Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors

Article abstract of DOI:10.1016/S0960-894X(99)00587-9

Tripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the cysteine- containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibition activ

Full text of DOI:10.1016/S0960-894X(99)00587-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 45±48
Structure-Based Design of Ketone-Containing, Tripeptidyl Human
Rhinovirus 3C Protease Inhibitors
Peter S. Dragovich,* Ru Zhou, Stephen E. Webber, Thomas J. Prins, Annette K. Kwok,
Koji Okano,y Shella A. Fuhrman, Leora S. Zalman, Fausto C. Maldonado,
Edward L. Brown, James W. Meador, III, Amy K. Patick, Cli€ord E. Ford,
Mary A. Brothers, Susan L. Binford, David A. Matthews, Rose Ann Ferre
and Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, CA 92121, USA
Received 25 August 1999; accepted 14 October 1999
AbstractÐTripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the
cysteine-containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibi-
tion activity (Ki=0.0045 mM) and in vitro antiviral properties (EC50=0.34 mM) when tested against HRV serotype-14. # 1999
Elsevier Science Ltd. All rights reserved.
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most signi®cant
cause of the common cold.1,2 The replication of these
viruses requires the proteolytic processing of a large
polyprotein produced by cellular translation of the viral
RNA genome. This processing is primarily accom-
plished by the human rhinovirus 3C protease (3CP),3,4 a
cysteine protease possessing minimal homology with
prevalent mammalian enzymes but which exhibits
structural similarity to the trypsin protein family.5 As a
critical component of the rhinovirus replication cycle,
the 3C protease is a potential target for the development
of novel antirhinoviral agents. Several examples of
reversible and irreversible 3CP inhibitors have recently
appeared in the literature, and some of these entities
display in vitro antiviral properties.6 Substrate-
derived3,4,7 peptide aldehydes, typi®ed by compound 18
(Ki=0.006 mM, EC50=2.4 mM), were among the ®rst
reversible 3CP inhibitors so described.8±12 These mole-
cules form covalent hemithioacetal adducts with the
active site 3CP cysteine residue8 in a manner analogous
to the interaction of related peptide aldehydes with
various serine proteases.13 However, concern over the
possible biological liabilities associated with the alde-
hyde functional group (e.g. toxicity, selectivity and sta-
bility) prompted us to identify reversible 3C protease
inhibitors which contained alternate electrophiles. In
this report, we describe the exploration of ketone-
containing molecules, some of which display potent 3CP
inhibition activity and antirhinoviral properties.
Replacement of the aldehyde group present in 1 with a
2-benzothiazole ketone moiety a€orded an active, albeit
signi®cantly less potent, reversible 3CP inhibitor 2
(Table 1). The combination of related ketone electro-
philes with appropriate peptidyl entities has also pro-
vided reversible inhibitors of several other cysteine14±16
and serine17±22 proteases. Unfortunately, compound 2
did not display in vitro antirhinoviral activity when tes-
ted to concentrations at which cytotoxicity (CC50) was
observed. However, replacement of the P1 glutamine
isostere of 2 with the natural Gln amino acid side-chain
a€orded a compound 3, which exhibited reduced 3CP
inhibition activity but improved antiviral properties with
less observed cytotoxicity. Importantly, reduction of the
ketone electrophile contained in 3 to the corresponding
alcohol 4 resulted in considerable loss in anti-3CP
activity. This result was consistent with the formation of
*Corresponding author. Tel.: +1-858-622-7918; fax: +1-858-622-
7918; e-mail: psd@agouron.com
yPermanent address: Central Pharmaceutical Research Institute, Japan
Tobacco, 1-1 Murasaki-cho, Takasuki, Osaka 569-11, Japan.
a covalent hemithioketal inhibitor-enzyme adduct
through addition of the 3CP active site cysteine residue
to the ketone moiety of 3. Introduction of a P1-lactam
0960-894X/00/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00587-9

