Uhlmann et al., 2000). All members are characterised bya catalytic dyad which is present in a His-Gly-spacer-Ala-Cys motif. Comparison of the crystal structures of cas-pase 1 and 3 with gingipain revealed a very similar activecentre topology where the active site residues occupyidentical positions. Intriguingly, the two active siteresidues are located on opposite sides of the scissilebond. A striking characteristic of the clan CD cysteinepeptidases is the lack of irreversible inhibition by E64, of-ten considered to be a general inhibitor of cysteine en-dopeptidases.

So far, synthetic inhibitors against clan CD peptidasesof the caspase, gingipain, and clostripain families havebeen reported. The benzoyloxymethyl ketone inhibitorCbz-L-Phe-L-Lys-CH2O-CO-2,4,6-Me3-Ph specificallyinhibits gingipain K. The chloromethylketones (CMK) H-D-Phe-L-Pro-L-Arg-CH2Cl and H-D-Phe-L-Phe-L-Arg-CH2Cl react irreversibly with Arg-specific gingipain andclostripain, allowing active site titration (Potempa et al.,1997). Chloromethylketones with the core-structure H-L-Phe-L-Ala-L-Lys-L-Arg having an ethyl residue andmethyl residue were shown to be potent irreversible in-hibitors of clostripain (Wikstrom et al., 1989). For the cas-pases, several different classes of inhibitors have beendescribed, including aldehydes (Chapman, 1992), non-peptidic pyridone aldehydes (Golec et al., 1997), pheny-lalkyl ketones (Mjalli et al., 1993), acyloxymethyl ketones(Revesz et al., 1994; Thornberry et al., 1994), 1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl-oxymethyl ketones (Dolleet al., 1994), fluoromethylketones (Revesz et al., 1994;Hara et al., 1997), and chloromethylketones (Schulz et al.,1996).

Legumain was first discovered in plants and the inver-tebrate animal, Schistosoma, but was found in humanplacenta by Chen et al. (1997). From all sources, legu-main shows strict specificity for cleavage after as-paragine residues, which suggests that the physiologicalfunction may involve limited proteolysis. So far, there isstrong evidence in plants that legumain might be in-volved in protein splicing (Min and Jones, 1994). An acti-vation of precursor proteins has been reported in plantsas well (Hara-Nishimura et al., 1995). The mammalianlysosomal enzyme was reported to play a key role in theprocessing of bacterial antigen for presentation in theMHC II class system (Manoury et al., 1998; Antoniouet al., 2000). In addition, by screening an osteoclastcDNA expression library, legumain has been identified in-hibiting osteoclast formation and bone resorption (Choiet al., 1999). The regulation of legumain activity is still un-clear, but its inhibition by some cystatins, the general ex-tracellular cysteine peptidase inhibitors, was reported

Biol. Chem., Vol. 383, pp.1205 – 1214, July /August 2002 · Copyright © by Walter de Gruyter · Berlin · New York

André J. Niestroj1, Kirstin Feußner1, UlrichHeiser1, Pam M. Dando2, Alan Barrett2, BerndGerhartz1 and Hans-Ulrich Demuth1,*1 Probiodrug AG, Weinbergweg 22 – Biocenter, D-06120Halle (Saale), Germany2 MRC Molecular Enzymology Laboratory, BabrahamInstitute, Cambridge CB2 4AT, UK

* Corresponding author

Legumain is a lysosomal cysteine peptidase specificfor an asparagine residue in the P1-position. It hasbeen classified as a member of clan CD peptidasesdue to predicted structural similarities to caspasesand gingipains. So far, inhibition studies on legumainare limited by the use of endogenous inhibitors suchas cystatin C. A series of Michael acceptor inhibitorsbased on the backbone Cbz-L-Ala-L-Ala-L-Asn (Cbz=benzyloxycarbonyl) has been prepared and resultedin an irreversible inhibition of porcine legumain. Vari-ation of the molecular size within the ‘war head’ re-vealed the best inhibition for the compound contain-ing the allyl ester (kobs/I=766 M– 1s– 1). To overcomecyclisation between the amide moiety of the Asnresidue and the ‘war head’, several asparagine ana-logues have been synthesised. Integrated in halo-methylketone inhibitors, azaasparagine is acceptedby legumain in the P1-position. The most potentinhibitor of this series, Cbz-L-Ala-L-Ala-AzaAsn-chloromethylketone, displays a kobs/I value of 139 000M– 1s– 1. Other cysteine peptidases, such as papainand cathepsin B, are not inhibited by this compoundat concentrations up to 100 µM. The synthetic in-hibitors described here represent useful tools for theinvestigation of the structural and physiological prop-erties of this unique asparagine-specific peptidase.Key words: α,β-Unsaturated esters /Azaasparagine /Benzoyloxymethylketone /Cysteine protease /Halomethylketone /Legumain /Michael acceptors.

Introduction

Cysteine peptidases grouped into the clan CD representa new emerging class of enzymes displaying a novel cat-alytic mechanism different to classical papain-like pepti-dases of clan CA. So far, five different families have beenplaced in the clan CD, namely family C11 clostripain,family C13 legumain, family C14 caspases, family C25gingipain, and family C50 separin (Chen et al., 1998a;

Inhibition of Mammalian Legumain by MichaelAcceptors and AzaAsn-Halomethylketones

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

(Chen et al., 1997). However, the inhibitory mechanism oflegumain by cystatins differs significantly from that of pa-pain by cystatins (Alvarez-Fernandez et al., 1999). Due totheir lack of specificity, cystatins are only of limited usein investigations of the physiological function of legu-main. The same is true for carboxy-terminally blockedtetrapeptides (Manoury et al., 1998), currently used to in-hibit legumain, but displaying only a weak inhibitory ac-tivity.

Here we report the synthesis of potent and selectiveinhibitors against legumain. The use of the stable as-paragine analogue, azaasparagine, allows the prepara-tion of halomethylketone inhibitors. Cbz-L-Ala-L-Ala-AzaAsn-CMK, inhibiting legumain with a kobs/I value of139 000 M– 1s– 1, represents a powerful tool to investigateenzymatic, structural, and physiological properties oflegumain.

Results and Discussion

During the past few years, clan CD cysteine endopepti-dases have become increasingly important in pharma-ceutical research. All of the enzymes belonging to thisclan show almost complete specificity for the amino acidin the P1-position of the substrates. This limited prote-olytic activity suits the enzymes for involvement in theregulation of physiological processes. Theoretically, thehigh specificity for the P1 amino acid should enable thedesign of highly specific inhibitors. Due to the therapeu-tic potential of caspase inhibitors, a wide variety of differ-ent compound classes have been tested against theseenzymes. Mammalian legumain is structurally related tocaspases, but is specific for Asn rather than Asp in theP1-position. Reports on its involvement in antigen pres-entation by the MHC II class system as well as in bone re-sorption and osteoclast formation have prompted inves-tigations on the physiological role of this protease.Recently, first data on legumain knock-out mice havebeen presented (Werber et al., 2001). Despite such a mo-lecular approach, physiological studies are limited by thelack of potent and selective synthetic inhibitors. Basedon the standard substrate of legumain, Cbz-L-Ala-L-Ala-L-Asn-AMC (Kembhavi et al., 1993), we used the dipep-

tide L-Ala-L-Ala as a backbone to prepare synthetic in-hibitors.

The use of electrophilic moieties with asparagine rais-es the problem of side reactions with the amide sidechain. This leads to instability of ordinary aldehydes andhalomethylketones as well as unstable intermediatesduring the synthesis. One way to overcome this is the useof a mild electrophile and protection of the asparagineamide by a trityl protecting group (Sieber and Riniker,1991). However, the trityl group has to be removed in or-der to obtain a potential substrate or inhibitor which canbe recognised by porcine legumain (data not shown). Re-moval of the trityl group requires harsh acidic conditionswhich affect the ‘war head’ of the inhibitor, resulting in de-composition. In the case of aldehydes and halomethylke-tones, which are well reported inhibitors for clan CD pep-tidases, all attempts failed to generate intact inhibitors.Another strategy to solve this problem involves the use ofasparagine analogues. The influence of the amino side-chain in asparagine was studied by a racemic substrate 4with an inversed amide (Figure 1). This compound is isos-teric to the reference substrate Cbz-L-Ala-L-Ala-L-Asn-AMC. However, no cleavage was observed at concentra-tions below 100 µM. An alternative approach forgenerating molecules binding competitive to legumainwas attempted in compound 5. The lack of the carboxyfunction in the substrate should lead to a substrate likebinding. The product was synthesised following classicalpeptide chemical methods starting with β-Ala-amide andsubsequent coupling with 6. Compound 5 showed no in-hibitory activity at all confirming the necessity of the α-function. Recently, quinone derivatives are reported to beinhibitors of caspases (Graczyk, 1999). Based on thesubstrate structure motif, a quinone containing com-pound was synthesised in order to apply this inhibitoryconcept to legumain. The synthesis was performed bycoupling 2-amino-1,4[(tert-butyldimethylsilyl)oxy]ben-zene with compound 6. Treatment of the obtained com-pound by ammonium cerium(IV) nitrate led to 7. Similar tocompound 5 this approach did not lead to any legumainbinding (Figure 2), strongly confirming the asparaginespecificity of the enzyme.

