20S Pro Tea Some Inhibitors

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564 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn

Design, synthesis and biological evaluation of pyrrolidinone analogs aspotential 20S proteasome inhibitors Yong-Jian Li, Feng-Rong Xu, Yan Niu, Xiao-Min Zou, Yue Yuan, Hai-Fei Gao, Chao Wang,Guan-Yu Yang, Qi Sun, Ping Xu *

Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Abstract: A novel series of pyrrolidinone analogs that are designed as Michael addition acceptors to react irreversibly with the proteasome active site Thr1O have been synthesized. Although biological evaluation results show that the compounds display poor inhibitory activity towards the proteasome active sites, pyrrolidinone analogs might still be modified to be potential 20S proteasome inhibitors.

Keywords: Pyrrolidinone; 20S proteasome; Peptidomimetic backbone

CLC number : R916.1 Document code : A Article ID : 10031057(2011)656408

Received date: 2011-05-24.

Foundation item: Beijing Natural Science Foundation (Grant No.7112088).*Corresponding author. Tel.: 86-10-82801505;E-mail: [email protected]

doi:10.5246/jcps.2011.06.072

1. Introduction

The 26S proteasome complex is a multifunctional proteolytic molecular machine, which is composedof the cylinder-shaped multimeric protein complexreferred to as the 20S proteasome core particle andone or two 19S regulatory caps [1]. The central 20Score contains three different active subunits and showsdiverse catalytic activities, including chymotrypsin-like (CT-L, 5 subunit), trypsin-like (T-L, 2 subunit),and caspase-like (PGPH, 1 subunit) activities. Theactive sites of each of these proteolytic subunitsutilize the N -terminus as the catalytic residues duringsubstrate hydrolysis. The 19S caps can recognizeubiquitinated protein substrates and promote their entry into the central catalytic chamber.

The proteasome plays an essential role in a number of proteolytically mediated processes such as thedestruction of misfolded and misassembled proteins,cell division, cell differentiation, and developmentof the immune response [2]. Thus, the proteasomerepresents a potential target for the developmentof therapeutic agents for the treatment of cancer,inflammation, immune diseases, and others. In recent

years, many types of electrophiles such as boronicacids, -lactones, epoxyketones, and cyclic thiono-carbonates [3] have been reported as proteasomeinhibitors. However, the dipeptidyl boronic acid borte-zomib was the only drug approved for the treatment

of relapsed and refractory multiple myeloma (MM)in 2003 and mantle cell lymphoma (MCL) in 2006 byFDA. Some anticancer candidates including peptide

boronic acid analogs MLN9708 and CEP18770, pep-tide epoxyketones carfilzomib, PR047, and -lactone

NPI0052 have entered clinical trials [4].Among these proteasome inhibitors, Syringolin A,

which was first isolated from the plant pathogen Pseudomonas syringae pv. Syringae in 1998, wasidentified as an irreversible inhibitor for all three

catalytic activities of eukaryotic proteasome via aMichael addition mechanism. In the past two years,three research groups reported the total synthesis of Syringolin A [57] . In this article, we designed a novelclass of C -terminal pyrrolidinone analogs containing-amino acid backbone. The unsaturated pyrrolidinoneis proposed as a new type of pharmacophore unitto react irreversibly with the proteasome active siteThr1O through a Michael addition mechanismin the same way as Syringolin A [8]. The crystal

structure of the yeast proteasome in complex withSyringolin A (PDB code: 2ZCY) was used to per-form a docking study. We compared the bindingmodes of pyrrolidinone analogs and Syringolin A by


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Y. J. Li et al. / Journal of Chinese Pharmaceutical Sciences 20 (2011) 564571 565

using GOLD software. Compound 6a represents thetypical binding mode of its kind. The result of dock-ing study showed that the substitutions of compound 6a can occupy the S1, S2 and S3 pockets very well(Fig. 1A) and the conformation of compound 6a when bound with the proteasome is similar to that of Syringolin A (Fig. 1B). The reasonable bindingaffinity suggests that the pyrrolidinone analogs mayrepresent a new class of potential proteasome inhibi-tors that can form covalent binding with N -terminalthreonine residue in the catalytic sites.

