₂b-Ala), 2.72 (m, 2H, -SCH₂-),

2.96 (m, 3H, a-CH₂hexadecyl, d-CH_2aPro), 3.20 (m, 4H, b-CH₂b-Ala,

b-CH₂Cys), 3.41 (m, 3H, -CH₂CH₂NH₂, d-CH_2bPro), 3.68 (s, 3H,

OCH₃), 4.76 (m, 1H, a-CH Cys), 4.92 (m, 1H, a-CH Pro), 5.04–5.29 (m,

3H, 3CH Far); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.3 (w-CH₃hexa-

decyl), 16.2 (CH₃Far), 16.4 (CH₃Far), 16.5 (CH₃Far), 17.9 (CH₃Far),

22.8 (CH₂), 22.9 (CH₂), 23.8 (CH₂), 24.7 (CH₂), 28.5 (CH₂), 28.6 (CH₂),

28.9 (CH₂), 29.1 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.8 (CH₂), 30.3 (CH₂),

30.5 (CH₂), 30.7 (CH₂), 31.5 (CH₂), 32.1 (CH₂), 32.3 (CH₂), 33.3 (CH₂),

33.4 (CH₂), 35.2 (g-CH Pro), 35.5 (CH₂), 36.7 (CH₂CH₂NH₂), 38.8 (b-

CH₂b-Ala), 39.2 (CH₂Far), 39.4 (CH₂CH₂NH₂), 39.8 (CH₂Far), 42.7 (a-

CH₂b-Ala), 52.5 (d-CH₂Pro), 52.9 (OCH₃), 57.4 (a-CH Cys), 59.1 (a-

CH₂hexadecyl), 59.2 (a-CH Pro), 119.7 (CH Far), 123.9 (CH Far), 124.5

(CH Far), 132.3 (C Far), 135.5 (C Far), 140.8 (C Far), 170.2 (C=O), 171.0

(C=O), 172.8 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₄₅H₈₂N₄NaO₆S₂: 861.5576, found: 861.5562 [M+Na]⁺.

N-[3-(Hexadecane-1-sulfonylamino)propionyl]-(4S)-(N’-allyloxycarbonyl-

aminoethyl)-l-prolyl-S-farnesyl-l-cysteine methyl ester(41)

: A 1m

NaOH solution (1.75 mL) was added to a solution of dipeptide 40

(539 mg, 0.88 mmol) in MeOH (3.5 mL). After stirring at room tempera-

ture for 3 h, the reaction mixture was diluted with CHCl₃(30 mL), acidi-

fied with 1n HCl to pH 3 and extracted with CHCl₃(230 mL). The

combined organic layers were dried over Na₂SO₄and the solvents were

removed under reduced pressure to yield the free acid (526 mg, 100%).

N-[(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionyl]-(4S)-

(N’-allyloxycarbonylaminoethyl)-l-proline methyl ester (43): At 08C,

EDC (81 mg, 0.41 mmol) was added to a solution of proline 38 (150 mg,

0.35 mmol), Ac-Dap(SO₂C₁₆H₃₃)-OH

4 (128 mg, 0.35 mmol), HOBt

(108 mg, 0.69 mmol) and NEtiPr₂in CH₂Cl₂(15 mL). The solution was

warmed to room temperature and stirred for 18 h. Then, the reaction

mixture was diluted with ethyl acetate (50 mL) and subsequently washed

with 1n HCl (15 mL) and Na₂CO₃solution (10%, 15 mL). The organic

layer was dried over Na₂SO₄, and, after removal of the solvent under re-

duced pressure, the residue was purified by chromatography (cyclohex-

ane/ethyl acetate 1:5) to yield 43 as colorless solid (176 mg, 76%). M.p.

788C; [a]²_D⁰= ꢀ9.2 (c=1.0 in CHCl₃); R_f=0.09 (cyclohexane/ethyl ace-

At 08C, EDC (205 mg, 1.05 mmol) was added to a solution of the above

acid (562 mg, 0.87 mmol), H-Cys(Far)-OMe 10 (356 mg, 1.05 mmol) and

R

HOBt (273 mg, 1.75 mmol) in CH₂Cl₂(50 mL). The solution was warmed

to room temperature and stirred for 20 h. Then, the reaction mixture was

diluted with ethyl acetate (100 mL) and subsequently washed with 1n

HCl (30 mL) and Na₂CO₃solution (10%, 30 mL). The organic layer was

dried over Na₂SO₄, and, after removal of the solvent under reduced pres-

4134

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

tate 1:5); ¹H NMR (CDCl₃, 400 MHz): d=0.81 (t, J=6.5 Hz, 3H, w-CH₃

hexadecyl), 1.18 (m, 24H, 12CH₂hexadecyl), 1.33 (m, 2H, g-CH₂hexa-

decyl), 1.55 (m, 3H, g-CH Pro, -CH₂CH₂NH-), 1.72 (m, 2H, b-CH₂hexa-

decyl), 1.94 (s, 3H, CH₃acetyl), 2.25 (m, 1H, b-CH_2aPro), 2.45 (m, 1H,

b-CH_2bPro), 2.93 (m, 2H, a-CH₂hexadecyl), 3.14 (m, 3H, d-CH_2aPro,

-CH₂CH₂NH-), 3.30 (m, 3H, d-CH_2bPro, b-CH₂Dap), 3.68 (s, 3H,

OCH₃), 3.99 (m, 1H, NH), 4.40 (m, 1H, a-CH Dap), 4.48 (m, 2H,

-OCH₂CH=CH₂), 4.86 (m, 1H, a-CH Pro), 5.12–5.25 (m, 2H,

-OCH₂CH=CH₂), 5.64 (m, 1H, NH), 5.85 (m, 1H, -OCH₂CH=CH₂), 6.70

(m, 1H, NH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.3 (w-CH₃hexadec-

yl), 22.8 (CH₃acetyl), 22.9 (CH₂), 24.5 (CH₂), 28.6 (CH₂), 28.8 (CH₂),

29.2 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.9 (CH₂), 30.2 (CH₂), 30.5 (CH₂),

30.6 (CH₂), 31.3 (CH₂), 32.0 (CH₂), 33.1 (CH₂), 35.1 (g-CH Pro), 36.7

(CH₂), 36.9 (CH₂CH₂NH), 39.6 (CH₂CH₂NH), 45.0 (b-CH₂Dap), 51.1 (d-

CH₂Pro), 52.8 (a-CH Dap), 52.9 (OCH₃), 59.3 (a-CH₂hexadecyl), 59.4

(a-CH Pro), 65.7 (OCH₂CH=CH₂), 117.8 (OCH₂CH=H₂), 133.0

(OCH₂CH=CH₂), 156.5 (C=O), 168.5 (C=O), 169.2 (C=O), 172.8 (C=O);

HRMS (FAB, 3-NBA): m/z: calcd for C₃₃H₆₀N₄NaO₈S: 695.4032, found:

695.4054 [M+Na]⁺.

residue was purified by chromatography (CH₂Cl₂/EtOH 10:1, then 5:1,

then 1:1, then 1:1 + 1% NEt₃) to yield 45 as a slightly yellow foam

(87 mg, 64%). [a]²_D⁰= ꢀ4.9 (c=0.5 in CHCl₃); R_f=0.04 (CH₂Cl₂/EtOH

1:1); ¹H NMR (CD₃OD, 400 MHz): d=0.81 (t, J=6.6 Hz, 3H, w-CH₃

hexadecyl), 1.18 (m, 24H, 12CH₂hexadecyl), 1.34 (m, 2H, g-CH₂hexa-

decyl), 1.50 (s, 6H, 2CH₃Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃

Far), 1.59 (m, 3H, g-CH Pro, -CH₂CH₂NH₂), 1.72 (m, 2H, b-CH₂hexa-

decyl), 1.94 (s, 3H, CH₃acetyl), 1.82–2.08 (m, 8H, 4CH₂Far), 2.25 (m,

1H, b-CH_2aPro), 2.45 (m, 1H, b-CH_2bPro), 2.72 (m, 2H, -SCH₂-), 2.96

(m, 3H, a-CH₂hexadecyl, d-CH_2aPro), 3.20 (m, 4H, b-CH₂Dap, b-CH₂

Cys), 3.41 (m, 3H, -CH₂CH₂NH₂, d-CH_2bPro), 3.71 (s, 3H, OCH₃), 4.76

(m, 1H, a-CH Cys), 4.92 (m, 1H, a-CH Pro), 5.04–5.29 (m, 3H, 3CH

Far); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.3 (w-CH₃hexadecyl), 16.2

(CH₃Far), 16.4 (CH₃Far), 16.5 (CH₃Far), 17.9 (CH₃Far), 22.8 (CH₃

acetyl), 22.8 (CH₂), 22.9 (CH₂), 23.8 (CH₂), 24.7 (CH₂), 28.5 (CH₂), 28.9

(CH₂), 29.1 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.8 (CH₂), 30.3 (CH₂), 30.5

(CH₂), 30.7 (CH₂), 31.5 (CH₂), 32.1 (CH₂), 32.3 (CH₂), 33.3 (CH₂), 33.4

(CH₂), 35.2 (g-CH Pro), 35.5 (CH₂), 36.7 (CH₂CH₂NH₂), 39.2 (CH₂Far),

39.4 (CH₂CH₂NH₂), 39.8 (CH₂Far), 45.7 (b-CH₂Dap), 52.5 (d-CH₂Pro),

52.8 (a-CH Dap), 53.0 (OCH₃), 57.4 (a-CH Cys), 59.1 (a-CH₂hexadec-

yl), 59.2 (a-CH Pro), 119.7 (CH Far), 123.9 (CH Far), 124.5 (CH Far),

132.3 (C Far), 135.5 (C Far), 140.8 (C Far), 170.2 (C=O), 171.0 (C=O),

171.2 (C=O), 172.8 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₄₇H₈₆N₅O₇S₂: 897.5969, found: 897.5938 [M+H]⁺.

N-[(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionyl]-(4S)-

(N’-allyloxycarbonylaminoethyl)-l-prolyl-S-farnesyl-l-cysteine

methyl

ester(44) : A 1m NaOH solution (416 mL) was added to a solution of di-

peptide 43 (140 mg, 0.21 mmol) in MeOH (832 mL). After stirring at

room temperature for 3 h, the reaction mixture was diluted with CHCl₃

(30 mL), acidified with 1n HCl to pH 3 and extracted with CHCl₃(2

20 mL). The combined organic layers were dried over Na₂SO₄and the

solvents were removed under reduced pressure to yield the free acid

(137 mg, 100%).

(2S)-[Bis(tert-butyloxycarbonyl)amino]butyrolactone (47): At 08C, a sol-

ution of Boc₂O (29.666 g, 131.85 mmol) in CH₂Cl₂(50 mL) was added to

a

solution of (2S)-aminobutyrolactone hydrobromide 46 (20 g,

109.88 mmol) and NEtiPr₂(56.43 mL, 329.63 mmol) in CH₂Cl₂(500 mL).

The solution was warmed to room temperature and stirred for 20 h.

Then, the reaction mixture was washed with 1n HCl (100 mL). The or-

ganic layer was dried over Na₂SO₄and the solvent was removed under

reduced pressure to yield the monoprotected lactone as a colorless solid

(22.1 g, 100%).

At 08C, EDC (49 mg, 0.25 mmol) was added to a solution of the above

acid (137 mg, 0.21 mmol), H-Cys(Far)-OMe 10 (85 mg, 0.25 mmol) and

N

HOBt (65 mg, 0.42 mmol) in CH₂Cl₂(25 mL). The solution was warmed

to room temperature and stirred for 20 h. Then, the reaction mixture was

diluted with ethyl acetate (50 mL) and subsequently washed with 1n HCl

(15 mL) and Na₂CO₃solution (10%, 15 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (cyclohexane/ethyl acetate

1:8, then CH₂Cl₂/EtOH 10:1) to yield 44 as colorless oil (169 mg, 83%).

[a]²_D⁰= ꢀ9.0 (c=0.5 in CHCl₃); R_f=0.13 (cyclohexane/ethyl acetate 1:8);

¹H NMR (CDCl₃, 400 MHz): d=0.81 (t, J=6.6 Hz, 3H, w-CH₃hexadec-

yl), 1.18 (m, 24H, 12CH₂hexadecyl), 1.34 (m, 2H, g-CH₂hexadecyl),

1.50 (s, 6H, 2CH₃Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃Far),

1.59 (m, 3H, g-CH Pro, -CH₂CH₂NH-), 1.72 (m, 2H, b-CH₂hexadecyl),

1.94 (s, 3H, CH₃acetyl), 1.82–2.08 (m, 8H, 4CH₂Far), 2.25 (m, 1H, b-

CH_2aPro), 2.45 (m, 1H, b-CH_2bPro), 2.72 (m, 2H, -SCH₂-), 2.96 (m, 3H,

a-CH₂hexadecyl, d-CH_2aPro), 3.20 (m, 4H, b-CH₂Dap, b-CH₂Cys),

3.41 (m, 3H, -CH₂CH₂NH-, d-CH_2bPro), 3.71 (s, 3H, OCH₃), 4.53 (m,

2H, -OCH₂CH=CH₂), 4.76 (m, 1H, a-CH Cys), 4.92 (m, 1H, a-CH Pro),

5.04–5.29 (m, 6H, 3CH Far, NH -OCH₂CH=CH₂), 5.54 (m, 1H, NH),

5.90 (m, 1H, -OCH₂CH=CH₂), 7.11 (m, 1H, NH); ¹³C NMR (CDCl₃,

100.6 MHz): d=14.3 (w-CH₃hexadecyl), 16.2 (CH₃Far), 16.4 (CH₃Far),

16.5 (CH₃Far), 17.9 (CH₃Far), 22.8 (CH₃Acetyl), 22.8 (CH₂), 22.9

(CH₂), 23.8 (CH₂), 24.7 (CH₂), 28.5 (CH₂), 28.6 (CH₂), 28.9 (CH₂), 29.1

(CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.8 (CH₂), 30.3 (CH₂), 30.5 (CH₂), 30.7

(CH₂), 31.5 (CH₂), 32.1 (CH₂), 32.3 (CH₂), 33.3 (CH₂), 33.4 (CH₂), 35.2

(g-CH Pro), 35.5 (CH₂), 36.7 (CH₂CH₂NH), 39.2 (CH₂Far), 39.4

(CH₂CH₂NH), 39.8 (CH₂Far), 45.7 (b-CH₂Dap), 52.5 (d-CH₂Pro), 52.8

(a-CH Dap), 53.0 (OCH₃), 57.4 (a-CH Cys), 59.1 (a-CH₂hexadecyl),

59.2 (a-CH Pro), 65.8 (OCH₂CH=CH₂), 117.9 (OCH₂CH=CH₂), 119.7

(CH Far), 123.9 (CH Far), 124.5 (CH Far), 132.3 (C Far), 133.0

(OCH₂CH=CH₂), 135.5 (C Far), 140.8 (C Far), 156.4 (C=O), 170.2 (C=

O), 171.0 (C=O), 171.2 (C=O), 172.8 (C=O); HRMS (FAB, 3-NBA):

m/z: calcd for C₅₁H₉₀N₅O₉S₂: 980.6181, found: 980.6188 [M+H]⁺.