Another possibility to increase the stability of the P1-Asn is the replacement of the chiral CH group by a nitro-

1206 A.J. Niestroj et al.

Fig. 1 Preparation of the Inverse Amide 4.Reagents and conditions: (a) TFA, 20°C, 15 min, after evaporation 1.5 equiv. of acetic anhydrid, 2.0 equiv. of N,N-diisopropylethylamine,DMF, 20°C, 30 min, 78%; (b) 1:4 piperidine:DMF, 20°C, 6 min, 100%; (c) 1.0 equiv. of 6, DMF, 20°C, 16 h, 78%.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

gen. Aza-analogues have been reported to increase sta-bility (Kati et al., 1999). Unfortunately, we did not succeedin synthesising the appropriate substrate, L-Ala-L-Ala-AzaAsn-AMC, which was designed to clarify the questionif the enzyme is able to recognise the aza-analogue.However, as will be reported later, the aza-analogues inhalomethylketones are recognised by the enzyme. Usingthe trityl protection strategy, a series of inhibitors com-prising a Michael acceptor moiety with different esterresidues (Table 1A) have been synthesised. The inhibitorswere generated by derivatisation of the aldehyde (10) viaWittig- or Horner-Emmons reaction (Figure 3). Com-pound 8 was prepared by a coupling reaction with thetrityl-protected asparagine in the presence of DCC start-ing from Cbz-L-Ala-L-Ala-OH. Compound 10 was syn-thesised by reduction of the corresponding Weinrebamide 9 (Fehrentz and Castro, 1983). The NMR-spectrarevealed only the trans olefin isomers occurred during thereaction. This is consistent with the preparation of otherMichael acceptor inhibitors (Thompson et al., 1986). Theprotecting group was removed by treatment with TFA to

provide the inhibitors 11–19 in moderate yields(Table 1B). The inhibition of the prepared inhibitors wascharacterised in a fluorogenic assay using Cbz-L-Ala-L-Ala-L-Asn-AMC as a substrate. As expected, the Michaelacceptor compounds were irreversible inhibitors of piglegumain. The irreversible inhibition is mediated by a co-valent binding of the active site cysteine to the inhibitorselectrophilic β-carbon (Hanzlik and Thompson, 1984;Palmer et al., 1995). The best inhibtion was achieved bythe α,ß-unsaturated ester 13 resulting in a second-orderrate constant of approx. 766 M– 1s– 1. The reactivity of theinhibitors is dependent on a steric fit into the active siteand on the reactivity determined by the electron with-drawing effect to the ß-carbon. Most likely, the variationof the R’ site does not clash with the active site topologyin case of the inhibitors 11–13 (Table 1A). However, anelectronic effect of the allyl containing compound seemsto favour the inhibition. Changing the R site from a hydro-gen to a methyl group resulted in a dramatic decrease inthe inhibitory activity indicating a steric conflict in the ac-tive site. This is most likely true for the phosphone con-

Synthetic Legumain Inhibitors 1207

Fig. 2 Structure of Compounds 5 and 7.

Table 1 Yields and Inhibition of Michael Acceptor Inhibitors.

(A)

(B)

Compound R R´ Preparation Yieldb kobs/I (M– 1s– 1)no. methoda

11 H CO2CH3 B 32% 54312 H CO2CH2CH3 B 30% 45613 H CO2CH2CH=CH2 A 21% 77614 H CO2H B → D 23% 415 CH3 CO2CH3 A → D → E 34% 216 CH3 CO2CH2CH3 A 65% 217 CH3 CO2H A → D 33% <118 H SO2CH3 C 36% 3019 H P(O)(OC2H5)2 C 23% <1

aMethod of preparation: A: Ph3P=CRCO2R´, R=H and R´= CH2CH=CH2 (13), R=CH3

and R´= CH2CH3 (15, 16, 17); B: (RO)2P(O)CH2CO2R, NaN(TMS)2, R=CH3 (11),R=CH2CH3 (12); C: CH3SO2CH2P(OCH3)2 (18), CH2[P(O)(OCH2CH3)2]2 (19), NaH; D: 1 MNaOH; E: diazomethane; bTFA, (i-Pr)3SiH.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

taining compound 19 displaying no inhibitory activity. Anattempt to increase the reactivity of the ‘war head’ by re-placing the carbon acid ester moiety by a sulfone did notresult in any increase of activity. This result is consistentwith altered activities of sulfones observed by Hanzlik(Thompson et al., 1986). The inhibitors carrying the α,β-unsaturated esters have been used to investigate thecrossreactivity to clan CA peptidases, namely papain andcathepsin B up to a concentration of 100 µM. The testedcompounds had no effect on the proteolytic activity ofpapain and cathepsin B. Halomethylketones are widelyused irreversible inhibitors of different types of proteases.Due to their high reactivity, the obtained second-orderrate constants of inhibition are often above 100 000M– 1s– 1. During the synthesis of a halomethylketone as alegumain inhibitor one has to overcome the problem ofinactivation by removing the trityl group. The replace-ment of the chiral CH group by a nitrogen atom in the aza-asparagine (Table 2A) enabled the synthesis of achloromethylketone (24), a bromomethylketone (26), anda benzoyloxymethylketone (28) without cyclisation (Fig-ure 4). The hydrazine tert.-butyl 2-(hydrazino)acetic acid(Niedrich, 1969) was coupled with Cbz-L-Ala-L-Ala-OSu

(6) to obtain the compound 20. Treatment of the t-butylester with TFA produced the compound 21, which wasconverted into the compound 22 via coupling reaction.Acylation with chloroacetyl chloride or bromoacetyl bro-mide gave the corresponding chloroacetyl and bro-moacetyl derivatives 23 and 25. The trityl protecting

1208 A.J. Niestroj et al.

Fig. 3 Preparation of the Compounds 6, 8–19.Reagents and conditions (Trt=CPh3, Ph=C6H5): (a) 1.0 equiv. of H-L-Asn(Trt)-OH, DMF, 20°C, 14 h, 92% ; (b) 1.1 equiv. of diisopropyl-carbodiimide, 1.1 equiv. of 1-hydroxybenzotriazole hydrate, 1.05 equiv. of HCl·HN(OCH3)CH3, 1.06 equiv. of Et3N, DMF, 0 to 20°C, 16 h,78%; (c) 1.25 equiv. of LiAlH4 (1 M in THF), THF, 0°C, 15 min, 86%; (d): Wittig- or Horner-Emmons reaction, method A, B or C; (e) 2.3equiv. of 1 M NaOH, EtOH, 3 h, 20°C; (f) 5.0 equiv. of diazomethane, THF, 20°C; (g) 5.0 equiv. of (i-Pr)3SiH, 7.5:10 TFA:CH2Cl2, 20°C, 30min; yields are given in Table 1.

Table 2 Second Order Rate Constant of the Inhibitors 24, 26and 28.

(A)

(B)

Compound no. Z kobs/I (M– 1s– 1)

24 COCH2Cl 13908826 COCH2Br 8400028 COCH2OC(O)C6H5 13

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

group was removed by TFA in the presence of triiso-propylsilane to give the inhibitors 24 and 26. Treatment of25 with benzoic acid in the presence of potassium fluo-ride (Krantz et al., 1991) was followed by treatment withtrifluoroacetic acid generated the benzoyloxymethylke-tone 28. Despite the replacement of the chiral CH groupby a nitrogen atom, the asparagine analogues wererecognised by the protease resulting in potent inhibition.The second-order rate constant for the inactivation oflegumain by the chloromethylketone, 139 088 M– 1s– 1 isapproximately 200-fold higher than that of the Michaelacceptor inhibitors (Table 2). Whereas the bromo-methylketone (26) is another potent inhibitor, the benzoy-loxymethylketone (28) displays only moderate inhibition.As shown for Michael acceptor inhibitors, neither papainnor cathepsin B is inhibited by these inhibitors. Such in-hibitory specificity will be essential to the use of a legu-main inhibitor in cell culture because of the co-localisa-tion of legumain with cathepsins in the lysosomes (Chenet al., 1998b). In conclusion, the synthetic inhibitors de-scribed here may well represent tools with which to in-vestigate and control the physiological functions of legu-main.

Materials and Methods

Materials

Solvents were distilled prior to use. Petroleum ether with a boil-ing range of 35 – 65°C was used. THF was distilled from sodiumdiphenyl ketyl immediately before use. All commercially avail-able reagents were used without further purification. Reactionssensitive to air were carried out under an atmosphere of argon.The buffer solution, pH 7.0 was prepared by dissolving potassi-

um dihydrogen phosphate (85.0 g) and sodium hydroxide(14.5 g) in water (1.0 l). The compound dimethyl methylsul-fonomethanephosphonate was prepared according to the pro-cedure of Almog and Shahak (Shahak and Almog, 1969). Legu-main was purified from porcine kidney as described earlier(Chen et al., 1997).