2. Results and discussion

2.1. Chemistry

The synthetic routes of our target pyrrolidinoneanalogs are described in Scheme 1 and Scheme 2.At first, Boc protected -amino acids were coupledwith L-amino acid methyl ester to give 1a c, whichwere hydrolyzed and treated with Meldrums acid togenerate 2a c. In Decicco and Grovers method [9],carboxylic acid is activated by IPCC, which is veryexpensive. We found that EDCl can be employed as acarboxyl activating agent to prepare 2ac in excellent

yields[10]

. Cyclization was carried out in acetonitrileto give tetramic acid 3ac from 2a c. The mechanismof cyclization was proposed as a nucleophilic attack of the nitrogen lone pair which was favored by NHhydrogen bonding to one of the carbonyl functions [11] .In general, any effort to purify products ( 2a c and 3a c) by column chromatography was unsuccessful.We obtained 4a c from 3a c after the reduction

by NaBH 4 without further purification. We triedtwo methods to obtain 5a c from 4a c. In the first

method, the hydroxyl group was transformed intomesylate or p-toluenesulfonate followed by elimina-tion to give 5a c. We found that the reaction can not

proceed completely. We gave up this method andtried to substitute hydroxyl group with iodine asour second method, which was followed by elimi-nation with base treatment to yield pyrrolidinones.Surprisingly, we found that the two steps can befinished in one step with the use of PPh 3, imidazoleand iodine [12] . The elimination reaction proceededsmoothly with a single product spot on TLC. Espe-cially, HPLC analysis showed that the reactionyielded high enantioselectivity and the enantiomeric

purity of compound 5a c was more than 90%. Weassigned the absolute stereochemistry of C -4 onthe pyrrolidinone ring as S -configuration as reported

by Decicco and Grover [9]. After the removal of theBoc protecting group and the coupling with com-

pounds 9a and 9b , pyrrolidinone analogs 6a d wereobtained. Compounds 9a b were successfully assem-

bled in two steps as shown in Scheme 2. We found partial racemization in compounds 6ad , which might be caused by the strong acid condition promoted1,4-H migration on the pyrrolidinone ring.

Figure 1. (A) Docking result showing the binding mode of compound 6a with proteasome 2ZCY 5 subunit; (B) The conformation of compound 6a (orange) and Syringolin A (blue) when bound with the

proteasome (proteasome removed).

A

B

NH

N

Ph O

O

O

H N

O

MeO 2C

HN

O

NH

O

H N

O

NH

NH

O

O

OH

Compound 6a

Syringolin A


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2.2. 20S proteasome inhibitory activity

Pyrrolidinone analogs 5ac and 6ad were screenedfor their proteasome inhibitory activity by usingthe Proteasome-Glo TM with human 20S proteasome(BML-PW8720-0050). The luminescence signal wasread after incubation for 15 min and 20 min with afinal concentration of 100 mol/L. The results showedthat none of the compounds has any inhibitory activitytowards the proteasome compared with positivecontrol MG132 and EGCG.

3. Conclusion

In summary, several pyrrolidinone analogs weresynthesized as potential proteasome inhibitors.However, evaluation of biological activities revealedthat these compounds are not active inhibitors of

proteasome. Lack of activity may be caused bythe decreased hydrogen bonding interaction of the

compounds with the amino acid residues of protea-some. Moreover, the partial racemization of compounds 6a d may be another reason for the poor inhibitoryactivity. Therefore, more structural modification isneeded to reach a definite conclusion on whether C -terminal pyrrolidinone is a useful pharmacophore

unit as a novel proteasome inhibitor.

4. Experimental

4.1. General methods

Unless otherwise noted, all reagents were purchasedfrom Acros, TCI, Sigma, Aldrich and used withoutfurther purification. Dry solvents were preparedaccording to standard procedures. Reaction progresswas monitored with thin-layer chromatography (TLC)carried out on GF 254 silica gel glass plates purchasedfrom Qingdao Haiyang Chemical Co. Melting pointswere taken with an X4 apparatus and were uncorrected.

Scheme 2. Reagents and conditions: (a) EDCl, HOBt, NMM, DMF, r.t., 12 h; (b) HCl in EtOAc, r.t., 4 h.

