Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics

Article abstract of DOI:10.1021/jm980537b

The structure-based design, chemical synthesis, and biological evaluation of various ketomethylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and an e

Full text of DOI:10.1021/jm980537b

J . Med. Chem. 1999, 42, 1203-1212
1203
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. 3. Str u ctu r e-Activity Stu d ies of
Ketom eth ylen e-Con ta in in g P ep tid om im etics
Peter S. Dragovich,* Thomas J . Prins, Ru Zhou, Shella A. Fuhrman, Amy K. Patick, David A. Matthews,
Clifford E. Ford, J ames W. Meador III, Rose Ann Ferre, and Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121
Received September 25, 1998
The structure-based design, chemical synthesis, and biological evaluation of various keto-
methylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described.
These compounds are comprised of a peptidomimetic binding determinant and an ethyl
propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active
site cysteine residue of the 3C enzyme. The ketomethylene-containing inhibitors typically
display slightly reduced 3CP inhibition activity relative to the corresponding peptide-derived
molecules, but they also exhibit significantly improved antiviral properties. Optimization of
the ketomethylene-containing compounds is shown to provide several highly active 3C protease
inhibitors which function as potent antirhinoviral agents (EC90 ) <1 µM) against multiple
virus serotypes in cell culture.
In tr od u ction
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most significant
cause of the common cold.1,2 Recently, we described the
discovery and development of novel HRV 3C protease
(3CP) inhibitors which display in vitro antiviral activity
against several rhinovirus serotypes.3,4 These inhibitors
are comprised of a substrate-derived tripeptide binding
determinant which provides affinity for the target
protease and a Michael acceptor moiety which irrevers-
ibly forms a covalent adduct with the active site cysteine
residue of the 3C enzyme (e.g., compound 1, Figure
1).5-8 Although optimization of these tripeptides af-
forded relatively active antirhinoviral agents, we also
sought to investigate related peptidomimetic 3CP in-
hibitors which might exhibit improved pharmacological
properties (e.g., greater metabolic stability, improved
oral bioavailability, etc.).9 The results of our efforts to
develop one series of peptidomimetic 3CP inhibitors are
described below.
In h ibitor Design a n d Str u ctu r e-Activity
Stu d ies
The replacement of a backbone amide moiety present
in peptide-derived enzyme inhibitors with a ketometh-
ylene isostere often affords peptidomimetic compounds
with inhibition activities similar to the parent molecules
(Chart 1).10 We therefore wished to introduce such an
isostere into a representative tripeptidyl 3CP inhibitor
F igu r e 1. Design of peptidomimetic HRV 3CP inhibitors.
(compound 1, Figure 1). Analysis of the HRV-2 3CP-1
crystal structure3 indicated that the backbone amide
NH linking the P2 Phe and P3 Leu amino acid residues
of 1 was highly solvent-exposed (Figure 2).6 In addition,
comparison with the uncomplexed 3CP crystal struc-
ture11 suggested that the 3CP serine residue which
formed a hydrogen bond with the P2-P3 amide NH of 1
was somewhat conformationally mobile (Ser-128, Figure
2). In contrast, the inhibitor P1-P2 and P3-P4 amide
NH’s were only slightly solvent-exposed and formed
hydrogen bonds with relatively stationary 3CP residues.
Replacement of the P2-P3 amide linkage with a keto-
methylene dipeptide isostere was therefore anticipated
to have less of an impact on inhibitor 3CP affinity than
substitution of either of the two other amide bonds
present in the tripeptidyl backbone of 1.
10.1021/jm980537b CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

1204 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Accordingly, an N-terminal S-cyclopentyl thiocarbam-
ate moiety, which afforded the most potent antiviral
activity in the tripeptidyl 3CP inhibitor series,4 was
evaluated with several different P2-P3 ketomethylene
dipeptide isosteres (Table 2). Inhibitors containing a
Leu-Phe mimetic (5-7) were highly active antirhinovi-
ral agents when tested against HRV-14 in cell culture,
but they displayed significantly less potent antiviral
properties against several other rhinovirus serotypes.
In contrast, compounds incorporating a Val-Phe dipep-
tide isostere (8-11) exhibited sub-micromolar EC50
antirhinoviral activity against all serotypes examined.14
Several inhibitors containing Phe-Phe peptidomimetics
were also studied (12 and 13), but these compounds
exhibited poorer antiviral activity than that displayed
by the Val-Phe isosteres described above. In addition,
a molecule containing a tBuGly-Phe dipeptide mimic
(14) displayed potent antiviral activity against multiple
rhinovirus serotypes. Similar to the 3CP inhibitors
depicted in Table 1, the N-terminal S-cyclopentyl thio-
carbamate-containing molecules did not exhibit cyto-
toxicity in cell culture up the limits of their solubility.13
F igu r e 2. Schematic diagram of 1 bound in the HRV-2 3CP
active site.3 Hydrogen bonds are represented as dashed lines,
and the residues which make up the enzyme binding subsites
are depicted.
Having completed the structure-activity studies de-
scribed above, a comparison was made between one of
the more potent ketomethylene-containing compounds
(9) and an analogous tripeptidyl inhibitor (15). Exami-
nation of EC90 antiviral activity against several HRV
serotypes clearly indicated that the peptidomimetic 9
was the superior antiviral agent and was at least 10-
fold more potent than 15 for each serotype studied
(Table 3).14 Similar improvements in antiviral activity
were noted when comparing other ketomethylene-
containing 3CP inhibitors with the corresponding pep-
tidyl compounds (data not shown). Replacement of the
N-terminal thiocarbamate moiety present in 9 with an
amide derived from 5-methylisoxazole-3-carboxylic acid15
afforded a molecule which retained sub-micromolar EC90
antiviral activity when tested against several HRV
serotypes (16, Table 3).14 The above data indicate that
the antirhinoviral activity displayed by tripeptidyl
Michael acceptors may be substantially increased by
reducing the number of amide NH’s present in the
molecules and suggest that further improvements in
such activity might be realized by additional amide
modifications.16
Ch a r t 1
In the event, the ketomethylene-containing peptido-
mimetic compound 2 (Figure 1) displayed slightly
reduced anti-3CP inhibitory activity but improved an-
tiviral properties when compared with the tripeptide 1
(Table 1). The improved antiviral activity of 2 relative
to 1 was consistent with increased cell membrane
permeability due to the reduced potential of 2 to form
hydrogen bonds with water.12 As was observed for
related tripeptidyl 3CP inhibitors, the ketomethylene-
containing molecule 2 did not exhibit cytotoxicity in cell
culture up to its solubility limit.13 These encouraging
findings prompted an extensive investigation of ir-
reversible 3CP inhibitors which incorporate a P2-P3
ketomethylene dipeptide isostere to better define their
potential for use as broad-spectrum antirhinoviral
agents.