NMR

NMR spectra were recorded on Varian Unity 500, Varian Gemini200 and Bruker AM 400 spectrometers. The following abbrevia-tions are used: s, singlet; d, doublet; t, triplet; q, quartet; br., broad.

Melting Point Analysis

Melting points were measured on a Leica Galen III melting pointapparatus and are uncorrected.

ESI-MS

Mass spectra were taken with an MDS Sciex API 365 massspectrometer equipped with an Ionspray™ interface (MDS Sci-ex; Thorn Hill, ON, Canada). The instrument settings, data ac-quisition and processing were controlled by the Applied Biosys-tems (Foster City, CA, USA) Analyst™ software for WindowsNT™. Fifty to one-hundred scans were performed by the posi-tive ionization Q1 scan mode to accumulate the peaks. Samplesolutions were diluted in 50% methanol containing 0.5% formicacid to reach concentrations of approximately 10 µg/ml. Eachsample solution was introduced directly by a microsyringe (1 ml)through an infusion pump (Havard 22; Apparatus Havard Instru-ments; Holliston, MA, USA) and fused silica capillary tubing at arate of 20 µl/min.

Thin Layer Chromatography

Thin layer chromatography (TLC) was performed withMacherey-Nagel Polygram SIL G/UV254. Visualisation was ac-complished by means of UV light at 254 nm, followed by stain-ing with potassium permanganate or ninhydrin.

Synthetic Legumain Inhibitors 1209

Fig. 4 Preparation of Compounds 20–28.Reagents and conditions: (a) 1.0 equiv. of H2NNHCH2CO2tBu, THF, 20°C, 14 h, 65% ; (b) 3.0 equiv. of PhSCH3, 1:1 TFA:CH2Cl2, 20°C,2 h, 73%; (c) 1.0 equiv. of HATU, 1.0 equiv. of 1-hydroxy-7-azabenzotriazole, 1.0 equiv. of TrtNH2, 2.0 equiv. of N,N-diisopropylethyl-amine, DMF, 0 to 20°C, 16 h, 8%; (d) 1.0 equiv. of chloroacetyl chloride or bromoacetyl bromide, 1.5 equiv. of Et3N, THF, 0°C, 15 min,82% (23), 80% (25); (e) 5.0 equiv. of (i-Pr)3SiH, 7.5:10 TFA:CH2Cl2, 20°C, 30 min, 58% (24), 54% (26), 16% (28); (f) 2.5 equiv. of KF, 1.2equiv. of PhCO2H, DMF, 14 h, 20°C, 100% crude yield.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

Preparative and Analytic HPLC

Preparative HPLC for purification was performed at a Merck-Hi-tachi device: acetonitrile-water (gradient: 5 – 95%, flow rate: 6 mlmin– 1), column: Nucleosil 7µ C18 100A, 250 × 21.2 mm (phe-nomenex), pump: L-6250. Analytic HPLC was performed at aMerck-Hitachi device: acetonitrile-water (gradient: 5 – 95%, flowrate: 1 ml min– 1), column: LUNA 5µ C18 (2), 125 × 4.0 mm (phe-nomenex), pump: L-6200.

Measurement of Enzyme Inhibition

Legumain activity was determined in a fluorogenic continuousrate assay using the substrate Cbz-L-Ala-L-Ala-L-Asn-AMC(AMC=7-amido-4-methylcoumarin) on a Kontron spectrofluo-rometer SFM25 (exitation 380 nm; emission 460 nm) equippedwith a four cell changer and controlled by an IBM-compatiblepersonal computer. The obtained data were analysed with thesoftware FLUCOL (Auerswald et al., 1995). The assay was per-formed at 37°C or 30°C, using a sodium citrate buffer (39.5 mM

citric acid/121 mM Na2HPO4) containing 1 mM dithiothreitol and1 mM EDTA. Additionally, 0.1% (w/v) Chaps or 0.015% (w/v) Brij35 was added. Kinetic constants were determined by non linearregression analysis using the software Prism (Graph Pad soft-ware, San Diego, USA). Inhibition of papain and cathepsin B hasbeen performed as reported earlier (Gerhartz et al., 1997).

Synthesis of the Inhibitors

The compounds 11–19 were prepared starting from the aldehyde10 using the Wittig reaction (method A) (Wittig, 1965) or the Horner-Emmons reaction with sodium bis(trimethylsilyl)amide (method B)or with sodium hydride as base (method C) (Shahak and Almog,1969). The cleavage of the trityl protecting group by TFA (TFA=tri-fluoroacetic acid) was carried out in the presence of triisopropylsi-lane (Pearson et al., 1989). Elemental analyses (C, H, N) of the com-pounds agreed to within ± 0.4% of theoretical values.

Preparation of the Compounds 1 – 28

2,2-[N-(tert.-butyloxycarbonyl)-N´-(fluorenylmethyloxycar-bonyl)]-diamino-N-(4-methyl-2-oxo-2H-chromen-7-yl)ac-etamide (1) NMM (62.3 µl, 0.567 mmol, 1.0 equiv.) wasadded to a stirred and cooled solution (– 15°C) of N-Boc-N’-Fmoc-diaminoacetic acid (234 mg, 0.567 mmol, 1.0 equiv.) indry THF (3 ml). Isobutylchloroformate (74.2 µl, 0.567 mmol, 1.0equiv.) was added slowly and stirring was continued for 15 minat – 15°C. 7-Amino-4-methylcoumarin (99.3 mg, 0.567 mmol,1.0 equiv.) was added and the whole mixture was stirred for 16 hduring which time it was allowed to warm to room temperature.The solvent was removed under reduced pressure and the ob-tained crude compound was dissolved in EE (10 ml), washedwith 1 N HCl, water and brine (5 ml per washing step). The or-ganic layer was dried over Na2SO4, filtered and concentrated un-der reduced pressure. The obtained residue was purified byflash chromatography, generating the desired compound(251 mg, 78%) as a white solid of m.p. 130 – 132°C. TLC(MeOH/CHCl3, 1:50): Rf=0.35. 1H NMR (200 MHz, CDCl3): δ=1.46[s, 9 H, C(CH3)3], 2.38 (d, 3 H, J=1.2 Hz, CH=CCH3), 4.21 (t, 1 H,J=7.0 Hz, CHCH2), 4.46 (d, 2 H, J=7.0 Hz, CHCH2), 5.62 (s, br., 1H, NH), 5.98 – 6.03 (m, 1 H, NHCHNH), 6.20 (d, 1 H, J=1.1 Hz,CH=CCH3), 7.26 – 7.32 (m, 2 H, aryl-H), 7.36 – 7.40 (m, 3 H, aryl-H), 7.48 – 7.64 (m, 4 H, aryl-H), 7.74 (d, 2 H, J=7.6 Hz, aryl-H),9.11 (s, br., 1 H, NH). MS (EI) m/z (%): 570 [M + H+], 592 [M + Na+].

2,2-[N-acyl-N´-(fluorenylmethyloxycarbonyl)]-diamino-N-(4-methyl-2-oxo-2H-chromen-7-yl)acetamide (2) Compound1 (100 mg, 0.176 mmol, 1.0 equiv.) was dissolved in TFA (1.5 ml)

and this solution was stirred for 15 min at room temperature. TFAwas removed under reduced pressure. Without further purifica-tion, the above oil was dissolved in DMF (0.25 ml). N,N-diiso-propylethylamine (60 µl, 0.35 mmol, 2.0 equiv.) and acetic anhy-dride (25 µl, 0.26 mmol, 1.5 equiv.) were added and this solutionwas stirred at room temperature for 30 min. The solvent wasevaporated under vacuum by using an oil pump. The crudecompound was dissolved in EE (10 ml) and washed with brine(5 ml). The organic layer was dried over Na2SO4, filtered and con-centrated under reduced pressure. The obtained residue waspurified by flash chromatography, generating the desired com-pound (70 mg, 78%) as a yellow solid of m.p. 255 – 257°C. TLC(MeOH/CHCl3, 1:30): Rf=0.11. 1H NMR (400 MHz, DMSO-d6):δ=1.89 [s, 3 H, C(O)CH3], 2.40 (d, 3 H, J=1.0 Hz, CH=CCH3),4.20 – 4.29 (m, 3 H, CHCH2, CHCH2), 5.75 (t, 1 H, J=7.6 Hz,NHCHNH), 6.27 (d, 1 H, J=1.1 Hz, CH=CCH3), 7.28 – 7.34 (m, 2H, aryl-H), 7.40 (t, 2 H, J=7.3 Hz, aryl-H), 7.56 (d, 1 H, J=7.0 Hz,aryl-H), 7.72 – 7.82 (m, 4 H, aryl-H), 7.88 (d, 2 H, J=7.6 Hz, aryl-H), 8.21 (d, 1 H, J=7.2 Hz, NH), 8.62 (d, 1 H, J=7.0 Hz, NH), 10.56(s, br., 1 H, NH). MS (EI) m/z (%): 512 [M + H+], 534 [M + Na+], 550[M + K+].

