Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure- activity studies

Article abstract of DOI:10.1021/jm9800696

The structure-based design, chemical synthesis, and biological evaluation of various peptidederived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (k(obs)/[I]) ranging from 60 to 280 000 M^-1 s^-1 and antiviral EC₅₀'s which approach 0.15 μM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (k(obs)/[I] = 800 000 M^-1 s^-1) and potent antiviral activity against HRV-14 in cell culture (EC₅₀ = 0.056 μM). A 1.9 A? crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.

Full text of DOI:10.1021/jm9800696

J . Med. Chem. 1998, 41, 2819-2834
2819
Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion of Ir r ever sible
Hu m a n Rh in ovir u s 3C P r otea se In h ibitor s. 2. P ep tid e Str u ctu r e-Activity
Stu d ies
Peter S. Dragovich,* Stephen E. Webber, Robert E. Babine, Shella A. Fuhrman, Amy K. Patick,
David A. Matthews, Siegfried H. Reich, J oseph T. Marakovits, Thomas J . Prins, Ru Zhou, J ayashree Tikhe,
Ethel S. Littlefield, Ted M. Bleckman, Michael B. Wallace, Thomas L. Little, Clifford E. Ford,
J ames W. Meador, III, Rose Ann Ferre, Edward L. Brown, Susan L. Binford, Dorothy M. DeLisle, and
Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121
Received J anuary 29, 1998
The structure-based design, chemical synthesis, and biological evaluation of various peptide-
derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds
are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding
determinant. The systematic modification of each amino acid residue present in the binding
determinant as well as the N-terminal functionality is described. Such modifications are shown
to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from
60 to 280 000 M-1 s-1 and antiviral EC50’s which approach 0.15 µM. An optimized inhibitor
which incorporates several improvements identified by the structure-activity studies is also
described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] )
800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 ) 0.056
µM). A 1.9 Å crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with
HRV-2 3CP is also detailed.
In tr od u ction
The preceding paper describes the discovery of ir-
reversible inhibitors of human rhinovirus (HRV) 3C
proteases (3CPs) which display in vitro antiviral activity
against several rhinovirus serotypes.1 These inhibitors
are comprised of a substrate-derived tripeptide binding
determinant which provides affinity for the target
proteases and a Michael acceptor moiety which forms
a covalent adduct with the active site cysteine residue
of the 3C enzymes (Figure 1).2,3 Efforts to optimize the
Michael acceptor portion of these molecules are detailed
in the preceding paper.1 In an attempt to further
improve and define this class of 3CP inhibitors, modi-
fication of the peptidyl binding determinant was also
undertaken. The results of this study are described
below.
Str u ctu r e-Activity Stu d ies
Exploration of peptidyl structure-activity relation-
ships began by truncation of the previously studied1
tripeptide inhibitor 3 (Table 1). A molecule derived from
a single amino acid (1) displayed poor 3CP inhibition
properties and no measurable antiviral activity (EC50).4
Similarly, a dipeptide inhibitor (2) exhibited signifi-
cantly reduced anti-3CP and antiviral properties rela-
tive to the tripeptide compound 3. These data indicated
that peptidyl inhibitors composed of at least three amino
acids were required for effective recognition of the target
protease, and subsequent structure-activity studies
were therefore conducted with tripeptide-derived mol-
ecules. A systematic approach was utilized in which one
amino acid component of 3 was modified while the other
two remained unchanged. In addition, alteration of the
F igu r e 1. Design of irreversible HRV 3CP inhibitors. EWG
) electron-withdrawing group.
N-terminal Cbz moiety present in 3 was examined upon
completion of the amino acid variations. Due to its ease
of preparation, the ethyl propenoate Michael acceptor
was employed extensively for such structure-activity
studies, although it was anticipated that this moiety
might undergo in vivo metabolism.
Tripeptidyl structure-activity studies commenced
with modification of the P1 glutamine residue present
in the irreversible 3CP inhibitor 3 (Table 2).3 Since
crystallographic analysis of the HRV-2 3CP-3 complex1
indicated that the glutamine amide formed several
hydrogen bonds with the protein, it was expected that
S0022-2623(98)00069-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/30/1998

2820 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Ta ble 1. Truncated 3C Protease Inhibitors
Dragovich et al.
c,d
compd no.
R
prepa
formulab
kobs/[I](M-1 s-1
)
EC50 (µM)c,d
CC50 (µM)d
1
2
3
Cbz
Cbz-L-Phe
Cbz-L-Leu-L-Phe
A
A
A
C17H22N2O5
C26H31N3O6
C32H42N4O7
4.5
400
25 000
>100
5.6
>100
>100
>320
0.54
a
b
Method of preparation: see Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14. See ref 1 for assay method and error.
d
Ta ble 2. Substitutions of the P1 Side Chain (Tr ) CPh3)
c,d
compd no.
R1
prepa
formulab
kobs/[I] (M-1 s-1
)
EC50 (µM)c,d
CC50 (µM)d
3
4
5
6
7
CH2CH2C(O)NH2
CH2CH2C(O)NHTr
CH2CH2C(O)NH(CH3)
CH2CH2C(O)N(CH3)2
CH2CH2CO2H
A
A
A
A
A
A
A
B
B
B
A
A
A
C32H42N4O7
C51H56N4O7
C33H44N4O7
C34H46N4O7
C32H41N3O8
C33H43N3O7
C33H45N3O7
C32H43N3O7S
C32H43N3O8S
C32H42N4O7
C31H41N5O7
C31H40N4O8
C30H39N3O7
25 000
0
0.54
100
5.6
4.0
14
>320
>320
>100
>100
>100
>100
>100
>100
>100
>320
>100
>320
190
750
60
500
1 400
0
2 200
60
800
3 500
5 600
0
8
9
CH2CH2C(O)CH3
1.6
e
CH2CH2CH(OH)CH3
>100
e
10
11
12
13
14
15
CH2CH2S(O)CH3
1.6
CH2CH2SO2CH3
CH2NHC(O)CH3
CH2NHC(O)NH2
CH2OC(O)NH2
CH2OH
>100
2.2
32
1.6
>190
a
b
Method of preparation: see Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14. See ref 1 for assay method and error. e 1:1 Mixture of diastereomers.
d
alteration of this moiety would result in reduced 3CP
inhibition. In the event, alkyl substitution of the
primary amide nitrogen led to significant or complete
loss of anti-3CP properties (4-6). Similar results were
obtained by replacement of the glutamine amide with
a variety of isosteres (7-12). The low activity displayed
by compounds 4, 6, 11, and 12 was somewhat surprising
since related peptide aldehydes are reported to exhibit
either good 3CP inhibition levels or measurable anti-
rhinoviral properties.5,6 Inclusion of various hetero-
atoms in the glutamine side chain also reduced activity
toward 3CP (13 and 14), but this reduction was not as
severe as that resulting from the amide modifications
described above. Replacement of the glutamine residue
with serine afforded a compound (15) which did not
inhibit 3CP and further confirmed that the presence of
a P1 glutamine was essential for potent 3CP inhibition
by peptidic Michael acceptors.
Variation of the P2 phenylalanine moiety present in
the tripeptidyl inhibitor 3 was also examined (Table 3).3
Removal or truncation of the amino acid side chain
resulted in significant loss of 3CP inhibitory activity
(16-22) while saturation of the aryl group afforded a
compound with relatively good anti-3CP properties (23).
Crystallographic analysis of the HRV-2 3CP-3 complex1
indicated that additional functionality could be incor-
porated at the 4-position of the aryl ring without
adversely affecting 3CP inhibition. Accordingly, many
compounds containing 4-substituted phenylalanine resi-
dues were prepared and evaluated as 3CP inhibitors
(24-35). Several of these molecules exhibited increased
inhibitory properties when tested against HRV-14 3CP.
However, in some cases this increase in activity was
accompanied by a decrease against proteases derived
from other HRV serotypes (compare 28 and 33 with 3).
The varied activity displayed by these inhibitors against
proteases derived from different HRV serotypes is
believed to be due to amino acid variations in this
portion of the enzyme.7,8 In addition, as illustrated by
compounds 27, 30, 34, and 35, not all 4-substituted Phe-
containing inhibitors showed increased activity toward
HRV-14 3CP. Substitution of other aryl moieties in
place of the phenyl ring present in 3 typically reduced
3CP inhibition properties somewhat (36 and 37), al-
though a thiophene-containing molecule (38) displayed
anti-3CP properties nearly identical to that of the parent
compound. Two other amino acids which occur at the
P2 position in 3CP substrates (Glu and Thr) were also
included in the inhibitor design.2 However, the result-
ing compounds (39 and 40) displayed significantly
reduced anti-3CP activity relative to the Phe-containing
inhibitor 3.
Modification of the P3 amino acid residue present in
tripeptidyl inhibitors such as 3 was undertaken as well
(Table 4).3 Analysis of the HRV-2 3CP-3 crystal struc-
ture1 indicated that the leucine side chain of 3 did not
appreciably contact the protein and was highly solvent-
exposed. A wide variety of functionality was therefore

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2821
Ta ble 3. Substitutions of the P2 Side Chain
compd
no.
kobs/[I]
EC50
CC50
d
R2
prepa
A
formulab
C32H42N4O7
serotypec
(M-1 s-1
)
(µM)d
(µM)d
3
CH2Ph
25 000
6 500
2 000
slow
1 300
4 500
2 300
1 200
600
2 300
2 900
3 000
16 300
3 500
700
46 000
9 200
2 400
59 400
5 300
1 400
11 300
1 200
29 000
3 400
1 100
4 200
1 200
82 300
7 100
43 100
1 300
700
0.54
2.3
1.6
>320
16
2
16
17
18
19
H
CH3
CH2CH3
CH2CH2CH3
A
A
C
C
C
25H36N4O7
141
20
6.0
5.0
ND
ND
5.4
8.9
10
1.9
ND
ND
1.8
ND
5.6
0.18
ND
ND
5.3
11
1.7
>320
>320
>320
>320
C26H38N4O7
C27H40N4O7
C28H42N4O7
16
2
20
21
22
23
CH2CH(CH3)2
CH2SCH3
CH2SCH2CH3
CH2Cyclohexyl
A
C
C
C
C29H44N4O7
C27H40N4O7S
C28H42N4O7S
C32H48N4O7
>320
>320
>320
>320
16
2
24
25
CH2Ph(4-F)
D
D
C32H41FN4O7‚1.25H2O
>320
>320
16
2
CH2Ph(4-CH3)
C33H44N4O7
16
2
26
27
28
CH2Ph(4-OH)
CH2Ph(4-OAc)
CH2Ph(4-OCH3)
D
D
C
C32H42N4O8
C34H44N4O9
C33H44N4O8‚0.25H2O
>320
>320
>320
16
2
ND
14
29
30
31
32
33
CH2Ph(4-OCH2CH3)
CH2Ph(4-OPO3H2)
CH2Ph(4-CH2OH)
CH2Ph(4-CH2OCH3)
CH2Ph(4-CH2CH2OH)
C
D
C
C
C
C34H46N4O8
>320
>320
>320
>320
>320
>320
C32H43N4O11
P
14
C33H44N4O8‚0.83H2O
C34H46N4O8
C34H46N4O8‚1.0H2O
0.55
39
3.5
ND
ND
16
2
34
35
36
37
38
CH2Ph(4-CN)
D
D
A
A
D
C33H41N5O7
C33H43N5O8
C31H41F3N6O9
C32H43F3N6O9
C30H40N4O7S
10 800
9 600
6 800
8 900
20 000
4 500
1 800
200
5.6
>320
177
>320
>320
>100
CH2Ph[4-C(O)NH2]
CH2(2-Imidazole)e
CH2(2-N-Methylimidazole)e
CH2(2-Thienyl)
>177
27
10
0.56
ND
ND
16
2
39
40
CH2CH2CO2H
CH(R-OH)CH3
A
C
C28H40N4O9
C27H40N4O8‚0.25H2O
>320
>320
>320
1 800
56
a
b
Method of preparation: see Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14 unless otherwise noted. See ref 1 for assay method and error. e TFA salt. ND ) not determined.
d
expected to be tolerated at this position within the
inhibitor design. As was observed for the P2 phenyl-
alanine residue, removal or truncation of the P3 amino
acid side chain reduced 3CP inhibitory properties (41
and 42). However, incorporation of several other ali-
phatic (43-50) and/or aromatic (51-55) moieties at the
P3 position often improved anti-3CP and antiviral
activity relative to the leucine-containing inhibitor
described above (compare to compound 3). Amino acids
containing hydroxylated side chains (56-59) also af-
forded active 3CP inhibitors when included at the P3
position, although such compounds exhibited antiviral
activities somewhat weaker than similar aliphatic
molecules (compare 43 with 57). Predictably, molecules
containing ionizable P3 residues (60-64) displayed
significantly reduced antiviral properties compared to
related nonionizable inhibitors, presumably due to
poorer membrane permeability.
In addition to alteration of the amino acid residues
described above, modification of the N-terminal (P4)
functionality contained in the tripeptidyl inhibitors was
also examined (Table 5).3 Replacement of the Cbz group
present in the parent compound 3 with other alkyl or
benzylic carbamates either increased or reduced 3CP
inhibitory properties depending on the structure of the
appended moiety (66-71). Crystallographic analysis of
the HRV-2 3CP-3 complex1 indicated that a significant
gap existed between the inhibitor and the protein
directly beneath the carbamate oxygen atom adjacent
to the benzyl group. Such analysis suggested that
incorporation of a larger moiety at this position would
improve the 3CP affinity of the resulting inhibitor.
Indeed, inclusion of a P4 S-alkyl thiocarbamate in the
inhibitor design (72-75) dramatically increased anti-
3CP properties relative to similar carbamate-containing
molecules (compare 3 with 75). This improvement was

