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Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory

Syndrome 3CL Protease

Jiun-Jie Shie,

Jim-Min Fang,*

,†,‡

Chih-Jung Kuo,

§

Tun-Hsun Kuo,

§

Po-Huang Liang,*

Hung-Jyun Huang,

Wen-Bin Yang,

Chun-Hung Lin,

§

Jiun-Ling Chen,

Yin-Ta Wu,

and Chi-Huey Wong

‡,|

Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan, Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan, and Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037

Received February 28, 2005

A diversified library of peptide anilides was prepared, and their inhibition activities against

the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The

most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline,

L

-phenylalanine and

4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL

protease with K

i

) 0.03 µM. The molecular docking experiment indicates that the P1 residue

of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.

Introduction

Severe Acute Respiratory Syndrome (SARS) first

occurred in Guandong (China) in November of 2002 and

spread through many countries in 2003. This illness is

caused by infection with a novel human coronavirus

(SARS-CoV).

1

The fatality rate of SARS-CoV infection

is rather high, estimated to be 14-15%. In only a few

months, nearly 1000 lives were claimed.

2

The natural

source of SARS-CoV is unclear, though studies on the

molecular evolution of SARS-CoV indicate that the virus

may have emerged from wild animals.

3

At present, no

efficacious therapy for SARS is available. Therefore, a

search for effective antivirals for the SARS-CoV is of

current interest.

SARS coronavirus is a positive-strand RNA virus,

4

that encodes two replicase polyproteins pp1a and pp1b.

Extensive proteolytic processing of these nonstructural

polyproteins is required to provide the functional

pro-teins for viral propagation. These processes are

medi-ated primarily by the main protease (M

pro

), which is also

known as dimeric chymotrypsin-like protease (3CL

pro

).

5

The active site of SARS-CoV 3CL protease contains

Cys145 and His41 to constitute a catalytic dyad, in

which cysteine functions as the common nucleophile in

the proteolytic process.

5

The 3CL protease cleaves pp1a

at no less than 11 conserved sites with a sequence of

(Leu,Met,Phe)-GlnV(Ser,Ala,Gly), in which a P1 glutamine

residue invariably occupies the S1 site.

5,6

The 3CL

protease is essential for the propagation of the virus and

thus serves as a key target for the discovery of

anti-SARS drugs.

So far, only a few inhibitors have been validated by

in vitro protease assays. These protease inhibitors

include C2 symmetric peptidomimetic compounds,

7

zinc-conjugated compounds,

8

bifunctional aryl boronic acids,

9

a quinolinecarboxylate derivative,

10

a

thiophenecar-boxylate,

11

and

phthalhydrazide-substituted

keto-glutamine analogues.

12

As a part of our efforts directed toward the

develop-ment of anti-SARS agents, we prepared several

chro-mogenic peptides (AA)x-Gln-pNA, e.g.

QTSITSAVLQ-pNA containing a p-nitroaniline moiety at the C-terminal

glutamine, to test their activities as substrates for the

SARS-CoV 3CL protease. Unlike typical

nitroanilide-based peptides which are readily hydrolyzed by serine

and cysteine proteases,

13

these (AA)x-Gln-pNA peptides

were not efficiently cleaved by the SARS-CoV 3CL

protease but displayed weak inhibition against the

enzyme. This observation, along with an earlier

cell-based assay showing the inhibitory activity of

N-(2-chloro-4-nitrophenyl)-5-chloro-2-hydroxybenzamide

(Niclosamide) against the replication of SARS-CoV,

14

led

us to explore peptide nitroanilides as inhibitors of the

SARS-CoV 3CL protease.

Results and Discussion

We chose to prepare a series of peptide anilides

having

L

-phenylalanine as the P1 residue, on the basis

* To whom correspondence should be addressed. Tel: 8862-23637812. Fax: 8862-23636359. E-mail: jmfang@ntu.edu.tw.

National Taiwan University.

