Design and synthesis of irreversible depsipeptidyl human rhinovirus 3C protease inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00542-X

Novel tripeptidyl C-terminal Michael acceptors with an ester replacement of the P₂-P₃ amide bond were investigated as irreversible inhibitors of the human rhinovirus (HRV) 3C protease (3CP). When screened against HRV serotype-14 the best compound was shown to have very good 3CP inhibition (k_obs/[I]=270,000 M^-1 s^-1) and potent in vitro antiviral activity (EC₅₀=7.0 nM).

Full text of DOI:10.1016/S0960-894X(01)00542-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2683–2686
Design and Synthesis of Irreversible Depsipeptidyl Human
Rhinovirus 3C Protease Inhibitors
Stephen E. Webber,* Joseph T. Marakovits, Peter S. Dragovich, Thomas J. Prins,
Ru Zhou, Shella A. Fuhrman, Amy K. Patick, David A. Matthews, Caroline A. Lee,
Babu Srinivasan, Terry Moran, Clifford E. Ford, Mary A. Brothers, James E. V. Harr,
James W. Meador, III, Rose Ann Ferre and Stephen T. Worland
Pfizer Global Research and Development, La Jolla, 3565 General Atomics Court, San Diego, CA 92121, USA
Received 18 June 2001; accepted 30 July 2001
Abstract—Novel tripeptidyl C-terminal Michael acceptors with an ester replacement of the P2–P3 amide bond were investigated as
irreversible inhibitors of the human rhinovirus (HRV) 3C protease (3CP). When screened against HRV serotype-14 the best com-
pound was shown to have very good 3CP inhibition (kobs/[I]=270,000 MÀ1 sÀ1) and potent in vitro antiviral activity
(EC50=7.0 nM). # 2001 Elsevier Science Ltd. All rights reserved.
Human rhinoviruses (HRVs),1 the primary cause of the
common cold, are small single stranded RNAviruses
belonging to the picornaviridae family.2 Proteins essen-
tial for HRV replication are generated by the proteo-
lysis of a large polyprotein. The virally encoded HRV
3C protease (3CP)3 is predominantly responsible for this
operation. 3CP is a cysteine protease that lacks
sequence homology to common host enzymes and
structurally resembles trypsin-like serine proteases.4,5
Inhibition of this enzyme has recently become the topic
of interesting research directed toward the development
of an antirhinoviral therapy.6 In particular, significant
attention has been devoted to a class of irreversible
inhibitors based on the peptide substrate binding deter-
minants incorporating C-terminal Michael acceptors
that are reactive with the 3CP catalytic cysteine.7 Recent
work describes structural modifications to this class of
molecules to remove their inherent peptidic character-
istics while improving physical properties without
adversely affecting activity.8À10 Inhibitory activity and/
or physical properties of several of these molecules were
improved by N-methylation of the P2–P3 amide linkage
or isosteric replacement of this amide with a keto-
methylene group.8,9 Close examination of the X-ray
crystal structures of irreversibly bound peptidic inhibi-
tors to HRV-2 3CP, specifically at the P2 site, reveals a
hydrogen bond between the P2-phenylalanine N–H and
the hydroxyl of residue Ser-128.7a,9 The hydroxymethyl
side chain of Ser-128 partially defines the S2 subsite and
is also exposed to solvent. The mobility of this side
chain allows for the design of P2–P3 amide modifica-
tions. Replacement of this linkage with an ester was
logical since modeling suggested the formation of a
hydrogen bond to Ser-128 with reversed ordering com-
pared to an amide. As illustrated in Figure 1, the serine
hydroxyl would now serve as the proton donor while
the alkoxy oxygen the acceptor.