2,2-N-acyl-diamino-N-(4-methyl-2-oxo-2H-chromen-7-yl)acetamide (3) Compound 2 (70.0 mg, 0.137 mmol) was dis-solved in a 1:4 mixture of piperidine and DMF (2 ml) and this so-lution was stirred at room temperature for 6 min. The solvent wasevaporated under vacuum by using an oil pump. The crudecompound was dissolved in EE (5 ml) and the solvent was evap-orated under vacuum again. This was repeated twice. The com-pound was isolated as a yellow solid (40 mg, 100% crude yield)of m.p. 95 – 100°C and was used without further purification.TLC (MeOH/CHCl3, 1:9): Rf=0.30. 1H NMR (200 MHz, DMSO-d6):δ=1.85 [s, 3 H, C(O)CH3], 2.39 (d, 3 H, J=0.9 Hz, CH=CCH3), 4.09(t, 1 H, J=8.2 Hz, NHCHNH), 5.02 (d, 1 H, J=7.3 Hz, NH), 6.26 (d,1 H, J=1.1 Hz, CH=CCH3), 7.25 – 7.94 (m, 4 H, aryl-H, NH), 8.41(d, 1 H, J=7.3 Hz, NH). MS (EI) m/z (%): 290 [M + H+].

N-[(benzyloxy)carbonyl]-L-alanyl-N1-{1-(acetylamino)-2-[(4-methyl-2-oxo-2H-chromen-7-yl)amino]-2-oxoethyl}-L-alan-inamide (4) Compound 6 (55 mg, 0.14 mmol) in dry DMF (1 ml)was added to a stirred solution of 3 (40 mg, 0.137 mmol, 1.0equiv.) also in dry DMF (2 ml). After the mixture was stirred for 16h at room temperature, the solvent was evaporated under vacu-um by using an oil pump. The obtained crude compound wastriturated with a small amount of water and filtered. The resultantresidue was purified by preparative HPLC, generating the prod-uct as a yellow solid (17 mg, 23%) of m.p. 260 – 262°C. TLC(MeOH/CHCl3, 1:9): Rf=0.45. 1H NMR (400 MHz, DMSO-d6):δ=1.19 (d, 3 H, J=7.2 Hz, CH3), 1.22 (d, 3 H, J=7.2 Hz, CH3), 1.88[s 3 H, C(O)CH3], 2.39 (d, 3 H, J=1.2 Hz, CH=CCH3), 4.04 – 4.10(m, 1 H, CH), 4.30 – 4.37 (m, 1 H, CH), 4.95 – 5.04 (m, 2 H, CH2O),5.80 – 5.87 (m, 1 H, NHCHNH), 6.26 (d, 1 H, J=1.2 Hz,CH=CCH3), 7.29 – 7.35 (m, 5 H, aryl-H), 7.45 (d, 1 H, J=6.6 Hz,NH), 7.54 – 7.60 (m, 1 H, aryl-H), 7.70 – 7.73 (m, 1 H, aryl-H),7.79 – 7.85 (m, 1 H, aryl-H), 8.01 (d, 1 H, J=7.4 Hz, NH), 8.08 (d,1 H, J=8.0 Hz, NH), 10.32 (s, 1 H, NH), 10.40 (s, 1 H, NH). MS (EI)m/z (%): 566 [M + H+].

Cbz-L-Ala-L-Ala-OSu (6) Compound 6 was prepared ac-cording to the method in the literature (Bodanszky and Bodan-szky, 1994). The crude compound was twice recrystallised fromisopropanol to give the active ester as a white solid (84%) of m.p.138 – 140°C. TLC (MeOH/CHCl3, 1:50): Rf=0.50. 1H NMR (400MHz, DMSO-d6): δ=1.20 (d, 3 H, J=7.0 Hz, CH3), 1.44 (d, 3 H,J=7.4 Hz, CH3), 2.79 (s, 4 H, 2 × CH2), 4.04 – 4.10 (m, 1 H, CH),4.63 – 4.69 (m, 1 H, CH), 4.96 – 5.04 (m, 2 H, CH2), 7.28 – 7.38 (m,

1210 A.J. Niestroj et al.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

5 H, aryl-H), 7.45 (d, 1 H, J=7.8 Hz, NH), 8.58 (d, 1 H, J=7.0 Hz,NH). MS (EI) m/z (%): 392 [M + H+], 414 [M + Na+], 430 [M + K+].

Cbz-L-Ala-L-Ala-L-Asn(Trt)-OH (8) Compound 8 was pre-pared starting from H-L-Asn(Trt)-OH and 6 in dry DMF. After usu-al work up the tripeptide was isolated as a white solid (86%) ofm.p. 195 – 197°C. The peptide was used without further purifi-cation. 1H NMR (500 MHz, DMSO-d6): δ=1.18 (d, 3 H, J=7.1 Hz,CH3), 1.22 (d, 3 H, J=7.0 Hz, CH3), 2.62 – 2.88 (m, 2 H, CHCH2),4.06 – 4.12 (m, 1 H, CH), 4.31 – 4.37 (m, 1 H, CH), 4.46 – 4.50 (m,1 H, CH), 4.98 – 5.04 (m, 2 H, CH2O), 7.15 – 7.35 (m, 20 H, aryl-H).MS (EI) m/z (%): 651 [M + H+], 673 [M + Na+], 689 [M + K+].

Cbz-L-Ala-L-Ala-L-Asn(Trt)-N(OCH3)CH3 (9) Compound 9was prepared according to the method of Yasuma (Yasumaet al., 1998). The Weinreb amide 9 was isolated as a white solid(92%) of m.p. 110 – 114°C and was used without further purifi-cation. TLC (MeOH/CHCl3, 1:30): Rf=0.49. – 1H NMR (400 MHz,CDCl3): δ=1.26 (d, 3 H, J=7.0 Hz, CH3), 1.29 (d, 3 H, J=7.0 Hz,CH3), 2.64 – 2.76 (m, 2 H, CHCH2), 2.86 (s, 3 H, NCH3), 3.66 (s, 3H, OCH3), 3.76 – 3.83 (m, 1 H, CH), 4.07 – 4.13 (m, 1 H, CH),4.33 – 4.40 (m, 1 H, CH), 5.02 – 5.09 (m, 2 H, CH2O), 7.13 – 7.34(m, 20 H, aryl-H). MS (EI) m/z (%): 694 [M + H+], 716 [M + Na+],732 [M + K+].

Cbz-L-Ala-L-Ala-L-Asn(Trt)-H (10) Aldehyde 10 was pre-pared according to the method of Fehrentz and Castro (1983).The aldehyde was isolated as a white solid of m.p. 110 – 114°Cwith a yield of 91% and was used without further purification.TLC (MeOH/CHCl3, 1:9): Rf=0.64. – 1H NMR (400 MHz, CDCl3):δ=1.33 (d, 3 H, J=7.0 Hz, CH3), 1.37 (d, 3 H, J=7.2 Hz, CH3),2.56 – 2.72 (m, 2 H, CHCH2), 4.08 – 4.13 (m, 1 H, CH), 4.28 – 4.34(m, 1 H, CH), 4.54 – 4.63 (m, 1 H, CH), 5.04 – 5.09 (m, 2 H, CH2O),7.24 – 7.32 (m, 20 H, aryl-H), 9.49 (s, 1 H, aldehyde). MS (EI) m/z(%): 635 [M + H+], 657 [M + Na+], 673 [M + K+].

Methyl (S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-6-oxo-2-hexenoate (11) Compound 11 was prepared by Method Bwith trimethyl phophonoacetate. After purification of the com-pound by flash chromatography the trityl protecting group wasremoved. The crude compound was purified by preparativeHPLC to generate the product as a white solid (43%) of m.p.242°C. TLC (MeOH/CHCl3, 1:9): Rf=0.34. 1H NMR (400 MHz,DMSO-d6): δ=1.20 (d, 3 H, J=7.1 Hz, CH3), 1.22 (d, 3 H, J=7.1 Hz,CH3), 2.38 (d, 2 H, J=6.8 Hz, CHCH2), 3.64 (s, 3 H, OCH3),4.01 – 4.14 (m, 1 H, CH), 4.16 – 4.25 (m, 1 H, CH), 4.73 – 4.78 (m,1 H, CH), 4.96 – 5.05 (m, 2 H, CH2O), 5.80 (d, 1 H, J=15.8 Hz,COCH=CH), 6.82 (dd, 1 H, J=15.8 Hz, J=4.8 Hz, COCH=CH),6.94 (s, br., 1 H, NH), 7.30 – 7.34 (m, 5 H, aryl-H), 7.46 (d, 1 H,J=7.2 Hz, NH), 8.01 (d, 1 H, J=7.4 Hz, NH), 8.08 (d, 1 H, J=8.0 Hz,NH). – 13C NMR (100 MHz, DMSO-d6): δ=17.89, 18.12 (CH3),46.64, 48.26, 50.03 (CH), 51.34 (OCH3), 65.37 (CH2C6H5), 119.70(COCH=CH), 127.79, 127.85, 128.41, 137.12 (aryl-C), 148.45(COCH=CH), 155.91, 166.14, 171.31, 171.63, 172.46 (C=O). MS(EI) m/z (%): 449 [M + H+], 471 [M + Na+], 487 [M + K+].