2822 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Ta ble 4. Substitutions of the P3 Side Chain
Dragovich et al.
compd
no.
kobs/[I]
EC50
CC50
c,d
R3
prepa
formulab
C32H42N4O7
(M-1 s-1
)
(µM)c,d
(µM)d
3
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
CH2CH(CH3)2
H
A
A
A
C
C
E
25 000
3 800
0.54
5.6
2.0
0.38
0.79
0.32
1.4
0.18
10
>320
>320
>320
>320
>100
178
>100
>100
>100
>100
>320
ND
>320
>320
>100
>320
>320
>320
>100
>100
>320
>100
>100
>320
>320
>320
C
28H34N4O7‚1.0H2O
CH3
CH(CH3)2
C29H36N4O7‚0.67H2O
C31H40N4O7
11 300
62 500
31 700
39 800
46 200
35 200
40 000
36 200
39 000
20 700
161 500
141 200
97 000
57 000
39 900
38 900
98 000
40 000
18 600
35 000
43 300
35 000
78 900
22 600
CH(S-CH3)CH2CH3
C(CH3)3
CH2CH2SCH3
CH2SCH3
CH(R-CH3)SCH(CH3)2
Cyclohexyl
CH2Cyclohexyl
Phe
C32H42N4O7
C32H42N4O7‚0.75H2O
C31H40N4O7S
C
E
C30H38N4O7S‚1.5H2O
C33H44N4O7S‚0.50H2O
C
C
D
D
B
D
D
D
C
C
C
A
C
C
C
C
C
C
C
34H44N4O7
1.0
1.2
C35H46N4O7‚0.50H2O
C34H38N4O7‚0.50H2O
C35H40N4O7
C35H40N4O7S
C36H42N4O7S
ND
0.56
0.12
0.20
0.25
1.8
1.8
0.66
1.3
205
5.1
7.1
2.4
5.5
CH2Ph
CH2SPh
CH2SCH2Ph
CH2CH2Ph
CH2OH
C36H42N4O7
C29H36N4O8‚0.60H2O
C30H38N4O8‚0.75H2O
C31H40N4O8‚0.25H2O
C32H42N4O8‚0.75H2O
C34H44 F3N5O9‚0.50H2O
C36H46F3N5O10‚0.75H2O
C37H48F3N5O10‚0.75H2O
C30H36N4O9
CH(R-OH)CH3
C(CH3)2OH
C(CH3)2CH2OH
f
(CH2)4NH2
CH2CH2Morpholinef
CH2(CH2)2Morpholinef
CH2CO2H
CH2CH2CO2H
CH2C(O)N(CH3)2
C31H38N4O9‚0.25H2O
C32H41N5O8‚1.0H2O
5.9
a
b
Method of preparation: see Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14. See ref 1 for assay method and error. e 9:1 mixture of diastereomers. f TFA salt. ND ) not determined.
d
the largest realized by a single modification during the
structure-activity studies described herein and was
subsequently shown crystallographically to result from
significantly increased recognition of the target protease
(see below). Inclusion of a variety of amides at the P4
position (76-84) in the inhibitor design was tolerated
but typically reduced anti-3CP and antiviral properties
relative to the Cbz-containing compound 3. One notable
exception was compound 82 which displayed properties
very similar to the parent molecule. Replacement of the
P4 carbamate group with a urea moiety significantly
reduced both anti-3CP and antiviral activity (85 and
86), while incorporation of several substrate-inspired2
X-r a y Str u ctu r e An a lysis
acylated amino acids afforded tetrapeptides with good
anti-3CP activity but poor antiviral properties (87-89).
Having completed the systematic structure-activity
studies described above, an attempt was made to
incorporate several modifications into a single com-
pound. Thus, an inhibitor was conceived which con-
tained functionalities shown above to increase both anti-
3CP and antiviral activity relative to the parent
compound 3. The resulting molecule (90) displayed very
rapid, irreversible inhibition of HRV-14 3CP and ex-
hibited potent antiviral activity when tested against
HRV-14 in cell culture. These results suggest that
improvements realized by the structure-activity studies
described above can be combined in an additive manner.
The activity of 90 against HRV serotypes other than 14
as well as the examination of other functional group
combinations will be described in future publications.
Several crystal structures of 3CP-inhibitor complexes
were obtained during the peptidyl structure-activity
studies described above. These structures confirmed the
binding geometries that the inhibitors adopted when
complexed with 3CP and suggested locations for peptide
side chain modifications. In particular, the previously
detailed crystal structure of the HRV-2 3CP-3 complex1
inspired several improvements to the tripeptide inhibi-
tor design. As illustrated above, the most significant
of these was the incorporation of an N-terminal S-alkyl
thiocarbamate moiety (compounds 72-75). The crys-
tallographic details of one such inhibitor complexed with
3CP are described below.
The 1.9 Å X-ray crystal structure of the covalent
adduct formed between compound 75 and HRV-2 3CP9
is shown in Figure 2. In general, the inhibitor bound

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2823
Ta ble 5. Substitutions of the P4 Moiety
compd
no.
kobs/[I]
EC50
CC50
d
R4
prepa
A
formulab
C32H42N4O7
serotypec
(M-1 s-1
)
(µM)d
(µM)d
3
CO2CH2Ph
25 000
6 500
3 400
0.54
2.3
1.6
>320
16
2
66
67
68
69
70
71
72
73
CO2CH2Ph(2-CH3)
CO2CH2Ph(2-Cl)
CO2CH2(4-Pyridine)
CO2CH3
CO2-Cyclohexyl
CO2C(CH3)3
C
C
F
C
C
A
C
C
C33H44N4O7‚0.50H2O
C32H41ClN4O7‚0.50H2O
C31H41N5O7
C26H38N4O7‚0.25H2O
C31H46N4O7
C29H44N4O7
C26H38N4O6S
C27H40N4O6S‚0.25H2O
41 800
19 000
12 000
9 100
15 500
700
69 800
91 300
20 000
8 900
1.0
0.63
56
1.3
7.6
4.5
1.1
0.46
>100
>100
>320
>320
>100
>100
>320
>320
C(O)SCH3
C(O)SCH2CH3
16
2
ND
1.0
74
75
C(O)S-Cyclopentyl
C(O)SCH2Ph
C
D
C
30H44N4O6S‚0.50H2O
114 000
280 000
75 000
28 400
21 000
9 600
8 000
3 700
18 800
8 200
23 700
2 100
4 500
6 900
3 200
26 700
43 400
4 600
0.18
0.27
ND
1.9
1.0
5.2
5.2
14
1.0
1.8
0.60
>100
>320
C32H42N4O6S
16
2
76
77
78
79
80
81
82
83
84
85
86
87
88
89
C(O)-2-Naphthalene
C(O)Ph
C(O)Ph(4-OPh)
C(O)CH3
C(O)CH(CH3)2
C(O)C(CH3)3
C(O)-Cyclopentyl
C(O)CH2OH
C(O)CH2CH2OH
C(O)NHCH2Ph
C(O)N(CH3)CH2Ph
Ac-L-Val
F
F
C
C
C
35H42N4O6
31H40N4O6
37H44N4O7‚1.0H2O
>320
>320
>320
>320
>320
>100
>320
>320
>320
>100
>100
>100
>100
>100
D
C
C
E
C
C
C
E
E
E
E
E
C26H38N4O6‚0.25H2O
C28H42N4O6‚0.25H2O
C29H44N4O6
C
30H44N4O6‚0.20H2O
C26H38N4O7‚0.50H2O
C27H40N4O7‚0.75H2O
C32H43N5O6‚0.25H2O
C33H45N5O6‚1.0H2O
C31H47N5O7‚0.50H2O
C29H43N5O7
30
19
>100
5.6
63
20
Ac-L-Ala
Ac-L-Thr
C30H45N5O8‚0.50H2O
>100
a
b
Method of preparation: see Scheme 1. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values.
c Serotype 14 unless otherwise noted. See ref 1 for assay method and error. ND ) not determined.
d
F igu r e 2. Stereoview of the crystal structure of 75 complexed with HRV-2 3CP. A portion of the water-accessible surface of the
protein is shown in gray. A fragment of compound 3 from the HRV-2 3CP-3 complex1 is superimposed and is shown in orange.
to the enzyme in a manner similar to that observed
previously for the related Cbz-containing molecule (3).1
A covalent bond was observed between the 3CP active
site cysteine residue (Cys-147) and the â-carbon of the
Michael acceptor of 75, and all hydrogen bonds observed
in the 3CP-3 complex were also present in the 3CP-
75 crystal structure. However, the sulfur atom present
in 75 was buried more deeply into the 3CP S4 binding
pocket than the related oxygen atom contained in the
Cbz moiety of 3 (Figure 2). The S atom placement also
slightly altered the location of the appended benzyl
group of 75 compared to that observed previously for

2824 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
Sch em e 1. Peptide Coupling Sequences Utilized to Prepare 3CP Inhibitors
Sch em e 2a
a
Reagents and conditions: (a) 1.0 equiv of isobutyl chloroformate, 1.0 equiv of HCl‚HN(OCH3)CH3, 2.0 equiv of NMM, CH2Cl2, 0 °C,
20 min f 23 °C, 30 min, 91%; (b) 2.25 equiv of DIBAL, THF, -78 °C, 1 h; (c) 1.0 equiv of (EtO)2POCH2CO2Et, 1.0 equiv of NaN(TMS)2,
THF, -78 °C, 15 min, then crude 92, -78 °C, 1 h f 0 °C, 10 min, 36% from 91; (d) HCl in 1,4-dioxane, 23 °C, 3 h; (e) 1.0 equiv Cbz-L-
Leu-L-Phe-OH, 1.2 equiv of HOBT, 4.0 equiv of NMM, 1.2 equiv of EDC, CH2Cl2, 23 °C, 14 h, 32%; (f) 1.5 equiv of (i-Pr)3SiH, 1:2 TFA:
CH2Cl2, 23 °C, 45 min, 72%.
compound 3. In contrast to the 3CP-3 complex, no gaps
or spaces were observed between protein and ligand in
the 3CP-75 crystal structure. This optimized recogni-
tion of 3CP by the thiocarbamate portion of 75 is
presumably responsible for the improved anti-3CP
activity exhibited by the thiocarbamate-containing com-
pounds described in this study.
including those contained in R-amino acids, were masked
as tert-butyl carbamates while carboxylic acids were
protected as the corresponding tert-butyl esters. The
latter derivatives usually survived acidic removal of the
Boc moieties during peptide syntheses (see below).
Hydroxyl groups were typically left unprotected during
peptide coupling reactions.11 Representative synthetic
examples of methods A and D are described below and
are detailed in the Experimental Section. The remain-
ing methods involved chemistries identical to those
described for A and D and altered only the sequence of
peptide/amino acid coupling reactions (cf. Scheme 1).
The preparations of several noncommercially available
amino acids are also included.12
Syn th esis
The tripeptide-derived Michael acceptors utilized in
this study were prepared by a variety of related syn-
thetic methods (A-F, Scheme 1). The particular method
used to synthesize a given compound is indicated in
Tables 1-5. Thus, an appropriately derivatized P1
amino acid was either coupled with a dipeptide frag-
ment (methods A and B) or iteratively modified with
two individual amino acids (methods C-F). Whenever
possible, commercially available dipeptides (e.g., Cbz-
L-Leu-L-Phe-OH) and amino acids were employed, al-
though some entities were prepared utilizing standard
peptide synthesis techniques. The N-terminal P4 moiety
was often incorporated into a given P3 amino acid
(methods A-D) and was also introduced by modification
of a tripeptidyl compound (methods E and F). As
previously detailed, the ethyl propenoate Michael ac-
ceptor could be incorporated by derivitization of an
appropriate P1 amino acid residue (methods A, C, E) or
by oxidation of a tripeptide alcohol with subsequent
olefination of the resulting aldehyde (methods B, D, F).1
In all cases, potentially reactive functionalities were
masked with acid-labile protecting groups which were
removed by treatment with trifluoroacetic acid (TFA)
in the final synthetic step. Accordingly, the glutamine
side chain present in many inhibitors was protected as
an N-trityl amide during synthesis.10 Primary amines,
An example of synthetic method A is provided by the
preparation of compound 7 (Scheme 2). Thus, N-R-Boc-
L-glutamic acid γ-tert-butyl ester was transformed into
aldehyde 92 by reduction of the corresponding Weinreb
amide (91).13,14 The crude aldehyde thus obtained was
converted to the desired Michael acceptor by reaction
with the sodium enolate of triethyl phosphonoacetate
to afford ethyl ester 93 in moderate yield. This material
was deprotected under acidic conditions (HCl), and the
resulting amine salt was subjected to carbodiimide-
mediated coupling with Cbz-L-Leu-L-Phe-OH to afford
tripeptide 94 in modest yield.15 The tert-butyl protect-
ing group was removed by short exposure of 94 to TFA
in the presence of triisopropylsilane to give compound
7 in good yield.16 As described previously,1 the tripep-
tide inhibitors prepared in this study were typically
isolated as white solids by removal of the volatiles from
the TFA reaction mixture, trituration of the resulting
oil with Et2O, and subsequent filtration.
An alternate version of preparative method A which
employed Fmoc-protected amino acid derivatives was

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2825
Sch em e 3a
a
Reagents and conditions: (a) 1.05 equiv of DCC, 2.0 equiv of BnSH, 0.10 equiv of DMAP, THF, 23 °C, 18 h, 60%; (b) 5.0 equiv of
Et3SiH, Pd/C, acetone, 23 °C, 15 min; (c) 1.5 equiv of Ph3PdCHCO2Et, THF, 23 °C, 24 h, 41% from 95; (d) 1:4 TFA:CH2Cl2, 23 °C, 2 h,
79%; (e) 3.0 equiv of isobutyl chloroformate, 8.0 equiv of (i-Pr)2NEt, 5.0 equiv of H2NCH3, CH2Cl2, 0 f 23 °C, 1 h, 67%; (f) 3.0 equiv of
isobutyl chloroformate, 8.0 equiv of (i-Pr)2NEt, 5.0 equiv of HCl‚HN(CH3)2, CH2Cl2, 0 f 23 °C, 40 min, 69%; (g) 9:1 piperidine:DMF, 23
°C, 30 min; (h) 2.0 equiv Cbz-L-Leu-L-Phe-OH, 3.0 of equiv HOBT, 5.0 equiv of NMM, 3.0 equiv of EDC, CH2Cl2, 23 °C, 24 h, 38%.
Sch em e 4a
2
a
Reagents and conditions (Tr ) CPh3): (a) 1.0 equiv of CDI, 1.0 equiv of Cbz-L-Tyr(OtBu)-OH, THF, 23 °C, 1 h, then 1.05 equiv of
L-Tr-glutaminol,6 overnight, 67%; (b) H2/Pd/C, CH3OH, 23 °C, 5 h; (c) 1.0 equiv of CDI, 1.0 equiv of Cbz-L-Leu-OH, THF, 23 °C, overnight,
58%; (d) 3.0 equiv of IBX, DMSO, 23 °C, 1.5 h, 93%; (e) 1.2 equiv of Ph3PdCHCO2Et, THF, 23 °C, overnight, 63%; (f) 1:10 TFA:CH2Cl2,
23 °C, 1-6 h, 24-95%; (g) 1.0 equiv of Ac2O, 1.0 equiv of pyridine, 5:1 CH2Cl2:DMF, 23 °C, 2-3 h, 72%; (h) 3.0 equiv of TiCl4, CH2Cl2,
0 °C, 1 h, 83%, (i) 2.0 equiv of tetrazole, 1.0 equiv of (EtN)2P[OC(CH3)3]2, THF, 23 °C, 2 h; (j) 1.2 equiv of m-CPBA, CH2Cl2, 0 °C, 30 min,
45%.
utilized for the synthesis of compounds 5 and 6 (Scheme
3). N-R-Fmoc-L-glutamic acid γ-tert-butyl ester was
converted into aldehyde 96 by reduction of the corre-
sponding S-benzyl thioester (95).17 Crude 96 was
condensed with (carbethoxymethylene)triphenylphos-
phorane to provide ethyl ester 97 in good yield after
purification on silica gel. The tert-butyl protecting group
was removed under acidic conditions, and the resulting
carboxylic acid (98) was independently coupled with
methylamine and dimethylamine hydrochloride to give
amides 99 and 100, respectively. Compound 99 was
subsequently transformed into inhibitor 5 by Fmoc
deprotection and coupling of the resulting amine with
Cbz-L-Leu-L-Phe-OH. Similar manipulation of amide
100 afforded inhibitor 6.
(Scheme 4). L-Tr-Glutaminol6 was transformed into
tripeptide 104 (via intermediates 101-103) utilizing
chemistries similar to those described previously for the
preparation of related compounds (see the Experimental
Section).1,6 Brief exposure of 104 to TFA afforded
compound 26 which was subsequently treated with
acetic anhydride to provide inhibitor 27 in good yield.
Alternatively, the tert-butyl protecting group present in
104 was selectively removed by treatment with TiCl4
to give phenol 105 in excellent yield.18 Coupling of 105
with di-tert-butyl diethylphosphoramidite and subse-
quent m-CPBA oxidation afforded phosphate ester 106.
Deprotection of 106 by treatment with TFA then
provided inhibitor 30 in excellent yield.
A second modification of preparative method D was
employed in the synthesis of compounds 34 and 35
(Scheme 5). L-Tr-Glutaminol6 was condensed with N-R-
A modified version of preparative method D was
utilized for the synthesis of inhibitors 26, 27, and 30