BocHN

O

BocHNCOOH

R 2

R 2

BocHN N

O

R 1OR 2

OH

BocHN

R 2

H N

O R 1

b, cOMe

O

NH

R 1

O

O

OO

O

BocHN N

O

R 1OR 2

OH BocHN N

O

R 1OR 2

d

a

e f

g, h

ClH 3 N CO 2Me

R 1

+

1a - c 2a - c

3a - c 4a - c 5a - c

NH

N

R 2 O R 1

O

O

H N

O

MeO 2C

R 6a - d

Scheme 1. Reagents and conditions: (a) EDCl, HOBt, NMM, DMF, r.t., 12 h; (b) 1 N NaOH, r.t., 2 h then 1 N HCl; (c) Meldrums acid, DMAP,EDCl, DCM, 5 C, 12 h; (d) CH 3CN, reflux, 1 h; (e) NaBH 4, HOAc, DCM, 5 C, 12 h; (f) I 2, PPh 3, imidazole, DCM, r.t., 6 h; (g) HCl inEtOAc, r.t., 4 h; (h) 9a b , EDCl, HOBt, NMM, DMF, r.t., 12 h.

5a R 1 = CH 2CH(CH 3)2 R 2 = Ph 5b R 1 = Cyclopropylmethyl R 2 = Ph 5c R 1 = CH 2CH(CH 3)2 R 2 = CH 2Ph

6a R 1 = CH 2CH(CH 3)2 R 2 = Ph R = Me 6b R 1 = Cyclopropylmethyl R 2 = Ph R = Me 6c R 1 = Cyclopropylmethyl R 2 = Ph R = H

6d R 1 = CH 2CH(CH 3)2 R 2 = CH 2Ph R = Me

8a R = Me 9a R = Me

8b R = H 9b R = H

CO 2 H

H N CO2 MeO

R

CO 2 Bu- t

COOH CO 2 Bu- t

OH

N CO2 Me

R a b

7

+ClH 3 N CO2 Me

R


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1H NMR spectra were recorded on a Bruker Avance III400 MHz system. 1H NMR spectra are reported inthe following manner: chemical shifts calculatedwith reference to solvent standards based on tetrame-thylsilane (TMS), multiplicity (s, singlet; d, doublet;t, triplet; q, quartet; sept, septuplet; m, multiplet),coupling constant ( J ) in Hz, number of protons, andthe corresponding attributions. MS data were obtainedwith MDS SCIEX QSTAR system.

4.2. Synthesis of pyrrolidinone analogs

4.2.1. ( S )-Methyl-2-(( R )-3-( tert -butoxycarbonylamino)-

3-phenylpropanamido)-4-methylpentanoate (1a) To a stirred solution of HClLeu-OMe (3.62 g,

20 mmol) in DMF (20 mL) was added N -methyl-morpholine (6.8 mL, 60 mmol). The suspension wasstirred for 30 min in ice bath and poured into thesolution of Boc--Phe-OH (5.31 g, 20 mmol), EDCl(4.60 g, 24 mmol), HOBt (3.24 g, 24 mmol) in DMF(20 mL). The reaction mixture was stirred overnightat room temperature. The mixture was partitioned

between ethyl acetate and water, and the organic

layer was then washed with 10% citric acid, saturatedaqueous NaHCO 3 and brine, dried over Na 2SO 4,concentrated in vacuo to give crude product, whichwas crystallized from EtOAc to yield 1a as whitecrystals (6.52 g, 83.1%). Mp: 142143 C. 1H NMR (400 MHz, CDCl 3) : 0.87 (d, J 6.0 Hz, 6H, 2C H 3),1.41 (s, 9H, Boc), 1.411.69 (m, 3H, C H 2, C H ),2.722.74 (m, 2H, C H 2CO), 3.67 (s, 3H, C H 3),4.524.55 (m, 1H, C H ), 5.025.04 (m, 1H, C H ),5.915.93 (m, 2H, 2N H ), 7.267.32 (m, 5H, Ph).

ESI-MS: 393.2 (M+H)+

, 415.2 (M+Na)+

.

4.2.2. ( S )-Methyl-2-(( R )-3-( tert -butoxycarbonylamino)-3-phenylpropanamido)-3-cyclopropyl propanoate(1b)

Using the same procedure as described for thesynthesis of 1a , but starting with Boc--Phe-OH(3.33 g, 12.6 mmol) and ( S )-2-amino-3-cyclopropyl-

propionic acid methyl ester hydrochloride (2.25 g,12.6 mmol), 1b was obtained in 88.2% yield aswhite solid. Mp: 146148 C. 1H NMR (400 MHz,CDCl 3) : 0.010.03 (m, 2H, C H 2), 0.360.39 (m,2H, C H 2), 0.410.52 (m, 1H, C H ), 1.41 (s, 9H,Boc), 1.591.68 (m, 2H, C H 2), 2.74 (d, J 4.8 Hz,

2H, C H 2CO), 3.70 (s, 3H, C H 3), 4.574.61 (m, 1H,C H ), 5.055.15 (m, 1H, PhC H ), 5.875.89 (m, 1H,

N H ), 6.166.18 (m, 1H, N H ), 7.217.39 (m, 5H,Ph). ESI-MS: 391.2 (M+H) +, 413.2 (M+Na) +.