Ethyl (S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-6-oxo-2-hexenoate (12) Compound 12 was prepared by method Bwith triethyl phophonoacetate and, after purification by flashchromatography, the trityl protecting group was removed. Thecrude compound was purified by preparative HPLC to generatethe product as a white solid (56%) of m.p. 194 – 196°C. TLC(MeOH/CHCl3, 1:9): Rf=0.38. 1H NMR (400 MHz, CD3OD): δ=1.25(t, 3 H, J=7.1 Hz, CH3), 1.35 (d, 3 H, J=7.1 Hz, CH3), 1.38 (d, 3 H,J=7.1 Hz, CH3), 2.55 (d, 2 H, J=6.7 Hz, CHCH2), 4.08 – 4.14 (m, 1H, CH), 4.16 (q, 2 H, J=7.1 Hz, CH2CH3), 4.30 – 4.36 (m, 1 H, CH),

4.86 – 4.90 (m, 1 H, CH), 5.06 – 5.12 (m, 2 H, CH2O), 5.94 (d, 1 H,J=15.7 Hz, COCH=CH), 6.89 (dd, 1 H, J=15.7 Hz, J=5.1 Hz,COCH=CH), 7.27 – 7.37 (m, 5 H, aryl-H). MS (EI) m/z (%): 463 [M+ H+], 485 [M + Na+], 501 [M + K+].

Allyl (S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-6-oxo-2-he-xenoate (13) Compound 13 was prepared by method A withallyl (triphenylphophoranylidene)acetate. After purification of thecompound by flash chromatography the trityl protecting groupwas removed by TFA in the presence of triisopropylsilane. Thecrude compound was purified by preparative HPLC to generatethe product as a white solid (62%) of m.p. 182 – 184°C. TLC(MeOH/CHCl3, 1:9): Rf=0.41. 1H NMR (400 MHz, DMSO-d6):δ=1.21 (t, 3 H, J=7.0 Hz, CH3), 1.22 (d, 3 H, J=7.0 Hz, CH3), 2.55(d, 2 H, J=6.8 Hz, CHCH2), 4.01 – 4.06 (m, 1 H, CH), 4.20 – 4.26(m, 1 H, CH), 4.59 (d, 2 H, J=5.3 Hz, CH2CH=CH2), 4.73 – 4.76 (m,1 H, CH), 4.98 – 5.05 (m, 2 H, CH2O), 5.19 – 5.32 (m, 2 H,CH2CH=CH2), 5.82 (d, 1 H, J=15.7 Hz, COCH=CH), 5.88 – 5.97(m, 1 H, CH2CH=CH2), 6.86 (dd, 1 H, J=15.7 Hz, J=4.7 Hz,COCH=CH), 6.95 (s, br., 1 H, NH), 7.30 – 7.42 (m, 5 H, aryl-H),7.46 (d, 1 H, J=7.4 Hz, NH), 8.01 (d, 1 H, J=7.2 Hz, NH), 8.09 (d,1 H, J=8.2 Hz, NH). MS (EI) m/z (%): 475 [M + H+], 492 [M + NH4

+],497 [M + Na+], 513 [M + K+].

(S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-6-oxo-2-hexenoic acid (14) Compound 14 was prepared by method Bwith triethyl phophonoacetate. After saponification and purifica-tion by flash chromatography the trityl protecting group was re-moved by TFA in the presence of triisopropylsilane. The crudecompound was purified by preparative HPLC to generate theproduct as a white solid (37%) of m.p. 205 – 208°C. TLC(MeOH/CHCl3, 1:9): Rf=0.19. 1H NMR (400 MHz, CD3OD): δ=1.34(t, 3 H, J=6.8 Hz, CH3), 1.37 (d, 3 H, J=6.8 Hz, CH3), 2.55 (d, 2 H,J=6.8 Hz, CHCH2), 4.10 – 4.14 (m, 1 H, CH), 4.31 – 4.36 (m, 1 H,CH), 4.84 – 4.88 (m, 1 H, CH), 5.04 – 5.11 (m, 2 H, CH2O), 5.92 (d,1 H, J=15.7 Hz, COCH=CH), 6.88 (dd, 1 H, J=15.7 Hz, J=5.1 Hz,COCH=CH), 7.28 – 7.35 (m, 5 H, aryl-H). MS (EI) m/z (%): 435 [M+ H+], 457 [M + Na+], 473 [M + K+].

Methyl (S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-2-methyl-6-oxo-2-hexenoate (15) Compound 15 was pre-pared by method A with (carbethoxyethylidene)triph-enylphophorane. After saponification, the compound wasesterified by a freshly prepared solution of diazomethane inether. The compound was purified by flash chromatography andthe trityl protecting group was removed by TFA in the presenceof triisopropylsilane. Purification by preparative HPLC generatedthe product as a white solid (65%) of m.p. 188 – 191°C. TLC(MeOH/CHCl3, 1:9): Rf=0.54. 1H NMR (400 MHz, CD3OD): δ=1.33(t, 3 H, J=7.1 Hz, CH3), 1.34 (d, 3 H, J=7.1 Hz, CH3), 1.91 [d, 3 H,J=1.4 Hz, COC(CH3)=CH], 2.43 – 2.57 (m, 2 H, CHCH2),4.06 – 4.10 (m, 1 H, CH), 4.27 – 4.29 (m, 1 H, CH), 5.01 – 5.06 (m,1 H, CH), 5.07 – 5.15 (m, 2 H, CH2O), 6.61 [dd, 1 H, J=9.0 Hz,J=1.4 Hz, COC(CH3)=CH], 7.29 – 7.38 (m, 5 H, aryl-H). MS (EI)m/z (%): 463 [M + H+], 485 [M + Na+], 501 [M + K+].

Ethyl (S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-2-methyl-6-oxo-2-hexenoate (16) Compound 16 was prepared bymethod A with (carbethoxyethylidene)triphenylphophorane. Theisolated crude product was purified by flash chromatographyand the trityl protecting group was removed by TFA in the pres-ence of triisopropylsilane. The crude compound was purified bypreparative HPLC, generating the product as a white solid (64%)of m.p. 200 – 204°C. TLC (MeOH/CHCl3, 1:9): Rf=0.33. 1H NMR(400 MHz, DMSO-d6): δ=1.15 – 1.21 (m, 9 H, 3 × CH3), 1.80 [d, 3H, J=1.2 Hz, COC(CH3)=CH], 2.31 – 2.35 (m, 2 H, CHCH2),

Synthetic Legumain Inhibitors 1211

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

4.02 – 4.08 (m, 1 H, CH), 4.15 – 4.21 (m, 3 H, CH, CH2CH3),4.83 – 4.87 (m, 1 H, CH), 4.97 – 5.05 (m, 2 H, CH2O), 6.46 [dd, 1H, J=9.0 Hz, J=1.4 Hz, COC(CH3)=CH], 6.86 (s, 1 H, NH),7.30 – 7.35 (m, 5 H, aryl-H), 7.44 (d, 1 H, J=7.2 Hz, NH), 7.93 (d,1 H, J=7.4 Hz, NH), 8.05 (d, 1 H, J=7.4 Hz, NH). MS (EI) m/z (%):477 [M + H+], 499 [M + Na+], 515 [M + K+].

(S)-(E)-4-[Cbz-L-Ala-L-Ala]amino-6-amino-2-methyl-6-oxo-2-hexenoic acid (17) Compound 17 was prepared by methodA with (carbethoxyethylidene)triphenylphophorane. After thesaponification and purification by flash chromatography, thetrityl protecting group was removed by TFA in the presence of tri-isopropylsilane. The crude compound was purified by prepara-tive HPLC, generating the product as a white solid (32%) of m.p.185 – 189°C. TLC (MeOH/CHCl3, 1:9): Rf=0.08. 1H NMR (400MHz, CD3OD): δ= 1.33 (t, 3 H, J=7.1 Hz, CH3), 1.34 (d, 3 H, J=7.1Hz, CH3), 1.89 [d, 3 H, J=1.2 Hz, COC(CH3)=CH], 2.42 – 2.58 (m,2 H, CHCH2), 4.07 – 4.10 (m, 1 H, CH), 4.28 – 4.31 (m, 1 H, CH),5.00 – 5.06 (m, 1 H, CH), 5.12 – 5.16 (m, 2 H, CH2O), 6.63 [dd, 1H, J=9.0 Hz, J=1.2 Hz, COC(CH3)=CH], 7.26 – 7.38 (m, 5 H, aryl-H). MS (EI) m/z (%): 449 [M + H+], 471 [M + Na+], 487 [M + K+].