2826 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
Sch em e 5a
a
Reagents and conditions (Tr ) CPh3): (a) 1.0 equiv of CDI, 1.0 equiv of Boc-L-Phe(4-I)-OH, THF, 23 °C, 1 h, then 1.1 equiv of L-Tr-
glutaminol,6 overnight, 73%; (b) 2.0 equiv of KCN, 0.015 equiv of Pd(PPh3)4, THF, reflux, overnight, 85%; (c) H2O2, 3:2 3.0 M Na2CO3:
EtOH, 23 °C, overnight, 91%; (d) HCl (gas), CH2Cl2, 23 °C, 5 min; (e) 1.0 equiv of CDI, 1.0 equiv of Cbz-L-Leu-OH, 1.05 equiv of Et3N,
THF, 23 °C, overnight, 47%; (f) 3.0 equiv of IBX, DMSO, 23 °C, 1.5 h, 86%; (g) 1.2 equiv of Ph3PdCHCO2Et, THF, 23 °C, overnight, 53%;
(h) 1:10 TFA:CH2Cl2, 23 °C, 6 h, 71%.
Sch em e 6a
a
Reagents and conditions [TBS ) Si(CH3)2tBu]: (a) 1.0 equiv of isobutyl chloroformate, 1.0 equiv of NMM, CH2Cl2, 0 °C, 15 min, then
1.0 equiv of CH3MgBr, THF, -78 °C, 45 min, 12%; (b) 1.25 equiv of NaBH4, 5:1 THF:H2O, 0 °C, 1.5 h, 74%; (c) 1.0 equiv of TBSOTf, 1.2
equiv of 2,6-lutidine, CH2Cl2, -78 °C, 1 h, 85%; (d) H2/Pd/C, EtOAc, 23 °C, 3 h, 89%.
Boc-L-4-iodophenylalanine to give dipeptide 107 in good
yield following silica gel chromatography. Palladium-
mediated coupling of 107 with KCN19 afforded nitrile
108 which was subsequently converted to amide 109 in
good yield by partial hydrolysis with H2O2.20 Nitrile 108
was transformed into inhibitor 34 (via intermediates
110-112) utilizing chemistries related to those de-
scribed above for the preparations of compounds 26 and
27 (see the Experimental Section). Similar elaboration
of amide 109 afforded compound 35 in good overall yield
(experimental not described).
The amino acid required for the preparation of
compounds 8 and 9 was prepared by the method
illustrated in Scheme 6. N-R-Boc-L-Glutamic acid R-ben-
zyl ester was converted to methyl ketone 113 by
sequential treatment with isobutyl chloroformate and
methylmagnesium bromide. A significant amount of
pyroglutamate 114 was also formed by this process, and
which was subsequently utilized in the syntheses of
compounds 8 and 9.23
The amino acids utilized in the synthesis of inhibitors
31-33 were prepared by the method depicted in Scheme
7. N-R-Boc-L-4-iodophenylalanine was converted to the
corresponding benzyl ester 118 in good yield. Pal-
ladium-mediated coupling of 118 with tributyl(vinyl)-
tin afforded intermediate 119 following purification on
silica gel.25 Hydroboration of 119 and subsequent
debenzylation of the resulting alcohol (120) gave car-
boxylic acid 121 in moderate overall yield. Alterna-
tively, osmium-catalyzed dihydroxylation of 119 fol-
lowed by periodate-mediated cleavage of the resulting
diol (not shown) provided aldehyde 122.26 This material
was reduced with NaBH4 and the alcohol thus obtained
(123) was deprotected to give carboxylic acid 124 in good
yield. Compounds 121 and 124 were subsequently
utilized in the preparation of inhibitors 33 and 31,
respectively.27
Con clu sion s
The peptidyl structure-activity studies described
above confirmed the ability of peptide-derived Michael
acceptors to function as potent inhibitors of human
rhinovirus 3C protease and further defined this class
of antirhinoviral agents. The systematic variation of a
lead tripeptidyl inhibitor (3) identified optimal amino
acid residues and N-terminal moieties for obtaining high
levels of anti-3CP and antiviral activity. The combina-
tion of several isolated improvements afforded a highly
active 3CP inhibitor (90) which displayed potent anti-
viral activity (EC50 ) 0.056 µM) against HRV-14 in cell
culture.
this entity may occur as an intermediate during the
grignard-assisted production of 113. Indeed, 114 could
be converted to 113 in moderate yield (51%) by treat-
ment with methylmagnesium bromide at low temper-
ature.21,22 Reduction of 113 with NaBH4 followed by
silylation of the resulting diastereomeric alcohols (115)
afforded a 1:1 mixture of silyl ethers (116) after puri-
fication by flash column chromatography. Removal of
the benzyl protecting group provided carboxylic acid 117

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2827
Sch em e 7a
a
Reagents and conditions: (a) 1.2 equiv of DCC, 1.2 equiv of BnOH, 0.40 equiv of DMAP, CH2Cl2, 23 °C, 6 h, 87%; (b) 1.1 equiv of
Bu3SnCHdCH2, 0.05 equiv of Pd(PPh3)4, 1,4-dioxane, 80 °C, 6 h, 83%; (c) 0.33 equiv of BH3‚THF, THF, 0 f 23 °C, 2 h, 49%; (d) 2.0 M
NaOH, CH3OH, 23 °C, 6 h, 89%; (e) 1.1 equiv of NMO, 0.03 equiv of OsO4, 8:1 acetone:H2O, 23 °C, 3 h, 61%; (f) 1.1 NaIO4, 5:1 Et2O:H2O,
23 °C, 2 h; (g) 1.0 equiv of NaBH4, EtOH, 0 °C, 20 min, 74%; (h) H2/Pd/C, EtOAc, 23 °C, 6 h, 93%.
2.0 equiv) in CH2Cl2 (80 mL) at 0 °C. The reaction mixture
was stirred at 0 °C for 15 min and then N,O-dimethylhydroxy-
lamine hydrochloride (0.688 g, 7.02 mmol, 1.0 equiv) was
added. The resulting solution was stirred at 0 °C for 20 min
and at 23 °C for 30 min and then was partitioned between
water (150 mL) and a 1:1 mixture of EtOAc and hexanes (2 ×
150 mL). The combined organic layers were dried over Na2-
SO4 and were concentrated. Purification of the residue by
flash column chromatography (40% EtOAc in hexanes) pro-
vided 91 (2.20 g, 91%) as a clear oil: Rf ) 0.43 (50% EtOAc in
hexanes); IR (cm-1) 3335, 2977, 1715, 1664; 1H NMR (CDCl3)
δ 1.44 (s, 9H), 1.45 (s, 9H), 1.79-1.89 (m, 1H), 1.96-2.07 (m,
1H), 2.29-2.34 (m, 2H), 3.21 (s, 3H), 3.78 (s, 3H), 4.68 (s, br,
1H), 5.22 (d, 1H, J ) 9.0). Anal. (C16H30N2O6) C, H, N.
[Boc-L-(OtBu )Glu ]-H (92). Diisobutylaluminum hydride
(9.53 mL of a 1.5 M solution in toluene, 14.3 mmol, 2.25 equiv)
was added to a solution of 91 (2.20 g, 6.35 mmol, 1 equiv) in
THF at -78 °C, and the reaction mixture was stirred at -78
°C for 1 h. Methanol (3 mL) and 1.0 M HCl (6 mL) were added
sequentially, and the mixture was warmed to 23 °C. The
resulting suspension was diluted with Et2O (150 mL) and was
washed with 1.0 M HCl (3 × 100 mL), half-saturated NaHCO3
(100 mL), and water (100 mL). The organic layer was dried
over MgSO4, filtered, and concentrated to give crude 92 which
was used immediately without further purification.
Exp er im en ta l Section
Gen er a l. All reactions were performed in septum-sealed
flasks under a slight positive pressure of argon unless other-
wise noted. All commercial reagents were used as received
from their respective suppliers with the following exceptions.
Tetrahydrofuran (THF) was distilled from sodium-benzophe-
none ketyl prior to use. Dichloromethane (CH2Cl2) was
distilled from calcium hydride prior to use. Flash column
chromatography28 was performed using silica gel 60 (Merck
Art 9385). 1H NMR spectra were recorded at 300 MHz
utilizing either a Varian UNITYplus 300 or a General Electric
QE-300 spectrometer equipped with Techmag operating soft-
ware. Chemical shifts are reported in ppm (δ) downfield
relative to internal tetramethylsilane, and coupling constants
are given in hertz. Infrared absorption spectra were recorded
using either a MIDAC Corp. or a Perkin-Elmer 1600 series
FTIR. Elemental analyses were performed by Atlantic Mi-
crolab, Inc., Norcross, GA. Melting points were determined
using a Mel-Temp II apparatus and are uncorrected. Experi-
mental conditions for enzyme and antiviral assays are detailed
elsewhere.1
A simplified naming system employing amino acid ab-
breviations is used to identify some intermediates and final
products. When utilizing this naming system, italicized amino
acid abbreviations represent modifications at the C-terminus
of that residue where acrylic acid esters are reported as “E”
(trans) propenoates. The amino acids required for the prepa-
ration of inhibitors 10-12,29 13,30 14-15,32 48,33 53,34 58,35 61-
62,36 66-67,37 69,38 70,37 72-75,39 83,38 and 8440 were syn-
thesized by standard techniques and/or literature methods.
Similarly, the dipeptides required for the synthesis of com-
pounds 36-37,41 39,42 and 5943 were prepared by standard
peptide chemistry methods and/or literature syntheses. In-
hibitor 71 was prepared utilizing general method A in which
the final detritylation step and penultimate peptide coupling
step were transposed.45
Eth yl 3-[Boc-L-(OtBu )Glu ]-(E)-p r op en oa te (93). So-
dium bis(trimethylsilyl)amide (6.35 mL of a 1.0 M solution in
THF, 6.35 mmol, 1.0 equiv) was added to a solution of triethyl
phosphonoacetate (1.55 g, 6.35 mmol, 1.0 equiv) in THF (150
mL) at -78 °C, and the resulting solution was stirred for 15
min at that temperature. Crude 92 (6.35 mmol, 1 equiv) in
THF (30 mL) was added via cannula, and the reaction mixture
was stirred for 1 h at -78 °C, warmed to 0 °C for 10 min, and
partitioned between 0.5 M HCl (150 mL) and a 1:1 mixture of
EtOAc and hexanes (2 × 150 mL). The combined organic
layers were dried over Na2SO4 and were concentrated. Puri-
fication of the residue by flash column chromatography (gradi-
ent elution, 10 f 20% EtOAc in hexanes) provided 93 (0.824
g, 36%) as a clear oil: Rf ) 0.50 (30% EtOAc in hexanes); IR
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od A.
Syn th esis of Eth yl 3-(Cbz-L-Leu -L-P h e-L-Glu )-(E)-p r op e-
n oa te (7). [Boc-L-(OtBu )Glu ]-N(OMe)Me (91). Isobutyl
chloroformate (0.910 mL, 7.02 mmol, 1.0 equiv) was added to
a solution of N-R-Boc-γ-trityl-L-glutamic acid (2.13 g, 7.02
mmol, 1 equiv) and 4-methylmorpholine (1.54 mL, 14.0 mmol,
1
(cm-1) 3357, 2979, 1723; H NMR (CDCl3) δ 1.29 (t, 3H, J )
7.2), 1.44 (s, 9H), 1.45 (s, 9H), 1.65-1.96 (m, 2H), 2.29-2.34
(m, 2H), 4.19 (q, 2H, J ) 7.2), 4.30 (s, br, 1H), 4.67 (s, br, 1H),