In order to test this hypothesis, and determine whether
depsipeptides possessing a C-terminal Michael acceptor
are useful as irreversible inhibitors of HRV 3CP, we
have prepared and evaluated some representative
examples. Three depsipeptidyl irreversible inhibitors of
HRV 3CP were prepared. This work was guided by our
*Corresponding author. Present address: Anadys Pharmaceuticals
Inc., 9050 Camino Santa Fe, San Diego, CA92121, USA. Fax: +1-
858-527-1540; e-mail: swebber@anadyspharma.com
Figure 1. Observed H-bond between P2–P3 amide and Ser-128 versus
postulated P2–P3 ester.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00542-X

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S. E. Webber et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2683–2686
initial protein-structure-based design and structure–
activity relationship (SAR) optimization of substrate-
derived tripeptides with incorporated C-terminal a,b-
unsaturated ethyl esters.7a,b These new P2–P3 ester deri-
vatives, compounds 1, 4, and 7, are directly compared
to their corresponding amide and ketomethylene analo-
gues as shown in Tables 1–3. The Michael acceptor, P2-
4-fluorophenylalanine and P3-valine groups are com-
mon structural features in the molecules.7a,b Derivatives
7–9 include the optimal P1-(S)-g-lactam and N-terminal
methylisoxazole carboxamide identified previously.10
14 3CP activity11 except in the case of 1 versus 2.
Excluding the data from compound 112 a possible
explanation for these results was made after inspection
of the HRV-2 3CP cocrystal structure of 4. As illu-
strated in Figure 2, the distance between Ser-128 and
the P2–P3 ester is 3.6 A indicating that our originally
hypothesized H-bond is not optimal in this case.
Again, with the exception of compound 1,12 depsipep-
tide inhibitors 4 and 7 displayed improved or compar-
able antiviral activity11 relative to the corresponding
peptides 5 and 8 and ketomethylene analogues 6 and 9.
These results imply that inhibitors 4 and 7 have membrane
Compared to their amide and ketomethylene counter-
parts, depsipeptides 1, 4, and 7 displayed reduced HRV-
Table 1.
Compd
X
kobs/[I]
(MÀ1 sÀ1 a
EC50
(mM)b
CC50
(mM)b
)
1
2
3
O
NH
CH2
99,800
59,900
293,000
1.0
0.19
0.02
>10
>100
>100
Figure 2. View of the P2 and S2 regions of the 1.9 A cocrystal structure
of compound 4 covalently bound to HRV-2 3CP. The distance
between Ser-128 and the P2 ester oxygen is 3.6 A. The isopropyl side
chain of the P3 Val is clipped out of view.
Table 2.
Compd
X
kobs/[I]
(MÀ1 sÀ1 a
EC50
(mM)b
CC50
(mM)b
)
4
5
6
O
NH
CH2
68,400
248,000
240,000
0.1
0.42
0.10
>10
>100
>100
Table 3.
Scheme 1. Reagents and conditions: (a) NaNO2, 1 N H2SO4, 79%; (b)
K2CO3, CH3I, acetone, 66%; (c) Ti(OiPr)4, allyl alcohol, 90 ꢀC, 68%;
(d) BOC-Val, DCC, DMAP, CH2Cl2, 90%; (e) Pd(PPh3)4, morpho-
line/THF, 86%; (f) HATU, (iPr)2NEt, DMF, 39%; (g) 1.3 M HCl/1,4-
dioxane, 83%; (h) cyclopentyl chlorothioformate, NEt3, CH2Cl2,
59%; (i) TFA, CH2Cl2, 60–63%; (j) 5-methylisoxazole-3-carbonyl
chloride, NEt3, CH2Cl2, 45%.
Compd
X
kobs/[I]
(MÀ1 sÀ1 a
EC50
(mM)b
CC50
(mM)b
)
7
8
9
O
NH
CH2
270,000
1,500,000
1,090,000
0.007
0.01
0.005
>100
>100
>100

S. E. Webber et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2683–2686
2685
the P2–P3 amide or ketomethylene unit of optimally
modified tripeptides with C-terminal Michael acceptors.
Depsipetide inhibitors may not be ideal therapeutic
candidates due to their lack of in vitro stability.
References and Notes
1. Couch, R. B. In Fields Virology; 3rd ed.; Field, B. N.,
Knipe, D. M., Howley, P. M., Eds.; Lippencott-Raven: Phila-
delphia, 1996; Vol. 1, Chapter 23, p 713.
2. Krausslich, H.-G.; Wimmer, E. Annu. Rev. Biochem. 1988,
57, 701.