4.2.3. ( S )-Methyl-2-(( S )-3-( tert -butoxycarbonylamino)-4-phenylbutanamido)-4-methylpentanoate (1c)

Using the same procedure as described for thesynthesis of 1a , but starting with Boc- L--homo-

phenylalanine (5.58 g, 20.0 mmol) and HClLeu-OMe(3.63 g, 20.0 mmol), 1c was obtained in 82.4% yieldas white solid. Mp: 9697 C. 1H NMR (400 MHz,CDCl 3) : 0.95 (d, J 5.6 Hz, 6H, 2C H 3), 1.42 (s, 9H,

Boc), 1.531.64 (m, 3H, C H , C H 2), 2.252.51 (m,2H, C H 2), 2.792.84 (m, 2H, C H 2), 3.78 (s, 3H,C H 3), 4.084.14 (m, 1H, C H ), 4.634.64 (m, 1H,C H ), 5.455.53 (m, 1H, N H ), 5.935.95 (m, 1H,

N H ), 7.217.38 (m, 5H, Ph). ESI-MS: 407.3 (M+H) +,429.2 (M+Na) +.

4.2.4. tert -Butyl-(1 R )-3-((2 S )-3-hydroxy-2-isobutyl-5-oxopyrrolidin-1-yl)-3-oxo-1-phenylpropylcarba-mate (4a)

To a stirred solution of 1a (2.42 g, 6.20 mmol) inTHF (20 mL) was added 1 N NaOH to pH 12. Thereaction mixture was stirred at room temperature for 4 h and then concentrated in vacuo. The solutionwas added 1 N HCl to pH 2 and partitioned betweenethyl acetate and additional water. Aqueous phasewas extracted with ethyl acetate (520 mL) and thecombined organic phases were dried over Na 2SO 4.After filtration, solvent was concentrated in vacuoand the resulting white solid was dissolved inCH2Cl2 (20 mL), followed by adding DMAP (1.13 g,9.3 mmol), Meldrums acid (1.34 g, 9.3 mmol) andEDCl (1.79 g, 9.3 mmol). The mixture was stirredat 5 C overnight and then washed with cold 10%citric acid, water and brine. The organic layer wasdried, evaporated in vacuo to give crude product,which was dissolved in MeCN (50 mL) and heatedat reflux for 1 h. The solvent was removed and theresidual solid was dissolved in a mixture of CH 2Cl 2(20 mL) and acetic acid (4.3 mL). The acidic solu-tion was cooled to 5 C and NaBH 4 (574 mg,15.5 mmol) was added over 0.5 h under vigorousstirring. The mixture was stirred overnight and washedwith brine, dried over Na 2SO 4, concentrated in vacuo


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to give crude product, which was separated by silicagel chromatography to yield 1.05 g 4a as colorlessoil. Yield: 41.9% for 4 steps. 1H NMR (400 MHz,CDCl 3) : 0.870.98 (m, 6H, 2C H 3), 1.40 (s, 9H,Boc), 1.271.65 (m, 3H, C H , C H 2), 2.642.68 (m,2H, C H 2), 2.983.22 (m, 2H, C H 2), 3.523.61 (m,1H, C H ), 4.324.41 (m, 1H, C H ), 4.434.46 (m,1H, O H ), 5.215.25 (m, 1H, C H ), 5.435.59 (m,1H, N H ), 7.287.39 (m, 5H, Ph). ESI-MS: 405.2(M+H) +, 427.2 (M+Na) +.