2828 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
5.92 (dd, 1H, J ) 15.6, 1.6), 6.82 (dd, 1H, J ) 15.6, 5.5). Anal.
reaction mixture was stirred at that temperature for 15 min,
then was filtered through Celite. The filtrate was concentrated
to afford crude 96 (8.40 g) which was used without further
purification.
(C18H31NO6) C, H, N.
Eth yl 3-[Cbz-L-Leu -L-P h e-L-(OtBu )Glu ]-(E)-p r op en oa te
(94). A solution of HCl in 1,4-dioxane (4.0 M, 9 mL) was added
to a solution of 93 (0.766 g, 2.14 mmol, 1 equiv) in the same
solvent (10 mL) at 23 °C. After 3 h, the volatiles were removed
under reduced pressure. The residue was dissolved in CH2-
Cl2 (30 mL), and Cbz-L-Leu-L-Phe-OH (0.883 g, 2.14 mmol, 1.0
equiv), 1-hydroxybenzotriazole hydrate (HOBT, 0.347 g, 2.57
mmol, 1.2 equiv), 4-methylmorpholine (0.941 mL, 8.56 mmol,
4.0 equiv), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiim-
ide hydrochloride (EDC, 0.492 g, 2.57 mmol, 1.2 equiv) were
added sequentially. The reaction mixture was stirred at 23
°C for 14 h and then was partitioned between water (150 mL)
and EtOAc (2 × 150 mL). The combined organic layers were
dried over Na2SO4 and were concentrated. Flash chromato-
graphic purification of the residue (3% CH3OH in CH2Cl2)
afforded 94 (0.439 g, 32%) as a white foam: Rf ) 0.51 (10%
CH3OH in CH2Cl2); IR (cm-1) 3289, 1722, 1646; 1H NMR
(CDCl3) δ 0.88-0.92 (m, 6H), 1.29 (t, 3H, J ) 7.2), 1.41 (s,
9H), 1.54-1.61 (m, 2H), 1.78-1.87 (m, 1H), 2.15-2.20 (m, 2H),
3.00-3.18 (m, 3H), 4.07-4.13 (m, 1H), 4.18 (q, 2H, J ) 7.2),
4.55-4.67 (m, 2H), 4.94-5.16 (m, 3H), 5.75 (d, 1H, J ) 15.7),
6.48 (d, 1H, J ) 7.5), 6.58 (d, 1H, J ) 8.1), 6.67 (dd, 1H, J )
15.7, 5.6), 7.16-7.39 (m, 11H). Anal. (C36H49N3O8) C, H, N.
Eth yl 3-(Cbz-L-Leu -L-P h e-L-Glu )-(E)-p r op en oa te (7).
Trifluoroacetic acid (6 mL) was added to a solution of 94 (0.420
g, 0.644 mmol, 1 equiv) and triisopropylsilane (0.20 mL, 0.976
mmol, 1.5 equiv) in CH2Cl2 (10 mL) at 23 °C. The reaction
mixture was stirred for 45 min at 23 °C, CCl4 (4 mL) was
added, and the volatiles were removed under reduced pressure.
The residue was triturated with Et2O (10 mL), and the
resulting white solid was collected by vacuum filtration,
washed with Et2O (3 × 10 mL), and air-dried to afford 7 (0.278
g, 72%): mp 180-183 °C; Rf ) 0.38 (10% CH3OH in CH2Cl2);
IR (cm-1) 3260, 1734, 1692, 1638; 1H NMR (DMSO-d6) δ 0.79
(d, 3H, J ) 10.3), 0.81 (d, 3H, J ) 10.3), 1.21 (t, 3H, J ) 7.2),
1.28-1.39 (m, 2H), 1.41-1.59 (m, 2H), 1.61-1.83 (m, 1H),
2.13-2.24 (m, 2H), 2.84 (dd, 1H, J ) 13.6, 8.4), 2.98 (dd, 1H,
J ) 13.6, 6.1), 3.95-3.99 (m, 1H), 4.10 (q, 2H, J ) 7.2), 4.42-
4.52 (m, 2H), 4.97 (d, 1H, J ) 12.6), 5.04 (d, 1H, J ) 12.6),
5.62 (d, 1H, J ) 15.6), 6.68 (dd, 1H, J ) 15.6, 5.3), 7.15-7.44
(m, 11H), 7.99 (d, 1H, J ) 8.1), 8.06 (d, 1H, J ) 8.1). Anal.
(C32H41N3O8) C, H, N.
Alter n a te Exa m p le of P r ep a r a tion Meth od A. Syn -
th esis of Eth yl 3-[Cbz-L-Leu -L-P h e-L-(N-Me)Gln ]-(E)-p r o-
p en oa t e (5). F m oc-L-(Ot Bu )Glu -SBn (95). 4-(Dimethyl-
amino)pyridine (0.574 g, 4.70 mmol, 0.10 equiv) and 1,3-
dicyclohexylcarbodiimide (10.2 g, 49.3 mmol, 1.05 equiv) were
added sequentially to a solution of N-R-Fmoc-L-glutamic acid
γ-tert-butyl ester (20.0 g, 47.0 mmol, 1 equiv) and benzyl
mercaptan (11.0 mL, 94.0 mmol, 2.0 equiv) in THF (300 mL)
at 23 °C. The cloudy reaction mixture was stirred at 23 °C
for 18 and then was filtered through a medium fritted funnel.
The pale yellow filtrate was concentrated and then was
partitioned between EtOAc (250 mL) and 1.0 M HCl (150 mL).
The organic layer was dried over Na2SO4 and concentrated,
and the resulting oil was triturated with Et2O (60 mL) and
again filtered through a medium fritted funnel. The filtrate
thus obtained was concentrated to provide a yellow oil which
solidified on standing. The resulting solid was slurried with
a 2:1 mixture of petroleum ether and Et2O (150 mL), collected
by vacuum filtration, washed with petroleum ether (4 × 100
mL), and air-dried to give 95 (15.0 g, 60%) as an off-white
solid: mp 114-116 °C; Rf ) 0.80 (50% EtOAc in hexanes); IR
(cm-1) 3333, 2982, 1725; 1H NMR (CDCl3) δ 1.44 (s, 9H), 1.91-
1.98 (m, 1H), 2.16-2.37 (m, 3H), 4.07-4.53 (m, 6H), 5.63 (d,
1H, J ) 8.4), 7.25-7.30 (m, 7H), 7.36-7.41 (m, 2H), 7.58-
7.61 (m, 2H) 7.75 (d, 2H, J ) 7.5). Anal. (C31H33NO5S) C, H,
N.
Eth yl 3-[F m oc-L-(OtBu )Glu ]-(E)-p r op en oa te (97). (Car-
bethoxymethylene)triphenylphosphorane (23.3 g, 68.7 mmol,
1.5 equiv) was added to a solution of 96 (18.3 g, 44.7 mmol, 1
equiv) in THF (200 mL) at 23 °C. The resulting mixture was
stirred at 23 °C for 24 h and then was concentrated under
reduced pressure. The residue was purified by flash column
chromatography (15% EtOAc in hexanes) to afford 97 (13.9 g,
65%) as a white foam: Rf ) 0.68 (50% EtOAc in hexanes); IR
1
(cm-1) 3339, 2979, 1721; H NMR (CDCl3) δ 1.26 (t, 3H, J )
7.2), 1.44 (s, 9H), 1.79-2.02 (m, 2H), 2.26-2.31 (m, 2H), 4.20
(q, 2H, J ) 7.2), 4.35-4.48 (m, 4H), 5.07 (d, 1H, J ) 8.1), 5.92
(d, 1H, J ) 15.5), 6.82 (dd, 1H, J ) 15.5, 5.4), 7.26-7.43 (m,
4H), 7.59 (d, 2H, J ) 7.2), 7.77 (d, 2H, J ) 7.5). Anal. (C28H33
NO6) C, H, N.
-
Eth yl 3-[F m oc-L-Glu ]-(E)-p r op en oa te (98). Trifluoro-
acetic acid (50 mL) was added to a solution of 97 (13.9 g, 28.9
mmol) in CH2Cl2 (200 mL) at 23 °C. The reaction mixture was
stirred for 2 h at 23 °C, and then the volatiles were removed
under reduced pressure. The residue was triturated with a
1:1 mixture of Et2O and hexanes (100 mL), and the resulting
solid was collected by filtration. Recrystallization from Et2O/
hexanes afforded 98 (9.62 g, 79%) as a off-white solid: Rf )
0.14 (50% EtOAc in hexanes); IR (cm-1) 3294, 2977, 1711; 1H
NMR (DMSO-d6) δ 1.20 (t, 3H, J ) 7.2), 1.64-1.82 (m, 2H),
2.22 (t, 2H, J ) 7.2), 4.07-4.15 (m, 3H), 4.22 (t, 1H, J ) 7.2),
4.31-4.33 (m, 2H), 5.82 (d, 1H, J ) 15.6), 5.76 (dd, 1H, J )
15.9, 6.0), 7.29-7.43 (m, 4H), 7.58 (d, 1H, J ) 7.8), 7.68-7.70
(m, 2H), 7.88 (d, 2H, J ) 7.2). Anal. (C24H25NO6‚0.25H2O) C,
H, N.
Eth yl 3-[F m oc-L-(N-Me)Gln ]-(E)-p r op en oa te (99). N,N-
Diisopropylethylamine (0.247 mL, 1.42 mmol, 3.0 equiv) and
isobutyl chloroformate (0.184 mL, 1.42 mmol, 3.0 equiv) were
added sequentially to a 0 °C slurry of 98 (0.200 g, 0.472 mmol,
1 equiv) in CH2Cl2 (5 mL). After the mixture was stirred for
40 min at 0 °C, additional N,N-diisopropylethylamine (0.411
mL, 2.36 mmol, 5.0 equiv) and a solution of methylamine in
THF (2.0 M, 1.18 mL, 2.36 mmol, 5.0 equiv) were added
sequentially. The reaction mixture was stirred for 40 min at
0 °C and for 20 min at 23 °C and then was partitioned between
1.0 M HCl (10 mL) and 10% CH3OH in CH2Cl2 (3 × 30 mL).
The combined organic layers were dried over Na2SO4 and were
concentrated. Flash chromatographic purification of the resi-
due (2% CH3OH in CHCl3) gave 99 (0.137 g, 67%) as a white
solid: mp 162 °C dec; Rf ) 0.46 (10% CH3OH in CHCl3); IR
1
(cm-1) 3306, 1716, 1692, 1643, 1539; H NMR (CDCl3) δ 1.29
(t, 3H, J ) 7.2), 1.82-2.05 (m, 2H), 2.17-2.26 (m, 2H), 2.79
(d, 3H, J ) 4.7), 4.14-4.23 (m, 3H), 4.26-4.48 (m, 3H), 5.57
(d, 1H, J ) 8.1), 5.74 (s, 1H), 5.93 (d, 1H, J ) 15.6), 6.83 (dd,
1H, J ) 15.6, 5.3), 7.28-7.43 (m, 4H), 7.60 (d, 2H, J ) 7.2),
7.76 (d, 2H, J ) 7.5). Anal. (C25H28N2O5‚0.50H2O) C, H, N.
Eth yl 3-[Fm oc-L-(N-Me2)Gln ]-(E)-pr open oate (100). N,N-
Diisopropylethylamine (0.197 mL, 1.13 mmol, 3.0 equiv) and
isobutyl chloroformate (0.147 mL, 1.13 mmol, 3.0 equiv) were
added sequentially to a 0 °C slurry of 98 (0.160 g, 0.378 mmol,
1 equiv) in CH2Cl2 (4 mL), and the resulting solution was
stirred 40 min. A solution of N,N-diisopropylethylamine (0.329
mL, 1.89 mmol, 5.0 equiv) and dimethylamine hydrochloride
(0.154 g, 1.89 mmol, 5.0 equiv) in CH3CN (4 mL) was added
dropwise, and the reaction mixture was allowed to warm to
23 °C, stirred for 40 min, and then partitioned between 1 M
HCl (10 mL) and 10% CH3OH in CH2Cl2 (3 × 30 mL). The
combined organic layers were washed with brine (50 mL),
dried over Na2SO4, and were concentrated. Flash chromato-
graphic purification of the residue (2% CH3OH in CHCl3) gave
100 (0.117 g, 69%) as a glass: Rf ) 0.62 (10% CH3OH in
1
CHCl3); IR (cm-1) 3284, 1713, 1631; H NMR (CDCl3) δ 1.29
F m oc-L-(Ot Bu )Glu -H (96). Triethylsilane (16.37 mL,
102.5 mmol, 5.0 equiv) was added slowly via addition funnel
to a degassed suspension of 95 (10.9 g, 20.5 mmol, 1 equiv)
and Pd/C (10%, 5.5 g) in acetone (500 mL) at 23 °C. The
(t, 3H, J ) 7.2), 1.95-2.03 (m, 2H), 2.35-2.50 (m, 2H), 2.97
(s, 3H), 2.98 (s, 3H), 4.15-4.24 (m, 3H), 4.27-4.43 (m, 3H),
5.90-6.00 (m, 2H), 6.87 (dd, 1H, J ) 15.9, 5.3), 7.27-7.43 (m,

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2829
4H), 7.56-7.63 (m, 2H), 7.76 (d, 2H, J ) 7.5). Anal.
(C26H30N2O5‚0.25H2O) C, H, N.
7.08 (d, 2H, J ) 7.7), 7.14-7.33 (m, 20H), 7.41 (d, 1H, J )
8.1), 7.63 (d, 1H, J ) 7.7), 7.85 (d, 1H, J ) 8.5), 8.51 (s, 1H).
Anal. (C51H60N4O7) C, H, N.
Eth yl 3-[Cbz-L-Leu -L-P h e-L-(N-Me)Gln ]-(E)-p r op en oa te
(5). A solution of 99 (0.115 g, 0.263 mmol, 1 equiv) in 9:1
piperidine:DMF (2 mL) was stirred for 30 min at 23 °C and
then was concentrated under reduced pressure. Toluene (25
mL) was added to the reside, the volatiles were subsequently
removed under vacuum, and the resulting oil was dissolved
in CH2Cl2 (8 mL). Cbz-L-Leu-L-Phe-OH (0.217 g, 0.526 mmol,
2.0 equiv), 4-methylmorpholine (0.145 mL, 1.32 mmol, 5.0
equiv), 1-hydroxybenzotriazole hydrate (0.107 g, 0.792 mmol,
3.0 equiv), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiim-
ide hydrochloride (0.152 g, 0.793 mmol, 3.0 equiv) were then
added sequentially. The reaction mixture was stirred for 24
h at 23 °C and then was loaded directly onto a flash column
and eluted with 2% CH3OH in CHCl3 to give a white solid
which smelled of 4-methylmorpholine. Evaporation from
toluene (2 × 30 mL) afforded 5 (0.060 g, 38%) as a white
solid: mp 224-225 °C; Rf ) 0.46 (10% CH3OH in CHCl3); IR
(cm-1) 3436, 3295, 1719, 16996, 1643, 1543; 1H NMR (DMSO-
d6) δ 0.78 (d, 3H, J ) 6.5), 0.82 (d, 3H, J ) 6.5), 1.21 (t, 3H, J
) 7.2), 1.25-1.38 (m, 2H), 1.43-1.56 (m, 1H), 1.59-1.82 (m,
2H), 2.02-2.10 (m, 2H), 2.52 (d, 3H, J ) 4.4), 2.84 (dd, 1H, J
) 14.0, 9.3), 2.97 (dd, 1H, J ) 14.0, 6.2), 3.93-4.03 (m, 1H),
4.10 (q, 2H, J ) 7.2), 4.31-4.52 (m, 2H), 4.97 (d, 1H, J ) 12.6),
5.04 (d, 1H, J ) 12.6), 5.63 (d, 1H, J ) 15.9), 6.68 (dd, 1H, J
) 15.9, 5.6), 7.13-7.24 (m, 5H), 7.27-7.37 (m, 5H), 7.43 (d,
1H, J ) 7.8), 7.59-7.66 (m, 1H), 8.00 (d, 1H, J ) 8.1), 8.04 (d,
1H, J ) 8.4). Anal. (C33H44N4O7) C, H, N.
Cbz-L-Leu -L-Tyr (OtBu )-L-(Tr )glu ta m in a l (103). o-Io-
doxybenzoic acid24 (1.32 g, 4.73 mmol, 3.0 equiv) was added
to a solution of 102 (1.32 g, 1.58 mmol, 1 equiv) in DMSO (16
mL) at 23 °C. After the mixture was stirred 1.5 h at 23 °C,
the DMSO was removed under reduced pressure. The residue
was twice diluted with CH2Cl2 (20 mL), and the volatiles were
evaporated to remove any residual DMSO. The resulting
residue was then triturated with EtOAc (80 mL), and the white
solid thus obtained was filtered off. The filtrate was washed
with a 1:1 mixture of 5% Na2S2O3 and 5% NaHCO3 solution
(80 mL), water (80 mL), and brine (80 mL), dried over Na2-
SO4, and concentrated to give 103 (1.23 g, 93%) as a white
glassy solid. This material was used immediately without
further purification: 1H NMR (DMSO-d6) δ 0.78-0.81 (m, 6H),
1.23 (s, 9H), 1.25-1.35 (m, 1H), 1.45-1.65 (m, 2H), 1.82-1.90
(m, 1H), 2.25-2.30 (m, 2H), 2.77-2.82 (m, 1H), 2.93-2.98 (m,
1H), 3.95-4.05 (m, 2H), 4.53-4.56 (m, 1H), 4.94-5.03 (m, 2H),
6.82 (d, 2H, J ) 8.5), 7.10-7.33 (m, 22H), 7.39 (d, 1H, J )
8.5), 7.97 (d, 1H, J ) 7.7), 8.38 (d, 1H, J ) 6.3), 8.59 (s, 1H),
9.20 (s, 1H).
Eth yl 3-[Cbz-L-Leu -L-Tyr (OtBu )-L-(Tr -Gln )]-(E)-p r op e-
n oa te (104). (Carbethoxymethylene)triphenylphosphorane
(0.61 g, 1.76 mmol, 1.2 equiv) was added to a solution of 103
(1.23 g, 1.47 mmol, 1 equiv) in THF (30 mL) at 23 °C, and the
reaction mixture was stirred overnight at that temperature.
The volatiles were then removed in vacuo, and the residue was
purified by flash column chromatography (gradient elution, 0
f 2% CH3OH in CHCl3) to provide 104 (0.84 g, 63%) as a white
glassy solid: Rf ) 0.20 (2% CH3OH in CHCl3); IR (cm-1) 3293,
1653, 1508; 1H NMR (DMSO-d6) δ 0.77-0.81 (m, 6H), 1.21 (t,
3H, J ) 7.0), 1.23 (s, 9H), 1.25-1.37 (m, 2H), 1.49-1.56 (m,
1H), 1.63-1.65 (m, 2H), 2.23-2.28 (m, 2H), 2.76-2.81 (m, 1H),
2.91-2.98 (m, 1H), 3.92-3.98 (m, 1H), 4.10 (q, 2H, J ) 7.0),
4.35-4.40 (m, 1H), 4.42-4.47 (m, 1H), 4.95 (d, 1H, J ) 12.5),
5.02 (d, 1H, J ) 12.5), 5.72 (d, 1H, J ) 15.4), 6.70 (dd, 1H, J
) 15.4, 5.5), 6.80 (d, 2H, J ) 8.1), 7.08 (d, 2H, J ) 8.5), 7.14-
7.32 (m, 20H), 7.41 (d, 1H, J ) 8.1), 7.93 (d, 1H, J ) 8.1), 8.06
(d, 1H, J ) 8.1), 8.58 (s, 1H). Anal. (C55H64N4O8) C, H, N.
E t h yl 3-(Cb z-L-Leu -L-Tyr -L-Gln )-(E)-p r op en oa t e (26).
Trifluoroacetic acid (1 mL) was added to a solution of 104 (0.43
g, 0.47 mmol) in CH2Cl2 (10 mL) at 23 °C, and the reaction
mixture was stirred at that temperature for 6 h. The volatiles
were then removed under reduced pressure, and the residue
was purified by flash column chromatography (gradient elu-
tion, 0 f 4% CH3OH in CHCl3) to give 26 (0.068 g, 24%) as a
white solid: mp 190-195 °C dec; IR (cm-1) 1643, 1539; 1H
NMR (DMSO-d6) δ 0.79 (d, 3H, J ) 6.6), 0.83 (d, 3H, J ) 6.6),
1.21 (t, 3H, J ) 7.0), 1.23-1.27 (m, 2H), 1.45-1.55 (m, 1H),
1.64-1.80 (m, 2H), 2.04-2.09 (m, 2H), 2.71-2.78 (m, 1H),
2.83-2.89 (m, 1H), 3.92-3.97 (m, 1H), 4.11 (q, 2H, J ) 7.0),
4.33-4.39 (m, 2H), 4.97 (d, 1H, J ) 12.5), 5.05 (d, 1H, J )
12.5), 5.71 (d, 1H, J ) 15.8), 6.60 (d, 2H, J ) 7.7), 6.72 (dd,
1H, J ) 15.8, 5.5), 6.75 (s, br, 1H), 6.96 (d, 2H, J ) 8.1), 7.20
(s, br, 1H), 7.29-7.34 (m, 5H), 7.45 (d, 1H, J ) 7.7), 7.91 (d,
1H, J ) 8.1), 8.04 (d, 1H, J ) 7.7), 9.04 (s, br, 1H). Anal.
(C32H42N4O8) C, H, N.
Eth yl 3-[Cbz-L-Leu -L-Tyr (OAc)-L-Gln ]-(E)-p r op en oa te
(27). Acetic anhydride (0.013 mL, 0.12 mmol, 1.0 equiv) was
added to a solution of 26 (0.075 g, 0.12 mmol, 1 equiv) and
pyridine (0.01 mL, 0.12 mmol, 1.0 equiv) in a 5:1 mixture of
CH2Cl2 and DMF (3.0 mL) at 23 °C. The reaction mixture was
stirred at 23 °C, and additional small amounts of pyridine and
acetic anhydride were added until TLC analysis indicated that
all starting material was consumed. The volatiles were then
removed in vacuo, and the residue was purified by flash
column chromatography (gradient elution, 0 f 3% CH3OH in
CHCl3) to give 27 (0.058 g, 72%) as a white solid: mp 233-
234 °C; Rf ) 0.29 (7% CH3OH in CHCl3); IR (cm-1) 1651, 1539,
1227; 1H NMR (DMSO-d6) δ 0.79 (d, 3H, J ) 6.6), 0.83 (d, 3H,
J ) 6.6), 1.21 (t, 3H, J ) 7.0), 1.28-1.36 (m, 2H), 1.43-1.55
(m, 1H), 1.66-1.74 (m, 2H), 2.04-2.07 (m, 2H), 2.23 (s, 3H),
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od D.
Syn th esis of Eth yl 3-(Cbz-L-Leu -L-Tyr -L-Gln )-(E)-p r op e-
n oa te (26), Eth yl 3-[Cbz-L-Leu -L-Tyr (OAc)-L-Gln ]-(E)-p r o-
p en oa te (27), a n d Eth yl 3-[Cbz-L-Leu -L-Tyr (OP O3H2)-L-
Gln ]-(E)-p r op en oa t e (30). Cb z-L-Tyr (Ot Bu )-L-(Tr )glu -
ta m in ol (101). Carbonyldiimidazole (0.74 g, 4.57 mmol, 1.0
equiv) was added to a solution of Cbz-L-Tyr(OtBu)-OH (1.69
g, 4.57 mmol, 1.0 equiv) in THF (45 mL) at 23 °C. After 1 h,
L-Tr-glutaminol6 (1.80 g, 4.79 mmol, 1.05 equiv) was added,
and the reaction mixture was stirred overnight at 23 °C. The
volatiles were removed under reduced pressure, and the
residue was purified by flash column chromatography (gradi-
ent elution, 0 f 3% CH3OH in CHCl3) to give 101 (2.24 g, 67%)
as a white glassy solid: Rf ) 0.21 (3% CH3OH in CHCl3); IR
1
(cm-1) 1666, 1506, 1238; H NMR (DMSO-d6) δ 1.25 (s, 9H),
1.40-1.52 (m, 1H), 1.68-1.82 (m, 1H), 2.23-2.35 (m, 2H),
2.67-2.75 (m, 1H), 2.86-2.94 (m, 1H), 3.11-3.15 (m, 1H),
3.22-3.26 (m, 1H), 3.63-3.73 (m, 1H), 4.15-4.20 (m, 1H),
4.62-4.66 (m, 1H), 4.86 (d, 1H, J ) 12.5), 4.91 (d, 1H, J )
12.5), 6.84 (d, 2H, J ) 8.1), 7.15-7.31 (m, 22H), 7.42 (d, 1H,
J ) 8.5), 7.69 (d, 1H, J ) 8.5), 8.48 (s, 1H). Anal. (C45H49N3O6)
C, H, N.
Cbz-L-Leu -L-Tyr (OtBu )-L-(Tr )glu ta m in ol (102). Carbo-
nyldiimidazole (0.45 g, 2.76 mmol, 1.0 equiv) was added to a
solution of Cbz-L-Leu-OH (0.73 g, 2.76 mmol, 1.0 equiv) in THF
(28 mL) at 23 °C, and the reaction mixture was stirred for 1 h
at that temperature. In a separate flask, a suspension of 101
(2.20 g, 3.03 mmol, 1.1 equiv) and Pd/C (10%, 0.22 g) in CH3-
OH (20 mL) was stirred under a hydrogen atmosphere (bal-
loon) until disappearance of the starting material was indi-
cated by TLC analysis (5 h). The reaction mixture was filtered
through Celite, and the filtrate was concentrated to afford a
white glassy solid. This material was dissolved in THF (5 mL)
and was added to the Cbz-L-Leu-OH solution prepared above.
The reaction mixture was stirred overnight at 23 °C, and then
the volatiles were removed under reduced pressure. Purifica-
tion of the residue by flash column chromatography (gradient
elution, 0 f 3% CH3OH in CHCl3) provided 102 (1.36 g, 58%)
as a white glassy solid: Rf ) 0.15 (3% CH3OH in CHCl3); IR
(cm-1) 1653, 1508, 1236; 1H NMR (DMSO-d6) δ 0.77-0.81 (m,
6H), 1.23 (s, 9H), 1.27-1.60 (m, 4H), 1.70-1.83 (m, 1H), 2.20-
2.24 (m, 2H), 2.72-2.78 (m, 1H), 2.88-2.93 (m, 1H), 3.04-
3.11 (m, 1H), 3.20-3.23 (m, 1H), 3.60-3.68 (m, 1H), 3.95-
4.02 (m, 1H), 4.43-4.48 (m, 1H), 4.59-4.62 (m, 1H), 4.97 (d,
1H, J ) 12.5), 5.02 (d, 1H, J ) 12.5), 6.80 (d, 2H, J ) 7.7),