Boc₂O (27.2 g, 120.86 mmol) was then added to a solution of the above

lactone (22.1 g, 109.88 mmol) and DMAP (2.74 g, 21.98 mmol) in CH₃CN

(380 mL). After stirring at room temperature for 15 h, the solvent was re-

moved under reduced pressure and the residue was purified by chroma-

tography (cyclohexane/ethyl acetate 1:1) to yield 47 as a colorless solid

(31.03 g, 94%). M.p. 758C; [a]²_D⁰= ꢀ42.5 (c=1.0 in CHCl₃); R_f=0.51

(cyclohexane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.52 (s,

18H, C

CH_2a), 4.48 (dt, J=5.7, 6.3 Hz, 1H, g-CH_2b), 5.12 (t, J=9.9 Hz, 1H, a-

CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=26.8 (b-CH₂), 28.2 (C

(CH₃)₃),

54.0 (a-CH), 65.4 (g-CH₂), 84.3 (C(CH₃)₃), 151.7 (C=O), 173.9 (C=O);

(CH₃)₃), 2.53 (m, 2H, b-CH₂), 4.26 (dt, J=8.4, 9.1 Hz, 1H, g-

ACHTREUNG

AHCTREUNG

HRMS (FAB, 3-NBA): m/z: calcd for C₁₄H₂₄NO₆: 302.1604, found:

302.1615 [M+H]⁺.

(2S)-[Bis(tert-butyloxycarbonyl)amino]-4-hydroxybutyric acid methyl

ester(48) : A 1m CsOH solution (102.59 mL) was added to a solution of

lactone 47 (30.915 g, 102.59 mmol) in MeOH (220 mL). After stirring at

room temperature for 2 h, the solvent was removed under reduced pres-

sure and the residue was coevaporated with toluene. The resulting salt

was dissolved in DMF (834 mL). Methyl iodide (7.67 mL, 123.11 mmol)

was added to this solution and the mixture was stirred at room tempera-

ture for 18 h. After removal of the solvent under reduced pressure, the

residue was dissolved in ethyl acetate (500 mL) and washed subsequently

with Na₂CO₃solution (10%, 100 mL) and saturated NaCl solution

(30 mL). The organic layer was dried over Na₂SO₄, and, after removal of

the solvent, the residue was purified by chromatography (cyclohexane/

ethyl acetate 4:1) to yield 48 as a colorless solid (27.019 g, 79%). M.p.

898C; [a]²_D⁰= ꢀ37.8 (c=1.0 in CHCl₃); R_f=0.36 (cyclohexane/ethyl ace-

tate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.51 (s, 18H, C

(CH₃)₃), 2.03

G

(m, 1H, b-CH_2a), 2.42 (m, 1H, b-CH_2b), 2.48 (brs, 1H, OH), 3.59 (m, 1H,

g-CH_2a), 3.73 (m, s, 4H, g-CH_2b, OCH₃), 5.01 (dd, J=4.7, 9.8 Hz, 1H, a-

N-[(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionyl]-(4S)-

aminoethyl-l-prolyl-S-farnesyl-l-cysteine methyl ester(45) : [Pd(PPh₃)₄]

A

CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.1 (C

ACHTREUNG

(18 mg, 0.02 mmol) was added to a degassed solution of tripeptide 44

(149 mg, 0.15 mmol) and N,N’-dimethylbarbituric acid (24 mg,

0.15 mmol) in THF (15 mL). The mixture was stirred at room tempera-

ture for 24 h. After removal of the solvent under reduced pressure, the

AHCTREUNG

171.5 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₅H₂₈NO₇: 334.1867,

found: 334.1886 [M+H]⁺.

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4135

H. Waldmann, A. Giannis et al.

(2S)-[Bis(tert-butyloxycarbonyl)amino]-4-cyano-4-hydroxybutyric

acid

(m, 2H, d-CH₂), 3.99 (m, 1H, g-CH), 4.57 (m, 1H, a-CH), 6.59 (m, 1H,

methyl ester(49) solution of DMSO (9.352 mL,

:

At ꢀ608C,

a

NH), 7.36–7.46 (m, 6H, arom. CH), 7.62–7.71 (m, 4H, arom. CH);

131.78 mmol) in CH₂Cl₂(28 mL) was added to a solution of oxalyl chlor-

ide (8.285 mL, 94.48 mmol) in CH₂Cl₂(189 mL). The reaction mixture

was stirred at ꢀ608C for 30 min. Then, a solution of alcohol 48 (13.5 g,

40.49 mmol) in CH₂Cl₂(57 mL) was added. After stirring at ꢀ608C for

4 h, NEtiPr₂(39.293 mL, 281.91 mmol) was added and the reaction mix-

ture was warmed to room temperature. The solution was poured into a

1m KH₂PO₄solution and extracted with ethyl acetate (100 mL). The or-

ganic layer was then subsequently washed with H₂O (50 mL) and saturat-

ed NaCl solution (50 mL). The organic layers were dried over Na₂SO₄

and the solvents were removed under reduced pressure to yield the crude

aldehyde as colorless oil (12.314 g, 92%).

¹³C NMR (CDCl₃, 100.6 MHz): d=19.3 (C

A

N

silyl), 28.2 (C

ACHTREUNG

AHCTREUNG

(arom. CH), 133.3 (arom. C), 133.5 (arom. C), 135.7 (arom. CH), 135.8

(arom CH), 151.9 (C=O), 169.5 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₃₁H₄₄N₂NaO₆Si: 591.2869, found: 591.2888 [M+Na]⁺.

(3S)-[Bis(tert-butyloxycarbonyl)amino]-5-hydroxypiperidine-2-on (52): A

1m TBAF solution in THF (9.7 mL, 9.7 mmol) was added to a solution of

lactam 51 (1.1 g, 1.93 mmol) in THF (30 mL). After stirring at room tem-

perature for 1.5 h, the solvent was removed under reduced pressure. The

residue was purified by chromatography (cyclohexane/ethyl acetate 1:1)

to yield 52 as a colorless solid (638 mg, quant.). M.p. 818C; R_f=0.19 (cy-

clohexane/ethyl acetate 1:10); ¹H NMR (CDCl₃, 400 MHz): d=1.47 (s,

TMSCN (9.3 mL, 72.43 mmol) was added to a solution of the above alde-

hyde (12.314 g, 37.25 mmol) in CH₂Cl₂(200 mL). The reaction mixture

was stirred at room temperature for 20 h. Then, the solvent was removed

under reduced pressure, the residue was dissolved in methanol and NH₄F

(2.7 g, 72.43 mmol) was added. After stirring for 5 min, the solvent was

removed under reduced pressure. The residue was dissolved in ethyl ace-

tate (500 mL) and subsequently washed with H₂O (100 mL) and saturat-

ed NaCl solution (100 mL). The organic layer was dried over Na₂SO₄,

and, after removal of the solvent under reduced pressure, the residue was

purified by chromatography (cyclohexane/ethyl acetate 6:1) to yield 49 as

a colorless oil (11.68 g, 90%). R_f=0.40 (cyclohexane/ethyl acetate 1:1);

18H, C

3.44–3.61 (brs, 1H, OH), 4.05 (m, 1H, g-CH), 4.81 (m, 1H, a-CH), 6.51

(m, 1H, NH); ¹³C NMR (CDCl₃, 100.6 MHz): d=27.9 (C

(CH₃)₃), 33.0 (b-

CH₂), 52.3 (a-CH), 53.3 (d-CH₂), 63.5 (g-CH), 82.9 (C(CH₃)₃), 152.1 (C=

A

AHCTREUNG

O), 169.4 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₅H₂₇N₂O₆:

331.1870, found: 331.1856 [M+H]⁺.

(3S)-[Bis(tert-butyloxycarbonyl)amino]-5-(cyanomethylene)piperidine-2-

on (53): A solution of Dess–Martin periodinane in CH₂Cl₂(15%, 8.2 mL,

3.86 mmol) was added to a solution of alcohol 52 (638 mg, 1.93 mmol) in

CH₂Cl₂(20 mL). The mixture was stirred at room temperature for 1 h.

The solution was diluted with ethyl acetate (100 mL) and a mixture of sa-

turated Na₂S₂O₃solution (10 mL) and saturated NaHCO₃solution

(30 mL) was added. After the two phases had become clear, the separat-

ed organic layer was washed with saturated NaHCO₃solution (30 mL).

The organic layer was dried over Na₂SO₄, and, after removal of the sol-

vents under reduced pressure, the residue was purified by chromatogra-

phy (cyclohexane/ethyl acetate 1:1) to yield the ketone as a colorless oil

(445 mg, 70%).

¹H NMR (CDCl₃, 400 MHz): d=1.53 (s, 18H, C

(CH₃)₃), 2.36 (m, 1H, b-

E

CH_2a), 2.73 (m, 1H, b-CH_2b), 3.76 (s, 3H, OCH₃), 4.51 (m, 2H, g-CH),

4.74 (brs, 1H, OH), 5.10 (m, 1H, a-CH); ¹³C NMR (CDCl₃, 100.6 MHz):

d=28.2 (C

A

84.7 (C(CH₃)₃), 119.3 (CN), 152.5 (C=O), 170.4 (C=O); HRMS (FAB, 3-

A

NBA): m/z: calcd for C₁₆H₂₇N₂O₇: 359.1819, found: 359.1846 [M+H]⁺.

(2S)-[Bis(tert-butyloxycarbonyl)amino]-4-(tert-butyldiphenylsilanyloxy)-

4-cyanobutyric acid methyl ester (50): TBDPSCl (11.661 mL,

43.95 mmol) was added to

a solution of cyanohydrin 49 (10.5 g,

29.3 mmol) and imidazole (6 g, 87.89 mmol) in DMF (30 mL). The solu-

tion was stirred at room temperature for 15 h. Then, the solvent was re-

moved under reduced pressure, the residue was dissolved in ethyl acetate

(500 mL) and washed subsequently with 1n HCl (100 mL) and saturated

NaCl solution (100 mL). The organic layer was dried over Na₂SO₄, and,

after removal of the solvent under reduced pressure, the residue was pu-

rified by chromatography (cyclohexane/ethyl acetate 10:1) to yield 50 as

a colorless oil (15.517 g, 90%). R_f=0.13 (cyclohexane/ethyl acetate 7:1);

Cyanomethyldiethylphosphonate 35 (746 mL, 28.01 mmol) was added to a

suspension of NaH (120 mg, 4.73 mmol) in THF (15 mL). After stirring

at room temperature for 1 h, a solution of the above ketone (445 mg,

1.35 mmol) in THF (15 mL) was added. The reaction mixture was stirred

for 1.5 h. After removal of the solvent under reduced pressure, the resi-

due was dissolved in ethyl acetate (50 mL) and washed subsequently with

H₂O (10 mL), 1n HCl (10 mL) and saturated Na₂CO₃solution (10 mL).

The organic layer was dried over Na₂SO₄, and, after removal of the sol-

vent under reduced pressure, the residue was purified by chromatography

(cyclohexane/ethyl acetate 2:1) to yield 53 as a colorless solid (428 mg,

90%). M.p. 1288C; [a]²_D⁰= +27.4 (c=1.0 in CHCl₃); R_f=0.18 (cyclohex-

ane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.45 (s, 18H, C-

¹H NMR (CDCl₃, 400 MHz): d=1.09 (s, 9H, C

(CH₃)₃Boc), 2.36 (m, 1H, b-CH_2a), 2.75 (m, 1H, b-CH_2b), 3.66 (s, 3H,

A

C

ACHTREUNG

OCH₃), 4.53 (dd, J=5.3, 7.4 Hz, 1H, g-CH), 5.05, 5.19 (dd, J=4.9,

8.4 Hz, 1H, a-CH), 7.37–7.49 (m, 6H, arom. CH), 7.63–7.73 (m, 4H,

arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=19.4 (C

ACHTREUNG

(CH₃)₃), 2.40 (dd, J=9.6, 16.8, 1H, b-CH_2a), 2.99 (m, 1H, b-CH_2b), 3.02

(C

A

ACHTREUNG

(m, 2H, d-CH₂), 5.13 (m, 1H, a-CH), 6.05 (m, 1H, -CHCN), 7.10 (m,

1H, NH); ¹³C NMR (CDCl₃, 100.6 MHz): d=22.1 (b-CH₂), 28.2 (C-

AHCTREUNG

128.2 (arom. CH), 128.2 (arom. CH), 130.5 (arom. C), 131.6 (arom. C),

135.9 (arom. CH), 136.1 (arom. CH), 151.7 (C=O), 170.4 (C=O); HRMS

(FAB, 3-NBA): m/z: calcd for C₃₂H₄₄N₂NaO₇Si: 619.2818, found:

619.2830 [M+Na]⁺.

A

G

(CN), 122.6 (C=CHCN), 152.1 (C=O), 167.2 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₁₇H₂₆N₃O₅: 352.1873, found: 352.1852 [M+H]⁺.