4.2.5. tert -Butyl-(1 R )-3-((2 S )-2-(cyclopropylmethyl)-3-hydroxy-5-oxopyrrolidin-1-yl)-3-oxo-1-phenyl-

propylcarbamate (4b) Using the same procedure as described for the

synthesis of 4a , but starting with 1b (3.90 g, 10 mmol), 4b was obtained in 47.5% yield as colorless oil.1H NMR (400 MHz, CDCl 3) : 0.110.16 (m, 2H,C H 2), 0.380.43 (m, 2H, C H 2), 0.760.85 (m, 1H,C H ), 1.42 (s, 9H, Boc), 1.451.58 (m, 1H, C H 2),1.651.78 (m, 1H, C H 2), 2.752.78 (m, 2H, C H 2),3.193.23 (m, 1H, COC H 2), 3.453.53 (m, 1H,COC H 2), 4.264.38 (m, 1H, O H ), 4.494.57 (m,

1H, C H ), 5.255.31 (m, 1H, C H ), 5.355.41 (m,1H, N H ), 7.267.36 (m, 5H, Ph). ESI-MS: 403.2(M+H) +, 425.2 (M+Na) +.

4.2.6. tert -Butyl-(2 S )-4-((2 S )-3-hydroxy-2-isobutyl-5-oxopyrrolidin-1-yl)-4-oxo-1-phenylbutan-2-ylcarbamate (4c)

Using the same procedure as described for thesynthesis of 4a , but starting with 1c (4.69 g, 11.6 mmol), 4c was obtained in 61.8% yield as colorless oil.1H NMR (400 MHz, CDCl 3) : 0.850.96 (m, 6H,

2C H 3), 1.34 (s, 9H, Boc), 1.601.76 (m, 3H, C H , C H 2),2.562.68 (m, 2H, C H 2), 2.722.88 (m, 2H, C H 2),3.053.18 (m, 2H, C H 2), 4.154.18 (m, 1H, C H ),4.254.27 (m, 1H, O H ), 4.374.39 (m, 1H, C H ),4.414.49 (m, 1H, C H ), 5.025.05 (m, 1H, N H ),7.187.28 (m, 5H, Ph). ESI-MS: 319.2 (M+H-Boc) +.

4.2.7. tert -Butyl-( R )-3-(( S )-2-isobutyl-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-3-oxo-1-phenylpropyl-carbamate (5a)

To a stirred solution of 4a (210 mg, 0.52 mmol) inCH 2Cl2 (15 mL) was added I 2 (171 mg, 0.68 mmol),imidazole (92 mg, 1.36 mmol), and PPh 3 (177 mg,0.68 mmol). The reaction mixture was stirred at room

temperature for 12 h after which it was filtered andthe filter was concentrated in vacuo to give crude

product, which was separated by silica gel chroma-tography to yield 138 mg 5a as white solid. Yield:68.7%. Mp: 7374 C. 1H NMR (400 MHz, CDCl 3) : 0.870.98 (m, 6H, 2C H 3), 1.261.29 (m, 1H,C H 2), 1.42 (s, 9H, Boc), 1.641.72 (m, 1H, C H ),1.851.96 (m, 1H, C H 2), 3.393.55 (m, 2H, C H 2),4.724.74 (m, 1H, C H ), 5.235.26 (m, 1H, PhC H ),5.565.62 (m, 1H, N H ), 6.116.15 (m, 1H, COC H =),7.227.39 (m, 6H, Ph, =C H ). HRMS calcd. for C17H23 N2O2+: 287.17540 (M+H-Boc) +; found 287.17525(M+H-Boc) +.

4.2.8. tert -Butyl-( R )-3-(( S )-2-(cyclopropylmethyl)-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-3-oxo-1-pheyl-propylcarbamate (5b)

Using the same procedure as described for thesynthesis of 5a, but starting with 4b (1.34 g, 3.33 mmol), 5b was obtained in 76.2% yield as white solid. Mp:6566 C. 1H NMR (400 MHz, CDCl 3) : 0.010.15(m, 2H, C H 2), 0.430.48 (m, 2H, C H 2), 0.520.58(m, 1H, C H ), 1.40 (s, 9H, Boc), 1.511.58 (m, 1H,C H 2), 1.791.82 (m, 1H, C H 2), 3.263.58 (m, 2H,C H 2), 5.235.26 (m, 1H, C H ), 5.425.50 (m, 1H,

N H ), 6.11 (dd, J 1 6.0 Hz, J 2 0.8 Hz, 1H, =C H ), 7.217.35 (m, 5H, Ph), 7.34 (d, J 6.0 Hz, 1H, =C H ). HRMScalcd. for C 17H21 N 2O 2+: 285.15975 (M+H-Boc) +;found 285.15969 (M+H-Boc) +.