Genomics Research Center, Academia Sinica. §Institute of Biological Chemistry, Academia Sinica. |The Scripps Research Institute.

10.1021/jm050184y CCC: $30.25 © 2005 American Chemical Society Published on Web 06/04/2005

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of another study on AG7088 analogues that the

inhibi-tory activity can be improved by using

L

-phenylalanine

to replace

L

-glutamine or its γ-lactam isostere (see Table

3 in Supporting Information). Anilide 1 was prepared

by condensation of 2-chloro-4-nitroaniline with the acyl

chloride derivative of Boc-Phe-OH. Using the previously

reported amide formation in a microtiter plate,

15

the

coupling reactions of a 60-member library of carboxylic

acids with the amine generated by removal of the Boc

group from anilide 1 afforded a 60-member library of

anilide 2. Tripeptide anilides 3a-x (24 members) and

tetrapeptide anilides 4a-x (24 members) were also

created by coupling of 1 with appropriate peptides. The

dimeric peptide anilides 5a-c and 6a-c were prepared

by using a diacid, e.g. succinic acid, S-malic acid, and

(2R,3R)-tartaric acid, as the core structure to link with

appropriate amino acids or peptides.

On the basis of the previously reported synthesis of

AG7088,

16

we also devised an expedient method for the

synthesis of anilides 7a-x, the ketomethylene isosteres

of tripeptides 3a-x (Scheme 1).

These anilide products 2-7 were characterized by

mass analyses and directly subjected, without isolation,

to the inhibition assay against the SARS-CoV 3CL

protease according to the previously described

fluoro-metric method.

7,17

The initial velocities of the inhibited

reactions using 50 nM of SARS-CoV 3CL protease and

6 µM of the fluorogenic substrate were plotted against

the different inhibitor concentrations to obtain the IC

50

values. On the basis of the preliminary results of assays,

some of the most promising inhibitor candidates were

selected for the scale-up synthesis. The IC

50

values and

inhibition constants (K

i

) of the pure samples were then

measured to validate their activities.

The results of preliminary assays indicated that the

2-chloro-4-nitroanilides 2-6 generally possessed good

inhibitory activities against the SARS-CoV 3CL

pro-tease, whereas the ketomethylene isosteres 7 were less

potent than the tripeptide counterpart 3. Surprisingly,

Niclosamide showed no inhibitory activity at a

concen-tration of 50 µM.

8

The IC

50

data for some anilide

inhibitors, either prepared in microtiter plate without

isolation or in pure form, are listed in Table 1. Anilide

2a (JMF1507) derived from 2-chloro-4-nitroaniline,

L

-phenylalanine, and 4-(dimethylamino)benzoic acid is the

most potent inhibitor, showing an IC

50

of 0.06 µM and

K

i

) 0.030 µM.

Lineweaver-Burk plots of kinetic data were fitted

with the computer program KinetAsyst II

(IntelliKinet-ics, PA) by nonlinear regression to obtain the K

i

values.

The double reciprocal plot of the initial rate vs substrate

concentration indicates that all these compounds are

competitive inhibitors. A representative example for

inhibition of the SARS-CoV 3CL protease by anilide 2a

is shown in Figure 1. It is worth to note that anilide 2a

functions as a potent inhibitor with K

i

) 0.03 µM, rather

than a substrate for the SARS-CoV 3CL protease. The

HPLC and absorption spectral analyses indicated that

no decomposition of anilide 2a occurred under the

enzymatic conditions for a period over 16 h (see the

Supporting Information). In hydrolysis of

N-nitrophen-ylamide, alignment of n,π-orbitals is required for the

facile leaving of nitroaniline. Due to the steric effect of

the chlorine atom at the ortho-position, the

2-chloro-4-nitrophenyl ring and amido group cannot be in a

coplanar conformation, thus making hydrolysis

unfavor-able. This speculation is in agreement with the

com-puter modeling shown in Figure 2.