3-tert-Butyldisulfanyl-(2R)-(4-methoxytetrahydropyran-4-yloxy)propionic

acid methyl ester(57) : A 0.2m NaOMe solution in MeOH (11.375 mL,

2.28 mmol) was added to a solution of thioester 55 (670 mg, 2.29 mmol)

in MeOH (27 mL). After stirring at room temperature for 30 min, the

solvent was removed under reduced pressure. The residue was dissolved

in ethyl acetate (50 mL) and washed subsequently with saturated NH₄Cl

solution (10 mL) and saturated NaCl solution (10 mL). The organic layer

was dried over Na₂SO₄, the solvent was removed under reduced pressure

and the residue was dissolved in DMF (86 mL). This solution was added

to a solution of 56 (2.200 g, 6.87 mmol) in DMF (43 mL). After stirring at

room temperature for 18 h, the solvent was removed under reduced pres-

sure. The residue was dissolved in ethyl acetate (100 mL) and washed

with saturated NaCl solution (20 mL). The organic layer was dried over

Na₂SO₄, and, after removal of the solvent under reduced pressure, the

residue was purified by chromatography (cyclohexane/ethyl acetate 8:1)

to yield 57 as a slightly yellow oil (682 mg, 88%). [a]²_D⁰= +23.8 (c=1.0

in CHCl₃); R_f=0.27 (cyclohexane/ethyl acetate 3:1); ¹H NMR (CDCl₃,

(3S)-[Bis(tert-butyloxycarbonyl)amino]-5-(tert-butyldiphenylsilanyloxy)-

piperidine-2-on (51): Pd (10% on charcoal, 1.176 g) was added to a de-

gassed solution of nitrile 50 (6.3 g, 19.56 mmol) in MeOH (630 mL) and

acetic acid (3.5 mL). The reaction mixture was hydrogenated under hy-

drogen atmosphere at room temperature for 15 h. Then the suspension

was filtered through a pad of Celite. After removal of the solvents under

reduced pressure, the residue was dissolved in CHCl₃(250 mL) and

washed with Na₂CO₃solution (10%, 50 mL). The organic layer was dried

over Na₂SO₄, and after removal of the solvent under reduced pressure,

the residue was dissolved in toluene (500 mL). DMAP (0.270 g,

2.21 mmol) was added to this solution and the reaction mixture was re-

fluxed for 42 h. After removal of the solvent under reduced pressure, the

residue was purified by chromatography (cyclohexane/ethyl acetate 2:1)

to yield 51 as a colorless foam (4.022 g, 67%). R_f=0.27 (cyclohexane/

ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.05 (s, 9H, C

ACHTREUNG

silyl), 1.48 (s, 18H, C

ACHTREUNG

4136

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

400 MHz): d=1.33 (s, 9H, C

G

176.0 (CF₃COOH); HRMS (FAB, 3-NBA): m/z: calcd for C₁₅H₂₄N₃O₃S₂:

358.1260, found: 358.1245 [MꢀC₂F₃O₂]⁺.

3.00 (d, J=6.6 Hz, 2H, b-CH₂), 3.23 (s, 3H, CH₃O-acetal), 3.61–3.78 (m,

4H, -CH₂C(O)CH₂-), 3.76 (s, 3H, OCH₃ester), 4.53 (t, J=6.6 Hz, 1H, a-

3-tert-Butyldisulfanyl-(2S)-{5-cyanomethylene-(3S)-[3-(hexadecane-1-sul-

CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=30.0 (C

fonylamino)-propionylamino]-2-oxopiperidine-1-yl}-propionic

acid

(CH₂C(O)₂CH₂), 34.7 (b-CH₂), 43.7 (C

methyl ester(61) : At 08C, EDC (20 mg, 0.1 mmol) was added to a solu-

tion of lactam 60 (39 mg, 0.08 mmol), C₁₆H₃₃SO₂-b-Ala-OH 39 (38 mg,

0.1 mmol), HOBt (26 mg, 0.16 mmol) and NEtiPr₂(17 mL, 0.1 mmol) in

CH₂Cl₂(6 mL). The solution was warmed to room temperature and stir-

red for 18 h. Then the reaction mixture was diluted with ethyl acetate

(50 mL) and subsequently washed with 1n HCl (10 mL) and Na₂CO₃sol-

ution (10%, 10 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent under reduced pressure, the residue was purified

by chromatography (cyclohexane/ethyl acetate 1:2) to yield 61 as color-

less foam (40 mg, 67%). [a]_D²⁰= 4.1 (c=1.0 in CHCl₃); R_f=0.16 (cyclo-

hexane/ethyl acetate 1:2); ¹H NMR (CDCl₃, 400 MHz): d=0.86 (t, J=

6.5 Hz, 3H, w-CH₃hexadecyl), 1.23 (m, 24H, 12CH₂hexadecyl), 1.33

(OCH₃ester), 65.0 (CH₂OCH₂), 69.1 (a-CH), 100.0 (C acetal), 172.5 (C=

O); GCMS (1008C, 3 min, then 208Cmin^ꢀ1to 3008C): t_R=9.8 min, MS:

m/z: 338 [M]⁺, 250 [M+Hꢀ

A

3-tert-Butyldisulfanyl-(2R)-(methanesulfonyloxy)propionic acid methyl

ester(58) : TsOH (251 mg, 1.29 mmol) was added to a solution of acetal

57 (435 mg, 1.29 mmol) in MeOH (35 mL). After stirring at room tem-

perature for 1 h, Na₂CO₃solution (10%, 5 mL) was added and the sol-

vent was removed under reduced pressure. The residue was dissolved in

ethyl acetate and washed with saturated NaHCO₃solution (10 mL). The

organic layer was dried over Na₂SO₄and the solvent was removed under

reduced pressure to yield the free alcohol as a colorless oil (288 mg,

quant.).

(s, 9H, C(CH₃)₃), 1.38 (m, 2H, g-CH₂hexadecyl), 1.76 (m, 2H, b-CH₂

G

hexadecyl), 2.55 (m, 3H, b-CH_2alactam, a-CH₂b-Ala), 2.71 (m, 1H, b-

CH_2blactam), 2.98 (m, 2H, a-CH₂hexadecyl), 3.16 (m, 2H, b-CH₂b-

Ala), 3.36 (m, 2H, d-CH₂lactam), 3.73 (s, 3H, OCH₃), 4.58 (m, 1H, a-

CH Lactam), 5.38 (m, 1H, NH), 5.48 (m, 1H, a-CH Cys), 6.18 (m, 1H,

-CHCN), 6.66 (m, 1H, NH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.3 (w-

CH₃hexadecyl), 22.1 (CH₂), 22.8 (CH₂), 23.8 (CH₂), 28.5 (CH₂), 28.7

(CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.3 (CH₂), 29.5 (CH₂), 29.7 (CH₂), 29.8

MsCl (120 mL, 1.54 mmol) was added to a solution of the above alcohol

(288 mg, 1.29 mmol) in pyridine (9 mL). After stirring at room tempera-

ture for 15 h, the solution was diluted with ethyl acetate (50 mL) and

washed with 1n HCl (10 mL). The organic layer was dried over Na₂SO₄,

and, after removal of the solvent under reduced pressure, the residue was

purified by chromatography (cyclohexane/ethyl acetate 3:1) to yield 58 as

a slightly yellow oil (388 mg, quant.). [a]²_D⁰= +12.9 (c=0.5, CHCl₃);

R_f=0.27 (cyclohexane/ethyl acetate 3:1); ¹H NMR (CDCl₃, 400 MHz):

(CH₂), 29.9 (CH₂), 29.9 (CH₂), 30.0 (CH₂), 30.1 (C

lactam), 32.1 (b-CH₂Cys), 36.7 (b-CH₂b-Ala), 39.6 (a-CH₂b-Ala), 48.8

(b-CH₂lactam), 49.1 (C(CH₃)₃), 49.4 (OCH₃), 52.3 (a-CH lactam), 52.8

A

d=1.33 (s, 9H, C(CH₃)₃), 3.00 (dd, J=8.4, 13.4 Hz, 1H, b-CH_2a), 3.15 (s,

A

AHCTREUNG

3H, CH₃SO₂-), 3.19 (dd, J=4.1, 13.9 Hz, 1H, b-CH_2b), 3.81 (s, 3H,

OCH₃), 5.24 (dd, J=4.1, 8.4 Hz, 1H, a-CH); ¹³C NMR (CDCl₃,

(a-CH₂hexadecyl), 52.8 (a-CH Cys), 108.1 (CHCN), 116.3 (CN), 128.5

(C=CHCN), 167.5 (C=O), 170.3 (C=O), 171.7 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₃₄H₆₁N₄O₆S₃: 718.3754, found: 718.3727 [M+H]⁺.

100.6 MHz): d=30.0 (C

(CH₃)₃), 48.6 (OCH₃), 69.7 (a-CH), 172.4 (C=O); GCMS (1008C, 3 min,

then 208Cmin^ꢀ1to 3008C): t_R=8.6 min, MS: m/z: 302 [M]⁺, 246

[M+Hꢀ

(CH₃)₃C)]⁺.

A

ACHTREUNG

(2S)-{(3S)-[(2S)-Acetylamino-3-(hexadecane-1-sulfonylamino)propionyl-

amino]-5-cyanomethylene-2-oxopiperidine-1-yl}-3-(tert-butyldisulfanyl)-

propionic acid methyl ester (62): At 08C, EDC (20 mg, 0.1 mmol) was

ACHTREUNG

(2S)-{(3S)-[Bis(tert-butyloxycarbonyl)amino]-5-cyanomethylene-2-oxopi-

peridine-1-yl}-3-tert-butyldisulfanylpropionic acid methyl ester (59): At

08C, a solution of lactam 53 (235 mg, 0.67 mmol) and mesylate 58

added to

a solution of lactam 60 (39 mg, 0.08 mmol), Ac-Dap-

(SO₂C₁₆H₃₃)-OH 4 (43 mg, 0.1 mmol), HOBt (26 mg, 0.16 mmol) and

NEtiPr₂(17 mL, 0.1 mmol) in CH₂Cl₂(3 mL). The solution was warmed

to room temperature and stirred for 18 h. Then the reaction mixture was

diluted with ethyl acetate (50 mL) and subsequently washed with 1n HCl

(10 mL) and Na₂CO₃solution (10%, 10 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (cyclohexane/ethyl acetate

1:10) to yield 62 as colorless foam (38 mg, 62%). [a]²_D⁰= +17.2 (c=1.0

in CHCl₃); R_f=0.07 (cyclohexane/ethyl acetate 1:3); ¹H NMR (CDCl₃,

400 MHz): d=0.81 (t, J=6.5 Hz, 3H, w-CH₃hexadecyl), 1.18 (m, 24H,

(326 mg, 1.08 mmol) was added to

a suspension of NaH (21 mg,

0.84 mmol) in THF (6 mL). The reaction mixture was warmed to room

temperature and stirred for 20 h. Then, the solution was diluted with

ethyl acetate (50 mL) and washed with H₂O (10 mL). The organic layer

was dried over Na₂SO₄, and, after removal of the solvents under reduced

pressure, the residue was purified by chromatography (cyclohexane/ethyl

acetate 4:1, then 1:2) to yield 59 as a colorless oil (213 mg, 57%). [a]_D²⁰

+46.8 (c=0.5 in CHCl₃); R_f=0.22 (cyclohexane/ethyl acetate 1:1);

¹H NMR (CDCl₃, 400 MHz): d=1.32 (s, 9H, C

(CH₃)₃StBu), 1.49 (s,

18H, C(CH₃)₃Boc), 2.38 (dd, J=9.6, 16.8 Hz, 1H, b-CH_2alactam), 2.99–

=

AHCTREUNG

12CH₂hexadecyl), 1.33 (s, 9H, C(CH₃)₃), 1.36 (m, 2H, g-CH₂hexadec-

U

ACHTREUNG

yl), 1.71 (m, 2H, b-CH₂hexadecyl), 2.01 (s, 3H, CH₃acetyl), 2.40 (m,

1H, b-CH_2alactam), 2.60 (m, 1H, b-CH_2blactam), 2.94 (m, 2H, a-CH₂

hexadecyl), 3.11 (m, 1H, d-CH_2alactam), 3.33 (m, 1H, d-CH_2blactam),

3.52 (m, 2H, b-CH₂Dap), 3.67 (s, 3H, OCH₃), 4.55 (m, 2H, a-CH

lactam, a-CH Dap), 5.24 (m, 1H, a-CH Cys), 5.97 (m, 1H, NH), 6.13 (m,

1H, -CHCN), 6.86 (m, 1H, NH), 6.97 (m, 1H, NH); ¹³C NMR (CDCl₃,

100.6 MHz): d=14.3 (w-CH₃hexadecyl), 22.1 (CH₂), 22.8 (CH₂), 22.9

(CH₃acetyl), 23.8 (CH₂), 28.5 (CH₂), 28.7 (CH₂), 28.9 (CH₂), 29.1 (CH₂),

29.3 (CH₂), 29.5 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 29.9 (CH₂),

3.27 (m, 5H, b-CH_2blactam, b-CH₂Cys, d-CH₂lactam), 3.72 (s, 3H,

OCH₃), 5.17 (dd, J=8.4, 15.6 Hz, 1H, a-CH lactam), 5.25 (dd, J=4.1,

8.4 Hz, 1H, a-CH Cys), 6.20 (m, 1H, -C=CHCN); ¹³C NMR (CDCl₃,

100.6 MHz): d=22.3 (b-CH₂lactam), 28.5 (C

StBu), 30.0 (d-CH₂lactam), 39.2 (b-CH₂Cys), 48.8 (C

(OCH₃), 52.7 (a-CH lactam), 69.7 (a-CH Cys), 83.4 (C

A

(CH₃)₃

AHCTREUNG

(CHCN), 116.5 (CN), 128.9 (C=CHCN), 152.2 (C=O), 166.4 (C=O),

171.3 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₂₅H₃₉N₃NaO₇S₂:

580.2129, found: 580.2104 [M+Na]⁺.

30.0 (CH₂), 30.1 (C

(b-CH₂Dap), 48.8 (b-CH₂lactam), 49.1 (C

CH lactam), 52.8 (a-CH₂hexadecyl), 52.8 (a-CH Cys), 53.3 (a-CH Dap),

107.1 (CHCN), 116.3 (CN), 128.5 (C=CHCN), 167.5 (C=O), 170.3 (C=

O), 170.6 (C=O), 171.7 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₃₆H₆₄N₅O₇S₃: 774.3969, found: 774.3989 [M+H]⁺.

ACHTREUNG

(2S)-[(3S)-Amino-5-cyanomethylene-2-oxopiperidine-1-yl]-3-tert-butyldi-

sulfanylpropionic acid methyl ester hydrotrifluoroacetate (60): TFA

(1.5 mL, 19.54 mmol) was added to a solution of lactam 59 (109 mg,

0.2 mmol) in CH₂Cl₂(6 mL). After stirring at room temperature for

30 min, the solvent was removed under reduced pressure and the residue

was coevaporated with toluene to yield 60 as a colorless oil (90 mg,

99%). [a]²_D⁰= +24.4 (c=1.0 in MeOH); R_f=0.01 (CH₂Cl₂/EtOH 10:1);

AHCTREUNG

7-Bromo-(2R)-hydroxy-1,2,3,11a-tetrahydro-10H-benzo[e]pyrrolo[1,2-

a,1,4]diazepine-5,11-dione (64): A solution of 5-bromoisatoic acid anhy-

dride 63 (5.219 g, 20.9 mmol) and (4R)-hydroxy-l-proline 31 (3.525 g,

26.8 mmol) in DMSO (30 mL) was stirred at 1408C for 5 h. The reaction

mixture was cooled down to room temperature, poured into ice cold

water (30 mL) and extracted with ethyl acetate (450 mL). The com-

bined organic layers were washed with ice cold water (330 mL) and

dried over Na₂SO₄. The solvent was removed under reduced pressure

¹H NMR (CDCl₃, 400 MHz): d=1.31 (s, 9H, C

(CH₃)₃), 2.62–2.85 (m, 2H,

G

b-CH₂lactam), 3.16–3.28 (m, 4H, b-CH₂Cys, d-CH₂lactam), 3.73 (s, 3H,

OCH₃), 4.19 (m, 1H, a-CH lactam), 5.23 (m, 1H, a-CH Cys), 6.20 (m,

1H, -CHCN); ¹³C NMR (CDCl₃, 100.6 MHz): d=30.1 (C

ACHTREUNG

AHCTREUNG

(OCH₃), 52.6 (a-CH lactam), 69.7 (a-CH Cys), 105.4 (CHCN), 116.6

(CN), 126.8 (CF₃COOH), 128.7 (C=CHCN), 166.5 (C=O), 171.11 (C=O),

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4137

H. Waldmann, A. Giannis et al.

and the residue was purified by chromatography (CH₂Cl₂/CH₃OH 10:1)

to yield 64 as a slightly yellow solid (6.043 g, 93%). M.p. 1288C; [a]_D²⁰

(67): At ꢀ408C, a solution of the azide 66 (3.99 g, 10.41 mmol) in THF

(20 mL) was added to a suspension of NaH (315 mg, 12.49 mmol) in THF

(20 mL). After stirring for 30 min, bromoacetonitrile (897 mL,

12.49 mmol) was added. The reaction mixture was warmed to room tem-

perature and stirred for 20 h. Then, the solvent was removed under re-

duced pressure, the residue was dissolved in CHCl₃(200 mL) and washed

with H₂O (50 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent, the residue was purified by chromatography

(CH₂Cl₂/EtOH 50:1) to yield 67 as a colorless solid (4.176 g, 95%). M.p.