2830 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
2.79-2.82 (m, 1H), 2.88-2.97 (m, 1H), 3.93-3.97 (m, 1H), 4.11
(q, 2H, J ) 7.0), 4.36-4.41 (m, 1H), 4.45-4.51 (m, 1H), 4.98
(d, 1H, J ) 12.5), 5.04 (d, 1H, J ) 12.5), 5.69 (d, 1H, J ) 15.4),
6.71 (dd, 1H, J ) 15.4, 5.5), 6.77 (s, 1H), 6.96 (d, 2H, J ) 8.1),
7.20 (s, 1H), 7.21 (d, 2H, J ) 7.4), 7.31-7.35 (m, 5H), 7.44 (d,
1H, J ) 7.7), 8.02-8.05 (m, 2H). Anal. (C34H44N4O9) C, H,
N.
bonyldiimidazole (1.02 g, 6.27 mmol, 1.0 equiv) was added to
a solution of Boc-L-Phe(4-I)-OH (2.45 g, 6.27 mmol, 1.0 equiv)
in THF (60 mL) at 23 °C. After 1 h, L-Tr-glutaminol6 (2.58 g,
6.90 mmol, 1.1 equiv) was added, and the reaction mixture
was stirred overnight at 23 °C. The volatiles were removed
under reduced pressure, and the residue was purified by flash
column chromatography (gradient elution, 0 f 3% CH3OH in
CHCl3) to give 107 (3.42 g, 73%) as a white glassy solid: Rf )
0.08 (3% CH3OH in CHCl3); IR (cm-1) 1665, 1491; 1H NMR
(DMSO-d6) δ 1.28 (s, 9H), 1.42-1.49 (m, 1H), 1.64-1.78 (m,
1H), 2.23-2.28 (m, 2H), 2.64-2.72 (m, 1H), 2.85-2.91 (m, 1H),
3.14-3.22 (m, 1H), 3.25-3.31 (m, 1H), 3.63-3.66 (m, 1H),
4.05-4.09 (m, 1H), 4.66-4.69 (m, 1H), 6.85 (d, 1H, J ) 8.5),
7.06 (d, 2H, J ) 8.1), 7.15-7.28 (m, 15H), 7.59-7.64 (m, 3H),
8.49 (s, 1H). Anal. (C38H42IN3O5) C, H, N.
E t h yl 3-[Cb z-L-Leu -L-Tyr -L-(Tr -Gln )]-(E)-p r op en oa t e
(105). Titanium(IV) chloride (0.19 mL, 1.71 mmol, 3.0 equiv)
was added dropwise to a solution of 104 (0.52 g, 0.57 mmol, 1
equiv) in CH2Cl2 (6 mL) at 0 °C. The reaction mixture was
stirred for 1 h at 0 °C and then was partitioned between CH2-
Cl2 (50 mL) and H2O (50 mL). The organic layer was washed
with brine (25 mL), dried over MgSO4, and filtered. The
filtrate was concentrated, and the residue was purified by flash
column chromatography (gradient elution, 0 f 2% CH3OH in
CHCl3) to give 105 (0.40 g, 83%) as a white glassy solid: Rf )
0.18 (7% CH3OH in CHCl3); IR (cm-1) 3325, 1707, 1655, 1516;
1H NMR (DMSO-d6) δ 0.77-0.81 (m, 6H), 1.20 (t, 3H, J ) 7.0),
1.31-1.36 (m, 2H), 1.44-1.53 (m, 1H), 1.60-1.70 (m, 2H),
2.20-2.26 (m, 2H), 2.69-2.74 (m, 1H), 2.82-2.87 (m, 1H),
3.94-3.97 (m, 1H), 4.10 (q, 2H, J ) 7.0), 4.34-4.41 (m, 2H),
4.94 (d, 1H, J ) 12.5), 5.03 (d, 1H, J ) 12.5), 5.69 (d, 1H, J )
15.8), 6.70 (dd, 1H, J ) 15.8, 5.5), 6.95 (d, 2H, J ) 7.5), 7.14-
7.35 (m, 20H), 7.42 (d, 1H, J ) 7.7), 7.85 (d, 1H, J ) 7.7), 8.04
(d, 1H, J ) 8.1), 8.58 (s, 1H), 9.13 (s, 1H). Anal. (C51H56N4O8‚
0.50H2O) C, H, N.
Eth yl 3-{Cbz-L-Leu -L-Tyr [OP (O)(OtBu )2]-L-(Tr -Gln )}-
(E)-p r op en oa te (106). Tetrazole (0.07 g, 0.95 mmol, 2.0
equiv) and di-tert-butyl diethylphosphoramidite (0.14 mL, 0.47
mmol, 1.0 equiv) were added sequentially to a solution of 105
(0.40 g, 0.47 mmol, 1 equiv) in THF (5 mL) at 23 °C. The
reaction mixture was stirred at 23 °C for 2 h and then was
cooled to 0 °C. A solution of m-CPBA (57-86%, 0.095 g, 0.55
mmol, 1.2 equiv) in CH2Cl2 (2 mL) was added via cannula,
and the reaction mixture was stirred for 30 min at 0 °C and
then was concentrated under reduced pressure. The residue
was partitioned between CH2Cl2 (25 mL) and 10% Na2S2O5
(25 mL), and the organic layer was washed with H2O (25 mL)
and brine (25 mL), dried over MgSO4, and concentrated. The
residue was purified by flash column chromatography (gradi-
ent elution, 0 f 2% CH3OH in CHCl3) to give 106 (0.22 g, 45%)
as a white glassy solid: Rf ) 0.15 (5% CH3OH in CHCl3); IR
(cm-1) 1717, 1669, 1508, 1267, 1007; 1H NMR (DMSO-d6) δ
0.76-0.81 (m, 6H), 1.20 (t, 3H, J ) 7.0), 1.25-1.50 (m, 3H),
1.41 (s, 18H), 1.70-1.80 (m, 2H), 2.29-2.39 (m, 2H), 2.81-
2.88 (m, 1H), 2.97-3.04 (m, 1H), 3.97-4.03 (m, 1H), 4.10 (q,
2H, J ) 7.0), 4.41-4.44 (m, 1H), 4.52-4.60 (m, 1H), 4.94 (d,
1H, J ) 12.5), 5.03 (d, 1H, J ) 12.5), 5.72 (d, 1H, J ) 15.8),
6.72 (dd, 1H, J ) 15.8, 5.5), 7.00 (d, 1H, J ) 8.1), 7.14-7.32
(m, 22H), 7.41 (d, 1H, J ) 7.7), 7.95 (d, 1H, J ) 7.7), 8.09 (d,
1H, J ) 7.7), 8.58 (s, 1H).
Boc-L-P h e(4-CN)-L-(Tr )glu ta m in ol (108). Potassium cya-
nide (0.13 g, 2.00 mmol, 2.0 equiv) and Pd(PPh3)4 (0.017 g,
0.015 mmol, 0.015 equiv) were added sequentially to a solution
of 107 (0.75 g, 1.00 mmol, 1 equiv) in THF (10 mL) at 23 °C.
The reaction mixture was then refluxed overnight, and the
solvent was subsequently removed in vacuo. The residue was
purified by flash column chromatography (gradient elution, 0
f 2% CH3OH in CHCl3) to give 108 (0.55 g, 85%) as a white
glassy solid: Rf ) 0.13 (5% CH3OH in CHCl3); IR (cm-1) 2228,
1663, 1491; 1H NMR (DMSO-d6) δ 1.26 (s, 9H), 1.40-1.53 (m,
1H), 1.696-1.69 (m, 1H), 2.23-2.30 (m, 2H), 2.77-2.85 (m,
1H), 2.98-3.05 (m, 1H), 3.17-3.24 (m, 1H), 3.26-3.33 (m, 1H),
3.64-3.70 (m, 1H), 4.14-4.20 (m, 1H), 4.68 (t, 1H, J ) 5.5),
6.93 (d, 1H, J ) 8.5), 7.15-7.28 (m, 15H), 7.44 (d, 1H, J )
8.5), 7.66 (d, 1H, J ) 8.5), 7.73 (d, 2H, J ) 8.1), 8.48 (s, 1H).
Anal. (C39H42N4O5) C, H, N.
Boc-L-P h e(4-ca r boxa m id e)-L-(Tr )glu ta m in ol (109). Hy-
drogen peroxide (30%, 0.45 mL) was added to a solution of 108
(0.49 g, 0.76 mmol) in a 1.5:1 mixture of 3.0 M Na2CO3 and
EtOH (2.5 mL) at 23 °C. The cloudy reaction mixture was
stirred for 24 h at 23 °C, and then additional H2O2 (30%, 0.50
mL) was added. After again stirring overnight at 23 °C, the
reaction mixture was concentrated under reduced pressure.
The residue was partitioned between EtOAc (30 mL) and H2O
(30 mL), and the organic layer was washed with brine (15 mL),
dried over MgSO4, and filtered. The filtrate was concentrated
to afford 109 (0.46 g, 91%) as a white solid which was used
1
without further purification: IR (cm-1) 1663, 1493; H NMR
(DMSO-d6) δ 1.27 (s, 9H), 1.42-1.53 (m, 1H), 1.68-1.76 (m,
1H), 2.23-2.29 (m, 2H), 2.73-2.82 (m, 1H), 2.95-3.04 (m, 1H),
3.14-3.22 (m, 1H), 3.25-3.33 (m, 1H), 3.64-3.73 (m, 1H),
4.12-4.18 (m, 1H), 4.66-4.69 (m, 1H), 6.88 (d, 1H, J ) 8.5),
7.15-7.28 (m, 16H), 7.30 (d, 2H, J ) 8.5), 7.65 (d, 1H, J )
8.5), 7.77 (d, 2H, J ) 8.1), 7.88 (s, br, 1H), 8.48 (s, 1H). Anal.
(C39H44N4O6) C, H, N.
Cbz-L-Leu -L-P h e(4-CN)-L-(Tr )glu ta m in ol (110). Anhy-
drous HCl gas was bubbled through a solution of 108 (0.84 g,
1.30 mmol) in CH2Cl2 (3 mL) at 23 °C for 5 min. The resulting
precipitate was filtered and washed with Et2O (2 × 10 mL) to
give a white crystalline solid. In a separate flask, carbonyl-
diimidazole (0.14 g, 0.84 mmol, 1.0 equiv) was added to a
solution of Cbz-L-Leu-OH (0.22 g, 0.84 mmol, 1.0 equiv) in THF
(9 mL) at 23 °C, and the reaction mixture was stirred for 1 h
at that temperature. A portion of the amine hydrochloride
salt prepared above (0.52 g, 0.89 mmol, 1.05 equiv) and Et3N
(0.13 mL, 0.89 mmol, 1.05 equiv) were added sequentially, and
the reaction mixture was stirred at 23 °C overnight. The
volatiles were removed under reduced pressure, and the
residue was purified by flash column chromatography (gradi-
ent elution, 0 f 2% CH3OH in CHCl3) to give 110 (0.31 g, 47%)
as a white glassy solid: Rf ) 0.18 (5% CH3OH in CHCl3); IR
(cm-1) 2228, 1647, 1520, 1238; 1H NMR (DMSO-d6) δ 0.76 (d,
3H, J ) 6.6), 0.80 (d, 3H, J ) 6.6), 1.22-1.30 (m, 2H), 1.41-
1.46 (m, 2H), 1.67-1.81 (m, 1H), 2.20-2.34 (m, 2H), 2.83-
2.91 (m, 1H), 3.04-3.09 (m, 1H), 3.17-3.24 (m, 1H), 3.28-
3.33 (m, 1H), 3.61-3.70 (m, 1H), 3.89-3.95 (m, 1H), 4.51-
4.59 (m, 1H), 4.65-4.72 (m, 1H), 4.97 (d, 1H, J ) 12.5), 5.02
(d, 1H, J ) 12.5), 7.14-7.33 (m, 16H), 7.39 (d, 2H, J ) 8.1),
E t h yl 3-[Cb z-L-Leu -L-Tyr (OP O3H 2)-L-Gln ]-(E)-p r op e-
n oa te (30). Trifluoroacetic acid (0.4 mL) was added to a
solution of 106 (0.19 g, 0.18 mmol) in CH2Cl2 (4 mL) at 23 °C,
and the reaction mixture was stirred at that temperature for
1 h. The volatiles were then removed under reduced pressure,
and the residue was triturated with a 2:1 mixture of Et2O and
EtOAc (15 mL). The resulting white precipitate was filtered,
washed with Et2O (2 × 10 mL), and then air-dried to give 30
1699, 1655, 1539; 1H NMR (DMSO-d6) δ 0.79 (d, 3H, J ) 6.6),
0.83 (d, 3H, J ) 6.6), 1.21 (t, 3H, J ) 7.0), 1.29-1.36 (m, 2H),
1.48-1.58 (m, 1H), 1.67-1.82 (m, 2H), 2.04-2.08 (m, 2H),
2.80-2.85 (m, 1H), 2.92-3.00 (m, 1H), 3.95-4.03 (m, 1H), 4.11
(q, 2H, J ) 7.0), 4.37-4.50 (m, 2H), 4.97 (d, 1H, J ) 12.5),
5.05 (d, 1H, J ) 12.5), 5.73 (d, 1H, J ) 15.8), 6.74 (dd, 1H, J
) 15.8, 5.5), 6.77 (s, br, 1H), 7.02 (d, 2H, J ) 8.5), 7.15 (d, 2H,
J ) 8.1), 7.21 (s, br, 1H), 7.28-7.34 (m, 5H), 7.45 (d, 1H, J )
7.7), 7.99 (d, 1H, J ) 8.1), 8.07 (d, 1H, J ) 7.7). Anal.
(C32H43N4O11P) C, H, N.
(0.12 g, 95%) as a white solid: mp 190-195 °C dec; IR (cm-1
)
Alter n a te Exa m p le of P r ep a r a tion Meth od D. Syn -
th esis of Eth yl 3-[Cbz-L-Leu -L-P h e(4-CN)-L-Gln ]-(E)-p r o-
p en oa te (34). Boc-L-P h e(4-I)-L-(Tr )glu ta m in ol (107). Car-