3. (a) Orr, D. C.; Long, A. C.; Kay, J.; Dunn, B. M.;
Cameron, J. M. J. Gen. Virol. 1989, 70, 2931. (b) Cordingly,
M. G.; Register, R. B.; Callahan, P. L.; Garsky, V. M.;
Colonno, R. J. J. Virol. 1989, 63, 5037.
4. (a) Werner, G.; Rosenwirth, B.; Bauer, E.; Siefert, J.-M.;
Werner, F.-J.; Besemer, J. J. Virol. 1986, 57, 1084. (b)
Aschauer, B.; Werner, G.; McCray, J.; Rosenwirth, B.; Bach-
mayer, H. Virology 1991, 184, 587. (c) Skern, T.; Sommer-
gruber, W.; Blaas, D.; Fraundorfer, F.; Pieler, C.; Fogy, I.;
Kuechler, E. Nucleic Acids Res. 1985, 13, 2111.
5. Matthews, D. A.; Smith, W. W.; Ferre, R. A.; Condon, B.;
Budahazi, G.; Sisson, W.; Villafranca, J. E.; Janson, C. A.;
McElroy, H. E.; Gribskov, C. L.; Worland, S. Cell 1994, 77,
761.
6. Matthews, D. A.; Dragovich, P. S.; Webber, S. E.; Fuhr-
man, S. A.; Patick, A. K.; Zalman, L. S.; Hendrickson, T. F.;
Love, R. A.; Prins, T. J.; Marakovits, J. T.; Zhou, R.; Tikhe,
J.; Ford, C. E.; Meador, J. W.; Ferre, R. A.; Brown, E. L.;
Binford, S. L.; Brothers, M. A.; DeLisle, D. M.; Worland,
S. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11000.
Scheme 2. Reagents and conditions: (DMB=2,4-dimethoxybenzyl)
(a) 1.3 M HCl/1,4-dioxane, satd K2CO3, 99%; (b) 5-methylisoxazole-
3-carbonyl chloride, pyr, CH2Cl2, 82%; (c) Pd(PPh3)4, morpholine/
THF, 95%; (d) HATU, (iPr)2NEt, DMF, 72%; (e) DDQ, CHCl3,
H2O, 50 ꢀC, 90%.
Table 4.
% Metabolism
Compd
ÀNADPH
+ NADPH
7
8
9
44
7
27
67
22
50
Incubation time=30 min; 25 mM compound; 1.0 mg/mL human liver
microsomes; 2.0 mM NADPH.
permeabilities similar to 6 and 9, respectively, and are
desolvated more readily than 5 and 8.9
7. (a) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhr-
man, S. A.; Patick, A. K.; Matthews, D. A.; Lee, C. A.; Reich,
S. H.; Prins, T. J.; Marakovits, J. T.; Littlefield, E. S.; Zhou,
R.; Tikhe, J.; Ford, C. E.; Wallace, M. B.; Meador, J. W., III;
Ferre, R. A.; Brown, E. L.; Binford, S. L.; Harr, J. E. V.;
DeLisle, D. M.; Worland, S. T. J. Med. Chem. 1998, 41, 2806.
(b) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman,
S. A.; Patick, A. K.; Matthews, D. A.; Reich, S. H.; Mar-
akovits, J. T.; Prins, T. J.; Zhou, R.; Tikhe, J.; Littlefield, E. S.;
Bleckman, T. M.; Wallace, M. B.; Little, T. L.; Ford, C. E.;
Meador, J. W., III; Ferre, R. A.; Brown, E. L.; Binford, S. L.;
DeLisle, D. M.; Worland, S. T. J. Med. Chem. 1998, 41, 2819.
(c) Kong, J.S.; Venkatraman, S.; Furness, K.; Nimkar, S.;
The HRV 3CP depsipeptide inhibitors 1, 4, and 7 were
synthesized from common intermediate 10, as outlined
in Schemes 1 and 2.13 Starting with l-4-F-Phe, (S)-
methyl-2-OH-3-(4-F-phenyl)propionate was prepared
according to the procedure of Hoffmann and Kim.14
The methyl ester was transformed to the more versatile
allyl ester without any loss of enantiomeric purity.