To know the structure-activity relationship, a series

of the 2a analogues was synthesized and the inhibitory

activity was examined. None of the analogues 8a-f

showed adequate activity (IC

50

> 10 µM). Deletion of

Scheme 1. Synthesis of Ketomethylene Isosteres of Tripeptides

a

aReagents and conditions: (i) NaH, THF, 0 °C to room temperature, 24 h. (ii) CF

3CO2H, CH2Cl2, rt, 24 h. (iii) H2, Pd/C, Boc2O, MeOH, rt, 10 h. (iv) allyl iodide, Cs2CO3, DMF, 45 °C, 5 h. (v) HCl, 1,4-dioxane, rt, 2 h. (vi) N-methylmorpholine, CH2Cl2, 0-25 °C, 2 h. (vii) Pd(PPh3)4, morpholine, THF, 25 °C, 3 h. (viii) HOBt, EDCI, (i-Pr)2NEt, CH2Cl2, 0 °C to room temperature, 20 h.

Figure 1. Lineweaver-Burk plot for inhibition of the SARS-CoV 3CL protease by anilide 2a (Ki) 0.03 µM).

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the chloro, nitro, or dimethylamino substituents from

anilide 2a significantly deteriorated potency as did

replacing the dimethylamino group in 2a with a nitro

group.

The crystal structure of SARS-CoV 3CL protease in

complex with a specific inhibitor of hexapeptidyl

chlo-romethyl ketone, Cbz-Val-Asn-Ser-Thr-Leu-Gln-CH

2

Cl,

has been reported (coded 1uk4 in the Protein Data Bank

deposition).

5b

On this basis, the docking experiment

(Autodock version 3.0.5)

18

using p-nitroaniline as the

core structure formed three main clusters (RMSD ) 2

Å). The clusters with lowest binding free energy occupies

Table 1. IC50Values for Some 2-Chloro-4-nitroanilide Inhibitors against the SARS-CoV 3CL Protease

structure type compd R R′ IC50(µM) Ki(µM)a

niclosamide >50a anilide 1 t-BuO >50a 2a Me2NC6H4 0.06a 0.03 ( 0.001 2b C14H29CH(Br) 3b 2c 3,4-(NH2)2C6H3 2b 2d (indol-3-yl)-CHdCH 3b 2e (2-NH2-1,3-thiazol-4-yl)-C(dNOCH3) 7b

tripeptide anilide 3a i-Bu Et 7b

3c i-Bu Ph 4b 3d i-Bu t-BuO >10a 3f i-Bu morpholino 19b 3h i-Bu thien-2-yl 5a 2.29 ( 1.01 3o PhCH2 5-Me-isoxazol-3-yl 7a 2.90 ( 1.27 3p PhCH2 Thien-2-yl 5a 4.3 ( 1.9

tetrapeptide anilide 4a i-Bu Et 7b

4f i-Bu morpholino 16b 4g PhCH2 t-Bu 2b 4j PhCH2 5-Me-isoxazol-3-yl 5a 1.61 ( 1.03 4k PhCH2 PhCH2O 6a 1.51 ( 0.95 4q 4-FC6H4CH2 Et 5b 4s 4-FC6H4CH2 Ph 2b dimeric anilide 5a H H >50b 5b (S)-OH H 4a 3.1 ( 0.4 5c (R)-OH (R)-OH 5b 6a H H 2b 6b (S)-OH H 2b 6c (R)-OH (R)-OH 2b

ketomethylene anilide 7a i-Bu Et 27b

7c i-Bu Ph 21b 7d i-Bu t-BuO 19b 7f i-Bu morpholino 29b 7h i-Bu thien-2-yl 22b 7o PhCH2 5-Me-isoxazol-3-yl 6b 7p PhCH2 Thien-2-yl 16b

aThe sample is in pure form.bThe sample is synthesized in a microtiter plate and assayed in situ without isolation.