Syn th esis
The development of such 3CP inhibitors commenced
with the inclusion of several beneficial modifications
discovered during previous studies of related tripeptide-
derived molecules.4 As before, the ethyl propenoate
Michael acceptor was extensively employed for keto-
methylene structure-activity studies due to its ease of
preparation. Introduction of an N-terminal S-benzyl
thiocarbamate moiety (3) into the ketomethylene inhibi-
tor design increased anti-3CP activity approximately
5-fold relative to the corresponding carbamate-contain-
ing molecule (compare 2 and 3, Table 1). Substitution
of a P3 valine amino acid residue in place of leucine
further improved anti-3CP activity along with antiviral
properties (compound 4, Table 1). These two improve-
ments paralleled those observed during the development
of tripeptidyl 3CP inhibitors and suggested that other
beneficial peptide modifications could also be directly
incorporated into the ketomethylene series.
The ketomethylene-containing 3CP inhibitors utilized
in this study were prepared by three different synthetic
methods (A-C). The particular method employed to
synthesize a given compound is indicated in Tables 1-3.
An example of synthetic method A, which incorporates
elements from previously described preparations of
hydroxyethylene dipeptide isosteres,17 is provided by the
synthesis of compound 9 (Schemes 1 and 2). Isobutyral-
dehyde was condensed with vinylmagnesium bromide
to provide allylic alcohol 17 in good yield.18 This material
was not purified but was instead subjected to a homolo-
gation protocol which involved transesterification with
diethyl malonate, Claisen rearrangement, and subse-
quent basic hydrolysis. The resulting carboxylic acid (18)
was transformed into acid chloride 19,19 and this
product was coupled with (1R,2R)-(-)-pseudoephedrine
to provide the corresponding N-acylated compound 20

SAR of Ketomethylene-Containing Peptidomimetics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1205
Ta ble 1
c,d
compd no.
R
X
Y
prep.a
formulab
kobs/[I] (M-1 s-1
)
EC50 (µM)c,d
CC50 (µM)d
1
2
3
4
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH(CH3)2
NH
O
O
S
ref 3
A
A
C32H42N4O7
C33H43N3O7
C33H43N3O6S
C32H41N3O6S
25 000
17 400
87 600
500 000
0.54
0.36
0.68
0.19
>320
>320
>100
>100
CH2
CH2
CH2
S
A
a
Method of preparation: see Experimental Section. b Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical
values. c Serotype-14. See ref 3 for assay method and error.
d
Ta ble 2
c
compd no.
R1
CH2Ph
R2
prep.a
formulab
C31H45N3O6S
serotype
kobs/[I] (M-1 s-1
)
EC50 (µM)c
CC50 (µM)c
5
6
CH2CH(CH3)2
CH2CH(CH3)2
A
B
14
14
1A
2
10
14
1A
2
55 700
67 200
ND
ND
ND
243 000
ND
ND
0.19
0.28
0.60
0.71
1.6
0.16
0.56
1.0
>100
>100
CH2Ph(4-F)
C31H44FN3O6S
7
CH2Ph(4-CH3)
CH2CH(CH3)2
B
C32H47N3O6S
>100
10
14
2
10
14
1A
2
10
14
1A
2
10
14
1A
2
10
14
14
14
1A
2
ND
255 000
ND
ND
293 000
ND
ND
ND
850 000
ND
ND
ND
150 000
ND
ND
ND
127 400
404 000
124 000
ND
ND
ND
1.4
8
9
CH2Ph
CH(CH3)2
CH(CH3)2
A
A
C30H43N3O6S
0.020
0.10
0.035
0.020
0.17
0.13
0.16
0.0060
0.16
0.13
0.060
0.050
0.18
0.18
0.16
0.48
0.14
0.050
0.43
0.36
0.50
>100
>100
CH2Ph(4-F)
C30H42FN3O6S‚0.25H2O
10
11
CH2Ph(4-CH3)
CH2Ph(4-CF3)
CH(CH3)2
CH(CH3)2
A
A
C31H45N3O6S
>100
>100
C31H42F3N3O6S
12
13
14
CH2Ph(4-F)
CH2Ph(4-CH3)
CH2Ph
CH2Ph
CH2Ph
C(CH3)3
B
B
C
C34H42FN3O6S
C35H45N3O6S
C31H45N3O6S
>100
>100
>100
10
a
Method of preparation: see Experimental Section. b Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical
values. c See ref 3 for assay method and error. ND ) not determined.
in accordance with literature precedent.20 Alkylation of
the dianion derived from 20 with 4-fluorobenzyl bromide
in the presence of excess lithium chloride then afforded
the benzylated product 21 in good yield.20,21 Exposure
of 21 to a slight excess of N-bromosuccinimide under
mildly acidic conditions at 0 °C followed by reflux for
45 min provided slightly impure bromo-γ-lactone 22
after careful flash column chromatography. This mate-
rial was then converted to amino-lactone 24 by azide
displacement and subsequent reduction in the presence
of di-tert-butyl dicarbonate.