Methyl [(S)-(E)-3-[Cbz-L-Ala-L-Ala]amino-5-amino-5-oxo-1-petenyl]-sulfonate (18) Compound 18 was prepared bymethod C (Shahak and Almog, 1969) with dimethyl methylsul-fonomethanephosphonate followed by purification of the com-pound by flash chromatography and removing the trityl protect-ing group. The crude compound was purified by preparativeHPLC, generating the product as a white solid (71%) of m.p.193 – 195°C. TLC (MeOH/CHCl3, 1:9): Rf=0.13. 1H NMR (400MHz, CD3OD): δ= 1.34 (t, 3 H, J=7.2 Hz, CH3), 1.38 (d, 3 H, J=7.2Hz, CH3), 2.58 – 2.61 (m, 2 H, CHCH2), 2.92 (s, 3 H, SO2CH3),4.09 – 4.16 (m, 1 H, CH), 4.24 – 4.34 (m, 1 H, CH), 5.04 – 5.17 (m,3 H, CH, CH2O), 6.68 (d, 1 H, J=15.2 Hz, COCH=CH), 6.85 (dd, 1H, J=15.2 Hz, J=4.3 Hz, COCH=CH), 7.27 – 7.35 (m, 5 H, aryl-H).MS (EI) m/z (%): 469 [M + H+], 491 [M + Na+], 507 [M + K+].

Diethyl [(S)-(E)-3-[Cbz-L-Ala-L-Ala]amino-5-amino-5-oxo-1-petenyl]-phosphate (19) Compound 19 was prepared bymethod C with tetraethyl methylendiphosphonate followed bypurification by flash chromatography and cleavage the trityl pro-tecting group. The crude compound was purified by preparativeHPLC, generating the product as a white solid (60%) of m.p.95 – 97°C. TLC (MeOH/CHCl3, 1:9): Rf=0.32. 1H NMR (400 MHz,CD3OD): δ= 1.26 – 1.38 (m, 12 H, 2 × CH2CH3, 2 × CHCH3),2.51 – 2.61 (m, 2 H, CHCH2), 3.98 – 4.15 (m, 5 H, CH, 2 ×CH2CH3), 4.28 – 4.34 (m, 1 H, CH), 5.04 – 5.15 (m, 3 H, CH,CH2O), 5.91 (dd, 1 H, J=35.4 Hz, J=17.4 Hz, COCH=CH), 6.85(ddd, 1 H, J=21.9 Hz, J=17.6 Hz, J=4.5 Hz, COCH=CH),7.28 – 7.37 (m, 5 H, aryl-H). MS (EI) m/z (%): 527 [M + H+], 549 [M+ Na+], 565 [M + K+].

tert-Butyl 2-[2-(Cbz-L-Ala-L-Ala)-hydrazino]acetic acid (20)To a stirred solution of 6 (7.98 g, 20.4 mmol, 1.0 equiv.) in dryTHF (50 ml) a solution of tert-butyl 2-(hydrazino)acetic acid(Niedrich, 1969) (2.98 g, 20.4 mmol, 1.0 equiv.) in dry THF (20 ml)was added and stirred for 14 h at room temperature. The solventwas evaporated under vacuum. The obtained crude compoundwas triturated with water and filtered. The resultant solid waswashed twice with a small amount of water, dried over P4O10 andused without further purification (5.6 g, 65%). TLC(MeOH/CHCl3, 1:9): Rf=0.53. 1H NMR (400 MHz, CDCl3): δ=1.35(d, 3 H, J=7.0 Hz, CH3), 1.36 (d, 3 H, J=7.1 Hz, CH3), 1.44 [s, 9 H,C(CH3)3], 3.73 (s, br., 2 H, NHCH2), 4.20 – 4.32 (m, 1 H, CH),4.42 – 4.50 (m, 1 H, CH), 5.06 – 5.13 (m, 2 H, CH2O), 5.39 (s, br., 1H, NH), 5.58 (s, br., 1 H, NH), 7.02 (s, br., 1 H, NH), 7.28 – 7.38 (m,

5 H, aryl-H). MS (EI) m/z (%): 423 [M + H+], 445 [M + Na+], 461 [M+ K+].

2-[2-(Cbz-L-Ala-L-Ala)-hydrazino]acetic acid (21) 20 (2.5 g,5.9 mmol, 1.0 equiv.) was dissolved in a mixture of TFA (40 ml),CH2Cl2 (40 ml) and methyl phenyl sulphide (2.08 ml, 17.7 mmol,3.0 equiv.). This solution was stirred for 2 h at room temperaturebefore it was diluted with toluol. The solvents were removed un-der reduced pressure and the residue obtained, was trituratedwith Et2O and filtered. The resultant solid was washed threetimes with Et2O and dried, generating the desired compound asa white solid (1.57 g, 73%) of m.p. 151 – 153°C which was usedwithout further purification. TLC (MeOH/CHCl3, 1:9): Rf=0.15. 1HNMR (200 MHz, DMSO-d6): δ=1.15 (d, 3 H, J=7.0 Hz, CH3), 1.17(d, 3 H, J=7.0 Hz, CH3), 3.40 (s, 2 H, NHCH2), 3.97 – 4.11 (m, 1 H,CH), 4.17 – 4.27 (m, 1 H, CH), 4.94 – 5.07 (m, 2 H, CH2O),7.26 – 7.39 (m, 5 H, aryl-H), 7.92 (d, 1 H, J=7.3 Hz, NH), 9.35 (s,br., 1 H, NH). MS (EI) m/z (%): 367 [M + H+], 389 [M + Na+], 405[M + K+].

2-[2-(Cbz-L-Ala-L-Ala)-hydrazino]triphenylmethylacet-amide (22) A solution of 21 (0.30 g, 0.82 mmol, 1.0 equiv.) indry DMF (5 ml) was cooled to 0°C with an ice-bath. The follow-ing additions were made to this stirred solution; HATU (0.31 g,0.82 mmol, 1.0 equiv.), 1-hydroxy-7-azabenzotriazole (0.11 g,0.82 mmol, 1.0 equiv.), triphenylmethylamine (0.32 g, 0.82 mmol,1.0 equiv.) and N,N-diisopropylethylamine (0.230 ml, 1.64 mmol,2.0 equiv.) and the entire mixture was stirred for 16 h duringwhich time it was allowed to warm to room temperature. The sol-vent was evaporated under vacuum by using an oil pump. Theobtained crude compound was dissolved in EE (25 ml), washedwith 1 N HCl, water, aqueous NaHCO3, and brine (5 ml per wash-ing step). The organic layer was dried over Na2SO4, filtered andconcentrated under reduced pressure. The obtained residuewas purified by flash chromatography to give the desired com-pound (40 mg, 8%) as a solid of m.p. 63 – 65°C. TLC(MeOH/CHCl3, 1:9): Rf=0.15. 1H NMR (500 MHz, DMSO-d6):δ=1.15 (d, 3 H, J=7.1 Hz, CH3), 1.16 (d, 3 H, J=7.1 Hz, CH3), 3.42(s, 2 H, NHCH2), 4.02 – 4.08 (m, 1 H, CH), 4.19 – 4.25 (m, 1 H,CH), 4.96 – 5.03 (m, 2 H, CH2O), 7.17 – 7.42 (m, 20 H, aryl-H),7.96 (d, 1 H, J=7.3 Hz, NH), 9.68 (s, br., 1 H, NH). MS (EI) m/z (%):608 [M + H+], 630 [M + Na+], 646 [M + K+].

2-[2-(Cbz-L-Ala-L-Ala)-1-(chloroacetyl)hydrazino]triphenyl-methylacetamide (23) To a stirred solution of 22 (40 mg,66 µmol, 1.0 equiv.) in dry THF (1.5 ml) at 0°C, triethylamine(14.0 µl, 100 µmol, 1.5 equiv.) and chloroacetyl chloride (5.3 µl,66 µmol, 1.0 equiv.) was added. After stirring the mixture for 15min, the solvent was removed under reduced pressure. The ob-tained crude compound was purified by flash chromatographyto generate the product (37 mg, 82%) as a white solid of m.p.85 – 92°C. TLC (MeOH/CHCl3, 1:9): Rf=0.70. 1H NMR (400 MHz,DMSO-d6): δ=1.16 (d, 3 H, J=7.0 Hz, CH3), 1.23 (d, 3 H, J=7.4 Hz,CH3), 4.02 – 4.08 (m, 1 H, CH), 4.12 – 4.17 (m, 1 H, CH), 4.22 [s, 2H, C(O)CH2Cl], 4.25 (s, 2 H, NHCH2), 4.97 – 5.03 (m, 2 H, CH2O),7.14 – 7.36 (m, 20 H, aryl-H), 7.43 (d, 1 H, J=7.8 Hz, NH), 8.16 (s,1 H, NH), 8.91 (d, 1 H, J=8.6 Hz, NH), 10.63 (s, br., 1 H, NH). MS(EI) m/z (%): 684 [M + H+, 35Cl], 686 [M + H+, 37Cl], 706 [M + Na+,35Cl], 708 [M + Na+, 37Cl], 722 [M + K+, 35Cl], 724 [M + K+, 37Cl].