Figure 2. Computer modeling of compound 2a binding to SARS-CoV 3CL protease (1uk4). Compound 2a is colored in yellow. The van der Waals filling space is generated by 1.0 scale and colored according to atom type. Clusters of possible docking modes were sorted by computed binding free energy and the docking mode with lowest docking energy (-9.1 kcal/ mol in this case) is generated by MGLTOOLS.

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the pocket formed by Cys145, Ser144, His163, and

Phe140 or the pocket formed by Thr25, His41, Cys44,

Thr45, and Ala46. The 2-chloro-4-nitroanilide moiety of

2a was found to occupy the second favorite pocket

described above (Figure 2). The nitro group in 2a is

predicted to be hydrogen bonded with the HN of Ala46,

while the chlorine atom is within 3 Å from γ-S atom of

Cys145 and -N2 atom of His41, providing a possible

key interaction with the catalytic dyad. The

(dimethyl-amino)phenyl group is fitted to the cleft formed by

Gln189-Gln192 and Met165-Pro68. The P1 phenyl

residue in 2a is positioned in the S1 pocket, which may

be modified to increase the interactions with Phe140,

His163, and Glu166.

The docking study showed that anilide 2a has the

lowest binding free energy of -9.1 kcal/mol in

compari-son with the anilides 8a-e (∆G* ) -7.5 to -8.7 kcal/

mol, see the Supporting Information). This docking

experiment supports the observation in the enzymatic

assay, which reveals the important roles of the

2-chloro-4-nitroanilide and (dimethylamino)phenyl moieties in

inhibition of the SARS-CoV 3CL protease. The docking

model also shows that the P1 site of anilide 2a is distant

(

∼4.95 Å) from the nucleophilic SH of Cys145. This

model is in agreement with the observation that anilide

2a is stable in the SARS 3CL protease.

Under the assay conditions similar to that for the

SARS-CoV 3CL protease, the IC

50

values of anilide 2a

against trypsin, chymotrypsin, and papain were

mea-sured to be 110, 200, and 220 µM, respectively. In

comparison, anilide 2a is a potent inhibitor for the

SARS-CoV 3CL protease with an IC

50

value of 0.06 µM.

Experimental Section

Materials and Methods. SARS-CoV 3CL protease was prepared according to the previously described procedure.17

Reactions requiring dry conditions were carried out under an inert atmosphere using standard techniques. All the reagents and solvents were reagent grade and were used without further purification unless otherwise specified. THF was distilled from sodium benzophenone ketyl under N2.

Representative Procedure of Coupling Reactions. A solution of N-Boc phenylalanine (2.65 g, 10 mmol) and 2-chloro-4-nitroaniline (1.73 g, 10 mmol) in pyridine (30 mL) was cooled to -15 °C, and phosphorus oxychloride (1 mL, 11 mmol) was added dropwise with vigorous stirring. After the mixture was stirred for 1.5 h at -15 °C, the reaction was quenched by pouring into ice-water (100 mL). The mixture was extracted with EtOAc (1× 50 mL and 3 × 30 mL). The combined organic phase was washed with saturated NaHCO3(2× 50 mL) and

brine (30 mL). The organic phase was dried over MgSO4and

filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography with elution of EtOAc/hexane (5:95) to give N-Boc phenylalanine 2-chloro-4-nitroanilide (1, 3.25 g, 78%) as white solids.

A solution of N-Boc phenylalanine 2-chloro-4-nitroanilide (0.42 g, 1 mmol) in 1,4-dioxane (3 mL) was treated with a solution of HCl in 1,4-dioxane (4 M, 2 mL) at 25 °C. The mixture was stirred for 1.5 h and concentrated under reduced pressure to give a crude aminium chloride salt. The material was dissolved in CH2Cl2(5 mL), cooled to 0 °C, and treated

with 4-methylmorpholine (0.3 mL, 2.5 mmol) and 4-(dimethyl-amino)benzoyl chloride (0.22 g, 1.2 mmol) sequentially. The ice bath was removed, and the mixture was stirred for 2 h at 25 °C. The reaction was quenched by addition of brine (15 mL). The mixture was extracted with CH2Cl2 (2 × 20 mL). The

organic phase was dried over Na2SO4and filtered, and the

filtrate was concentrated under reduced pressure. The residue

was purified by flash column chromatography with elution of EtOAc/hexane (1:9) to provide N-[4-(dimethylamino)phenyl]-phenylalanine 2-chloro-4-nitroanilide (2a, 0.29 g, 61%) as light yellow solids.