obtained (25) was condensed with the known trityl-
protected glutamine derivative 263 to afford coupling
product 27 in moderate yield. Removal of the N-terminal
Boc protecting group from 27 under acidic conditions
and derivatization of the resulting amine hydrochloride
with cyclopentyl chlorothiolformate23 then provided
thiocarbamate 28 in good yield. The trityl protecting
group present in 28 was removed by short exposure to
trifluoroacetic acid (TFA) in the presence of triisopro-
pylsilane to give inhibitor 9 in good yield.24 Several
different ketomethylene-containing molecules could be
prepared utilizing method A by variation of the starting
aldehyde (P3), the benzylic halide employed to alkylate
the γ,δ-unsaturated pseudoephedrine amide (P2), and
the derivatizing agent used for N-terminal modification
The desired ketomethylene dipeptide isostere was
prepared from amino-lactone 24 by basic hydrolysis and
TPAP-mediated oxidation22 of the resulting hydroxy-
acid (not shown, Scheme 2). The crude keto-acid thus

1206 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Ta ble 3
a
b
Method of preparation: see Experimental Section. See ref 3 for assay method and error.
Sch em e 1a
a
Reagents and conditions: (a) ref 18; (b) 1.1 equiv of diethyl malonate, 0.1 equiv of Ti(OEt)4, 80f125 °C, 3.5 h, then 190 °C, 9.5 h, then
EtOH, 6.0 M KOH, reflux, 5 h, 98%; (c) 1.2 equiv of SOCl2, CHCl3, reflux, 30 min, 41%; (d) 0.87 equiv of (1R,2R)-(-)-pseudoephedrine,
1.13 equiv of Et3N, THF, 0 °C, 15 min, 56%; (e) 3.1 equiv of LDA, 10.0 equiv of LiCl, THF, -78 °C, 1.8 h, then 3.0 equiv of (4-F)PhCH2Br,
0 °C, 30 min, 94%; (f) 1.05 equiv of NBS, 5.0 equiv of HOAc, 4:1 THF:H2O, 0 °C, 15 min, then reflux, 45 min, 80%; (g) 2.5 equiv of NaN3,
DMF, 50 °C, 67 h, 62%; (h) 1.4 equiv of (Boc)2O, H2/Pd/C, CH3OH, 23 °C, 16 h, 62%.
(P4). The ketomethylene-containing inhibitors prepared
in this study were typically isolated as white solids by
trituration and filtration as described previously for the
synthesis of analogous tripeptides.3,4 However, in sev-
eral cases, flash column chromatography was utilized
to further increase the purity of the desired compounds
(see the Experimental Section).
An example of preparation method B, which also
incorporates elements from previously described syn-
theses of hydroxyethylene dipeptide isosteres,25 is pro-
vided by the preparation of compound 12 (Scheme 3).
Thus, the optically active epoxide 2926 was coupled with
the lithium enolate of amide 30 to afford alcohol 31 in
moderate yield. Subsequent transformation of 31 into
amino-lactone 32 was then accomplished by exposure
to mild acid. Compound 32 was converted into inhibitor
12 (via intermediates 33 and 34) by a process analogous
to the 24f9 conversion depicted in Scheme 2 (see the
Experimental Section). As with synthetic method A,
several different ketomethylene-containing inhibitors
could be prepared via method B by altering the epoxide
(P3) or amide (P2) components employed.
The final synthetic method used to prepare the
peptidomimetic compounds described in this study is
illustrated in Scheme 4 (method C).27 This method was
utilized to prepare the tBuGly-Phe-containing isostere
14 for which the azide displacement reaction analogous
to the 22f23 conversion failed (equation 1, cf., Scheme
1). Cbz-protected L-tert-butylglycine was transformed
into â-keto-ester 35 in moderate yield by activation with
carbonyldiimidazole and subsequent displacement with
the lithium enolate of tert-butyl acetate. Condensation

SAR of Ketomethylene-Containing Peptidomimetics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1207
Sch em e 2a
a
Reagents and conditions (Tr ) CPh3): (a) 5.0 equiv of LiOH, DME, 23 °C, 30 min; (b) 2.0 equiv of NMO, 0.10 equiv of TPAP, 4 Å
sieves, CH2Cl2, 23 °C, 1.3 h; (c) 1.2 equiv of 26, 1.5 equiv of HOBt, 4.0 equiv of NMM, 1.5 equiv of EDC, CH2Cl2, 23 °C, 19 h, 53% from
24; (d) HCl, 1,4-dioxane, 23 °C, 2 h; (e) 1.7 equiv of cyclopentyl chlorothiolformate, 3.0 equiv of (i-Pr)2NEt, CH2Cl2, 23 °C, 2 h, 59%; (f) 3.0
equiv of (i-Pr)3SiH, 1:2 TFA:CH2Cl2, 23 °C, 30 min, 82%.
Sch em e 3a
Sch em e 4a
a
Reagents and conditions: (a) 2.0 equiv of n-BuLi, THF, -78f0
a
Reagents and conditions (Tf ) SO2CF3): (a) 1.1 equiv of CDI,
°C, 1 h, 49%; (b) 1.0 equiv of TsOH‚H2O, 5:1 toluene:CH2Cl2, 23
°C, 13 h, 59%; (c) 5.0 equiv of LiOH, DME, 23 °C, 20 min; (d) 2.0
equiv of NMO, 0.10 equiv of TPAP, 4 Å sieves, 1:1 CH3CN:CH2Cl2,
23 °C, 3 h; (e) 1.2 equiv of 26, 1.3 equiv of HOBt, 4.0 equiv of
NMM, 1.3 equiv of EDC, CH2Cl2, 23 °C, 22 h, 43% from 32; (f)
HCl, 1,4-dioxane, 23 °C, 1.5 h; (g) 1.5 equiv of cyclopentyl
chlorothiolformate, 3.0 equiv of NMM, CH2Cl2, 0 °C, 30 min, 52%;
(h) 2.7 equiv of (i-Pr)3SiH, 3:5 TFA:CH2Cl2, 23 °C, 30 min, 71%.