2-[2-(Cbz-L-Ala-L-Ala)-1-(chloroacetyl)hydrazino]acet-amide (24) Compound 24 was prepared starting from 23(37 mg, 54 µmol) by the cleavage of the trityl protecting group.The crude compound was purified by preparative HPLC, gener-ating the product (14 mg, 58%) as a white solid of m.p.95 – 97°C. TLC (MeOH/CHCl3, 1:9): Rf=0.20. 1H NMR (500 MHz,

1212 A.J. Niestroj et al.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

DMSO-d6): δ=1.18 (d, 3 H, J=6.8 Hz, CH3), 1.23 (d, 3 H, J=6.8 Hz,CH3), 4.02 – 4.08 (m, 1 H, CH), 4.15 – 4.23 (m, 1 H, CH), 4.28 [s,br., 2 H, C(O)CH2Cl], 4.40 (s, br., 2 H, NHCH2), 4.96 – 5.03 (m, 2 H,CH2O), 7.20 (s, 1 H, NH), 7.28 – 7.37 (m, 5 H, aryl-H), 7.47 (d, 1 H,J=7.3 Hz, NH), 8.22 (s, br., 1 H, NH), 10.58 (s, br., 1 H, NH). MS(EI) m/z (%): 442 [M + H+, 35Cl], 444 [M + H+, 37Cl], 464 [M + Na+,35Cl], 466 [M + Na+, 37Cl], 480 [M + K+, 35Cl], 482 [M + K+, 37Cl].

2-[2-(Cbz-L-Ala-L-Ala)-1-(bromoacetyl)hydrazino]triphenyl-methylacetamide (25) To a stirred solution of 22 (57 mg,94 µmol, 1.0 equiv.) in dry THF (3 ml) at 0°C was added triethy-lamine (20.0 µl, 140 µmol, 1.5 equiv.) and bromoacetyl bromide(8.2 µl, 94 µmol, 1.0 equiv.). After stirring the mixture for 15 min,the solvent was removed under reduced pressure. The obtainedcrude compound was purified by flash chromatography, gener-ating the product (55 mg, 80%) as a white solid of m.p.93 – 100°C. TLC (MeOH/CHCl3, 1:9): Rf=0.68. 1H NMR (400MHz, DMSO-d6): δ=1.16 (d, 3 H, J=7.0 Hz, CH3), 1.23 (d, 3 H,J=7.4 Hz, CH3), 4.04 – 4.10 (m, 1 H, CH), 4.13 – 4.18 (m, 1 H, CH),4.25 [s, 2 H, C(O)CH2Br], 4.29 (s, 2 H, NHCH2), 4.94 – 5.03 (m, 2H, CH2O), 7.15 – 7.34 (m, 20 H, aryl-H), 7.43 (d, 1 H, J=7.4 Hz,NH), 8.17 (d, 1 H, J=8.6 Hz, NH), 8.92 (s, 1 H, NH), 10.71 (s, br., 1H, NH). MS (EI) m/z (%): 728 [M + H+, 79Br], 730 [M + H+, 81Br], 750[M + Na+, 79Br], 752 [M + Na+, 81Br], 766 [M + K+, 79Br], 768 [M +K+, 81Br].

2-[2-(Cbz-L-Ala-L-Ala)-1-(bromoacetyl)hydrazino]acet-amide (26) Compound 26 was prepared starting from 25(55 mg, 76 µmol) by the cleavage of the trityl protecting group.The crude compound was purified by preparative HPLC, gener-ating the product (20 mg, 54%) as a white solid. TLC(MeOH/CHCl3, 1:50): Rf=0.22. 1H NMR (500 MHz, DMSO-d6):δ=1.18 (d, 3 H, J=7.0 Hz, CH3), 1.24 (d, 3 H, J=7.0 Hz, CH3),4.00 – 4.10 (m, 1 H, CH), 4.08 – 4.12 (m, 1 H, CH), 4.26 [s, br., 2 H,C(O)CH2Br], 4.38 (s, br., 2 H, NHCH2), 4.96 – 5.03 (m, 2 H, CH2O),7.16 (s, 1 H, NH), 7.29 – 7.37 (m, 5 H, aryl-H), 7.43 (d, 1 H, J=7.3Hz, NH), 8.18 (d, J=7.3 Hz, 1 H, NH), 10.59 (s, br., 1 H, NH). MS(EI) m/z (%): 486 [M + H+, 79Br], 488 [M + H+, 81Br], 508 [M + Na+,79Br], 510 [M + Na+, 81Br], 524 [M + K+, 79Br], 526 [M + K+, 81Br].

2-[2-(Cbz-L-Ala-L-Ala)-1-(benzoyloxyacetyl)hydrazino]triphenylmethylacetamide (27) Dry KF (15 mg, 257 µmol, 2.5equiv.) was added to a stirred solution of 25 (75 mg, 103 µmol,1.0 equiv.) in dry DMF (4 ml) at room temperature. After stirringthe mixture for 3 min, benzoic acid was added (15 mg, 123 µmol,1.2 equiv.) and the whole mixture was stirred for 16 h. The sol-vent was evaporated under vacuum by using an oil pump. Theobtained crude compound (80 mg, 100% crude yield) was usedwithout further purification. TLC (MeOH/CHCl3, 1:9): Rf=0.44.MS (EI) m/z (%): 770 [M + H+], 792 [M + Na+], 808 [M + K+].

2-[2-(Cbz-L-Ala-L-Ala)-1-(benzoyloxyacetyl)hydrazino]ac-etamide (28) Compound 28 was prepared starting from 27(80 mg, 103 µmol) by the cleavage of the trityl protecting group.The crude compound was purified by preparative HPLC, gener-ating the product as a white solid (8.7 mg, 16%). TLC(MeOH/CHCl3, 1:9): Rf=0.24. 1H NMR (400 MHz, DMSO-d6):δ=1.20 (d, 3 H, J=7.1 Hz, CH3), 1.27 (d, 3 H, J=7.1 Hz, CH3),4.04 – 4.10 (m, 1 H, CH), 4.21 – 4.26 (m, 1 H, CH), 4.38 (s, br., 2 H,NHCH2), 4.78 [s, br., 2 H, C(O)CH2O], 4.95 – 5.03 (m, 2 H, CH2O),7.18 (s, 1 H, NH), 7.28 – 7.36 (m, 4 H, aryl-H), 7.43 (d, 1 H, J=7.6Hz, NH), 7.50 – 7.56 (m, 3 H, aryl-H), 7.65 – 7.70 (m, 1 H, aryl-H),7.97 – 7.99 (m, 2 H, aryl-H), 8.22 (s, br., 1 H, NH), 10.60 (s, br., 1H, NH). MS (EI) m/z (%): 528 [M + H+], 545 [M + NH4

+], 550 [M +Na+], 566 [M + K+].

Acknowledgements

This work was supported by a grant from the BMBF, no.0312302. The authors wish to thank A. Hamann for her technicalassistance. Furthermore, the authors wish to thank Dr. F. Roscheand Dr. R. Wolf for the generation of the Mass spectra. In addi-tion, we wish to thank Dr. S. Manhart and C. Schnittka for the pu-rification of the compounds.

References

Alvarez-Fernandez, M., Barrett, A.J., Gerhartz, B., Dando, P.M.,Ni, J., and Abrahamson, M. (1999). Inhibition of mammalianlegumain by some cystatins is due to a novel second reactivesite. J. Biol. Chem. 274, 19195 – 19203.

Antoniou, A.N., Blackwood, S.L., Mazzeo, D., and Watts, C.(2000). Control of antigen presentation by a single proteasecleavage site. Immunity 12, 391 – 398.

Auerswald, E.A., Nagler, D.K., Assfalg-Machleidt, I., Stubbs,M.T., Machleidt, W., and Fritz, H. (1995). Hairpin loop muta-tions of chicken cystatin have different effects on the inhibitionof cathepsin B, cathepsin L and papain. FEBS Lett. 361,179 – 184.

Bodanszky, M. and Bodanszky, A. (1994). The practice of pep-tide synthesis (Berlin, Heidelberg, Germany: Springer-Verlag).

Chapman, K.T. (1992). Synthesis of a potent, reversible inhibitorof interleukin-1ß converting enzyme. Bioorg. Med. Chem.Lett. 2, 613 – 618.

Chen, J.M., Dando, P.M., Rawlings, N.D., Brown, M.A., Young,N.E., Stevens, R.A., Hewitt, E., Watts, C., and Barrett, A.J.(1997). Cloning, isolation, and characterization of mammalianlegumain, an asparaginyl endopeptidase. J. Biol. Chem. 272,8090 – 8098.