46
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 10 (2000) 45±48
glutamine replacement into the inhibitor design (com-
pound 5) greatly improved 3CP inhibition activity
relative to 3 in a manner anticipated from studies of
related Michael acceptor-containing 3CP inhibitors.23,24
Curiously, compound 5 displayed only moderately
improved in vitro antirhinoviral properties relative to 3.
As was observed for compound 3 above, reduction of 5
to the corresponding alcohol 6 resulted in signi®cant
loss of anti-3CP activity and supported the formation of
a 3CP-5 hemithioketal adduct.
potency (compare 7 with 5, Table 2). In contrast, repla-
cement of the 2-benzothiazole moiety present in 5 with a
2-pyridine only slightly diminished 3CP inhibition
properties (compound 8). However, a molecule con-
taining
a 2-benzothiophene electrophile displayed
drastically reduced anti-3CP activity and showed no
measurable antiviral properties when tested to the 10
mM level (compare 9 with 5). This result paralleled those
obtained with related serine protease inhibitors and was
consistent with the formation of a hydrogen bond
between the benzothiazole nitrogen atom and a proto-
nated His residue in the 3CP active site.20,22 Subsequent
analysis of the 3CP-5 X-ray crystal structure con®rmed
such an interaction as well as the formation of an inhi-
bitor enzyme hemithioketal adduct.26 A similar reduc-
tion in anti-3CP activity was exhibited by a compound
which incorporated a phenyl ketone electrophile in
lieu of a 2-pyridyl ketone moiety (compare 10 with 8,
Table 2).
Having determined that tripeptidyl molecules contain-
ing P1-lactam and C-terminal 2-benzothiazole ketone
moieties could function as potent, reversible 3CP inhi-
bitors, we then examined modi®cation of the ketone
electrophile. Incorporation of a 2-thiazole into the inhi-
bitor design resulted in signi®cant loss of anti-3CP
activity and a somewhat lesser reduction in antiviral
Table 1. 25
Table 2. 25
Compd
R1
R2 R3 Ki (mM)a EC50 (mM)a CC50 (mM)
2
1.7
>25
25
>100
80
Compd
R
Ki (mM)a
EC50 (mM)a
CC50 (mM)
5
0.065
3.2
>320
3
3.5
17
7
8
0.70
0.17
7.9
4.0
240
200
4b
OH
H
NIc
32
5
0.065
3.2
>320
9
4.7
3.2
>10
>10
>10
>10
6b
OH
H
34
7.9
63
10
aSerotype-14.
b1:1 mixture of diastereomers.
cNI=no inhibition to 10 mM.
aSerotype-14.

P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 10 (2000) 45±48
47
The structure±activity results illustrated in Table 2
identi®ed the 2-benzothiazole as an ideal fragment for
incorporation into C-terminal ketone-containing 3CP
inhibitors. Accordingly, this moiety was combined with
a tripeptidyl binding element that was previously shown
to impart high levels of anti-3CP activity to related
Michael acceptor-containing molecules.23 The resulting
compound 11 displayed very potent levels of reversible
3CP inhibition along with sub-micromolar antiviral
activity against several rhinovirus serotypes. In addi-
tion, the molecule did not exhibit signi®cant cytotoxicity
when tested in cell culture. Compound 11 therefore,
ranks as one of the most potent reversible 3CP inhibitors
reported to date and its antirhinoviral activity compares
quite favorably with that displayed by related peptide
aldehydes.8±11
As illustrated in Schemes 1 and 2, the 3CP inhibitors
described in this work were prepared by methods rela-
ted to those utilized previously to synthesize tripeptidyl
Michael acceptor-containing compounds.23,27 In gen-
eral, an appropriate organolithium species was con-
densed with an amino acid or peptide-derived Weinreb
amide28 entity to form the desired ketone electrophiles.
For the preparation of compound 11, the initially
formed ketone moiety (21, Ar=2-benzothiazole) was
reduced to the corresponding alcohol 23 prior to pep-
tide coupling in order to minimize racemization of the
ꢀ
ꢀ
.
Scheme 1. Reagents and conditions (Tr=CPh3): (a) EDC, HCl HN(CH3)OCH3, NMM, CH2Cl, 23 C, 18 h, 78%; (b) H2, Pd on C, CH3OH, 23 C,
2 h; (c) Cbz-l-Leu-l-Phe-OH, N-hydroxysuccinimide, DCC, CH2Cl2, 23 ꢀC, 12 h, 89%; (d) 10 equiv 2-lithio-benziothiazole, THF, 78 to 23 ꢀC,
18 h, 21%; (e) 10 equiv 2-lithio-benzothiazole, THF, 78 ꢀC, 2 h, 35%; (f) HCl, 1,4-dioxane, 23 ꢀC, 3 h; (g) Cbz-l-Leu-l-Phe-OH, HATU, DIEA,
DMF, 0 ꢀC, 45 min, 74%; (h) TFA, CH2Cl2, 23 ꢀC, 49%.
Scheme 2. Reagents and conditions (Ar=see Table 2, DMB=2,4-dimethoxybenzyl): (a) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH, 23 ꢀC, 18 h,
66%; (b) isobutyl chloroformate, NMM, HCl HN(CH3)OCH3, CH2Cl2, 20 to 23 ꢀC, 2 h, 71%; (c) 3±10 equiv ArLi, THF, 78 ꢀC, 2±4 h, 20±80%;
.
(d) HCl, 1,4-dioxane, 23 ꢀC, 3 h; (e) Cbz-l-Leu-l-Phe-OH, HATU, DIEA, DMF, 0 ꢀC, 45 min, 37%; (f) DDQ, CH3CN, 60 ꢀC, 5±8 h, 50±80%; (g)
NaBH4, EtOH, 0 ꢀC, 68%; (h) Boc-l-Val-l-Phe(4-F)-OH, HATU, NMM, CH3CN, 0 ꢀC, 45 min, 94%; (i) 5-methylisoxazole-3-carbonyl chloride,
2,4,6-collidine, CH2Cl2, 0 ꢀC, 30 min, 65%; (j) Dess±Martin peroiodinane, CH2Cl2, 23 ꢀC, 2 h, 94%.