THF, 23 °C, 1 h, then 2.1 equiv of LDA, 2.1 equiv of t-BuOAc,
THF, -78 °C, 1 h, 44%; (b) 1.05 equiv of NaH, THF, 0 °C, 10 min,
then 3.1 equiv of 36, THF, 23 °C, 24 h; (c) 1:5 TFA:CH2Cl2, 23 °C,
24 h, 54% from 35; (d) 1.3 equiv of (Boc)2O, H2/Pd/C, CH3OH, 23
°C, 24 h, 70%; (e) 8.0 equiv of NaOH, CH3OH, 0 °C, 2 h, 99%.
42) by a method analogous to the 25f9 conversion
depicted in Scheme 2 above (see the Experimental
Section).
of the sodium salt of 35 with triflate 36 then provided
the coupling product 37 which, without purification, was
subsequently converted into Cbz-protected γ-keto-ester
38 in moderate overall yield. The N-terminal Cbz
protecting group present in 38 was exchanged for a Boc
moiety by hydrogenation in the presence of di-tert-butyl
dicarbonate, and the resulting Boc-protected γ-keto-
ester (39) was hydrolyzed under basic conditions to
provide γ-keto-acid 40 in good yield.28 Compound 40 was
then converted to isostere 14 (via intermediates 41 and
Con clu sion s
The structure-activity studies described above con-
firmed the ability of Michael acceptor-containing mol-
ecules which incorporate ketomethylene dipeptide isos-
teres to function both as irreversible inhibitors of the
human rhinovirus 3C protease and as potent antirhi-
noviral agents in cell culture. Such compounds typically
display slightly reduced 3C protease inhibition activity
relative to the corresponding peptide-derived molecules,
but they also exhibit significantly improved antiviral

1208 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
over Na2SO4 and was concentrated. The residue was purified
by flash column chromatography (gradient elution, 40f60%
EtOAc in hexanes) to give 20 (10.8 g, 56%) as a clear, colorless
oil: Rf ) 0.35 (50% EtOAc in hexanes); IR (cm-1) 3382, 1622;
1H NMR (CDCl3, mixture of isomers) δ 0.96 (d, J ) 6.8), 0.97
(d, J ) 6.5), 1.11 (d, J ) 6.9), 2.18-2.59 (m), 2.82 (s), 2.92 (s),
3.99-4.04 (m), 4.32-4.42 (m), 4.44-4.49 (m), 4.55-4.62 (m),
5.32-5.49 (m), 7.24-7.42 (m). Anal. (C18H27NO2) C, H, N.
properties. Optimization of the ketomethylene-contain-
ing compounds afforded several highly active 3C pro-
tease inhibitors which display potent antiviral activity
(EC90 ) <1 µM) against multiple rhinovirus serotypes
in cell culture. The pharmacological properties of the
inhibitors described in this study will be detailed in
subsequent publications.
tr a n s-(1′R,2S,2′R)-6-Meth yl-2-(4′′-flu or oben zyl)h ep t-4-
en oic Acid (2′-Hyd r oxy-1′-m eth yl-2-p h en yleth yl)m eth yl
Am id e (21). n-Butyllithium (32.5 mL of a 1.6 M solution in
hexanes, 52.0 mmol, 3.1 equiv) was added to a suspension of
anhydrous lithium chloride (7.18 g, 169 mmol, 10 equiv) and
diisopropylamine (7.80 mL, 55.7 mmol, 3.3 equiv) in THF (250
mL) at -78 °C. The reaction mixture was stirred for 30 min
at -78 °C, then was maintained at 0 °C for 5 min, and was
subsequently cooled again to -78 °C. A solution of 20 (4.91 g,
17.0 mmol, 1 equiv) in THF (50 mL) was added via cannula,
and the resulting mixture was stirred at -78 °C for 1.75 h,
maintained at 0 °C for 20 min, stirred at 23 °C for 5 min, and
then was cooled again to 0 °C. A solution of 4-fluorobenzyl
bromide (6.34 mL, 50.9 mmol, 3.0 equiv) in THF (15 mL) was
added, and the reaction mixture was stirred at 0 °C for 30
min and then was partitioned between half-saturated NH4Cl
(230 mL) and a 1:1 mixture of EtOAc and hexanes (2 × 150
mL). The combined organic layers were dried over Na2SO4 and
were concentrated. Purification of the residue by flash column
chromatography (gradient elution, 20f40% EtOAc in hexanes)
provided 21 (6.33 g, 94%) as a viscous oil: Rf ) 0.38 (40%
EtOAc in hexanes); IR (cm-1) 3378, 1614; 1H NMR (CDCl3,
mixture of isomers) δ 0.85-0.95 (m), 0.96 (d, J ) 6.8), 2.10-
2.32 (m), 2.34-2.46 (m), 2.58 (s), 2.67-2.79 (m), 2.82-2.94 (m),
3.00-3.18 (m), 3.94 (br), 4.37-4.52 (m), 5.24-5.42 (m), 5.44-
5.56 (m), 6.89-7.01 (m), 7.08-7.14 (m), 7.19-7.38 (m). Anal.
(C25H32FNO2) C, H, N.
Exp er im en ta l Section
General descriptions of experimental procedures, reagent
purifications, and instrumentation along with conditions for
enzyme and antiviral assays are provided elsewhere.3 1H NMR
chemical shifts are reported in ppm (δ) downfield relative to
internal tetramethylsilane, and coupling constants are given
in hertz. A simplified naming system employing amino acid
abbreviations 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. In addition, the terminology “AA1Ψ-
[COCH2]-AA2” indicates that, for any peptide sequence, two
amino acids (AA1 and AA2) usually linked by an amide bond
are replaced by a ketomethylene dipeptide isostere. The
following abbreviations also apply: HOBt (1-hydroxybenzo-
triazole hydrate), EDC (1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride), and CDI (1,1′-carbonyldiimida-
zole).