4.2.9. tert -Butyl-( S )-4-(( S )-2-isobutyl-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-4-oxo-1-phenylbutan-2-ylcarbamate (5c)

Using the same procedure as described for the

synthesis of 5a , but starting with 4c (3.00 g, 7.18 mmol), 5c was obtained in 72.8% yield as white solid. Mp:9293 C. 1H NMR (400 MHz, CDCl 3) : 1.011.19(m, 6H, 2C H 3), 1.321.36 (m, 1H, C H ), 1.37 (s, 9H,Boc), 1.651.78 (m, 1H, C H 2), 2.082.16 (m, 1H,C H 2), 2.852.92 (m, 2H, C H 2), 3.093.24 (m, 2H,C H 2), 4.264.38 (m, 1H, C H ), 4.824.96 (m, 1H,

N H ), 4.985.12 (m, 1H, C H ), 6.06 (dd, J 1 6.0 Hz, J 2 1.6 Hz, 1H, =C H ), 7.31 (m, 5H, Ph), 7.36 (d, J 6.0 Hz, 1H, =C H ). HRMS calcd. for C 23H33 N 2O4+:

401.24348 (M+H)+

, C23H 32 N 2O 4 Na+

: 423.22543(M+Na) +, C23H32 N 2O4K +: 439.19937 (M+K) +; found401.24459 (M+H) +, 423.22688 (M+Na) +, 439.20099(M+K) +.


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4.2.10. ( S )-Methyl-2-(4-(( R )-3-(( S )-2-isobutyl-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-3-oxo-1-phenyl-propylcarbamoyl)benzamido)propanoate (6a)

i) To a solution of 5a (438 mg, 1.13 mmol) inCH 2Cl2 (5 mL) was added 1.1 mL of TFA. The solu-tion was stirred at room temperature for 2 h and thenconcentrated in vacuo. N -Methylmorpholine (2 mL)was added to neutralize the residual in situ and theresulting solution was used for the following stepwithout further purification. ii) To a stirred solutionof EDCl (260 mg, 1.4 mmol), HOBt (183 mg,1.4 mmol) and 9a (284 mg, 1.13 mmol) in DMFin an ice bath was added the crude product of stepone. The reaction mixture was warmed to roomtemperature and stirred over night. The mixture was

partitioned between ethyl acetate and water, and theorganic layer was then washed with 10% citric acid,saturated aqueous NaHCO 3 and brine, dried over

Na 2SO4, concentrated in vacuo to give crude product,which was separated by silica gel chromatographyto yield 6a as white solid (446 mg, 76.0%). Mp:203204 C. 1H NMR (400 MHz, CDCl 3) : 0.84(d, J 6.0 Hz, 3H, C H 3), 0.96 (d, J 6.0 Hz, 3H, C H 3),1.321.48 (m, 1H, C H 2), 1.55 (d, J 6.8 Hz, 3H,C H 3), 1.651.76 (m, 1H, C H (CH 3)2), 1.952.02 (m,1H, C H 2), 3.52 (dd, J 1 15.2 Hz, J 2 4.8 Hz, 1H,C H 2CO), 3.76 (dd, J 1 15.2 Hz, J 2 4.8 Hz, 1H,C H 2CO), 3.82 (s, 3H, OC H 3), 4.724.78 (m, 1H,C H ), 4.804.84 (m, 1H, C H ), 5.775.82 (m, 1H,PhC H ), 6.12 (d, J 5.6 Hz, 1H, =C H CO), 6.91 (d,

J 6.8 Hz, 1H, N H ), 7.297.39 (m, 6H, Ph, =C H ),7.58 (d, J 8.0 Hz, 1H, N H ), 7.86 (s, 4H, Ph). HRMScalcd. for C 29H34 N 3O6+: 520.24421 (M+H) +; found520.24494 (M+H) +.

4.2.11. ( S )-Methyl-2-(4-(( R )-3-(( S )-2-(cyclopropyl-methyl)-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-3-oxo-1-phenylpropylcarbamoyl)benzamido)pro-panoate(6b)

Using the same procedure as described for thesynthesis of 6a , but starting with 5b (205 mg,0.53 mmol) and 9a (134 mg, 0.53 mmol), 6b wasobtained in 81.2% yield as white solid. Mp: 195

197 C.1

H NMR (400 MHz, CDCl 3) : 0.010.06(m, 2H, C H 2), 0.330.45 (m, 2H, C H 2), 0.530.62(m, 1H, C H ), 1.40 (d, J 7.2 Hz, 3H, C H 3), 1.681.72(m, 2H, C H 2), 3.413.43 (m, 1H, C H 2), 3.443.48