Chen, J.M., Dando, P.M., Stevens, R.A., Fortunato, M., and Bar-rett, A.J. (1998a). Cloning and expression of mouse legumain,a lysosomal endopeptidase. Biochem. J. 335, 111 – 117.

Chen, J.M., Rawlings, N.D., Stevens, R.A., and Barrett, A.J.(1998b). Identification of the active site of legumain links it tocaspases, clostripain and gingipains in a new clan of cysteineendopeptidases. FEBS Lett. 441, 361 – 365.

Choi, S.J., Reddy, S.V., Devlin, R.D., Menaa, C., Chung, H.,Boyce, B.F., and Roodman, G.D. (1999). Identification of hu-man asparaginyl endopeptidase (legumain) as an inhibitor ofosteoclast formation and bone resorption. J. Biol. Chem. 274,27747 – 27753.

Dolle, R.E., Singh, J., Rinker, J., Hoyer, D., Prasad, C.V., Graybill,T.L., Salvino, J.M., Helaszek, C.T., Miller, R.E., and Ator, M.A.(1994). Aspartyl α-[(1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy]methyl ketones as interleukin-1ß converting enzyme in-hibitors. Significance of the P1 and P3 amido nitrogens for en-zyme-peptide inhibitor binding. J. Med. Chem. 37, 3863 – 3866.

Fehrentz, J.A. and Castro, A. (1983). An efficient synthesis of op-tically active α-(t-butoxycarbonylamino)-aldehydes from α-amino acids. Synthesis 8, 676 – 678.

Gerhartz, B., Engh, R.A., Mentele, R., Eckerskorn, C., Torquato,R., Wittmann, J., Kolb, H.J., Machleidt, W., Fritz, H., and Auer-swald, E.A. (1997). Quail cystatin: isolation and characterisa-tion of a new member of the cystatin family and its hypotheti-cal interaction with cathepsin B. FEBS Lett. 412, 551 – 558.

Golec, J.M.C., Mullican, M.D., Murcko, M.A., Wilson, K.P., Kay,D.P., Jones, S.D., Murdoch, R., Bemis, G.W., Raybuck, S.A.,Yu-Ping Luong, and Livingston, D.J. (1997). Structure-baseddesign of non-peptidic pyridone aldehydes as inhibitors of in-terleukin-1β converting enzyme. Bioorg. Med. Chem. Lett. 7,2181 – 2186.

Synthetic Legumain Inhibitors 1213

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25

Graczyk, P.P. (1999). Caspase inhibitors – a chemist’s perspec-tive. Restor. Neurol. Neurosci. 14, 1 – 23.

Hanzlik, R.P. and Thompson, S.A. (1984). Vinylogous amino acidesters: a new class of inactivators for thiol proteases. J. Med.Chem. 27, 711 – 712.

Hara, H., Friedlander, R.M., Gagliardini, V., Ayata, C., Fink, K.,Huang, Z., Shimizu-Sasamata, M., Yuan, J., and Moskowitz,M.A. (1997). Inhibition of interleukin 1β converting enzymefamily proteases reduces ischemic and excitotoxic neuronaldamage. Proc. Natl. Acad. Sci. USA 94, 2007 – 2012.

Hara-Nishimura, I., Shimada, T., Hiraiwa, N., and Nishimura, M.(1995). Vacuolar processing enzyme responsible for matura-tion of seed proteins. J. Plant. Physiol. 145, 632 – 640.

Kati, W.M., Sham, H.L., McCall, J.O., Montgomery, D.A., Wang,G.T., Rosenbrook, W., Miesbauer, L., Buko, A., and Norbeck,D.W. (1999). Inhibition of 3C protease from human rhinovirusstrain 1B by peptidyl bromomethylketonehydrazides. Arch.Biochem. Biophys. 362, 363 – 375.

Kembhavi, A.A., Buttle, D.J., Knight, C.G., and Barrett, A.J.(1993). The two cysteine endopeptidases of legume seeds:purification and characterization by use of specific fluoromet-ric assays. Arch. Biochem. Biophys. 303, 208 – 213.

Krantz, A., Copp, L.J., Coles, P.J., Smith, R.A., and Heard, S.B.(1991). Peptidyl (acyloxy)methyl ketones and the quiescentaffinity label concept: the departing group as a variable struc-tural element in the design of inactivators of cysteine pro-teinases. Biochemistry 30, 4678 – 4687.

Manoury, B., Hewitt, E.W., Morrice, N., Dando, P.M., Barrett,A.J., and Watts, C. (1998). An asparaginyl endopeptidaseprocesses a microbial antigen for class II MHC presentation.Nature 396, 695 – 699.

Min, W. and Jones, D.H. (1994). In vitro splicing of concanavalinA is catalyzed by asparaginyl endopeptidase. Nature Struct.Biol. 1, 502 – 504.

Mjalli, A.M.M., Chapman, K.T., MacCoss, M., and Thornberry,N.A. (1993). Phenylalkyl ketones as potent reversible in-hibitors of interleukin-1ß converting enzyme. Bioorg. Med.Chem. Lett. 3, 2689 – 2692.

Niedrich, H. (1969). Synthese von substituierten 2.4-Bis-car-boxymethyl-1-acyl-semicarbaziden, den α-Aza-asparagin-Peptiden. In: Hydrazinverbindungen als Heterobestandteil inPeptiden, XI. Chem. Ber. 102, 1557 – 1569.

Palmer, J.T., Rasnick, D., Klaus, J.L., and Brömme, D. (1995).Vinyl sulfones as mechanism-based cysteine protease in-hibitors. J. Med. Chem. 38, 3193 – 3196.

Pearson, D.A., Blanchette, M., Baker, M.L., and Guidon, C.A.(1989). Trialkylsilane as scavangers for the trifluoroacetic aciddeblocking of protecting groups in peptide synthesis. Tetrahe-dron Lett. 30, 2739 – 2742.

Potempa, J., Pike, R., and Travis, J. (1997). Titration andmapping of the active site of cysteine proteinases from Por-phyromonas gingivalis (gingipains) using peptidyl chloro-methanes. Biol. Chem. 378, 223 – 230.

Revesz, L., Briswalter, C., Heng, R., Leutwiler, A., Mueller, R.,and Wuetherich, H.-J. (1994). Synthesis of P1 aspartate-based peptide acyloxymethyl and fluoromethyl ketones as in-hibitors of interleukin-1β-converting enzyme. TetrahedronLett. 35, 9693 – 9696.

Schulz, J.B., Weller, M., and Klockgether, T. (1996). Potassiumdeprivation-induced apoptosis of cerebellar granule neurons:a sequential requirement for new mRNA and protein synthe-sis, ICE-like protease activity, and reactive oxygen species. J.Neurosci. 16, 4696 – 4706.

Shahak, I. and Almog, J. (1969). Horner-Wittig Syntesis of α,β-unsaturated sulfides and sulfones. Synthesis 170 – 172.

Sieber, P. and Riniker, B. (1991). Protection of carboxamide func-tions by the trityl residue. Application to peptide synthesis.Tetrahedron Lett. 32, 739 – 742.

Thompson, S.A., Andrews, P.R., and Hanzlik, R.P. (1986). Car-boxyl-modified amino acids and peptides as protease in-hibitors. J. Med. Chem. 29, 104 – 111.

Thornberry, N.A., Peterson, E.P., Zhao, J.J., Howard, A.D., Grif-fin, P.R., and Chapman, K.T. (1994). Inactivation of interleukin-1β-converting enzyme by peptide (acyloxy)methyl ketones.Biochemistry 33, 3934 – 3940.

Uhlmann, F., Wernic, D., Poupart, M.A., Koonin, E.V., and Nas-myth, K. (2000). Cleavage of cohesin by the CD clan proteaseseparin triggers anaphase in yeast. Cell 103, 375 – 386.

Werber, I., Chen, J.M., Barrett, A.J., Deussing, J., and Peters, C.(2001). Genomic structure, chromosomal localization andgene targeting of murine legumain. Proceedings of the 2nd

General Meeting of the International Proteolysis Society, 2001Munich-Freising, Germany, p. 61.

Wikstrom, P., Kirschke, H., Stone, S., and Shaw, E. (1989). Theproperties of peptidyl diazoethanes and chloroethanes asprotease inactivators. Arch. Biochem. Biophys. 270, 286 –293.

Wittig, G. (1965). Variationen zu einem Thema von Staudinger;ein Beitrag zur Geschichte der phosphororganischen Car-bonylolefinierung. Pure Appl. Chem. 9, 245 – 254.

Yasuma, T., Oi, S., Choh, N., Nomura, T., Furuyama, N., Nishimu-ra, A., Fujisawa, Y., and Sohda, T. (1998). Synthesis of peptidealdehyde derivatives as selective inhibitors of human cathep-sin L and their inhibitory effect on bone resorption. J. Med.Chem. 41, 4301 – 4308.

Received January 14, 2002; accepted March 20, 2002

1214 A.J. Niestroj et al.

Bereitgestellt von | SUB GöttingenAngemeldet

Heruntergeladen am | 22.01.15 10:25