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-{cyclop en tylSCO-L-Va lΨ[COCH2]-
L-P h e(4-F )-L-Gln }-E-p r op en oa te (9). tr a n s-6-Meth ylh ep t-
4-en oic Acid (18). Diethyl malonate (25.2 mL, 166 mmol, 1.1
equiv), 1718 (15.1 g, 151 mmol, 1 equiv), and titanium(IV)
ethoxide (3.16 mL, 15.1 mmol, 0.1 equiv) were combined in a
100 mL pear-shaped flask equipped with a stir bar and a short
path distillation head (with receiving flask). The reaction
vessel was placed in an 80 °C oil bath, and the bath temper-
ature was gradually raised to 125 °C over 2 h, keeping the
distillation head temperature below 75 °C. After the mixture
was stirred for 1.5 h at 125 °C, the bath temperature was
gradually raised to 190 °C over 5 h, maintained at that
temperature for 4.5 h, and then allowed to cool to room
temperature overnight. EtOH (75 mL) and 6.0 M KOH (75 mL)
were added to the reaction mixture which was warmed to
reflux for 5 h and then allowed to cool. The resulting mixture
was filtered through a medium fritted funnel, and the filtrate
was concentrated, diluted with water (50 mL), and washed
with Et2O (50 mL). The aqueous phase was acidified to pH 1
with 12 M HCl and extracted with Et2O (3 × 100 mL). The
combined organic layers were washed with brine (25 mL),
dried over Na2SO4, and concentrated to give 18 (21 g, 98%) as
a brown oil. This material was used without further purifica-
tion: 1H NMR (CDCl3) δ 0.96 (d, 6H, J ) 6.5), 2.18-2.45 (m,
5H), 5.31-5.50 (m, 2H).
(1′R,3R,5S)-5-(1′-Br om o-2′-m et h ylp r op yl)-3-(4′′-flu o-
r oben zyl)d ih yd r ofu r a n -2-on e (22). N-Bromosuccinimide
(2.93 g, 16.5 mmol, 1.05 equiv) was added in small portions
over 10 min to a solution of 21 (6.24 g, 15.7 mmol, 1 equiv)
and glacial acetic acid (4.49 mL, 78.4 mmol, 5.0 equiv) in a
4:1 mixture of THF and H2O (165 mL) at 0 °C. The resulting
yellow solution was stirred for 15 min at 0 °C, then was
warmed to 23 °C, and subsequently refluxed for 45 min. After
cooling to 23 °C, the reaction mixture was partitioned between
half-saturated NaHCO3 (200 mL) and a 1:1 mixture of EtOAc
and hexanes (2 × 200 mL). The combined organic layers were
dried over Na2SO4 and were concentrated. Flash chromato-
graphic purification of the residue (gradient elution, 5f10%
EtOAc in hexanes) gave 22 (4.14 g, 80%) as a pale yellow oil
(containing approximately 5-10% unidentified impurities by
1H NMR): Rf ) 0.56 (25% EtOAc in hexanes); IR (cm-1) 1772;
1H NMR (CDCl3) δ 0.94 (d, 3H, J ) 6.5), 1.00 (d, 3H, J ) 6.8),
2.05-2.35 (m, 3H), 2.83 (dd, 1H, J ) 13.6, 8.4), 2.92-3.03 (m,
1H), 3.11 (dd, 1H, J ) 13.6, 4.7), 3.90 (dd, 1H, J ) 9.0, 3.7),
4.33-4.40 (m, 1H), 6.98-7.06 (m, 2H), 7.14-7.20 (m, 2H).
Anal. (C15H18BrFO2) C, H.
tr a n s-6-Meth ylh ep t-4-en oic Acid Ch lor id e (19). Thionyl
chloride (5.54 mL, 75.9 mmol, 1.2 equiv) and 18 (9.0 g, 63
mmol, 1 equiv) were dissolved in CHCl3 (120 mL) and warmed
to reflux for 30 min. Additional thionyl chloride (1.0 mL, 14
mmol, 0.22 equiv) was added, and the reflux was continued
for 15 min more. The reaction mixture was concentrated, and
the residue was distilled at vacuum line pressure (approxi-
mately 1 Torr), collecting the distillate between 32 and 36 °C
(1′S,3R,5S)-5-(1′-Azido-2′-m eth ylpr opyl)-3-(4′′-flu or oben -
zyl)d ih yd r ofu r a n -2-on e (23). A suspension of sodium azide
(1.90 g, 29.2 mmol, 2.5 equiv) and 22 (3.85 g, 11.7 mmol, 1
equiv) in DMF (40 mL) was heated at 50 °C for 67 h. The
reaction mixture was cooled to 23 °C and was partitioned
between half-saturated NaCl (200 mL) and a 1:1:1 mixture of
EtOAc, hexanes, and acetone (2 × 200 mL). The combined
organic layers were dried over Na2SO4 and concentrated, and
the residue was purified by flash column chromatography
(gradient elution, 10f20% EtOAc in hexanes) to give 23 (2.10
g, 62%) as a white solid (containing approximately 5-10%
unidentified impurities by 1H NMR): mp 91-96 °C; Rf ) 0.44
(25% EtOAc in hexanes); IR (cm-1) 2097, 1772; 1H NMR
(CDCl3) δ 0.99 (d, 3H, J ) 6.5), 1.02 (d, 3H, J ) 6.8), 1.95-
2.20 (m, 3H), 2.78-2.88 (m, 1H), 2.94 (dd, 1H, J ) 7.0, 4.2),
3.03-3.17 (m, 2H), 4.37-4.43 (m, 1H), 6.97-7.09 (m, 2H),
7.14-7.21 (m, 2H).