1738C; [a]²_D⁰= +468 (c=1.0 in CHCl₃); R_f=0.18 (CH₂Cl₂/EtOH 40:1);

=

+415 (c=1.0 in CHCl₃); R_f=0.22 (CH₂Cl₂/CH₃OH 10:1); ¹H NMR

(CD₃OD, 400 MHz): d=2.00 (m, 1H, -CH_2a-), 2.83 (m, 1H, -CH_2b-), 3.61

(dd, J=4.9, 12.5 Hz, 1H, -CH_2aN-), 3.76 (ddd, J=1.4, 3.7, 12.3 Hz, 1H,

-CH_2bN-), 4.31 (dd, J=5.9, 8.2 Hz, 1H, -CHOH), 4.50 (m, 1H, -CHC(O)-),

7.05 (d, J=8.6 Hz, 1H, arom. CH), 7.67 (dd, J=2.3, 8.6 Hz, 1H, arom.

CH), 7.99 (d, J=2.3 Hz, 1H, arom. CH); ¹³C NMR (CD₃OD,

100.6 MHz): d=34.2 (CH₂), 54.0 (CH₂N), 55.8 (CHC(O)), 68.2 (CHOH),

117.1 (arom. C), 123.3 (arom. CH), 127.8 (arom. C), 132.9 (arom. CH),

135.4 (arom. CH), 135.7 (arom. C), 165.6 (C=O), 170.5 (C=O); HRMS

(EI, 70 eV): m/z: calcd for C₁₂H₁₁BrN₂O₃: 309.9953, found: 309.9943

[M]⁺; elemental analysis calcd (%) for C₁₂H₁₁BrN₂O₃: C 46.32, H 3.56, N

9.00; found: C 46.11, H 3.54, N 8.93.

¹H NMR (CDCl₃, 400 MHz): d=1.54 (s, 9H, C

(CH₃)₃), 2.42 (m, 1H, -

A

CH_2a-), 3.08 (m, 1H, -CH_2b-), 3.70 (d, J=12.9 Hz, 1H, -CH_2aN-), 3.83

(dd, J=5.3, 12.9 Hz, 1H, -CH_2bN-), 4.25 (m, 1H, -CHN₃), 4.30 (d, J=

17.2 Hz, 1H, -CH_2aCN), 4.39 (m, 1H, -CHC(O)-), 4.96 (d, J=17.2 Hz,

1H, -CH_2bCN), 6.44 (d, J=16.0 Hz, 1H, -CH=CHC(O)-), 7.45 (d, J=

8.4 Hz, 1H, arom. CH), 7.56 (d, J=16.0 Hz, 1H, -CH=CHC(O)-), 7.75

(dd, J=2.2, 8.4 Hz, 1H, arom. CH), 8.19 (d, J=2.2 Hz, 1H, arom. CH);

3-[(2R)-Hydroxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyr-

rolo[1,2-a,1,4]diazepine-7-yl]-acrylic acid tert-butyl ester(65)

(OAc)₂] (145 mg, 0.64 mmol) was added to a solution of benzodiazepine

64 (4 g, 12.86 mmol), P(o-tol)₃(404 mg, 1.29 mmol), NEt₃(5.375 mL,

: [Pd-

ACHTREUNG

¹³C NMR (CDCl₃, 100.6 MHz): d=28.2 (C

(CH₂CN), 52.6 (CH₂N), 56.5 (CHN₃), 58.2 (CHC(O)), 81.2 (C

ACHTREUNG

AHCTREUNG

ACHTREUNG

38.57 mmol) and tert-butyl acrylate (5.655 mL, 38.57 mmol) in CH₃CN

(16 mL). The solution was stirred at 1008C in a sealed tube for 20 h. The

reaction mixture was cooled down to room temperature and the solvent

was removed under reduced pressure. The residue was purified by chro-

matography (CH₂Cl₂/EtOH 20:1) to yield 65 as a yellowish solid (5.588 g,

97%). M.p. 888C; [a]_D²⁰= +238.8 (c=1.0 in CHCl₃); R_f=0.11 (CH₂Cl₂/

115.4 (CN), 121.3 (CH=CHC(O)), 122.9 (arom. CH), 129.6 (arom. C),

130.8 (arom. CH), 132.0 (arom. CH), 133.7 (arom. CH), 139.0 (arom.C),

140.6 (CH=CHC(O)), 164.7 (C=O), 165.7 (C=O), 169.7 (C=O); HRMS

(EI, 70 eV): m/z: calcd for C₂₁H₂₂N₆O₄: 422.1703, found: 422.1707 [M]⁺.

(2S)-Azido-10-cyanomethyl-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-

benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-carboxylic acid tert-butyl ester

(68): A solution of RuCl₃in 1 mm HCl (2 mg RuCl₃per mL 1 mm HCl,

73.56 mL) was added to a solution of NaIO₄(10.229 g, 47.34 mmol) in

H₂O/CH₃CN/CCl₄(2:1:1, 150 mL). After stirring at room temperature

for 5 min, a solution of benzodiazepine 67 (2 g, 4.73 mmol) in CH₃CN/

CCl₄(1:1, 150 mL) was added. After stirring for 18 h, the solvent was re-

moved under reduced pressure, the residue was dissolved in 1n HCl

(50 mL) and extracted with CHCl₃(50 mL). The organic layer was dried

over Na₂SO₄and the solvent was removed under reduced pressure to

yield the free acid as a colorless solid (1.604 g, quant.).

EtOH 20:1); ¹H NMR (CDCl₃, 400 MHz): d=1.52 (s, 9H, -C

(CH₃)₃),

H

2.20 (m, 1H, -CH_2a-), 2.92 (m, 1H, -CH_2b-), 3.50 (brs, 1H, OH), 3.63 (dd,

J=4.5, 12.7 Hz, 1H, -CH_2aN-), 4.02 (d, J=12.7 Hz, 1H, -CH_2bN-), 4.32

(m, 1H, -CHOH-), 4.61 (m, 1H, -CHC(O)-), 6.35 (d, J=16.0 Hz, 1H,

-CH=CHC(O)-), 7.12 (d, J=8.4 Hz, 1H, arom. CH), 7.48 (d, J=16.0 Hz,

1H, -CH=CHC(O)-), 7.58 (dd, J=2.2, 8.6 Hz, 1H, arom CH), 8.00 (d,

J=2.0 Hz, 1H, arom. CH), 9.27 (s, 1H, NH); ¹³C NMR (CDCl₃,

100.6 MHz): d=28.3 (C

ACHTREUNG

(CH₂C(O)), 68.9 (CH₂OH), 81.0 (C

AHCTREUNG

(arom. CH), 126.6 (arom. C), 131.3 (arom. CH), 131.6 (arom. C), 131.7

(arom. CH), 136.5 (arom. C), 141.6 (CH=CHC(O)), 165.9 (C=O), 166.2

(C=O), 169.2 (C=O); HRMS (EI, 70 eV): m/z: calcd for C₁₉H₂₂N₂O₅:

358.1529, found: 358.1547 [M]⁺.

Then, K₂CO₃(17.1 g, 122.29 mmol), and Me₃CBr (26.2 mL, 225.69 mmol)

were subsequently added to

a solution of the above acid (1.604 g,

4.73 mmol) and Et₃(PhCH₂)NCl (1.09 g, 4.7 mmol). The reaction mixture

AHCTREUNG

3-[(2S)-Azido-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrro-

was stirred at 558C for 16 h. Then, the solvent was removed under re-

duced pressure, the residue was dissolved in H₂O (50 mL) and extracted

with CHCl₃(100 mL). The organic layer was dried over Na₂SO₄, and

after removal of the solvent, the residue was purified by chromatography

(cyclohexane/ethyl acetate 1:1) to yield 68 as a colorless solid (1.758 g,

90%). M.p. 1408C; [a]²_D⁰= +287.7 (c=1.0 in CHCl₃); R_f=0.28 (cyclo-

hexane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.59 (s, 9H,

lo[1,2-a,1,4]diazepine-7-yl]-acrylic acid tert-butyl ester(66)

: At 08C,

MsCl (570 mL, 7.37 mmol) was added to a solution of alcohol 65 (2.2 g,

6.14 mmol) in pyridine (20 mL). The reaction mixture was warmed to

room temperature and stirred for 18 h. Then, the solution was diluted

with CH₂Cl₂(100 mL) and washed with 1n HCl (330 mL). The organic

layer was dried over Na₂SO₄and the solvent was removed under reduced

pressure to yield the mesylate as a brownish solid (2.494 g, 93%).

C(CH₃)₃), 2.40 (m, 1H, -CH_2a-), 3.14 (d, J=12.9 Hz, 1H, -CH_2b-), 3.68 (d,

A

J=12.9 Hz, 1H, -CH_2aN-), 3.77 (dd, J=5.5, 12.9 Hz, 1H, -CH_2bN-), 4.20

(dd, J=2.3, 9.0 Hz, 1H, -CHN₃), 4.28 (d, J=17.4 Hz, 1H, -CH_2aCN), 4.36

(m, 1H, -CHC(O)-), 4.95 (d, J=17.4 Hz, 1H, -CH_2bCN), 7.48 (d, J=

8.8 Hz, 1H, arom.CH), 8.18 (dd, J=2.2, 8.6 Hz, 1H, arom. CH), 8.49 (d,

J=2.2 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.3 (C-

NaN₃(8.9 g, 137.46 mmol) was then added to a solution of the above me-

sylate (2.494 g, 5.71 mmol) in DMF (40 mL). The reaction mixture was

stirred at 458C for 4 d. Then, the solvent was removed under reduced

pressure, the residue was dissolved in H₂O (50 mL) and extracted with

CHCl₃(350 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent under reduced pressure, the residue was purified

by chromatography (cyclohexane/ethyl acetate 1:1, then CHCl₃) to yield

66 as a colorless solid (1.95 g, 89%). M.p. 2118C; [a]²_D⁰= +305.0 (c=1.0

in CHCl₃); R_f=0.53 (CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃, 400 MHz):

AHCTREUNG

C), 130.8 (arom. CH), 132.6 (arom. CH), 134.0 (arom. C), 141.2 (arom.

C), 163.8 (C=O), 164.6 (C=O), 169.3 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₁₉H₂₁N₆O₄: 397.1625, found: 397.1622 [M+H]⁺; elemental

analysis calcd (%) for C₁₉H₂₀N₆O₄: C 57.57, H 5.09, N 21.20; found: C

57.33, H 5.00, N 20.97.

d=1.53 (s, 9H, -C(CH₃)₃), 2.38 (m, 1H, -CH_2a-), 3.12 (m, 1H, -CH_2b-),

N

3.72 (d, J=12.9 Hz, 1H, -CH_2aN-), 3.82 (dd, J=5.3, 12.9 Hz, 1H, -

CH_2bN-), 4.23 (dd, J=2.0, 9.0 Hz, 1H, -CHN₃), 4.39 (m, 1H, -CHC(O)-),

6.40 (d, J=15.8 Hz, 1H, -CH=CHC(O)-), 7.16 (d, J=8.4 Hz, 1H, arom.

CH), 7.56 (d, J=15.8 Hz, 1H, -CH=CHC(O)-), 7.62 (dd, J=2.2, 8.4 Hz,

1H, arom CH), 8.15 (d, J=2.0 Hz, 1H, arom. CH), 9.63 (s, 1H, NH);

(2S)-Amino-10-cyanomethyl-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-

benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-carboxylic acid tert-butyl ester

(69): Pd on BaSO₄(354 mg) was added to a degassed solution of azide 68

(660 mg, 1.67 mmol) in MeOH (132 mL) and CHCl₃(4.4 mL). The reac-

tion mixture was hydrogenated under hydrogen atmosphere at room tem-

perature for 4 h. Then the suspension was filtered through a pad of

Celite and the solvent was removed under reduced pressure to yield 69

as a colorless solid (615 mg, quant.). M.p. 1488C; [a]²_D⁰= 216.2 (c=1.0 in

¹³C NMR (CDCl₃, 100.6 MHz): d=28.3 (C

(CH₂N), 56.2 (CHN₃), 58.0 (CHC(O)), 80.9 (C

ACHTREUNG

AHCTREUNG

CHC(O)), 122.0 (arom. CH), 125.8 (arom. C), 131.5 (arom. CH), 131.8

(arom. C), 131.9 (arom. CH), 136.7 (arom. C), 141.5 (CH=CHC(O)),

165.5 (C=O), 166.1 (C=O), 171.0 (C=O); HRMS (EI, 70 eV): m/z: calcd

for C₁₉H₂₁N₅O₄: 383.1594, found: 383.1603 [M]⁺.

1

CHCl₃); R_f=0.38 (CH₂Cl₂/EtOH 1:1); H NMR (CD₃OD, 400 MHz): d=

3-[(2S)-Azido-10-cyanomethyl-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-

benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-yl)-acrylic acid tert-butyl ester

1.63 (s, 9H, C

A

-CH_2b-), 3.86 (dd, J=6.3, 13.5 Hz, 1H, -CH_2aN-), 4.02 (dd, J=6.3,

4138

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

13.5 Hz, 1H, -CH_2bN-), 4.13 (m, 1H, -CHC(O)-), 4.53 (brd, J=6.3 Hz,

1H, -CHNH₂), 4.94 (d, J=17.6 Hz, 1H, -CH_2aCN), 5.13 (d, J=17.6 Hz,

1H, -CH_2bCN), 7.62 (d, J=8.6 Hz, 1H, arom. CH), 8.21 (dd, J=2.2,

8.6 Hz, 1H, arom. CH), 8.43 (d, J=2.2 Hz, 1H, arom. CH); ¹³C NMR

10-(2-Aminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-5,11-dioxo-

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-car-

boxylic acid (72): A saturated solution of HCl in Et₂O (20 mL) was

added to a solution of benzodiazepine 71 (178 mg, 0.27 mmol) in CH₂Cl₂.