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2831
7.66 (d, 2H, J ) 8.1), 7.72 (d, 1H, J ) 8.8), 7.94 (d, 1H, J )
Na2SO4 and were concentrated. Purification of the residue by
flash column chromatography (gradient elution, 30 f 50%
EtOAc in hexanes) provided 113 (1.24 g, 12%) and 11446 (3.35
g, 35%) both as clear oils: 113: Rf ) 0.65 (50% EtOAc in
hexanes); IR (cm-1) 3364, 1740, 1713; 1H NMR (CDCl3) δ 1.43
(s, 9H), 1.84-1.96 (m, 2H), 2.09 (s, 3H), 2.38-2.59 (m, 2H),
4.30-4.32 (m, 1H), 5.11-5.15 (m, 2H), 5.20 (d, 1H, J ) 12.1),
7.27-7.36 (m, 5H). Anal. (C18H25NO5) C, H, N.
Alcoh ol 115. Sodium borohydride (0.175 g, 4.63 mmol, 1.25
equiv) was added to a solution of 113 (1.24 g, 3.70 mmol, 1
equiv) in a 5:1 mixture of THF and H2O (80 mL) at 0 °C. After
1.5 h, the reaction mixture was partitioned between 0.5 M HCl
(150 mL) and a 1:1 mixture of EtOAc and hexanes (2 × 150
mL). The combined organic layers were dried over Na2SO4
and were concentrated. The residue was purified by flash
column chromatography (gradient elution, 40 f 50% EtOAc
in hexanes) to provide 115 (0.93 g, 74%) as a colorless oil: Rf
) 0.43 (50% EtOAc in hexanes); IR (cm-1) 3371, 2972, 1710;
1H NMR (CDCl3, 1:1 mixture of diastereomers) δ 1.13-1.16
(m), 1.32-1.55 (m), 1.43 (s), 1.64-1.99 (m), 3.73-3.81 (m), 4.38
(s, br), 5.13 (d, J ) 12.1), 5.22 (d, J ) 12.1), 7.32-7.40 (m).
Anal. (C18H27NO5) C, H, N.
8.5), 8.53 (s, 1H).
Cbz-L-Leu -L-P h e(4-CN)-L-(Tr )glu ta m in a l (111). o-Io-
doxybenzoic acid24 (0.31 g, 1.10 mmol, 3.0 equiv) was added
to a solution of 110 (0.29 g, 0.37 mmol, 1 equiv) in DMSO (4
mL) at 23 °C. After 1.5 h of stirring at 23 °C, the reaction
mixture was concentrated under reduced pressure. The
residue was twice diluted with CH2Cl2 (10 mL), and the
volatiles were evaporated to remove any residual DMSO. The
residue was then triturated with EtOAc (50 mL), and the
resulting white solid was filtered off. The filtrate was washed
with a 1:1 mixture of 5% Na2S2O3 and 5% NaHCO3 solution
(50 mL), water (50 mL), and brine (50 mL), dried over Na2-
SO4, and concentrated to afford 111 (0.25 g, 86%) as a white
glassy solid. This material was used immediately without
further purification: 1H NMR (DMSO-d6) δ 0.77 (d, 3H, J )
6.6), 0.80 (d, 3H, J ) 6.6), 1.21-1.34 (m, 2H), 1.41-1.50 (m,
1H), 1.58-1.68 (m, 1H), 1.87-1.93 (m, 1H), 2.28-2.38 (m, 2H),
2.88-2.96 (m, 1H), 3.10-3.16 (m, 1H), 3.91-4.00 (m, 2H),
4.61-4.67 (m, 1H), 4.94-5.03 (m, 2H), 7.14-7.38 (m, 21H),
7.42 (d, 2H, J ) 8.1), 7.68 (d, 2H, J ) 8.1), 8.05 (d, 1H, J )
8.5), 8.42 (d, 1H, J ) 7.0), 8.63 (s, 1H), 9.28 (s, 1H).
Eth yl 3-[Cbz-L-Leu -L-P h e(4-CN)-L-(Tr -Gln )]-(E)-p r op e-
n oa te (112). (Carbethoxymethylene)triphenylphosphorane
(0.13 g, 0.37 mmol, 1.2 equiv) was added to a solution of 111
(0.25 g, 0.31 mmol, 1 equiv) in THF (6 mL) at 23 °C, and the
reaction mixture was stirred at that temperature overnight.
The volatiles were then removed under reduced pressure, and
the residue was purified by flash column chromatography
(gradient elution, 0 f 0.75% CH3OH in CHCl3) to give 112
(0.14 g, 53%) as a white glassy solid: Rf ) 0.15 (3% CH3OH in
CHCl3); IR (cm-1) 2228, 1649, 1516; 1H NMR (DMSO-d6) δ 0.77
(d, 3H, J ) 6.6), 0.79 (d, 3H, J ) 6.6), 1.21 (t, 3H, J ) 7.0),
1.24-1.32 (m, 2H), 1.42-1.30 (m, 1H), 1.54-1.75 (m, 2H),
2.20-2.35 (m, 2H), 2.88-2.92 (m, 1H), 2.95-3.08 (m, 1H),
3.92-3.98 (m, 1H), 4.12 (q, 2H, J ) 7.0), 4.32-4.40 (m, 1H),
4.55-4.59 (m, 1H), 4.96 (d, 1H, J ) 12.5), 5.02 (d, 1H, J )
12.5), 5.54 (d, 1H, J ) 15.8), 6.68 (dd, 1H, J ) 15.8, 5.5), 7.13-
7.34 (m, 21H), 7.51 (d, 2H, J ) 8.1), 7.66 (d, 2H, J ) 8.1), 8.07-
8.08 (m, 2H), 8.58 (s, 1H).
Eth yl 3-[Cbz-L-Leu -L-P h e(4-CN)-L-Gln ]-(E)-p r op en oa te
(34). Trifluoroacetic acid (0.4 mL) was added to a solution of
112 (0.12 g, 0.14 mmol) in CH2Cl2 (4 mL) at 23 °C, and the
reaction mixture was stirred at room temperature for 6 h and
then was concentrated under reduced pressure. The residue
was triturated with a 2:1 mixture of Et2O and EtOAc (15 mL),
and the white solid thus obtained was collected by vacuum
filtration, washed with Et2O (2 × 20 mL), and air dried to give
34 (0.063 g, 71%) as a white solid: mp 225-227 °C dec; IR
(cm-1) 2230, 1653, 1535; 1H NMR (DMSO-d6) δ 0.78 (d, 3H, J
) 6.6), 0.82 (d, 3H, J ) 6.6), 1.24 (t, 3H, J ) 7.0), 1.41-1.53
(m, 1H), 1.61-1.80 (m, 4H), 2.02-2.07 (m, 2H), 2.88-2.95 (m,
1H), 3.02-3.07 (m, 1H), 3.95-3.99 (m, 1H), 4.13 (q, 2H, J )
7.0), 4.39-4.42 (m, 1H), 4.53-4.60 (m, 1H), 4.98 (d, 1H, J )
12.5), 5.04 (d, 1H, J ) 12.5), 5.55 (d, 1H, J ) 15.8), 6.71 (dd,
1H, J ) 15.8, 5.5), 6.76 (s, br, 1H), 7.19 (s, br, 1H), 7.24-7.43
(m, 8H), 7.67 (d, 2H, J ) 8.1), 8.08 (d, 1H, J ) 8.5), 8.12 (d,
1H, J ) 8.1). Anal. (C33H41N5O7) C, H, N.
P r ep a r a tion of Am in o Acid 117. Meth yl Keton e 113.
Isobutyl chloroformate (3.94 mL, 30.4 mmol, 1.0 equiv) was
added to a solution of N-R-Boc-L-glutamic acid R-benzyl ester
(10.3 g, 30.4 mmol, 1 equiv) and 4-methylmorpholine (3.34 mL,
30.4 mmol, 1.0 equiv) in CH2Cl2 (150 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 15 min and then was
partitioned between water (150 mL) and CH2Cl2 (2 × 100 mL).
The combined organic layers were dried over Na2SO4 and were
concentrated to provide a colorless oil.
This material was dissolved in THF (200 mL), and the
resulting solution was cooled to -78 °C. Methylmagnesium
bromide (21.7 mL of a 1.4 M solution in toluene/THF, 30.4
mmol, 1.0 equiv) was added, and the reaction mixture was
stirred at -78 °C for 45 min and then was partitioned between
0.5 M HCl (150 mL) and a 1:1 mixture of EtOAc and hexanes
(2 × 150 mL). The combined organic layers were dried over
Silyl Eth er 116. tert-Butyldimethylsilyl trifluoromethane-
sulfonate (0.633 mL, 2.76 mmol, 1.0 equiv) was added to a
solution of alcohol 115 (0.93 g, 2.76 mmol, 1 equiv) and 2,6-
lutidine (0.385 mL, 3.31 mmol, 1.2 equiv) in CH2Cl2 (150 mL)
at -78 °C. The reaction mixture was stirred at -78 °C for 1
h and then was partitioned between 0.5 M HCl (150 mL) and
a 1:1 mixture of EtOAc and hexanes (2 × 150 mL). The
combined organic layers were dried over Na2SO4 and were
concentrated. Flash chromatographic purification of the resi-
due (20% EtOAc in hexanes) afforded 116 (1.07 g, 85%) as a
colorless oil: Rf ) 0.68 (30% EtOAc in hexanes); IR (cm-1
)
1
3367, 2958, 1717; H NMR (CDCl3, 1:1 mixture of diastereo-
mers) δ -0.11 to -0.08 (m), 0.76 (s), 0.96-0.99 (m), 1.20-
1.38 (m), 1.33 (s), 1.48-1.93 (m), 3.61-3.71 (m), 4.23 (s, br),
5.02 (d, J ) 12.1), 5.11 (d, J ) 12.1), 7.16-7.25 (m). Anal.
(C24H41NO5Si) C, H, N.
Am in o Acid 117. A suspension of 116 (0.240 g, 0.531
mmol) and Pd/C (10%, 0.040 g) in EtOAc (30 mL) was stirred
at 23 °C under a hydrogen atmosphere (balloon) for 3 h. The
mixture was filtered through Celite, and the filtrate was
concentrated to afford 117 (0.172 g, 89% crude yield) as a
colorless oil: Rf ) 0.10 (30% EtOAc in hexanes); IR (cm-1) 3321
(br), 2958, 1714; 1H NMR (CDCl3, 1:1 mixture of diastereo-
mers) δ 0.05-0.06 (m), 0.89 (s), 1.13-1.15 (m), 1.45 (s), 1.48-
1.57 (m), 1.66-1.98 (m), 3.82-3.87 (m), 4.28 (s, br), 5.06-5.18
(m). Anal. (C17H35NO5Si) C, H, N.
P r ep a r a tion of Am in o Acid s 121 a n d 124. Boc-L-P h e-
(4-I)-OBn (118). 1,3-Dicyclohexylcarbodiimide (1.20 g, 5.92
mmol, 1.2 equiv) and 4-(dimethylamino)pyridine (0.239 g, 1.96
mmol, 0.40 equiv) were added sequentially to a solution Boc-
L-Phe(4-I)-OH (1.93 g, 4.9 mmol, 1 equiv) and benzyl alcohol
(0.61 mL, 5.92 mmol, 1.2 equiv) in CH2Cl2 (100 mL) at 23 °C.
The reaction mixture was stirred at 23 °C for 6 h and then
was filtered. The filtrate was partitioned between H2O (100
mL) and CH2Cl2 (2 × 100 mL), and the combined organic
layers were dried over Na2SO4 and were concentrated. The
residue was purified by flash column chromatography (10%
EtOAc in hexanes) to afford 118 (2.05 g, 87%) as a white
solid: mp 111-112 °C; Rf ) 0.72 (50% EtOAc in hexanes); IR
(cm-1) 2975, 1712, 1487, 1124; 1H NMR (CDCl3) δ 1.42 (s, 9H),
2.95-3.06 (m, 2H), 4.57-4.63 (m, 1H), 4.98 (d, 1H, J ) 7.5),
5.06-5.20 (m, 2H), 6.75 (d, 2H, J ) 7.8), 7.26-7.29 (m, 2H),
7.35-7.40 (m, 3H), 7.52 (d, 2H, J ) 8.1). Anal. (C21H24INO4)
C, H, N.
Boc-L-P h e(4-CHdCH2)-OBn (119). Tributyl(vinyl)tin (1.60
mL, 5.32 mmol, 1.1 equiv), several crystals of 2,6-di-tert-butyl-
4-methylphenol, and Pd(PPh3)4 (0.280 g, 0.242 mmol, 0.05
equiv) were added sequentially to a solution of 118 (2.33 g,
4.84 mmol, 1 equiv) in 1,4-dioxane (40 mL) at 23 °C. The
reaction mixture was stirred at 80 °C for 6 h, cooled to 23 °C,
and partitioned between H2O (100 mL) and CH2Cl2 (2 × 100
mL). The combined organic layers were dried over Na2SO4