1
to yield 19 (4.18 g, 41%) as a clear, colorless liquid: H NMR
(CDCl3) δ 0.96 (d, 6H, J ) 6.8), 2.18-2.32 (m, 1H), 2.34-2.43
(m, 2H), 2.95 (t, 2H, J ) 7.3), 5.26-5.38 (m, 1H), 5.49 (dd,
1H, J ) 15.6, 6.5).
tr a n s-(1′R,2′R)-6-Meth ylh ep t-4-en oic Acid (2′-Hyd r oxy-
1′-m eth yl-2-p h en yleth yl)m eth yl Am id e (20). A solution of
19 (12.2 g, 76.2 mmol, 1.15 equiv) in THF (40 mL) was added
dropwise over 10 min to a 0 °C solution of (1R,2R)-(-)-
pseudoephedrine (10.95 g, 66.27 mmol, 1 equiv) and triethyl-
amine (12.0 mL, 86.1 mmol, 1.3 equiv) in THF (200 mL). After
being stirred for 15 min at 0 °C, the reaction mixture was
poured into a separatory funnel and was washed with water
(20 mL) and brine (2 × 40 mL). The organic layer was dried

SAR of Ketomethylene-Containing Peptidomimetics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1209
(1S,2′S,4′S)-{2-Meth yl-1-[4′-(4′′-flu or oben zyl)-5′-oxo-tet-
r a h yd r ofu r a n -2′-yl]p r op yl}ca r ba m ic Acid ter t-Bu tyl Es-
ter (24). A suspension of 23 (2.02 g, 6.93 mmol, 1 equiv), di-
tert-butyl dicarbonate (2.12 g, 9.71 mmol, 1.4 equiv), and Pd/C
(10%, 0.20 g) in CH3OH (100 mL) was stirred at 23 °C under
a hydrogen atmosphere (balloon) for 16 h. The reaction mixture
was vacuum-filtered through Whatman No. 3 paper and was
concentrated. Purification of the residue by flash column
chromatography (15% EtOAc in hexanes) provided 24 (1.58 g,
62%) as a white foam: Rf ) 0.80 (5% MeOH in CH2Cl2); IR
6.92-7.00 (m, 2H), 7.03-7.12 (m, 3H), 7.18-7.32 (m, 15H).
Anal. (C49H56FN3O6S‚0.5H2O) C, H, N.
E t h yl-3-{cyclop en t ylSCO-L-Va lΨ[COCH 2]-L-P h e(4-F )-
L-Gln }-E-p r op en oa te (9). Triisopropylsilane (0.097 mL, 0.47
mmol, 3.0 equiv) and trifluoroacetic acid (3 mL) were added
sequentially to a solution of 28 (0.132 g, 0.158 mmol, 1 equiv)
in CH2Cl2 (6 mL), producing a bright yellow solution. This
solution was stirred for 30 min at 23 °C, during which time it
became colorless, then was concentrated. The residue was
triturated with Et2O (6 mL), and the resulting solid was
collected by filtration, washed with Et2O (2 × 5 mL), and then
dried under vacuum to give 9 (0.077 g, 82%) as a white solid:
mp ) 215 °C (dec); Rf ) 0.45 (10% CH3OH in CH2Cl2); IR
(cm-1) 3413, 3296, 1715, 1649; 1H NMR (DMSO-d6) δ 0.75 (d,
3H, J ) 6.5), 0.82 (d, 3H, J ) 6.5), 1.21 (t, 3H, J ) 6.9), 1.36-
1.75 (m, 8H), 1.92-2.14 (m, 5H), 2.52-2.85 (m, 4H), 2.87-
2.99 (m, 1H), 3.47-3.58 (m, 1H), 4.06-4.18 (m, 1H), 4.09 (q,
2H, J ) 6.9), 4.25-4.36 (m, 1H), 5.41 (d, 1H, J ) 15.6), 6.61
(dd, 1H, J ) 15.6, 5.1), 6.74 (s, 1H), 6.98-7.23 (m, 5H), 8.01
(d, 1H, J ) 8.4), 8.28 (d, 1H, J ) 8.1). Anal. (C30H42FN3O6S‚
0.25H2O) C, H, N.
1
(cm-1) 3331, 1766, 1702; H NMR (CDCl3) δ 0.93 (d, 3H, J )
6.8), 0.95 (d, 3H, J ) 6.5), 1.41 (s, 9H), 1.71-1.83 (m, 1H),
1.95-2.06 (m, 1H), 2.16-2.27 (m, 1H), 2.80 (dd, 1H, J ) 13.5,
8.6), 2.88-2.99 (m, 1H), 3.09 (dd, 1H, J ) 13.5, 4.4), 3.32-
3.40 (m, 1H), 4.42-4.48 (m, 2H), 6.95-7.03 (m, 2H), 7.11-
7.18 (m, 2H). Anal. (C20H28FNO4) C, H, N.
Eth yl-3-{Boc-L-Va lΨ[COCH2]-L-P h e(4-F )-L-(Tr -Gln )}-E-
p r op en oa te (27). Lithium hydroxide (9.62 mL of a 1.0 M
aqueous solution, 9.62 mmol, 5.0 equiv) was added to a solution
of 24 (0.703 g, 1.92 mmol, 1 equiv) in DME (25 mL) at 23 °C.
The resulting suspension was stirred at 23 °C for 30 min and
then was partitioned between 10% KHSO4 (50 mL) and CH2-
Cl2 (3 × 100 mL). The combined organic layers were dried over
Na2SO4 and concentrated, and the residue was dissolved in
CH2Cl2 (30 mL). Powdered 4 Å molecular sieves (0.70 g),
4-methylmorpholine N-oxide (0.451 g, 3.85 mmol, 2.0 equiv),
and tetrapropylammonium perruthenate (0.068 g, 0.19 mmol,
0.10 equiv) were added sequentially. The resulting dark
reaction mixture was stirred for 1.33 h at 23 °C and then was
vacuum-filtered (twice) through Whatman No. 3 and No. 5
papers. The filtrate was concentrated under reduced pressure
to provide a dark residue (intermediate 25) which was dis-
solved in CH2Cl2 (30 mL). Crude ethyl-3-[H2N-L-(Tr-Gln)]-E-
propenoate‚HCl3 (26, 2.30 mmol, 1.2 equiv), 4-methylmorpho-
line (0.846 mL, 7.69 mmol, 4.0 equiv), HOBt (0.390 g, 2.89
mmol, 1.5 equiv), and EDC (0.553 g, 2.88 mmol, 1.5 equiv) were
added sequentially. The reaction mixture was stirred for 19 h
at 23 °C and then was partitioned between brine (100 mL)
and CH2Cl2 (3 × 100 mL). The combined organic layers were
dried over Na2SO4 and were concentrated. Purification of the
residue by flash column chromatography (gradient elution,
30f40% EtOAc in hexanes) provided 27 (0.820 g, 53%) as a
tan foam: Rf ) 0.50 (50% EtOAc in hexanes); IR (cm-1) 3307,
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od B.