After stirring at room temperature for 4 h, the solvent was removed

under reduced pressure and the residue was coevaporated with toluene

(CD₃OD, 100.6 MHz): d=27.1 (C

ACHTREUNG

49.4 (CH₂N), 56.8 (CHNH₂), 60.0 (CHC(O)), 82.3 (C

AHCTREUNG

to yield 72 as a colorless solid (164 mg, quant.). M.p. 1668C; [a]_D²⁰

=

122.3 (arom. CH), 128.5 (arom. C), 130.3 (arom. CH), 131.6 (arom. CH),

133.5 (arom. C), 141.6 (arom. C), 164.0 (C=O), 165.2 (C=O), 169.8 (C=

O); HRMS (FAB, 3-NBA): m/z: calcd for C₁₉H₂₃N₄O₄: 371.1720, found:

371.1702 [M+H]⁺.

1

+260.2 (c=0.5 in MeOH); H NMR (CD₃OD, 400 MHz): d=0.88 (t, J=

6.6 Hz, 3H, w-CH₃hexadecyl), 1.22–1.37 (m, 24H, 12CH₂hexadecyl),

1.42 (m, 2H, g-CH₂hexadecyl), 1.83 (m, 2H, b-CH₂hexadecyl), 2.41 (m,

1H, -CH_2a-), 2.74 (m, 1H, -CH_2b-), 3.13 (m, 2H, a-CH₂hexadecyl), 3.35

(m, 1H, -CH_2bN-), 3.54 (dd, J=5.5, 13.0 Hz, 1H, -CH_2aN-), 4.04–4.44 (m,

6H, -NCH₂CH₂NH₂, NCH₂CH₂NH₂, -CHNH-, -CHC(O)-), 7.60 (d, J=

8.6 Hz, 1H, arom. CH), 8.21 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.49 (d,

J=2.0 Hz, 1H, arom. CH); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.3 (w-

CH₃hexadecyl), 22.9 (CH₂), 23.7 (CH₂), 28.5 (CH₂), 29.3 (CH₂), 29.5

(CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 29.9

(CH₂), 30.0 (CH₂), 30.0 (CH₂), 32.1 (CH₂), 33.0 (CH₂), 51.8 (CH₂N), 54.1

(a-CH₂hexadecyl), 55.2 (CHNH), 57.2 (CHC(O)), 58.1 (NCH₂CH₂NH₂),

60.5 (NCH₂CH₂NH₂), 121.0 (arom. CH), 129.3 (arom. C), 131.6 (arom.

CH), 132.5 (arom. CH), 134.2 (arom. C), 141.2 (arom. C), 164.4 (C=O),

170.6 (C=O), 173.5 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

10-Cyanomethyl-(2S)-(hexadecane-1-sulfonylamino)-5,11-dioxo-

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-car-

boxylic acid tert-butyl ester(70) : At 08C, NEtiPr₂(568 mL, 3.32 mmol)

and hexadecanesulfonic acid chloride (674 mg, 1.99 mmol) were subse-

quently added to a solution of amine 69 (615 mg, 1.66 mmol) in DMF

(20 mL). The solution was warmed to room temperature and stirred for

22 h. Then, the solvent was removed under reduced pressure, the residue

was dissolved in ethyl acetate (50 mL) and washed with 1n HCl (30 mL).

The organic layer was dried over Na₂SO₄, and, after removal of the sol-

vent under reduced pressure, the residue was purified by chromatography

(n-hexane/ethyl acetate 2:1) to yield 70 as a colorless solid (887 mg,

82%). M.p. 968C; [a]²_D⁰= +298.1 (c=1.0 in CHCl₃); R_f=0.32 (cyclohex-

ane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz): d=0.88 (t, J=

6.6 Hz, 3H, w-CH₃hexadecyl), 1.22–1.37 (m, 24H, 12CH₂hexadecyl),

C₃₁H₅₁N₄O₆S:

607.3530,

found:

607.3553

[M+H]⁺.

10-(2-Allyloxycarbonylaminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-

5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diaze-

pine-7-carboxylic acid tert-butyl ester(73) : AlocCl (127 mL, 1.15 mmol)

was added to a solution of amine 71 (510 mg, 0.77 mmol) and NEt₃

(214 mL, 1.54 mmol) in CH₂Cl₂(20 mL). After stirring at room tempera-

ture for 18 h, the solution was diluted with ethyl acetate (100 mL) and

washed with 1n HCl (30 mL). The organic layer was dried over Na₂SO₄,

and, after removal of the solvent under reduced pressure, the residue was

purified by chromatography (cyclohexane/ethyl acetate 1:1) to yield 73 as

a colorless solid (483 mg, 84%). M.p. 908C; [a]²_D⁰= +269.38C (c=1.0 in

CHCl₃); R_f=0.31 (cyclohexane/ethyl acetate 1:1); ¹H NMR (CDCl₃,

400 MHz): d=0.88 (t, J=6.6 Hz, 3H, w-CH₂hexadecyl), 1.22–1.37 (m,

24H, 12CH₂hexadecyl), 1.42 (m, 2H, g-CH₂hexadecyl), 1.58 (s, 9H, C-

1.43 (m, 2H, g-CH₂hexadecyl), 1.60 (s, 9H, C(CH₃)₃), 1.84 (m, 2H, b-

R

CH₂hexadecyl), 2.50 (m, 1H, -CH_2a-), 2.76 (d, J=14.5 Hz, 1H, -CH_2b-),

3.07 (m, 2H, a-CH₂hexadecyl), 3.86 (dd, J=5.5, 13.1 Hz, 1H, -CH_2aN-),

3.97 (d, J=13.1 Hz, 1H, -CH_2bN-), 4.27 (d, J=8.8 Hz, 1H, -CHNH-),

4.29 (m, 1H, -CHC(O)-), 4.31 (d, J=17.2, 1H, -CH_2aCN), 5.05 (d, J=

17.2, 1H, -CH_2bCN), 5.40 (d, J=7.4 Hz, 1H, NH), 7.51 (d, J=8.6 Hz, 1H,

arom. CH), 8.23 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.53 (d, J=2.1 Hz,

1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.3 (w-CH₃hexa-

decyl), 22.9 (CH₂), 23.8 (CH₂), 28.3 (C(CH₃)₃), 28.5 (CH₂), 29.3 (CH₂),

T

29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂),

29.9 (CH₂), 29.9 (CH₂), 29.9 (CH₂), 32.1 (CH₂), 33.0 (CH₂), 37.4

(CH₂CN), 51.9 (CH₂N), 54.2 (a-CH₂hexadecyl), 55.4 (CHNH), 57.0

A

J=14.5 Hz, 1H, -CH_2b-), 3.04 (m, 2H, a-CH₂hexadecyl), 3.37 (m, 1H,

-NCH₂CH_2aNH-), 3.63 (m, 1H, -NCH₂CH_2bNH-), 3.82 (dd, J=5.8,

13.0 Hz, 1H, -CH_2aN-), 3.93 (m, 2H, -CH_2bN-, -NCH_2aCH₂NH-), 4.14 (m,

2H, -CHNH-, -NCH_2bCH₂NH-), 4.27 (m, 1H, -CHC(O)-), 4.50 (m, 2H,

-OCH₂CH=CH₂), 5.00 (m, 1H, NH), 5.14–5.28 (m, 2H, -OCH₂CH=CH₂),

5.86 (m, 1H, -OCH₂CH=CH₂), 5.93 (m, 1H, NH), 7.44 (d, J=8.6 Hz,

1H, arom. CH), 8.15 (dd, J=2.0, 8.6 Hz, 1H, arom. CH), 8.48 (d, J=

2.1 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.2 (w-CH₃

(CHC(O)), 82.6 (C(CH₃)₃), 114.9 (CN), 121.1 (arom. CH), 129.4 (arom.

G

C), 131.4 (arom. CH), 132.7 (arom. CH), 134.1 (arom. C), 141.0 (arom.

C), 163.7 (C=O), 164.1 (C=O), 170.3 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₃₅H₅₅N₄O₆S: 659.3843, found: 659.3863 [M+H]⁺.

10-(2-Aminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-5,11-dioxo-

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-car-

boxylic acid tert-butyl ester(71) : PtO₂·H₂O (25 mg, 0.1 mmol) was added

to a degassed solution of nitrile 70 (670 mg, 1.02 mmol) in EtOH (45 mL)

and CHCl₃(900 mL). The reaction mixture was hydrogenated under hy-

drogen atmosphere at room temperature for 4.5 h. Then, the suspension

was filtered through a pad of Celite. After removal of the solvents under

reduced pressure, the residue was purified by chromatography (CH₂Cl₂/

EtOH 10:1, then 1:1) to yield 71 as a colorless solid (543 mg, 81%). M.p.

1098C; [a]²_D⁰= +287.4 (c=1.0 in MeOH); R_f=0.51 (CH₂Cl₂/EtOH 1:1);

¹H NMR (CDCl₃, 400 MHz): d=0.88 (t, J=6.6 Hz, 3H, w-CH₃hexadec-

yl), 1.22–1.37 (m, 24H, 12CH₂hexadecyl), 1.42 (m, 2H, g-CH₂hexadec-

hexadecyl), 22.9 (CH₂), 23.7 (CH₂), 28.3 (C(CH₃)₃), 28.4 (CH₂), 29.4

A

(CH₂), 29.5 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9

(CH₂), 29.9 (CH₂), 30.0 (CH₂), 30.1 (CH₂), 32.1 (CH₂), 33.1 (CH₂), 51.8

(CH₂N), 54.0 (a-CH₂hexadecyl), 55.1 (CHNH), 57.2 (CHC(O)), 58.1

(NCH₂CH₂NH₂), 60.4 (NCH₂CH₂NH₂), 66.1 (OCH₂CH=CH₂), 82.3 (C-

(CH₃)₃), 118.0 (OCH₂CH=CH₂), 121.2 (arom. CH), 129.3 (arom. C),

131.5 (arom. CH), 132.4 (arom. CH), 132.6 (OCH₂CH=CH₂), 134.2

(arom. C), 141.2 (arom. C), 156.7 (C=O), 163.4 (C=O), 164.4 (C=O),

170.4 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₃₉H₆₃N₄O₈S:

747.4367, found: 747.4379 [M+H]⁺.

yl), 1.58 (s, 9H, C(CH₃)₃), 1.83 (m, 2H, b-CH₂hexadecyl), 2.41 (m, 1H,

A

-CH_2a-), 2.74 (brd, J=14.5 Hz, 1H, -CH_2b-), 2.92–3.13 (m, 4H, a-CH₂

hexadecyl, -NCH₂CH₂NH₂), 3.84 (dd, J=5.5, 13.0 Hz, 1H, -CH_2aN-), 3.91

(m, 2H, -CH_2bN-, NCH_2aCH₂NH₂), 4.17 (d, J=8.8 Hz, 1H, -CHNH-),

4.24 (m, 2H, -CHC(O)-, NCH_2bCH₂NH₂), 7.48 (d, J=8.6 Hz, 1H, arom.

CH), 8.14 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.49 (d, J=2.0 Hz, 1H,

arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.3 (w-CH₃hexadecyl),

10-(2-Allyloxycarbonylaminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-

5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diaze-

pine-7-carboxylic acid (74): TFA (8 mL) was added to a solution of ben-

zodiazepine 73 (405 mg, 0.54 mmol) in CH₂Cl₂(8 mL). After stirring at

room temperature for 4 h, the solvent was removed under reduced pres-

sure and the residue was coevaporated with toluene to yield 74 as a col-

orless solid (374 mg, quant.). M.p. 1658C; [a]_D²⁰

= +229.8 (c=1.0,

22.9 (CH₂), 23.7 (CH₂), 28.3 (C(CH₃)₃), 28.5 (CH₂), 29.3 (CH₂), 29.5

A

MeOH); R_f=0.64 (CH₂Cl₂/EtOH 1:1); ¹H NMR (CDCl₃, 400 MHz): d=

0.87 (t, J=6.6 Hz, 3H, w-CH₂hexadecyl), 1.22–1.36 (m, 24H, 12CH₂

(CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 29.9

(CH₂), 30.0 (CH₂), 30.0 (CH₂), 32.1 (CH₂), 33.0 (CH₂), 51.8 (CH₂N), 54.1

(a-CH₂hexadecyl), 55.2 (CHNH), 57.2 (CHC(O)), 58.1 (NCH₂CH₂NH₂),

hexadecyl), 1.44 (m, 2H, g-CH₂hexadecyl), 1.56 (s, 9H, C(CH₃)₃), 1.84

G

60.5 (NCH₂CH₂NH₂), 82.4 (C(CH₃)₃), 121.0 (arom. CH), 129.3 (arom. C),

A

(m, 2H, b-CH₂hexadecyl), 2.42 (m, 1H, -CH_2a-), 2.73 (brd, J=14.5 Hz,

1H, -CH_2b-), 3.03 (m, 2H, a-CH₂hexadecyl), 3.35 (m, 1H,

-NCH₂CH_2aNH-), 3.64 (m, 1H, -NCH₂CH_2bNH-), 3.82 (dd, J=5.8,

13.0 Hz, 1H, -CH_2aN-), 3.93 (m, 2H, -CH_2bN-, -NCH_2aCH₂NH-), 4.13 (m,

131.6 (arom. CH), 132.5 (arom. CH), 134.2 (arom. C), 141.2 (arom. C),

163.5 (C=O), 164.4 (C=O), 170.6 (C=O); HRMS (FAB, 3-NBA): m/z:

calcd for C₃₅H₅₉N₄O₆S: 663.4156, found: 663.4177 [M+H]⁺.