2832 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
and were concentrated. The residue was purified by flash
column chromatography (10% EtOAc in hexanes) to afford 119
(1.53 g, 83%) as a dark brown oil: Rf ) 0.72 (50% EtOAc in
hexane); IR (cm-1) 2969, 1711, 1495, 1165; 1H NMR (CDCl3) δ
1.41 (s, 9H), 3.06-3.08 (m, 2H), 4.60-4.64 (m, 1H), 4.96-4.99
(d, 1H, J ) 7.8), 5.07-5.19 (m, 2H), 5.22 (d, 1H, J ) 5.7), 5.68-
5.74 (d, 1H, J ) 17.4), 6.67 (dd, 1H, J ) 17.4, 10.8), 6.99 (d,
2H, J ) 7.8), 7.26-7.29 (m, 4H), 7.32-7.37 (m, 3H).
Boc-L-P h e(4-CH2CH2OH)-OBn (120). A solution of BH3‚
THF (1.0 M in THF, 0.40 mL, 0.40 mmol, 0.33 equiv) was
added dropwise to a solution of 119 (0.467 g, 1.21 mmol, 1
equiv) in THF (2 mL) at 0 °C. The reaction mixture was
stirred at 23 °C for 2 h and then H2O (3 mL) and NaBO3‚4H2O
(0.186 g, 1.21 mmol, 1.0 equiv) were added. The resulting
mixture was stirred at 23 °C for 1 h and then was partitioned
between H2O (10 mL) and Et2O (4 × 10 mL). The combined
organic layers were washed with brine (50 mL), dried over Na2-
SO4, and concentrated. The residue was purified by flash
column chromatography (50% EtOAc in hexanes) to afford 120
(0.238 g, 49%) as a yellow oil: Rf ) 0.30 (50% EtOAc in
hexanes); IR (cm-1) 3376, 1709, 1165; 1H NMR (CDCl3) δ 1.37
(s, 9H), 2.82 (t, 2H, J ) 6.6), 3.05 (m, 2H), 3.83 (q, 2H, J )
6.3), 4.58-4.63 (m, 1H), 4.97 (d, 1H, J ) 8.7), 5.08-5.20 (m,
2H), 6.98 (d, 2H, J ) 7.8), 7.09 (d, 2H, J ) 7.5), 7.30-7.38 (m,
5H). Anal. (C23H29NO5‚0.75H2O) C, H, N.
Boc-L-P h e(4-CH 2CH2OH)-OH (121). Sodium hydroxide
(2.0 M in H2O, 1.76 mL, 3.52 mmol, 8.0 equiv) was added to a
solution of 120 (0.176 g, 0.44 mmol, 1 equiv) in CH3OH (5 mL)
at 23 °C. The reaction mixture was stirred at 23 °C for 6 h
and then was partitioned between 10% KHSO4 (50 mL) and
CH2Cl2 (3 × 50 mL). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4, and concen-
trated to give crude 121 (0.121 g, 89%). This material was
used without further purification: 1H NMR (CDCl3) δ 1.41 (s,
9H), 2.81 (t, 2H, J ) 6.6), 3.05-3.17 (m, 2H), 3.80-3.87 (m,
2H), 4.57-4.60 (m, 1H), 4.96-4.99 (d, 1H, J ) 8.7), 7.05-7.16
(m, 2H), 7.26-7.37 (m, 2H).
dried over Na2SO4 and were concentrated. Purification of the
residue by flash column chromatography (25% EtOAc in
hexanes) provided 123 (0.298 g, 74%) as a white foam: Rf )
0.88 (50% EtOAc in hexanes); IR (cm-1) 3378, 1699, 1506, 1184;
1H NMR (CDCl3) δ 1.41 (s, 9H), 1.68 (m, 1H), 3.07-3.09 (m,
2H), 4.58-4.60 (m, 1H), 4.64 (d, 2H, J ) 4.5), 4.97 (d, 1H, J )
8.4), 5.08-5.20 (m, 2H), 7.02 (d, 2H, J ) 7.8), 7.22 (d, 2H, J )
7.8), 7.28-7.37 (m, 5H). Anal. (C22H27NO5) C, H, N.
Boc-L-P h e(4-CH2OH)-OH (124). A suspension of 123 (0.28
g, 0.73 mmol) and Pd/C (0.060 g) in EtOAc (10 mL) was stirred
at 23 °C under an H2 atmosphere (balloon) for 6 h. The
reaction mixture was filtered thought Celite, and the filtrate
was concentrated to afford crude 124 (0.20 g, 93%) as a
colorless oil. This material was used without further puri-
fication: 1H NMR (CDCl3) δ 1.39 (s, 9H), 3.13 (m, 2H), 4.59
(m, 1H), 4.66 (s, 2H), 4.97-5.00 (d, 1H J ) 8.1), 7.19 (d, 2H,
J ) 8.7), 7.28 (d, 2H, J ) 8.7).
P r otein -Liga n d Cr ysta l Str u ctu r e Deter m in a tion .
Serotype 2 human rhinovirus 3C protease was incubated with
a 3-fold molar excess of compound 75, and the complex was
concentrated to a final protein concentration of 10 mg/mL.
Equal volumes of the protein/ligand stock solution and a 2.0
M solution of ammonium sulfate buffered with 100 mM ADA
pH 6.5 were mixed and allowed to incubate at 21 °C for 1 h.
This solution was then passed through a Centrex 0.45 µM filter
and used immediately for crystallization experiments. Crys-
tallization was carried out at 13 °C using a hanging drop vapor
diffusion method in which 3 µL drops of the protein/inhibitor
complex were mixed with an equal volume of reservoir solution
on plastic coverslips and sealed over individual reservoir wells
containing a solution of 0.5 M ammonium sulfate, 1.5 M Na/K
phosphate, and 100 mM ADA pH 6.6 buffer. Following a 4 h
equilibration at 13 °C, a microcrystal of HRV-2 3CP-31 was
added to each drop using a Hampton microneedle to induce
growth of HRV-2 3CP-75 crystals. Crystals of the complex
typically reached dimensions of 0.5 × 0.35 × 0.15 mm in 7-10
days.
A single crystal measuring 0.3 × 0.2 × 0.1 mm (space group
P21212; a ) 61.56, b ) 77.68, c ) 33.95 Å) was prepared for
low-temperature data collection by serial transfer to artificial
mother liquor solutions of increasing glycerol concentration.
When fully equilibrated against an artificial mother liquor
containing 25% glycerol, the crystal was flash frozen. X-ray
diffraction data were collected at -170 °C using a MAR
imaging plate and processed with DENZO.47 The resulting
data were 95% complete to a resolution of 1.9 Å with R(sym)
) 3.9%.
Protein atomic coordinates from the HRV-2 3CP-3 structure
determination1 were used to initiate rigid body refinement in
X-PLOR48 followed by simulated annealing and conjugate
gradient minimization protocols. Placement of the inhibitor
75, addition of ordered solvent, and further rounds of refine-
ment proceeded as described for the HRV-2 3CP-3 complex.1
The final R factor was 21.2% (11 618 reflections with F >2σ-
(F)). The root-mean-square deviations from ideal bond lengths
and angles were 0.017 Å and 2.9°, respectively. The final
model consisted of all atoms for residues 1-180 (excluding side
chains for residues 12, 55, and 65) plus 111 water molecules.
Boc-L-P h e(4-CHO)-OBn (122). 4-Methylmorpholine N-
oxide (0.22 g, 1.88 mmol, 1.1 equiv) and OsO4 (0.02 M solution
in H2O, 2.57 mL, 0.051 mmol, 0.03 equiv) were added
sequentially to a solution of 119 (0.654 g, 1.71 mmol, 1 equiv)
in an 8:1 mixture of acetone and H2O (9 mL) at 23 °C. The
reaction mixture was stirred at 23 °C for 3 h, and then Na2S2O5
(1 g) and H2O (50 mL) were added carefully. The resulting
mixture was stirred at 23 °C for 20 min and then was
partitioned between EtOAc (2 × 100 mL) and H2O (50 mL).
The combined organic layers were dried over Na2SO4 and were
concentrated. The residue was purified by flash column
chromatography (10% EtOAc in hexanes) to afford the corre-
sponding diol (0.44 g, 61%) as an off-white foam: Rf ) 0.12
(50% EtOAc in hexanes); IR (cm-1) 3376, 1706, 1500, 1165;
1H NMR (CDCl3) δ 1.40 (s, 9H), 2.21 (s, br, 1H), 2.61 (s, br,
1H), 3.00-3.13 (m, 2H), 3.56-3.64 (m, 2H), 3.68-3.75 (m, 1H),
4.58-4.64 (m, 1H), 4.74-4.77 (m, 1H), 5.00 (d, 2H, J ) 7.8),
5.08-5.19 (m, 2H), 7.02 (d, 2H, J ) 7.5), 7.09-7.26 (m, 3H),
7.29-7.39 (m, 4H).
This material (0.44 g, 1.05 mmol, 1 equiv) was dissolved in
Et2O (10 mL) at 23 °C, and a solution of NaIO4 (0.247 g, 1.15
mmol, 1.1 equiv) in H2O (2 mL) was added. The reaction
mixture was stirred at 23 °C for 2 h and then was partitioned
between H2O (50 mL) and a 1:1 mixture of EtOAc and hexanes
(2 × 50 mL). The combined organic layers were dried over
Na2SO4 and were concentrated to give crude 122 as a colorless
oil. This material was used without further purification: IR
(cm-1) 1699, 1507, 1167; 1H NMR (CDCl3) δ 1.41 (s, 9H), 3.08-
3.28 (m, 2H), 4.63-4.68 (m, 1H), 5.02-5.04 (d, 1H, J ) 7.8),
5.07-5.21 (m, 2H), 7.18 (d, 2H, J ) 8.1), 7.30-7.41 (m, 5H),
7.72 (d, 2H, J ) 8.1), 9.96 (s, 1H).
Ack n ow led gm en t. We are grateful for many helpful
discussions throughout the course of this work with
Prof. Larry Overman. We also thank Dr. Kim Albizati,
Dr. Srinivasan Babu, and Terence Moran for the large-
scale preparation of several intermediates, Dr. Donald
Skalitzky for optimizing the synthesis of compound 98,
Sogole Bahmanyar for the synthesis of compound 78,
and Dr. Xinjun Hou for assistance with the preparation
of Figure 2.
Boc-L-P h e(4-CH2OH)-OBn (123). Sodium borohydride
(0.040 g, 1.05 mmol, 1.0 equiv) was added to a solution of 122
(0.40 g, 1.05 mmol, 1 equiv) in EtOH (10 mL) at 0 °C. The
reaction mixture was stirred at 0 °C for 20 min and then was
partitioned between H2O (50 mL) and a 1:1 mixture of EtOAc
and hexanes (2 × 50 mL). The combined organic layers were
Refer en ces
(1) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman, 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.;