48
P. S. Dragovich et al. / Bioorg. Med. Chem. Lett. 10 (2000) 45±48
P1 side-chain.29 The alcohol functionality was later oxi-
dized to a€ord the desired product 11.30
M.; Oh, H.-J.; Erhard, K. F.; Desjarlais, R. L.; Head, M. S.;
Smith, W. W.; Zhao, B.; Janson, C. A.; Abdel-Maguid, S. S.;
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1998, 41, 3563.
15. Tao, M.; Bihovsky, R.; Kauer, J. C. Bioorg. Med. Chem.
Lett. 1996, 6, 3009.
Acknowledgements
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We thank Dr. Kim Albizati, Dr. Srinivasan Babu, Dr.
Naresh Nayyar and Terence Moran for the large-scale
preparation of several synthetic intermediates.
References and Notes
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23. Dragovich, P. S.; Prins, T. J.; Zhou, R.; Webber, S. E.;
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24. Note that the P1-lactam moiety contained in 5 may also
attenuate intramolecular hemiaminoketal formation relative
to 3. The extent to which such hemiaminoketal formation (if
any) a€ects 3CP inhibition is currently not known.
25. Enzyme and antiviral assays were performed as described
in Webber, S. E.; Tikhe J.; Worland, S. T.; Fuhrman, S. A.;
Hendrickson, T. F.; Matthews, D. A.; Love, R. A.; Patick, A.
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26. Matthews, D. A., unpublished results.
27. Intermediate 12 was prepared as described in ref 8. Inter-
mediate 15 was prepared as described in Dragovich, P. S.;
Webber, S. E.; Babine, R. E.; Fuhrman, S. E.; Patick, A. K.;
Matthews, D. A.; Lee, C. A.; Reich, S. H.; Prins, T. J.; Mar-
akovits, J. T.; Little®eld, 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. Intermediate 18 was pre-
pared as described in ref 23.
5. Matthews, D. A.; Smith, W. W.; Ferre, R. A.; Condon, B.;
Budahazi, G.; Sisson, W.; Villafranca, J. E.; Janson, C. A.;
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7. The nomenclature used for describing the individual amino
0
0
acid residues of a peptide substrate (P2, P1, P1 ,0 P2 ,0etc.) and
the corresponding enzyme subsites (S2, S1, S1 , S2 , etc.) is
described in: Schechter, I.; Berger, A. Biochem. Biophys. Res.
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12. Note that the most potent peptide aldehyde 3CP inhibitors
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molecular hemiaminal formation. See refs 8±11 for additional
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1
29. Signi®cant racemization (>10%) was not detected by H
NMR during the preparation of other tripeptidyl ketones.
30. In several instances, the DMB group was removed from
intermediate 21 prior to conversion to ®nal compounds 5±10.
Either synthetic sequence a€orded the desired products in
acceptable overall yields.