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4139

H. Waldmann, A. Giannis et al.

2H, -CHNH-, -NCH_2bCH₂NH-), 4.26 (m, 1H, -CHC(O)-), 4.50 (m, 2H, -

OCH₂CH=CH₂), 5.28 (m, 1H, NH), 5.14–5.26 (m, 2H, -OCH₂CH=CH₂),

5.86 (m, 1H, -OCH₂CH=CH₂), 5.95 (m, 1H, NH), 7.45 (d, J=8.6 Hz,

1H, arom. CH), 8.14 (dd, J=2.0, 8.6 Hz, 1H, arom. CH), 8.46 (d, J=

2.1 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=14.2 (w-CH3

hexadecyl), 22.8 (CH₂), 23.6 (CH₂), 28.4 (CH₂), 29.3 (CH₂), 29.5 (CH₂),

29.7 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 29.9 (CH₂), 29.9 (CH₂),

29.9 (CH₂), 30.1 (CH₂), 32.1 (CH₂), 33.3 (CH₂), 51.8 (CH₂N), 54.2 (a-CH₂

hexadecyl), 55.4 (CHNH), 57.5 (CHC(O)), 58.1 (NCH₂CH₂NH₂), 60.3

(NCH₂CH₂NH₂), 66.1 (OCH₂CH=CH₂), 118.2 (OCH₂CH=CH₂), 121.4

(arom. CH), 129.4 (arom. C), 131.6 (arom. CH), 132.5 (arom. CH), 132.6

(OCH₂CH=CH₂), 134.0 (arom. C), 141.0 (arom. C), 156.5 (C=O), 164.3

(C=O), 170.3 (C=O), 175.1 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₃₅H₅₅N₄O₈S: 691.3741, found: 691.3755 [M+H]⁺.

-NCH_2bCH₂NH-), 4.12–4.30 (m, 2H, -CHC(O)-, -CHNH-), 4.80 (m, 1H,

a-CH Cys), 5.01 (m, 2H, 2CH Far), 5.15 (m, 1H, CH Far), 7.49 (d, J=

8.6 Hz, 1H, arom. CH), 8.08 (dd, J=2.1, 8.5 Hz, 1H, arom. CH), 8.30 (d,

J=2.1 Hz, 1H, arom. CH); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.3 (w-

CH₃hexadecyl), 16.2 (CH₃Far), 16.5 (CH₃Far), 16.5 (CH₃Far), 17.9

(CH₃Far), 22.7 (CH₂), 23.7 (CH₂), 25.9 (CH₂), 26.5 (CH₂), 26.8 (CH₂),

27.1 (CH₂), 27.3 (CH₂), 27.4 (CH₂), 27.7 (CH₂), 27.7 (CH₂), 28.2 (CH₂),

28.2 (CH₂), 28.3 (CH₂), 28.6 (CH₂), 29.0 (CH₂), 29.3 (CH₂), 29.9 (CH₂),

32.1 (CH₂), 33.2 (CH₂), 39.1 (CH₂Far), 39.8 (CH₂Far), 39.9 (CHNH),

43.5 (NCH₂CH₂NH₂), 49.7 (CH₂N), 51.9 (OCH₃), 52.7 (NCH₂CH₂NH₂),

52.7 (CHC(O), 54.3 (a-CH₂hexadecyl), 57.4 (a-CH Cys), 119.5 (CH

Far), 123.5 (arom. CH), 123.9 (CH Far), 124.5 (CH Far), 129.4 (arom. C),

129.7 (arom. CH), 131.5 (arom. CH), 131.6 (arom. C), 132.4 (C Far),

135.5 (C Far), 140.3 (C Far), 142.5 (arom. C), 164.4 (C=O), 165.4 (C=O),

170.4 (C=O), 171.6 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₅₀H₈₂N₅O₇S₂: 928.5656, found: 928.5638 [M+H]⁺.

10-(2-Allyloxycarbonylaminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-

5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diaze-

pine-7-yl-S-farnesyl-l-cysteine methyl ester(75) : At 08C, EDC (125 mg,

0.64 mmol) was added to a solution of acid 74 (367 mg, 0.53 mmol), H-

10-Cyanomethyl-(2S)-(methanesulfonylamino)-5,11-dioxo-2,3,5,10,11,11a-

hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-carboxylic acid tert-

butyl ester(77) : At 08C, MsCl (37 mL, 0.48 mmol) was added to a solu-

tion of amine 71 (118 mg, 0.32 mmol) and NEtiPr₂(161 mL, 0.64 mmol) in

DMF (15 mL). The reaction mixture was warmed to room temperature

and stirred for 22 h. Then, the solvent was removed under reduced pres-

sure, the residue was dissolved in ethyl acetate (50 mL) and washed with

1n HCl (30 mL). The organic layer was dried over Na₂SO₄, and, after re-

moval of the solvent under reduced pressure, the residue was purified by

chromatography (cyclohexane/ethyl acetate 1:5) to yield 77 as a colorless

solid (132 mg, 92%). M.p. 1148C; [a]²_D⁰= +378.4 (c=1.0 in CHCl₃);

R_f=0.34 (cyclohexane/ethyl acetate 1:5); ¹H NMR (CDCl₃, 400 MHz):

Cys(Far)-OMe 10 (216 mg, 0.63 mmol) and HOBt (166 mg, 1.06 mmol) in

A

CH₂Cl₂(20 mL). The solution was warmed to room temperature and stir-

red for 18 h. Then the reaction mixture was diluted with ethyl acetate

(50 mL) and subsequently washed with 1n HCl (30 mL) and Na₂CO₃sol-

ution (10%, 30 mL). The organic layer was dried over Na₂SO₄, and, after

removal of the solvent under reduced pressure, the residue was purified

by chromatography (cyclohexane/ethyl acetate 1:1) to yield 75 as color-

less solid (423 mg, 89%). M.p. 778C; [a]²_D⁰= +194.1 (c=0.5 in CHCl₃);

R_f=0.13 (cyclohexane/ethyl acetate 1:1); ¹H NMR (CDCl₃, 400 MHz):

d=0.88 (t, J=6.7 Hz, 3H, w-CH₃hexadecyl), 1.20–1.35 (m, 24H, 12

CH₂hexadecyl), 1.43 (m, 2H, g-CH₂hexadecyl), 1.50 (s, 6H, 2CH₃

Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃Far), 1.73 (m, 2H, b-CH₂

hexadecyl), 1.82–2.08 (m, 8H, 4CH₂Far), 2.33 (m, 1H, -CH_2a-), 2.62

(m, 1H, -CH_2b-), 2.90–3.24 (m, 6H, a-CH₂hexadecyl, b-CH₂Cys, -SCH₂-

), 3.38 (m, 1H, -NCH₂CH_2aNH-), 3.62 (m, 1H, -NCH₂CH_2bNH-), 3.76 (s,

3H, OCH₃), 3.83–3.99 (m, 3H, -CH₂N-, -NCH_2aCH₂NH-), 4.12–4.30 (m,

3H, -CHNH-, -CHC(O)-, -NCH_2bCH₂NH-), 4.50 (m, 2H, -OCH₂CH=

CH₂), 4.96 (m, 1H, a-CH Cys), 5.04–5.29 (m, 6H, 3CH Far,

-OCH₂CH=CH₂, NH), 5.84 (m, 1H, OCH₂CH=CH₂), 5.96 (m, 1H, NH),

7.23 (m, 1H, NH), 7.49 (d, J=8.6 Hz, 1H, arom. CH), 8.08 (dd, J=2.1,

8.6 Hz, 1H, arom. CH), 8.30 (d, J=2.1 Hz, 1H, arom. CH); ¹³C NMR

(CDCl₃, 100.6 MHz): d=14.3 (w-CH₃hexadecyl), 16.2 (CH₃Far), 16.4

(CH₃Far), 16.5 (CH₃Far), 17.9 (CH₃Far), 22.9 (CH₂), 23.8 (CH₂), 25.9

(CH₂), 26.6 (CH₂), 26.9 (CH₂), 27.0 (CH₂), 27.3 (CH₂), 27.5 (CH₂), 27.7

(CH₂), 27.9 (CH₂), 28.1 (CH₂), 28.2 (CH₂), 28.4 (CH₂), 28.7 (CH₂), 29.0

(CH₂), 29.4 (CH₂), 29.9 (CH₂), 32.1 (CH₂), 33.3 (CH₂), 39.2 (CH₂Far),

39.8 (CH₂Far), 39.9 (CHNH), 43.3 (NCH₂CH₂NH), 49.8 (CH₂N), 51.9

(OCH₃), 52.5 (NCH₂CH₂NH), 52.9 (CHC(O), 54.3 (a-CH₂hexadecyl),

57.4 (a-CH Cys), 66.0 (OCH₂CH=CH₂), 118.08 (OCH₂CH=CH₂), 119.6

(CH Far), 123.4 (arom. CH), 123.9 (CH Far), 124.5 (CH Far), 129.2

(arom. C), 129.8 (arom. CH), 131.5 (arom. CH), 131.7 (arom. C), 132.4

(C Far), 132.9 (OCH₂CH=CH₂), 135.5 (C Far), 140.3 (C Far), 142.5

(arom. C), 156.7 (C=O), 164.5 (C=O), 165.1 (C=O), 170.2 (C=O), 171.6

(C=O); HRMS (FAB, 3-NBA): m/z: calcd for C₅₄H₈₅N₅NaO₉S₂:

1034.5689, found: 1034.5699 [M+Na]⁺.

d=1.52 (s, 9H, C(CH₃)₃), 2.42 (m, 1H, -CH_2a-), 2.61 (m, 1H, -CH_2b-),

G

2.95 (s, 3H, CH₃SO₂-), 3.70 (dd, J=2.5, 12.9 Hz, 1H, -CH_2aN-), 3.90 (dd,

(5.9, 12.9 Hz, 1H, -CH_2bN-), 4.18 (m, 2H, -CHNH-, -CHC(O)-), 4.34 (d,

J=17.4, 1H, -CH_2aCN), 4.93 (d, J=17.4, 1H, -CH_2bCN), 7.42 (d, J=

8.6 Hz, 1H, arom. CH), 8.16 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.42 (d,

2.1 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.1 (C-

AHCTREUNG

129.0 (arom. C), 132.0 (arom. CH), 132.4 (arom. CH), 134.0 (arom. C),

141.1 (arom. C), 163.9 (C=O), 164.6 (C=O), 169.8 (C=O); HRMS (FAB,

3-NBA): m/z: calcd for C₂₀H₂₅N₄O₆S: 449.1496, found: 449.1486 [M+H]⁺.

10-(2-Aminoethyl)-(2S)-(methanesulfonylamino)-5,11-dioxo-

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-car-

boxylic acid tert-butyl ester(78) : PtO₂·H₂O (8 mg, 0.03 mmol) was added

to a degassed solution of nitrile 77 (123 mg, 0.27 mmol) in EtOH (9 mL)

and CHCl₃(180 mL). The reaction mixture was hydrogenated under hy-

drogen atmosphere at room temperature for 4.5 h. Then, the suspension

was filtered through a pad of Celite. After removal of the solvents under

reduced pressure, the residue was purified by chromatography (CH₂Cl₂/

EtOH 10:1, then 1:1

+ 1% NEt₃) to yield 78 as a colorless solid

(105 mg, 85%). M.p. 1238C; [a]²_D⁰= +310.1 (c=1.0 in CHCl₃); R_f=0.39

(CH₂Cl₂/EtOH 1:1); ¹H NMR (CDCl₃, 400 MHz): d=1.58 (s, 9H, C-

A

3H, CH₃SO₂-), 3.03 (m, 2H, -NCH₂CH₂NH₂), 3.84 (dd, J=5.5, 13.0 Hz,

1H, -CH_2aN-), 3.91 (m, 2H, -CH_2bN-, NCH_2aCH₂NH₂), 4.17 (d, J=

8.8 Hz, 1H, -CHNH-), 4.24 (m, 2H, -CHC(O)-, NCH_2bCH₂NH₂), 7.48 (d,

J=8.6 Hz, 1H, arom. CH), 8.14 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.49

(d, J=2.0 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=28.3

10-(2-Aminoethyl)-(2S)-(hexadecane-1-sulfonylamino)-5,11-dioxo-

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-yl-S-

farnesyl-l-cysteine methyl ester(76) : [Pd(PPh₃)₄] (23 mg, 0.02 mmol) was

A

added to a degassed solution of benzodiazepine 75 (200 mg, 0.2 mmol)

and N,N’-dimethylbarbituric acid (31 mg, 0.2 mmol) in THF (20 mL). The

mixture was stirred for 3 h at room temperature. After removal of the

solvent under reduced pressure, the residue was purified by chromatogra-

phy (CH₂Cl₂/EtOH 30:1, then 15:1, then 5:1 + 1% NEt₃) to yield 76 as

a slightly yellow foam (147 mg, 80%). [a]²_D⁰= +165.2 (c=0.5 in CHCl₃);

R_f=0.16 (CH₂Cl₂/EtOH); ¹H NMR (CD₃OD, 400 MHz): d=0.88 (t, J=

6.7 Hz, 3H, w-CH₃hexadecyl), 1.20–1.35 (m, 24H, 12CH₂hexadecyl),

1.43 (m, 2H, g-CH₂hexadecyl), 1.50 (s, 6H, 2CH₃Far), 1.55 (s, 3H,

CH₃Far), 1.57 (s, 3H, CH₃Far), 1.73 (m, 2H, b-CH₂hexadecyl), 1.82–

2.08 (m, 8H, 4CH₂Far), 2.33 (m, 1H, -CH_2a-), 2.62 (m, 1H, -CH_2b-),

2.90–3.20 (m, 8H, a-CH₂hexadecyl, b-CH₂Cys, -SCH₂-, -NCH₂CH₂NH₂),

3.70 (m, 1H, -NCH_2aCH₂NH₂), 3.74 (s, 3H, OCH₃), 3.99 (m, 1H,

(C

ACHTREUNG

(CHC(O)), 58.1 (NCH₂CH₂NH₂), 60.5 (NCH₂CH₂NH₂), 82.4 (C

ACHTREUNG

121.0 (arom. CH), 129.3 (arom. C), 131.6 (arom. CH), 132.5 (arom. CH),

134.2 (arom. C), 141.2 (arom. C), 163.5 (C=O), 164.4 (C=O), 170.6 (C=

O); HRMS (FAB, 3-NBA): m/z: calcd for C₂₀H₂₉N₄O₆S: 453.1809, found:

453.1793 [M+H]⁺.