Human Rhinovirus 3C Protease Inhibitors. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2833
Brown, E. L.; Binford, S. L.; Harr, J . E. V.; DeLisle, D. M.;
Worland, S. T. Structure-Based Design, Synthesis, and Biological
Evaluation of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 1. Michael Acceptor Structure-Activity Studies. J .
Med. Chem. 1998, 41, 2806-2818.
(14) Smith, R. A.; Copp, L. J .; Donnelly, S. L.; Spencer, R. W.; Krantz,
A. Inhibition of Cathepsin B by Peptidyl Aldehydes and Ke-
tones: Slow-Binding Behavior of a Trifluoromethyl Ketone.
Biochemistry 1988, 27, 6568-6573.
(15) Gibson, F. S.; Bergmeier, S. C.; Rapoport, H. Selective Removal
of an N-BOC Protecting Group in the Presence of a tert-Butyl
Ester and Other Acid-Sensitive Groups. J . Org. Chem. 1994, 59,
3216-3218.
(16) Triisopropylsilane could be omitted from the deprotection reac-
tions without adversely affecting the product yields.
(17) Ho, P. T.; Ngu, K.-y. An Effective Synthesis of N-(9-Fluorenyl-
methyloxy-carbonyl) R-Amino Aldehydes from S-Benzyl
Thioesters. J . Org. Chem. 1993, 58, 2313-2316.
(18) Schlessinger, R. H.; Nugent, R. A. Total Synthesis of Racemic
Verrucarol. J . Am. Chem. Soc. 1982, 104, 1116-1118.
(19) Sekiya, A.; Ishikawa, N. Facile Synthesis of Aryl Cyanides From
Iodides Catalyzed by Palladium Triphenylphosphine Complex.
Chem. Lett. 1975, 277-278.
(20) Sakamoto, T.; Kondo, Y.; Yamanaka, H. Condensed Heteroaro-
matic Ring Systems. XIV. Cyclization of ortho-Substituted
R-Ethoxycinnamates to Some Heteroaromatics. Heterocycles
1988, 27, 453-456.
(21) Ohta, T.; Hosoi, A.; Kimura, T.; Nozoe, S. Direct Chain Elonga-
tion of N-Carbamoylpyroglutamate. An Efficient Synthesis of
(-)-Pyrrolidine-2,5-Dicarboxylic Acid. Chem. Lett. 1987, 2091-
2094.
(2) (a) Cordingley, M. G.; Callahan, P. L.; Sardana, V. V.; Garsky,
V. M.; Colonno, R. J . Substrate Requirements of Human Rhi-
novirus 3C Protease for Peptide Cleavage in Vitro. J . Biol. Chem.
1990, 265, 9062-9065. (b) Orr, D. C.; Long, A. C.; Kay, J .; Dunn,
B. M.; Cameron, J . M. Hydrolysis of a Series of Synthetic Peptide
Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned
and Expressed in Escherichia coli. J . Gen. Virol. 1989, 70, 2931-
2942. (c) Cordingley, M. G.; Register, R. B.; Callahan, P. L.;
Garsky, V. M.; Colonno, R. J . Cleavage of Small Peptides In Vitro
by Human Rhinovirus 14 3C Protease Expressed in Escherichia
coli. J . Virol. 1989, 63, 5037-5045.
(3) The nomenclature used for describing the individual amino acid
residues of a peptide substrate (P2, P1, P1′, P2′, etc.) and the
corresponding enzyme subsites (S2, S1, S1′, S2′, etc.) is described
in the following: Schechter, I.; Berger, A. On the Size of the
Active Site in Proteases. I. Papain. Biochem. Biophys. Res.
Commun. 1967, 27, 157-162.
(4) In the discussion of structure-activity relationships, the notation
“3CP” indicates 3C protease derived from HRV-14 unless
otherwise specified.
(5) Shepherd, T. A.; Cox, G. A.; McKinney, E.; Tang, J .; Wakulchik,
M.; Zimmerman, R. E.; Villarreal, E. C. Small Peptidic Aldehyde
Inhibitors of Human Rhinovirus 3C Protease. Bioorg. Med.
Chem. Lett. 1996, 6, 2893-2896.
(6) Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin, Y.;
Fuhrman, S. A.; Matthews, D. A.; Hendrickson, T. F.; Love, R.
A.; Patick, A. K.; Meador, J . W., III; Ferre, R. A.; Brown, E. L.;
Ford, C. E.; Binford, S. L.; Worland, S. T. Tripeptide Aldehyde
Inhibitors of Human Rhinovirus 3C Protease: The Design,
Synthesis, Biological Evaluation and Cocrystal Structure Solu-
tion of P1 Glutamine Isosteric Replacements. J . Med. Chem.
1998, 41, 2786-2805.
(7) (a) Lee, W.-M.; Wang, W.; Rueckert, R. R. Complete Sequence
of the RNA Genome of Human Rhinovirus 16, a Clinically Useful
Common Cold Virus Belonging to the ICAM-1 Receptor Group.
Virus Genes 1994, 9, 177-181. (b) Aschauer, B.; Werner, G.;
McCray, J .; Rosenwirth, B.; Bachmayer, H. Biologically Active
Protease 3C of Human Rhinovirus 1A is Expressed from a
Cloned cDNA Segmant in Escherichia coli. Virology 1991, 184,
587-594. (c) Hughes, P. J .; North, C.; J ellis, C. H.; Minor, P.
D.; Stanway, G. The Nucleotide Sequence of Human Rhinovirus
1B: Molecular Relationships within the Rhinovirus Genus. J .
Gen. Virol. 1988, 69, 49-58. (d) Duechler, M.; Skern, T.;
Sommergruber, W.; Neubauer, C.; Gruendler, P.; Fogy, I.; Blaas,
D.; Kuechler, E. Evolutionary Relationships Within the Human
Rhinovirus Genus: Comparison of Serotypes 89, 2, and 14. Proc.
Natl. Acad. Sci. U.S.A. 1987, 84, 2605-2609. (e) Werner, G.;
Rosenwirth, B.; Bauer, E.; Siefert, J .-M.; Werner, F.-J .; Besemer,
J . Molecular Cloning and Sequence Determination of the Ge-
nomic Regions Encoding Protease and Genome-Linked Protein
of Three Picornaviruses. J . Virol. 1986, 57, 1084-1093. (f) Skern,
T.; Sommergruber, W.; Blaas, D.; Gruendler, P.; Fraundorfer,
F.; Pieler, C.; Fogy, I.; Kuechler, E. Human Rhinovirus 2:
Complete Nucleotide Sequence and Proteolytic Processing Sig-
nals in the Capsid Protein Region. Nucleic Acids Res. 1985, 13,
2111-2126. (g) Callahan, P. L.; Mizutani, S.; Colonno, R. J .
Molecular Cloning and Complete Sequence Determination of
RNA Genome of Human Rhinovirus Type 14. Proc. Natl. Acad.
Sci. U.S.A. 1985, 82, 732-736. (h) Stanway, G.; Hughes, P. J .;
Mountford, R. C.; Minor, P. D.; Almond, J . W. The complete
Nucleotide Sequence of a Common Cold Virus: Human Rhi-
novirus 14. Nucleic Acids Res. 1984, 12, 7859-7875.
(22) The preparation of ketone 113 from N-R-Boc-L-glutamic acid
R-benzyl ester has not been optimized.
(23) Compound 8 was prepared from 9 by oxidation with IBX.24
(24) Frigerio, M.; Santagostino, M.; Sputore, S.; Palmisano, G.
Oxidation of Alcohols with o-Iodoxybenzoic Acid (IBX) in
DMSO: A New Insight into an Old Hypervalent Iodine Reagent.
J . Org. Chem. 1995, 60, 7272-7276.
(25) (a) McKean, D. R.; Parrinello, G.; Renaldo, A. F.; Stille, J . K.
Synthesis of Functionalized Styrenes via Palladium-Catalyzed
Coupling of Aryl Bromides with Vinyl Tin Reagents. J . Org.
Chem. 1987, 52, 422-424. (b) Mitchell, T. N. Palladium-
Catalysed Reactions of Organotin Compounds. Synthesis 1992,
803-815,
(26) (a) VanRheenen, V.; Kelly, R. C.; Cha, D. Y. An Improved
Catalytic OsO4 Oxidation of Olefins to cis-1,2-Glycols Using
Tertiary Amine Oxides as the Oxidant. Tetrahedron Lett. 1976,
1973-1976. (b) Pappo, R.; Allen, D. S., J r.; Lemieux, R. U.;
J ohnson, W. S. Osmium Tetroxide-Catalyzed Periodate Oxida-
tion of Olefinic Bonds. J . Org. Chem. 1956, 21, 478-479.
(27) Methylation of 123 (CH3I, Ag2O) and subsequent debenzylation
provided the carboxylic acid (not shown) required for the
preparation of compound 32.
(28) Still, W. C.; Kahn, M.; Mitra, A. Rapid Chromatographic
Technique for Preparative Separations with Moderate Resolu-
tion. J . Org. Chem. 1978, 43, 2923-2925.
(29) The tripeptide aldehydes required for the synthesis of compounds
10 and 12 were prepared as described in ref 6. The tripeptide
aldehyde required for the synthesis of compound 11 was
prepared from Cbz-L-Leu-L-Phe-L-(SO)methioninol6 by the fol-
lowing sequence: (i) m-CPBA; (ii) IBX.24
(30) Compound 13 was prepared from Cbx-L-Leu-L-Phe-L-(N-Boc-
aminoalaninal) (ref 6) by the following sequence: (i)
Ph3PdCHCO2Et, (ii) TFA, (iii) [(4-NO2)PhO]2CO, NH3.31
(31) Izdebski, J .; Pawlak, D. A New Convenient Method for the
Synthesis of Symmetrical and Unsymmetrical N,N′-Disubsti-
tuted Ureas. Synthesis 1989, 423-425.
(32) The olefin intermediate required for the preparation of compound
15 was prepared from tert-butyl (S)-(-)-4-formyl-2,2-dimethyl-
3-oxazolidine-carboxylate by reaction with (carbethoxymethyl-
ene)triphenylphosphorane. Inhibitor 14 was prepared from
compound 15 by the following sequence: (i) chloroacetyl isocy-
anate; (ii) NaHCO3, H2O, THF, EtOH. See: Richter, H. G. F.;
Angehrn, P.; Hubschwerlen, C.; Kania, M.; Page, M. G. P.;
Specklin, J .-L.; Winkler, F. K. Design, Synthesis, and Evaluation
of 2â-Alkenyl Penam Sulfone Acids as Inhibitors of â-Lacta-
mases. J . Med. Chem. 1996, 39, 3712-3722.
(33) The benzyl ester of the desired N-Cbz-protected amino acid was
obtained by a combination of the following: (a) Nakajima, K.;
Oda, H.; Okawa, K. Studies on 2-Aziridinecarboxylic Acid. IX.
Convenient Synthesis of Optically Active S-Alkylcysteine, threo-
S-Alkyl-â-methylcysteine, and Lanthionine Derivatives via the
Ring-opening Reaction of Aziridine by Several Thiols. Bull.
Chem. Soc. J pn. 1983, 56, 520-522. (b) Willems, J . G. H.;
Hersmis, M. C.; de Gelder, R.; Smits, J . M. M.; Hammink, J . B.;
Dommerholt, F. J .; Thijs, L.; Zwanenburg, B. Synthesis and
Crystal Structure of Enantiopure N-Tritylaziridin-2-yl-metha-
nols from L-Serine and L-Threonine. J . Chem. Soc., Perkin Trans.
1 1997, 963-967. The free carboxylic acid was prepared by the
following sequence: (i) CH2dCHCH2OH, cat. Ti(O-i-Pr)4, 100 °C;
(ii) Morpholine, Pd(PPh3)4.
(8) Matthews, D. A.; Smith, W. W.; Ferre, R. A.; Condon, B.;
Budahazi, G.; Sisson, W.; Villafranca, J . E.; J anson, C. A.;
McElroy, H. E.; Gribskov, C. L.; Worland, S. Structure of Human
Rhinovirus 3C Protease Reveals a Trypsin-like Polypeptide Fold,
RNA-Binding Site, and Means for Cleaving Precursor Polypro-
tein. Cell 1994, 77, 761-771.
(9) In the discussion of the X-ray crystal structure, the notation
“3CP” indicates 3C protease derived from HRV-2 unless other-
wise specified. This protein provided diffraction quality crystals
more routinely than the serotype 14-derived protease. The
inhibitor-binding regions of the two enzymes do not differ
significantly: Matthews, D. A.; et al. manuscript in preparation.
(10) Sieber, P.; Riniker, B. Protection of Carboxamide Functions by
the Trityl Residue. Application to Peptide Synthesis. Tetrahe-
dron Lett. 1991, 32, 739-742.
(11) The hydroxyl groups present in the amino acid residues required
for the preparation of compounds 26 and 56 were protected as
tert-butyl ether derivatives during peptide synthesis.
(12) The synthesis of compound 3 is described in ref 1.
(13) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as Ef-
fective Acylating Agents. Tetrahedron Lett. 1981, 22, 3815-3818.

2834 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Dragovich et al.
(34) Marzoni, G.; Kaldor, S. W.; Trippe, A. J .; Shamblin, B. M.; Fritz,
J . E. A Convenient, Large Scale Synthesis of N-CBZ-(S-Phenyl)-
L-Cysteine. Synth. Commun. 1995, 25, 2475-2482.
(35) The free amino acid was obtained as described in: Shao, H.;
Goodman, M. An Enantiomeric Synthesis of allo-Threonines and
â-Hydroxyvalines. J . Org. Chem. 1996, 61, 2582-2583. The
N-Cbz-protected amino acid was prepared as follows: CbzCl,
NaHCO3, i-PrOH.
(36) The tert-butyl esters of the amino acids required for the
preparation of 61 and 62 were synthesized by the reductive
coupling of morpholine with the aldehydes derived from N-R-
Cbz-L-aspartic acid R-tert-butyl ester and N-R-Cbz-L-glutamic
acid R-tert-butyl ester, respectively. See: (a) Valerio, R. M.;
Alewood, P. F.; J ohns, R. B. Synthesis of Optically Active 2-(tert-
Butyloxycarbonylamino)-4-dialkoxyphosphorylbutanoate Pro-
tected Isosteres of O-Phosphonoserine for Peptide Synthesis.
Synthesis 1988, 786-789. (b) Faust, J .; Schreiber, K. Der
Normalisierungsfaktor fu¨r die Tomatenmutante chloronerva;
Synthese des (S)-Piperidin-2-carbonsa¨ureanalogons von Nicoti-
anamin. Z. Chem. 1989, 29, 20-21. The free carboxylic acids
were prepared by treatment of the tert-butyl esters with TFA.
(37) The amino acids required for the synthesis of compounds 66,
67, and 70 were prepared by addition of 2-methylbenzyl alcohol,
2-chlorobenzyl alcohol, or cyclohexanol, respectively, to (S)-(-)-
2-isocyanato-4-methylvaleric acid methyl ester (benzene, 50 °C)
followed by basic hydrolysis (LiOH, CH3OH, H2O).
D. R.; Horwell, D. C.; Hunter, J . C.; Martin, K.; Pritchard, M.
C.; Rahman, S. S.; Richardson, R. S.; Roberts, E. Rationally
Designed ‘Dipeptoid’ Analogues of Cholecystokinin (CCK): N-
Terminal Structure-Affinity Relationships of R-Methyl-Tryp-
tophan Derivatives. Eur. J . Med. Chem. 1993, 28, 37-45.
(40) The amino acid utilized in the preparation of compound 84 was
synthesized by coupling of H2N-L-Leu-OtBu‚HCl with â-propi-
olactone (AlMe3) and subsequent acidic deprotection (TFA).
(41) Cbz-L-Leu-OH was coupled with H2N-L-His-OMe‚2HCl or H2N-
L-His(1-Me)-OMe‚HCl (EDC, HOBt) and the resulting dipeptides
were hydrolyzed under basic conditions (NaOH, CH3OH, H2O).
(42) N-R-Cbz-L-glutamic acid γ-tert-butyl ester was esterified with
2-(trimethylsilyl)ethanol (EDC). The Cbz group present in the
resulting di-ester was removed (H2, Pd/C), and the free amine
was coupled with Cbz-L-Leu-OH (EDC, HOBt, 4-methylmorpho-
line). Removal of the 2-(trimethylsilyl)ethyl moiety (Bu4NF)
provided the desired dipeptide.
(43) (S)-3-Azido-4,4-dimethyldihydrofuran-2-one44 was coupled with
H2N-L-Phe-OtBu‚HCl (AlMe3), and the product thus obtained
was converted to the desired dipeptide by the following se-
quence: (i) H2, Pd/C; (ii) CbzCl, 4-methylmorpholine; (iii) TFA.
(44) Freskos, J . N. Use of R-Pantolactone in the Synthesis of L-tert-
Leucine Derivatives. Synth. Commun. 1994, 24, 557-563.
(45) The dipeptide required for the preparation of 71 was synthesized
by coupling Boc-L-Leu-OSu with H2N-L-Phe-OBn‚HCl [(i-Pr)2-
NEt] and subsequent debenzylation (H2, Pd/C).
(38) The amino acids required for the preparation of compounds 69
and 83 were synthesized by reaction of methyl chloroformate
(Et3N) or glycolic acid (EDC, HOBt, 4-methylmorpholine),
respectively, with H2N-L-Leu-OtBu‚HCl and subsequent acidic
deprotection (TFA).
(39) The amino acids required for the synthesis of compounds 72-
75 were prepared by reaction of the appropriate chlorothiolfor-
mate with H2N-L-Leu-OtBu‚HCl (Et3N) and subsequent acidic
deprotection (TFA). Benzyl chlorothiolformate and cyclopentyl
chlorothiolformate were prepared in a manner similar to that
described in the following: Eden, J . M.; Higginbottom, M.; Hill,
(46) Carpino, L. A.; Mansour, E.-L. M. E. Protected â- and γ-Aspartic
and Glutamic Acid Fluorides. J . Org. Chem. 1992, 57, 6371-
6373.
(47) Otwinowski, Z. DENZO in Data Collection and Processing. In
Proceedings of the CCP4 Study Weekend; Sawyer, L., Isaacs, N.,
Bailey, S., Eds.; SERC Daresbury Laboratory: Warrington, UK,
1993; pp 55-62.
(48) X-PLOR, version 3.1. Bru¨nger A. T. X-PLOR v3.1 Manual; Yale
University Press: New Haven, CT, 1992.
J M9800696