(m, 1H, C H 2), 3.64 (s, 3H, OC H 3), 4.484.56 (m,1H, C H ), 4.754.86 (m, 1H, C H ), 5.615.76 (m,1H, PhC H ), 6.246.28 (m, 1H, =C H CO), 7.227.38(m, 5H, Ph), 7.687.74 (m, 1H, =C H ), 7.927.96(m, 4H, Ph), 8.968.98 (m, 1H, N H ), 9.019.04 (m,1H, N H ). HRMS calcd. for C 29H32 N 3O6+: 518.22856(M+H) +, C29H31 N 3O6 Na +: 540.21051 (M+Na) +,C29H31 N3O6K +: 556.18444 (M+K) +; found 518.22787(M+H) +, 540.20965 (M+Na) +, 556.18363 (M+K) +.

4.2.12. Methyl-2-(4-(( R )-3-(( S )-2-(cyclopropylmethyl)-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-3-oxo-1-phenyl-propylcarbamoyl)benzamido)acetate (6c)

Using the same procedure as described for thesynthesis of 6a , but starting with 5c (180 mg,0.47 mmol) and 9b (111 mg, 0.47 mmol), 6c wasobtained in 68.2% yield as white solid. Mp: 167168 C. 1H NMR (400 MHz, CDCl 3) : 0.030.05(m, 2H, C H 2), 0.350.38 (m, 2H, C H 2), 0.480.56(m, 1H, C H ), 1.511.54 (m, 1H, C H 2), 1.952.02(m, 1H, C H 2), 3.493.56 (m, 2H, C H 2), 3.813.90(m, 1H, C H ), 3.85 (s, 3H, OC H 3), 4.254.36 (m,2H, C H 2), 4.804.86 (m, 1H, C H ), 5.735.80 (m,1H, N H ), 6.136.16 (m, 1H, N H ), 6.776.82 (m,1H, =C H CO), 7.457.56 (m, 5H, Ph), 7.607.66 (m,1H, =C H ), 7.857.92 (m, 4H, Ph). HRMS calcd. for C28H29 N 3O6+: 504.21291 (M+H) +; found 504.21291(M+H) +.

4.2.13. ( S )-Methyl-2-(4-(( S )-4-(( S )-2-isobutyl-5-oxo-2,5-dihydro-1 H -pyrrol-1-yl)-4-oxo-1-phenylbutan-2-ylcarbamoyl)benzamido)propanoate (6d)

Using the same procedure as described for thesynthesis of 6a , but starting with 5c (200 mg,

0.50 mmol) and 9a (126 mg, 0.50 mmol), 6d wasobtained in 85.2% yield as white solid. Mp: 134135 C. 1H NMR (400 MHz, CDCl 3) : 0.090.96(m, 6H, 2C H 3), 1.52 (d, J 7.2 Hz, 3H, C H 3), 1.671.70 (m, 1H, C H (CH 3)2), 1.962.02 (m, 2H, C H 2),3.253.32 (m, 4H, 2C H 2), 3.79 (s, 3H, C H 3), 4.784.82 (m, 3H, 3C H ), 6.07 (d, J 6.0 Hz, 1H, =C H CO),6.90 (d, J 7.2 Hz, 1H, N H ), 7.107.16 (m, 1H, N H ),7.287.38 (m, 5H, Ph), 7.72 (d, J 8.0 Hz, 2H, Ph- H ),7.80 (d, J 8.0 Hz, 2H, Ph- H ). HRMS calcd. for

C30H36 N 3O6+

: 534.25986 (M+H)+

, C30H35 N 3O6 Na+

:556.24181 (M+Na) +, C30H 35 N 3O 6K + : 572.21574(M+K) +; found 534.25895 (M+H) + , 556.24085(M+Na) +, 572.21447 (M+K) +.