Intermediate 13 was generated via amide formation
with carboxylic acid 11 and the P1 amino-ester 127a
using HATU.15 Removal of the BOC group, followed
by N-terminus modification and deprotection of the S1
amide readily afforded products 1 and 4. In a similar
fashion inhibitor 7 was synthesized from carboxylic
acid 14 and the P1 amino-ester 15.10 The synthesis of
the peptidyl and ketomethylene 3CP inhibitors used
for comparative purposes were described pre-
viously.7a,7b,9,10,13
´
Shepherd, T. A.; Wang, Q. M.; Aube, J.; Hanzlik, R. P. J.
Med. Chem. 1998, 41, 2579.
8. Dragovich, P. S.; Webber, S. E.; Prins, T. J.; Zhou, R.;
Marakovits, J. T.; Tikhe, J. G.; Fuhrman, S. A.; Patick, A. K.;
Matthews, D. A.; Ford, C. E.; Brown, E. L.; Binford, S. L.;
Meador, J. W., III; Ferre, R. A.; Worland, S. T. Bioorg. Med.
Chem. Lett. 1999, 9, 2189.
9. Dragovich, P. S.; Prins, T. J.; Zhou, R.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Ford, C. E.; Meador, J. W.,
III; Ferre, R. A.; Worland, S. T. J. Med. Chem. 1999, 42,
1203.
10. Dragovich, P. S.; Prins, T. J.; Zhou, R.; Webber, S. E.;
Marakovits, J. T.; Fuhrman, S. A.; Patick, A. K.; Matthews,
D. A.; Lee, C. A.; Ford, C. E.; Burke, B. J.; Rejto, P. A.;
Hendrickson, T. F.; Tuntland, T.; Brown, E. L.; Meador,
J. W., III; Ferre, R. A.; Harr, J. E. V.; Kosa, M. B.; Worland,
S. T. J. Med. Chem. 1999, 42, 1213.
One possible drawback of depsipetide inhibitors may be
their potential to behave as better substrates for pro-
teolytic hydrolysis relative to their corresponding pep-
tides.16 Other than esterases, another concern is their
possible susceptibility toward hepatic metabolism. As
shown in Table 4, depsipeptide 7 was the least stable of
the three analogues when exposed to human liver micro-
somes either in the presence or absence of cofactor.17
11. Enzymatic and antiviral assays were performed as descri-
bed in ref 7a.
12. When comparing the inhibition data for depsipeptides 1,
4, and 7 directly to that of peptides 2, 5, and 8, it is unclear
why compound 1 is more potent than 2 as an HRV-14 3CP
In conclusion, we have prepared potent irreversible
HRV 3CP inhibitors where an ester is substituted for

2686
S. E. Webber et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2683–2686
inhibitor but a poorer antiviral agent against HRV-14 in cell
culture.
13. (a) Dragovich, P. S.; Marakovits, J. T.; Prins, T. J.; Tikhe,
J. G.; Webber, S. E.; Zhou, R.; Johnson, T. O. Patent Appl.
17. Metabolism was determined as follows: 25 mM of com-
pound was incubated with 1.0 mg/mL HLMs with or without
20 mM NADPH for 30 min. A 200 mL aliquot of the incuba-
tion mixture was then precipitated with 2.0 mL of CH3CN.
Separation was performed using an Agilent 110 HPLC with a
MetaChem Metasil Basic reverse phase column (5 m,
4.6Â150 mm) at rt. Sample volume=100 mL. Gradient solvent
system: CH3CN/CH3OH/0.01% TFA, pH 3.5 adjusted with
NEt3 from 5:20:75 to 60:20:20 over 20 min and held for 10 min
at a flow rate=1.0 mL/min. The compounds were monitored
by UV; l=230 nm. % Metabolism=100À(peak area at
30 min/peak area at time 0)Â100.
WO 99/57135, 1999. (b)
All new compounds described
herein have been fully characterized using 1H NMR, IR,
HRMS and/or elemental analysis.
14. Hoffman, R. V.; Kim, H.-O. Tetrahedron 1992, 48, 3007.
15. Carpino, L. A.; El-Faham, A. J. Org. Chem. 1995, 60,
3561.
16. For example, see: Holmquist, B.; Vallee, B. L. Biochem.
1976, 15, 101.