10-(2-Allyloxycarbonylaminoethyl)-(2S)-(methanesulfonylamino)-5,11-

dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-

7-carboxylic acid tert-butyl ester(79) : AlocCl (40 mL, 0.36 mmol) was

added to a solution of amine 78 (107 mg, 0.24 mmol) and NEt₃(66 mL,

0.47 mmol) in CH₂Cl₂(4 mL). After stirring at room temperature for

18 h, the solution was diluted with ethyl acetate (50 mL) and washed

4140

www.chemeurj.org

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

with 1n HCl (10 mL). The organic layer was dried over Na₂SO₄, and,

after removal of the solvent under reduced pressure, the residue was pu-

rified by chromatography (cyclohexane/ethyl acetate 1:5) to yield 79 as a

colorless solid (105 mg, 83%). M.p. 1118C; [a]²_D⁰= +288.3 (c=1.0 in

CHCl₃); R_f=0.28 (cyclohexane/ethyl acetate 1:5); ¹H NMR (CDCl₃,

a slightly yellow foam (21 mg, 65%). [a]²_D⁰= +180.5 (c=0.5 in CHCl₃);

R_f=0.16 (CH₂Cl₂/EtOH); ¹H NMR (CD₃OD, 400 MHz): d=1.50 (s, 6H,

2CH₃Far), 1.55 (s, 3H, CH₃Far), 1.57 (s, 3H, CH₃Far), 1.82–2.08 (m,

8H, 4CH₂Far), 2.33 (m, 1H, -CH_2a-), 2.62 (m, 1H, -CH_2b-), 2.95 (s, 3H,

CH₃SO₂), 2.90–3.20 (m, 6H, b-CH₂Cys, -SCH₂-, -NCH₂CH₂NH₂), 3.70

(m, 1H, -NCH_2aCH₂NH₂), 3.74 (s, 3H, OCH₃), 3.99 (m, 1H,

-NCH_2bCH₂NH₂), 4.12–4.30 (m, 2H, -CHC(O)-, -CHNH-), 4.80 (m, 1H,

a-CH Cys), 5.01 (m, 2H, 2CH Far), 5.15 (m, 1H, CH Far), 7.49 (d, J=

8.6 Hz, 1H, arom. CH), 8.08 (dd, J=2.1, 8.5 Hz, 1H, arom. CH), 8.30 (d,

J=2.1 Hz, 1H, arom. CH); ¹³C NMR (CD₃OD, 100.6 MHz): d=16.2

(CH₃Far), 16.5 (CH₃Far), 16.5 (CH₃Far), 17.9 (CH₃Far), 22.9 (CH₂

Far), 23.9 (CH₂Far), 33.2 (CH₂), 32.1 (CH₂S), 39.1 (CH₂Far), 39.4

(CH₂), 39.8 (CH₂Far), 39.9 (CHNH), 43.5 (NCH₂CH₂NH₂), 49.7 (CH₂N),

51.4 (CH₃SO₂), 51.9 (OCH₃), 52.7 (NCH₂CH₂NH₂), 52.7 (CHC(O), 57.4

(a-CH Cys), 119.5 (CH Far), 123.5 (arom. CH), 123.9 (CH Far), 124.5

(CH Far), 129.4 (arom. C), 129.7 (arom. CH), 131.5 (arom. CH), 131.6

(arom. C), 132.4 (C Far), 135.5 (C Far), 140.3 (C Far), 142.5 (arom. C),

164.4 (C=O), 165.4 (C=O), 170.4 (C=O), 171.6 (C=O); HRMS (FAB, 3-

NBA): m/z: calcd for C₃₅H₅₁N₅NaO₇S₂: 740.3130, found: 740.3144

[M+Na]⁺.

400 MHz): d=1.58 (s, 9H, C(CH₃)₃), 2.41 (m, 1H, -CH_2a-), 2.74 (brd, J=

A

14.5 Hz, 1H, -CH_2b-), 2.95 (s, 3H, CH₃SO₂-), 3.37 (m, 1H,

-NCH₂CH_2aNH-), 3.63 (m, 1H, -NCH₂CH_2bNH-), 3.82 (dd, J=5.8,

13.0 Hz, 1H, -CH_2aN-), 3.93 (m, 2H, -CH_2bN-, -NCH_2aCH₂NH-), 4.14 (m,

2H, -CHNH-, -NCH_2bCH₂NH-), 4.27 (m, 1H, -CHC(O)-), 4.50 (m, 2H,

-OCH₂CH=CH₂), 5.00 (m, 1H, NH Urethan), 5.14–5.28 (m, 2H,

-OCH₂CH=CH₂), 5.86 (m, 1H, -OCH₂CH=CH₂), 5.93 (m, 1H, NH), 7.44

(d, J=8.6 Hz, 1H, arom. CH), 8.15 (dd, J=2.0, 8.6 Hz, 1H, arom. CH),

8.48 (d, J=2.1 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=

28.3 (C

57.2 (CHC(O)), 58.1 (NCH₂CH₂NH₂), 60.4 (NCH₂CH₂NH₂), 66.1

(OCH₂CH=CH₂), 82.3 (C(CH₃)₃), 118.0 (OCH₂CH=CH₂), 121.2 (arom.

A

AHCTREUNG

CH), 129.3 (arom. C), 131.5 (arom. CH), 132.4 (arom. CH), 132.6

(OCH₂CH=CH₂), 134.2 (arom. C), 141.2 (arom. C), 156.7 (C=O), 163.4

(C=O), 164.4 (C=O), 170.4 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₂₄H₃₃N₄O₈S: 537.2020, found: 537.2007 [M+H]⁺.

Synthesis of the pentapeptide substrate for the inhibition studies of

APT1

10-(2-Allyloxycarbonylaminoethyl)-(2S)-(methanesulfonylamino)-5,11-

dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2a,1,4]diazepine-

G

Bis-[(N-Acetyl-l-methionyl)-l-seryl]-l-cystine-di-tert-butyl ester(82) : At

08C, EDC (253 mg, 1.29 mmol) was added to a solution of Ac-Met-Ser-

OH 16 (300 mg, 1.08 mmol), (HCl·H-Cys-OtBu)₂(252 mg, 0.59 mmol),

HOBt (337 mg, 2.16 mmol) and NEtiPr₂(221 mL, 1.29 mmol) in DMF

(25 mL). The reaction mixture was warmed to room temperature and

stirred for 18 h. After removal of the solvent under reduced pressure, the

residue was dissolved in CHCl₃(100 mL) and subsequently washed with

1n HCl (20 mL) and Na₂CO₃solution (10%, 20 mL). The organic layer

was dried over Na₂SO₄, and, after removal of the solvent under reduced

pressure, the residue was purified by chromatography (CH₂Cl₂/EtOH

10:1, then 5:1) to yield 82 as a colorless oil (336 mg, 65%). [a]²_D⁰= ꢀ16.4

(c=1.0 in MeOH); R_f=0.10 (CH₂Cl₂/ethanol 10:1); ¹H NMR (CD₃OD,

7-yl-S-farnesyl-l-cysteine methyl ester(80) : TFA (5 mL) was added to a

solution of benzodiazepine 79 (50 mg, 0.09 mmol) in CH₂Cl₂(8 mL).

After stirring at room temperature for 4 h, the solvent was removed

under reduced pressure and the residue was coevaporated with toluene

to yield the acid as a colorless solid (45 mg, quant.).

At 08C, EDC (22 mg, 0.11 mmol) was added to a solution of the above

acid (45 mg, 0.09 mmol), H-Cys(Far)-OMe 10 (32 mg, 0.09 mmol) and

A

HOBt (29 mg, 0.19 mmol) in CH₂Cl₂(10 mL). The solution was warmed

to room temperature and stirred for 18 h. Then the reaction mixture was

diluted with ethyl acetate (50 mL) and subsequently washed with 1n HCl

(30 mL) and Na₂CO₃solution (10%, 30 mL). The organic layer was dried

over Na₂SO₄, and, after removal of the solvent under reduced pressure,

the residue was purified by chromatography (cyclohexane/ethyl acetate

400 MHz): d=1.48 (s, 18H, 2C(CH₃)₃), 2.01 (s, 6H, 2CH₃acetyl),

G

2.09 (s, 6H, 2CH₃Met), 1.91–2.14 (m, 4H, 2b-CH₂Met), 2.55 (m,

4H, 2g-CH₂Met), 3.06 (dd, J=7.8, 13.8 Hz, 2H, 2b-CH_2aCys), 3.20

(dd, J=5.3, 13.9 Hz, 2H, 2b-CH_2bCys), 3.82 (m, 4H, 2b-CH₂Ser),

4.51 (m, 4H, 4a-CH Ser, Cys), 4.68 (m, 2H, 2a-CH Met); ¹³C NMR

(CD₃OD, 100.6 MHz): d=14.2 (CH₃acetyl), 21.4 (CH₃Met), 27.1 (C-

1:5) to yield 80 as colorless solid (56 mg, 75%). M.p. 928C; [a]_D²⁰

=

+196.6 (c=0.5 in CHCl₃); R_f=0.13 (cyclohexane/ethyl acetate 1:5);

¹H NMR (CDCl₃, 400 MHz): d=1.50 (s, 6H, 2CH₃Far), 1.55 (s, 3H,

CH₃Far), 1.57 (s, 3H, CH₃Far), 1.82–2.08 (m, 8H, 4CH₂Far), 2.33 (m,

1H, -CH_2a-), 2.62 (m, 1H, -CH_2b-), 2.95 (s, 3H, CH₃SO₂-), 2.90–3.24 (m,

4H, b-CH₂Cys, -SCH₂-), 3.38 (m, 1H, -NCH₂CH_2aNH-), 3.62 (m, 1H,

-NCH₂CH_2bNH-), 3.76 (s, 3H, OCH₃), 3.83–3.99 (m, 3H, -CH₂N-,

AHCTREUNG

-NCH_2aCH₂NH-),

4.12–4.30

(m,

3H,

-CHNH-,

-CHC(O)-,

(FAB, 3-NBA): m/z: calcd for C₃₄H₆₁N₆O₁₂S₄: 873.3231, found: 873.3268

[M+H]⁺.

-NCH_2bCH₂NH-), 4.50 (m, 2H, -OCH₂CH=CH₂), 4.96 (m, 1H, a-CH

Cys), 5.04–5.29 (m, 6H, 3CH Far, -OCH₂CH=CH₂, NH), 5.84 (m, 1H,

OCH₂CH=CH₂), 5.96 (m, 1H, NH), 7.23 (m, 1H, NH), 7.49 (d, J=

8.6 Hz, 1H, arom. CH), 8.08 (dd, J=2.1, 8.6 Hz, 1H, arom. CH), 8.30 (d,

J=2.1 Hz, 1H, arom. CH); ¹³C NMR (CDCl₃, 100.6 MHz): d=16.2 (CH₃

Far), 16.4 (CH₃Far), 16.5 (CH₃Far), 17.9 (CH₃Far), 22.9 (CH₂Far), 23.3

(CH₂Far), 32.1 (CH₂S), 33.3 (CH₂), 39.2 (CH₂Far), 39.4 (CH₂), 39.8

(CH₂Far), 39.9 (CHNH), 43.3 (NCH₂CH₂NH), 49.8 (CH₂N), 51.4

(CH₃SO₂), 51.9 (OCH₃), 52.5 (NCH₂CH₂NH), 52.9 (CHC(O), 57.4 (a-

CH Cys), 66.0 (OCH₂CH=CH₂), 118.0 (OCH₂CH=CH₂), 119.6 (CH Far),

123.4 (arom. CH), 123.9 (CH Far), 124.5 (CH Far), 129.2 (arom. C), 129.8

(arom. CH), 131.5 (arom. CH), 131.7 (arom. C), 132.4 (C Far), 132.9

(OCH₂CH=CH₂), 135.5 (C Far), 140.3 (C Far), 142.5 (arom. C), 156.7

(C=O), 164.5 (C=O), 165.1 (C=O), 170.2 (C=O), 171.6 (C=O); HRMS

(FAB, 3-NBA): m/z: calcd for C₃₉H₅₆N₅O₉S₂[M+H]⁺: 802.3520, found:

802.3527.

N-Acetyl-l-methionyl-l-seryl-S-palmitoyl-l-cysteine-tert-butyl ester(83) :

1,4-Dithiothreitol (208 mg, 1.35 mmol) and NEt₃(75 mL, 0.54 mmol) were

added to

a degassed solution of (Ac-Met-Cys-OtBu)₂82 (0.236 g,

0.27 mmol) in CH₂Cl₂(10 mL). After stirring at room temperature for

2 h, the solution was washed with 1n HCl (22 mL). The organic layer

was dried over Na₂SO₄and cooled down to 08C. NEt₃(75 mL, 054 mmol)

and palmitoyl chloride (418 mL, 1.35 mmol) were added to this solution.

The solution was warmed to room temperature and stirred for 3.5 h.

Then, the reaction mixture was diluted with ethyl acetate (100 mL) and

washed with 1n HCl (20 mL). The organic layer was dried over Na₂SO₄,

and, after removal of the solvents under reduced pressure, the residue

was purified by chromatography (CH₂Cl₂/EtOH 30:1, then, 20:1, then,

10:1) to yield 83 as a colorless solid (273 mg, 75%). M.p. 538C; [a]_D²⁰

=

ꢀ15.0 (c=1.0 in CHCl₃); R_f=0.31 (CH₂Cl₂/EtOH); ¹H NMR (CDCl₃,

400 MHz): d=0.88 (t, J=6.6 Hz, 3H, w-CH₃Pal), 1.20–1.40 (m, 24H,

10-(2-Aminoethyl)-(2S)-(methanesulfonylamino)-5,11-dioxo-

12CH₂Pal), 1.48 (s, 9H, C(CH₃)₃), 1.65 (m, 2H, b-CH₂Pal), 2.05 (s,

A

2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a,1,4]diazepine-7-yl-S-

3H, CH₃acetyl), 2.12 (s, 3H, CH₃Met), 1.94–2.18 (m, 2H, b-CH₂Met),

2.59 (m, 4H, a-CH₂Pal, g-CH₂Met), 3.30 (dd, J=7.8, 13.9 Hz, 1H, b-

CH_2aCys), 3.48 (dd, J=5.3, 13.9 Hz, 1H, b-CH_2bCys), 3.50 (dd, J=5.4,

11.5 Hz, 1H, b-CH_2aSer), 4.03 (dd, J=3.9, 11.7 Hz, 1H, b-CH_2bSer),

4.52 (m, 1H, a-CH Ser), 4.68 (m, 2H, a-CH Cys, a-CH Met), 6.62 (m,

1H, NH), 7.21 (m, 1H, NH), 7.25 (m, 1H, NH); ¹³C NMR (CDCl₃,

100.6 MHz): d=14.3 (w-CH₃Pal), 22.9 (CH₃acetyl), 23.2 (CH₃Met),

farnesyl-l-cysteine methyl ester(81) : [Pd(PPh₃)₄] (11 mg, 0.01 mmol) was

A

added to a degassed solution of benzodiazepine 80 (37 mg, 0.05 mmol)

and N,N’-dimethylbarbituric acid (7 mg, 0.05 mmol) in THF (5 mL). The

mixture was stirred for 3 h at room temperature. After removal of the

solvent under reduced pressure, the residue was purified by chromatogra-

phy (CH₂Cl₂/EtOH 20:1, then 15:1, then 5:1 + 1% NEt₃) to yield 81 as

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4141

H. Waldmann, A. Giannis et al.

23.3 (CH₂), 25.7 (C

A

reduced pressure, the residue was purified by chromatography (CH₂Cl₂/

EtOH 10:1, then 5:1, then 1:1 + 1% NEt₃) to yield 4 as a colorless solid

(CH₂), 28.9 (CH₂), 29.1 (CH₂), 29.3 (CH₂), 29.4 (CH₂), 29.5 (CH₂), 29.6

(CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 30.1 (CH₂), 30.2 (CH₂), 30.3