4-(Dimethylamino)benzoyl-L -Phe-(2-chloro-4-nitro-anilide) (2a). Light yellow solid; mp 205-207 °C; λmax) 320

nm; TLC (EtOAc/hexane, 1:1) Rf) 0.5; IR (KBr) 3304, 2926, 1710, 1607, 1512, 1344, 1279, 1185 cm-1;1H NMR (CDCl 3, 400 MHz) δ 9.17 (1 H, s), 8.67 (1 H, d, J ) 9.2 Hz), 8.24 (1 H, d, J ) 2.4 Hz), 8.14 (1 H, dd, J ) 9.2, 2.4 Hz), 7.60 (2 H, d, J ) 8.9 Hz), 7.35-7.31 (3 H, m), 7.30-7.27 (2 H, m), 6.65 (2 H, d, J ) 8.9 Hz), 6.41 (1 H, d, J ) 7.1 Hz), 5.08-5.06 (1 H, m), 3.34 (2 H, d, J ) 7.1 Hz), 3.04 (6 H, s);13C NMR (CDCl 3, 100 MHz) δ 170.5 (C), 168.1 (C), 152.9 (C), 143.0 (C), 140.4 (C), 136.2 (CH), 129.3 (C), 129.0 (CH, 2× ), 128.7 (CH, 2 × ), 127.4 (CH, 2 × ), 124.7 (CH), 123.3 (CH), 122.9 (C), 120.4 (C), 119.2 (CH), 111.0 (CH, 2× ), 55.6 (CH), 40.0 (CH3, 2× ), 36.7 (CH2); FAB MS m/z 467.1 (M+ + H); HRMS calcd for C24H24ClN4O4:

467.1486 (M++ H); found: 467.1488; Anal. Calcd for C 24H23

-ClN4O4: C 61.74, H 4.97, N 12.00; found: C 61.71, H 5.01, N

11.96.

Inhibition Assay against the SARS-CoV 3CL Protease. A fluorometric assay17was utilized to determine the inhibition

constants of the prepared samples. Briefly, a fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edans is used as the substrate, and the enhanced fluorescence due to cleavage of this substrate catalyzed by the protease was monitored at 538 nm with excitation at 355 nm. The IC50value of individual

sample was measured in a reaction mixture containing 50 nM of the SARS 3CL protease and 6 µM of the fluorogenic substrate in 20 mM Bis-Tris (pH 7.0). The enzyme stock solution was kept in 12 mM Tris-HCl (pH 7.5) containing 120 mM NaCl, 0.1 mM EDTA, and 1 mM DTT plus 7.5 mM β-ME before adding to the assay solution. The Kimeasurements were

performed at two fixed inhibitor concentrations and various substrate concentrations.

Acknowledgment. We thank National Science

Coun-cil (Taiwan) for financial support, and Shih-Jia Shiao

and Su-Lung Tang for preparation of some anilide

inhibitors.

Supporting Information Available: Physical and spec-troscopic properties of new compounds, inhibition assay, HPLC and UV-vis analyses, molecular modeling, and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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數據

Figure 1. Lineweaver-Burk plot for inhibition of the SARS- SARS-CoV 3CL protease by anilide 2a (K i ) 0.03 µM).
Table 1. IC 50 Values for Some 2-Chloro-4-nitroanilide Inhibitors against the SARS-CoV 3CL Protease

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