Syn th esis of Eth yl-3-{cyclop en tylSCO-L-P h eΨ[COCH2]-
L-P h e(4-F )-L-Gln }-E-p r op en oa te (12). 3-(4′-F lu or op h en yl)-
p r op ion ic Acid cis-(1′′S,2′′R)-1′′-Am in o-2′′-in d a n ola ce-
ton id e Am id e (30). Oxalyl chloride (6.14 mL, 70.4 mmol, 1.05
equiv) was added to a solution of 3-(4′-fluorophenyl)propionic
acid (11.3 g, 67.2 mmol, 1 equiv) and DMF (0.03 mL, 0.39
mmol, 0.006 equiv) in benzene (150 mL) at 23 °C. The reaction
mixture was stirred at 23 °C for 1.5 h and then was concen-
trated. The resulting oil was dissolved in THF (30 mL) and
was added to a 0 °C solution of cis-(1S,2R)-1-amino-2-indanol
(10.0 g, 67.0 mmol, 1.0 equiv) and Et3N (10.3 mL, 73.9 mmol,
1.1 equiv) in THF (250 mL). After being stirred for 20 min at
0 °C, the reaction mixture was partitioned between half-
saturated NH4Cl (150 mL) and EtOAc (2 × 150 mL). The
organic layers were dried over Na2SO4 and were concentrated
to afford a white solid. This material was dissolved in a
mixture of CH2Cl2 (400 mL) and 2-methoxypropene (30 mL),
and the resulting solution was treated with methanesulfonic
acid (0.20 mL). After being stirred for 15 min at 23 °C, the
reaction mixture was partitioned between half-saturated
NaHCO3 (150 mL) and CH2Cl2 (2 × 150 mL). The combined
organic layers were dried over MgSO4 and gravity-filtered. The
filtrate was concentrated, and the residue was purified by flash
column chromatography (gradient elution, 10f20% EtOAc in
hexanes) to provide 30 (18.2 g, 83%) as a pale yellow oil: Rf )
0.52 (50% EtOAc in hexanes); IR (cm-1) 2934, 1645; 1H NMR
(CDCl3) δ 1.34 (s, 3H), 1.60 (s, 3H), 2.91-2.95 (m, 2H), 3.04-
3.13 (m, 4H), 4.68-4.71 (m, 1H), 5.06 (d, 1H, J ) 4.7), 6.94-
7.00 (m, 2H), 7.02-7.30 (m, 6H). Anal. (C21H22FNO2) C, H, N.
1
1708, 1666; H NMR (CDCl3) δ 0.67 (d, 3H, J ) 6.8), 0.92 (d,
3H, J ) 6.8), 1.28 (t, 3H, J ) 7.2), 1.40 (s, 9H), 1.53-1.67 (m,
1H), 1.91-2.04 (m, 2H), 2.32-2.41 (m, 2H), 2.46-2.55 (m, 1H),
2.63 (dd, 1H, J ) 12.1, 5.9), 2.69-2.80 (m, 1H), 2.83 (dd, 1H,
J ) 12.1, 8.2), 3.03 (dd, 1H, J ) 17.7, 10.0), 4.05-4.11 (m,
1H), 4.17 (q, 2H, J ) 7.2), 4.40-4.50 (m, 1H), 4.84 (d, 1H, J )
8.4), 5.38 (d, 1H, J ) 15.7), 6.01 (d, 1H, J ) 8.4), 6.60 (dd, 1H,
J ) 15.7, 5.0), 6.92-6.99 (m, 2H), 7.03-7.12 (m, 3H), 7.17-
7.30 (m, 15H). Anal. (C48H56FN3O7) C, H, N.