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4.2.14. ( S )- tert -Butyl-4-(1-methoxy-1-oxopropan-2-ylcarbamoyl)benzoate (8a)

To a stirred solution of HClAla-OMe (140 mg,1 mmol) in DCM (10 mL) was added N -methyl-morpholine (0.34 mL, 3 mmol). The suspension wasstirred for 30 min in ice bath and poured into thesolution of 4-( tert -butoxycarbonyl)benzoic acid (222 mg,1 mmol), EDCl (230 mg, 1.2 mmol), HOBt (162 mg,1.2 mmol) in DCM (10 mL). The reaction mixturewas stirred overnight at room temperature. Themixture was partitioned between ethyl acetate andwater, and the organic layer was then washed with10% citric acid, saturated aqueous NaHCO

3and

brine, dried over Na 2SO 4, concentrated in vacuo togive crude product, which was crystallized fromEtOAc to yield 8a as colorless oil (293 mg, 95.4%).1H NMR (400 MHz, CDCl 3) : 1.53 (d, J 7.2 Hz,3H, C H 3), 1.61 ( s, 9H, C(C H 3)3), 3.80 (s, 3H, C H 3),4.804.84 (m, 1H, C H ), 6.846.88 (m, 1H, N H ),7.83 (d, J 8.4 Hz, 2H, Ph- H ), 8.08 (d, J 8.4 Hz, 2H,Ph- H ). ESI-MS: 308.2 (M+H) +.

4.2.15. tert -Butyl-4-(2-methoxy-2-oxoethylcarbamoyl)benzoate (8b)

Using the same procedure as described for thesynthesis of 8a , but starting with HClGly-OMe(375 mg, 3.00 mmol), 8b was obtained in 86.3%yield as white solid. Mp: 9394 C. 1H NMR (400 MHz, CDCl 3) : 1.64 (s, 9H, C(C H 3)3), 3.85(s, 3H, C H 3), 4.30 (d, J 5.2 Hz, 2H, C H 2), 6.736.76(m, 1H, N H ), 7.88 (d, J 8.8 Hz, 2H, Ph- H ), 8.08 (d,

J 8.8 Hz, 2H, Ph- H ). ESI-MS: 294.1 (M+H) +, 316.1(M+Na) +.

4.2.16. ( S )-4-(1-Methoxy-1-oxopropan-2-ylcarbamoyl)benzoic acid (9a)

8a (307 mg, 1 mmol) was dissolved in HCl/EtOAcand the solution was stirred at room temperaturefor 6 h. The reaction solution was filtered to obtaincrude product, which was crystallized from EtOActo yield 9a as white crystals (202 mg, 80.6%). Mp:156158 C. 1H NMR (400 MHz, CDCl 3) : 1.57 (d,

J 7.2 Hz, 3H, C H 3), 3.83 (s, 3H, C H 3), 4.844.92

(m, 1H, C H ), 6.85 (d, J 7.6 Hz, 1H, N H ), 7.88 (d, J 8.4 Hz, 2H, Ph- H ), 8.14 (d, J 8.4 Hz, 2H, Ph- H ).ESI-MS: 252.1 (M+H) +, 274.1 (M+Na) +.

4.2.17. 4-(2-Methoxy-2-oxoethylcarbamoyl)benzoicacid (9b)

Using the same procedure as described for thesynthesis of 9a , but starting with 8b (753 mg,2.56 mmol), 9b was obtained in 84.8% yield aswhite solid. Mp: 186188 C. 1H NMR (400 MHz,CDCl 3) : 3.84 (s, 3H, C H 3), 4.29 (d, J 5.2 Hz, 2H,C H 2), 6.746.80 (m, 1H, N H ), 7.89 (d, J 8.0 Hz, 2H,Ph- H ), 8.15 (d, J 8.0 Hz, 2H, Ph- H ). ESI-MS: 238.1(M+H) +; 260.1 (M+Na) +.

4.3. 20S proteasome inhibitory assay

The Proteasome-GloTM

3-substrate System wasused for all 3 subunit activity test with human 20S

proteasome from ENZO LIFE SCIENCES INTLINC (BML-PW8720-0050). MG132 and EGCG wereused as positive control. Inhibitors were resuspendedin DMSO, serially diluted in 10 mM HEPES (pH 7.6)and combined with 1 g/mL human 20S proteasomewith a final concentration of 100 mol/L. The corre-sponding Proteasome-Glo TM substrates (Suc-LLVY-Glo TM Substrate for CT-L, Z-LRR-Glo TM Substrate

for T-L and Z-nLPnLD-Glo Substrate for PGPH)were added after incubation for 15 min. Luminescencewas recorded after incubation for 20 min on aGloMax 96 Microplate Luminometer. Values arecalculated as relative luminescence units (RLU) andexpressed as percent of control.

Acknowledgements

This work was supported by Beijing Natural

Science Foundation (Grant No. 7112088).

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