(CH₂), 32.1 (CH₂), 44.3 (a-CH₂Pal), 52.7 (a-CH Cys), 53.4 (a-CH Met),

(34 mg, 75%). M.p. 698C; [a]_D²⁰

=

ꢀ9.0 (c=0.5 in MeOH); R_f=0.01

(CH₂Cl₂/EtOH 10:1); ¹H NMR (CD₃OD, 400 MHz): d=0.78 (t, J=

6.6 Hz, 3H, w-CH₃Pal), 1.15 (m, 24H, 12CH₂Pal), 1.49 (s, 6H, 2CH₃

Far), 1.57 (s, 6H, 2CH₃Far), 1.26–1.68 (m, 8H, 2CH₂Lys, 2 CH₂

Pal), 1.93 (s, 3H, CH₃acetyl), 2.00 (s, 3H, CH₃Met), 1.76–2.05 (m, 12H,

b-CH₂Met, CH₂Lys, 4 CH₂Far), 2.46 (m, 2H, g-CH₂Met), 2.68–3.17

(m, 8H, a-CH₂Pal, 2b-CH₂Cys, -SCH₂-), 3.26–3.46 (m, 2H, e-CH₂

Lys), 3.64 (s, 3H, OCH₃), 3.68 (m, 1H, b-CH_2aSer), 3.87 (m, 1H, b-CH_2b

Ser), 4.25–4.54 (m, 5H, 5a-CH), 5.00 (m, 2H, 2CH Far), 5.14 (m, 1H,

CH Far); ¹³C NMR (CD₃OD, 100.6 MHz): d=14.0 (w-CH₃Pal), 16.5

(CH₃acetyl), 16.8 (CH₃Met), 17.0 (CH₃Far), 17.2 (CH₃Far), 19.3 (CH₃

Far), 19.3 (CH₂), 21.3 (CH₂), 24.0 (CH₂), 24.3 (CH₂), 25.3 (CH₃Far), 28.6

(CH₂), 29.2 (CH₂), 29.3 (CH₂), 30.0 (CH₂), 30.0 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3

(CH₂), 30.9 (CH₂), 31.8 (CH₂), 32.5 (CH₂), 33.8 (CH₂), 34.4 (CH₂), 34.5

(CH₂), 39.8 (CH₂Far), 40.5 (CH₂Far), 44.6 (e-CH₂Lys), 50.4 (OCH₃),

53.5 (a-CH), 54.2 (a-CH), 54.5 (a-CH₂Pal), 56.0 (a-CH), 56.1 (a-CH),

57.1 (a-CH), 64.8 (b-CH₂Ser), 117.1 (CH Far), 121.7 (CH Far), 122.5

(CH Far), 133.9 (C Far), 138.8 (C Far), 139.3 (C Far), 170.9 (C=O), 172.0

(C=O), 174.7 (C=O), 175.4 (C=O), 197.6 (C=O); HRMS (FAB, 3-NBA):

m/z: calcd for C₅₄H₉₇N₆O₉S₃: 1069.6480, found: 1069.6503 [M+H]⁺.

54.8 (a-CH Ser), 63.0 (b-CH₂Ser), 83.6 (C(CH₃)₃), 169.1 (C=O), 170.3

T

(C=O), 170.6 (C=O), 171.9 (C=O), 197.6 (C=O); HRMS (FAB, 3-NBA):

m/z: calcd for C₃₃H₆₂N₃O₇S₂: 676.4030, found: 676.4008 [M+H]⁺.

N-Acetyl-l-methionyl-l-seryl-S-palmitoyl-l-cysteine (84): TFA (10 mL)

was added to

a

solution of Ac-Met-Ser-Cys(Pal)-OtBu 83 (100 mg,

A

015 mmol) in CH₂Cl₂(10 mL). After stirring at room temperature for

2 h, the solvent was removed under reduced pressure and the residue was

coevaporated with toluene to yield 84 as a colorless solid (92 mg, quant.).

M.p. 648C; [a]²_D⁰= ꢀ17.0 (c=1.0 in MeOH); R_f=0.04 (CH₂Cl₂/EtOH

10:1); ¹H NMR (CD₃OD, 400 MHz): d=0.88 (t, J=6.6 Hz, 3H, w-CH₃

Pal), 1.20–1.40 (m, 24H, 12CH₂Pal), 1.65 (m, 2H, b-CH₂Pal), 2.05 (s,

3H, CH₃acetyl), 2.12 (s, 3H, CH₃Met), 1.94–2.18 (m, 2H, b-CH₂Met),

2.59 (m, 4H, a-CH₂Pal, g-CH₂Met), 3.30 (dd, J=7.8, 13.9 Hz, 1H, b-

CH_2aCys), 3.48 (dd, J=5.3, 13.9 Hz, 1H, b-CH_2bCys), 3.50 (dd, J=5.4,

11.5 Hz, 1H, b-CH_2aSer), 4.03 (dd, J=3.9, 11.7 Hz, 1H, b-CH_2bSer),

4.38 (m, 1H, a-CH Ser), 4.49 (m, 2H, a-CH Cys, a-CH Met); ¹³C NMR

(CD₃OD, 100.6 MHz): d=14.3 (w-CH₃Pal), 22.9 (CH₃acetyl), 23.2 (CH₃

Met), 23.3 (CH₂), 28.1 (CH₂), 28.8 (CH₂), 28.4 (CH₂), 28.6 (CH₂), 28.9

(CH₂), 29.1 (CH₂), 29.3 (CH₂), 29.6 (CH₂), 29.7 (CH₂), 29.8 (CH₂), 29.9

(CH₂), 30.1 (CH₂), 30.2 (CH₂), 30.3 (CH₂), 32.1 (CH₂), 44.3 (a-CH₂Pal),

52.7 (a-CH Cys), 53.4 (a-CH Met), 54.8 (a-CH Ser), 63.0 (b-CH₂Ser),

169.1 (C=O), 170.3 (C=O), 170.6 (C=O), 174.9 (C=O), 197.6 (C=O);

HRMS (FAB, 3-NBA): m/z: calcd for C₂₉H₅₃N₃O₇S₂Na: 642.3225, found:

642.3218 [M+Na]⁺.

Assay forinhibition of APT1 : The assay for inhibition of APT1 is based

on the free fatty acid indicator Acrylodan labeled Intestinal Fatty Acid

Binding Protein (ADIFAB). Our system has been adapted from an assay

described in the literature.^[37,38]

ADIFAB storage solution: 200 mg ADIFAB (from Molecular Probes,

Oregon) were dissolved in 1000 mL storage buffer (50 mm HEPES, 1 mm

EDTA, 0.5 mm phenylmethylsulfonylfluoride, 0.05% NaN₃, pH 8.0).

N-Acetyl-l-methionyl-l-seryl-S-palmitoyl-l-cysteyl-N^e-(allyloxycar-

bonyl)-l-lysyl-S-farnesyl-l-cysteine methyl ester(85)

(23 mg, 0.12 mmol) was added to a solution of Ac-Met-Ser-Cys

84 (60 mg, 0.10 mmol), freshly prepared H-Lys(Aloc)-Cys(Far)-OMe 19

:

At 08C, EDC

Substrate solution in sample buffer: Ac-Met-Ser-Cys

N

AHCTREUNG

OMe (86; 4 mg, 3.74 mmol) was dissolved in DMSO (500 mL). 5 mL of this

solution were added to 50 mL with sample buffer (20 mm HEPES, 150 mm

NaCl, 5 mm KCl, 1 mm Na₂HPO₄, pH 7.4).

A

ACHTREUNG

(53 mg, 0.10 mmol) and HOBt (30 mg, 0.19 mmol) in CH₂Cl₂(10 mL).

The solution was warmed to room temperature and stirred for 16 h. Then

the reaction mixture was diluted with ethyl acetate (30 mL) and subse-

quently washed with 1n HCl (10 mL) and Na₂CO₃solution (10%,

10 mL). The organic layer was dried over Na₂SO₄, and, after removal of

the solvent under reduced pressure, the residue was purified by chroma-

tography (CH₂Cl₂/EtOH 20:1, then 10:1) to yield 85 as colorless solid

(55 mg, 65%). M.p. 718C; [a]²_D⁰= ꢀ11.4 (c=0.5 in CHCl₃); R_f=0.31

(CH₂Cl₂/EtOH 10:1); ¹H NMR (CDCl₃/CD₃OD 1:1, 400 MHz): d=0.78

(t, J=6.6 Hz, 3H, w-CH₃Pal), 1.15 (m, 24H, 12CH₂Pal), 1.49 (s, 6H,

2CH₃Far), 1.57 (s, 6H, 2CH₃Far), 1.26–1.68 (m, 8H, 2CH₂Lys, 2

CH₂Pal), 1.93 (s, 3H, CH₃acetyl), 2.00 (s, 3H, CH₃Met), 1.76–2.05 (m,

12H, b-CH₂Met, CH₂Lys, 4 CH₂Far), 2.46 (m, 2H, g-CH₂Met), 2.68–

3.17 (m, 8H, a-CH₂Pal, 2b-CH₂Cys, -SCH₂-), 3.26–3.46 (m, 2H, e-CH₂

Lys), 3.64 (s, 3H, OCH₃), 3.68 (m, 1H, b-CH_2aSer), 3.87 (m, 1H, b-CH_2b

Ser), 4.25–4.54 (m, 7H, 5a-CH, -OCH₂CH=CH₂), 5.00 (m, 2H, 2CH

Far), 5.08–5.22 (m, 3H, CH Far, -OCH₂CH=CH₂), 5.74 (m, 1H,

-OCH₂CH=CH₂); ¹³C NMR (CDCl₃/CD₃OD 1:1, 100.6 MHz): d=14.0

(w-CH₃Pal), 16.5 (CH₃acetyl), 16.8 (CH₃Met), 17.0 (CH₃Far), 17.2

(CH₃Far), 19.3 (CH₃Far), 19.3 (CH₂), 21.3 (CH₂), 24.0 (CH₂), 24.3

(CH₂), 25.3 (CH₃Far), 28.6 (CH₂), 29.2 (CH₂), 29.3 (CH₂), 30.0 (CH₂),

30.0 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂),

30.3 (CH₂), 30.3 (CH₂), 30.3 (CH₂), 30.9 (CH₂), 31.8 (CH₂), 32.5 (CH₂),

33.8 (CH₂), 34.4 (CH₂), 34.5 (CH₂), 39.8 (CH₂Far), 40.5 (CH₂Far), 44.6

(e-CH₂Lys), 50.4 (OCH₃), 53.5 (a-CH), 54.2 (a-CH), 54.5 (a-CH₂Pal),

56.0 (a-CH), 56.1 (a-CH), 57.1 (a-CH), 64.8 (b-CH₂Ser), 67.3

(OCH₂CH=CH₂), 115.1 (OCH₂CH=CH₂), 117.1 (CH Far), 121.7 (CH

Far), 122.5 (CH Far), 133.9 (C Far), 137.5 (OCH₂CH=CH₂), 138.8 (C

Far), 139.3 (C Far), 157.5 (C=O), 170.9 (C=O), 172.0 (C=O), 174.7 (C=

O), 175.4 (C=O), 197.6 (C=O); HRMS (FAB, 3-NBA): m/z: calcd for

C₅₈H₁₀₀N₆O₁₁S₃Na: 1175.6512, found: 1175.6524 [M+Na]⁺.

Inhibitorsolutions in sample buffer : According to their molecular mass

all inhibitors were treated like the above substrate leading to the desired

concentrations of the inhibitor solutions.

APT1 solution: The applied APT1 solution was the result of the isolation

of the enzyme from rat liver following a protocol of Duncan et al.^[24]and

consisted of 30 mg APT1 in 10 mL buffer (50 mm HEPES, 2 mm MgCl₂,

1 mm EDTA, 7.5 mm CHAPS, pH 8.0).

Determination of the maximal palmitate concentration: Substrate solu-

tion (2 mL) were pipetted into

a quarz cuvette with sample buffer

(1.5 mL) resulting a substrate concentration of 1 mM. Then, after excita-

tion at 386 nm the fluorescence emissions at 432 and 505 nm were meas-

ured at 258C (I₅₀₅^blank, I₄₃₂^blank). Subsequently, ADIFAB solution (10 mL)

was added, and again the fluorescence emissions at 432 and 505 nm were

measured (I₅₀₅⁰, I₄₃₂⁰). The R₀value was then calculated as follows:

0

blank

I₅₀₅ꢀI₅₀₅

R₀

¼

0

I₄₃₂ꢀI₄₃₂

Now, APT1 solution (20 mL) was added, and, after incubation at 258C for

15 min, the fluorescence emissions at 432 and 505 nm were measured at

least eight times and averaged (I₅₀₅, I₄₃₂). The R value was then calculated

as follows:

blank

I₅₀₅ꢀI₅₀₅

R ¼ R₀

¼

blank

I₄₃₂ꢀI₄₃₂

The free palmitate concentration was calculated as follows:

RꢀR₀

½ADIFAB_totalÂ 19:5 Â ðRꢀR₀Þ

11:5ꢀR þ 19:5 Â ðRꢀR₀

N-Acetyl-l-methionyl-l-seryl-S-palmitoyl-l-cysteyl-l-lysyl-S-farnesyl-l-

½Pal ¼ K_dÂ 19:5 Â

þ

11:5ꢀR

cysteine methyl ester(86) : [Pd(PPh₃)₄] (7 mg, 0.01 mmol) was added to a

A

degassed solution of pentapeptide 85 (50 mg, 0.04 mmol) and N,N’-dime-

thylbarbituric acid (7 mg, 0.04 mmol) in THF (6 mL). The mixture was

stirred for 4 h at room temperature. After removal of the solvent under

In this equation K_d=0.28 and [ADIFAB]_total=100 nm according to the lit-

erature.^[37]

4142

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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2006, 12, 4121 – 4143

Thioesterase Inhibitors

FULL PAPER

Determination of the IC₅₀values: The free palmitate concentration when

inhibitors are present, was measured with the method described above.

The determination of the blank values (I₅₀₅^blank, I₄₃₂^blank) was measured

after the appropriate amount of inhibitor was added. In order to calcu-

late the IC₅₀values, the relative palmitate concentration in percent (pal-

mitate with inhibitor/palmitate without inhibitor) were used in a curve

fitting with the program Origin.

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Received: September 12, 2005

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Published online: March 10, 2006

Chem. Eur. J. 2006, 12, 4121 – 4143

ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

4143

Article Doi

Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors

DOI: 10.1002/chem.200501128

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