(1S,2′S,4′R)-{1-[4′-(4′′-F lu or oben zyl)-5′-oxo-tetr a h yd r o-
fu r a n -2′-yl]-2-p h en yleth yl}ca r ba m ic Acid ter t-Bu tyl Es-
ter (32). n-Butyllithium (13.5 mL of a 1.6 M solution in
hexanes, 21.6 mmol, 2.0 equiv) was added to a solution of
(1R,2S)-1-oxiranyl-2-phenylethyl)carbamic acid tert-butyl ester
2926 (2.85 g, 10.8 mmol, 1 equiv) and 30 (3.67 g, 10.8 mmol,
1.0 equiv) in THF (150 mL) at -78 °C. The reaction mixture
was stirred for 5 min at -78 °C, maintained at 0 °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 organic
layers were dried over Na2SO4 and were concentrated. Flash
chromatographic purification of the residue (gradient elution,
25f40% EtOAc in hexanes) gave the coupling product 31 (3.19
g, 49%) as a yellow oil contaminated with several minor
impurities. This material was dissolved in a 5:1 mixture of
toluene and CH2Cl2 (180 mL) and was treated with p-
toluenesulfonic acid monohydrate (1.01 g, 5.31 mmol, 1.0
equiv) at 23 °C. After being stirred for 13 h at 23 °C, the
reaction mixture was filtered through a medium fritted funnel,
and the filtrate was partitioned between half-saturated NaH-
CO3 (150 mL) and a 1:1 mixture of EtOAc and hexanes (2 ×
150 mL). The organic layers were dried over Na2SO4 and were
concentrated. The residue was purified by flash column
E t h yl-3-{cyclop en t ylSCO-L-Va lΨ[COCH 2]-L-P h e(4-F )-
L-(Tr -Gln )}-E-p r op en oa te (28). A solution of HCl in 1,4-
dioxane (4.0 M, 3 mL) was added to a solution of 27 (0.273 g,
0.339 mmol, 1 equiv) in the same solvent (3 mL) at 23 °C. The
reaction mixture was stirred at 23 °C for 2 h and then was
concentrated. The residue was dissolved in CH2Cl2 (5 mL), and
N,N-diisopropylethylamine (0.177 mL, 1.02 mmol, 3.0 equiv)
and cyclopentyl chlorothiolformate23 (0.095 mL, 0.576 mmol,
1.7 equiv) were added sequentially. The reaction mixture was
stirred for 2 h at 23 °C and then was partitioned between brine
(30 mL) and CH2Cl2 (3 × 30 mL). The combined organic layers
were dried over Na2SO4 and concentrated, and the residue was
chromatographed on silica gel (40% EtOAc in hexanes) to
afford 28 (0.166 g, 59%) as a white foam: Rf ) 0.24 (40%
EtOAc in hexanes); IR (cm-1) 3307, 1713, 1654; 1H NMR
(CDCl3) δ 0.69 (d, 3H, J ) 6.8), 0.93 (d, 3H, J ) 6.8), 1.29 (t,
3H, J ) 7.2), 1.47-1.75 (m, 7H), 1.91-2.12 (m, 4H), 2.30-
2.41 (m, 2H), 2.51 (dd, 1H, J ) 17.2, 2.3), 2.63 (dd, 1H, J )
12.3, 5.9), 2.69-2.80 (m, 1H), 2.84 (dd, 1H, J ) 12.3, 8.4), 3.01
(dd, 1H, J ) 17.2, 10.0), 3.57-3.67 (m, 1H), 4.17 (q, 2H, J )
7.2), 4.33-4.50 (m, 2H), 5.39 (dd, 1H, J ) 15.7, 1.7), 5.61 (d,
1H, J ) 7.8), 6.11 (d, 1H, J ) 8.1), 6.60 (dd, 1H, J ) 15.7, 4.8),

1210 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
chromatography (20% EtOAc in hexanes) to provide 32 (1.28
g, 59%) as a white foam: Rf ) 0.46 (30% EtOAc in hexanes);
1H NMR (DMSO-d6) δ 1.21 (t, 3H, J ) 6.8), 1.33-1.72 (m, 8H),
1.90-2.07 (m, 4H), 2.49-3.07 (m, 7H), 3.43-3.47 (m, 1H), 4.09
(q, 2H, J ) 6.8), 4.33-4.35 (m, 2H), 5.37-5.46 (m, 1H), 6.59-
6.67 (m, 1H), 6.77 (s, br, 1H), 7.00-7.28 (m, 9H), 8.04 (d, 1H,
J ) 7.8), 8.46 (d, 1H, J ) 7.5), 8.53 (d, 1H, J ) 7.5). Anal.
(C32H42FN3O6S) C, H, N.
1
IR (cm-1) 3332, 2976, 1767, 1702; H NMR (CDCl3) δ 1.36 (s,
9H), 1.88-1.97 (m, 1H), 2.19-2.29 (m, 1H), 2.75-2.99 (m, 4H),
3.05 (dd, 1H, J ) 13.5, 4.5), 3.93 (q, 2H, J ) 8.5), 4.13-4.18
(m, 1H), 4.54 (d, 1H, J ) 9.7), 6.91-6.98 (m, 2H), 7.08-7.32
(m, 7H). Anal. (C24H28FNO4) C, H, N.
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od C.
Syn th esis of Eth yl-3-{cyclopen tylSCO-L-tBu GlyΨ[COCH2]-
L-P h e(4-F )-L-Gln }-E-p r op en oa te (14). Cbz-L-tBu Gly-OH.
Benzyl chloroformate (6.42 mL, 43.68 mmol, 1.12 equiv) and
freshly prepared aqueous NaOH solution (1.0 M, 39.0 mL, 39.0
mmol, 1 equiv) were added simultaneously to a solution of
H2N-L-tBuGly-OH (5.12 g, 39.0 mmol, 1 equiv) in 1,4-dioxane
(80 mL) at 0 °C. The reaction mixture was warmed to 23 °C
and was stirred at that temperature for 18 h. The resulting
solution was concentrated under reduced pressure to ∼40 mL
volume and then was partitioned between 0.5 M HCl (100 mL)
and CH2Cl2 (2 × 100 mL). The combined organic layers were
dried over Na2SO4 and were concentrated to give Cbz-L-
tBuGly-OH (10.34 g, 100%) as a colorless oil. This material
was used without further purification: IR (cm-1) 3336, 1521,
1232; 1H NMR (CDCl3) δ 1.02 (s, 9H), 1.26 (s, 1H), 4.21 (d,
1H, J ) 9.9), 5.13 (s, 2H), 5.35 (d, 1H, J ) 9.0), 7.33-7.34 (m,
5H).
(S)-4-Ben zyloxycar bon ylam in o-5,5-dim eth yl-3-oxo-h ex-
a n oic Acid ter t-Bu t yl E st er (35). To a solution of Cbz-L-
tBuGly-OH (20.53 g, 77.4 mmol, 1 equiv) in THF (150 mL)
was added CDI (13.81 g, 85.14 mmol, 1.1 equiv) at 23 °C. The
resulting mixture was stirred at 23 °C for 1 h. In a separate
flask, n-butyllithium (101.6 mL of a 1.6 M solution in hexanes,
162.5 mmol, 2.1 equiv) was added to a solution of diisopropyl-
amine (22.78 mL, 162.5 mmol, 2.1 equiv) in THF (100 mL) at
-78 °C. The reaction mixture was stirred for 15 min at -78
°C, warmed to 0 °C for 5 min, and then cooled back to -78 °C.
A solution of tert-butyl acetate (21.9 mL, 162.5 mmol, 2.1
equiv) in THF (10 mL) was added via cannula, and the
resulting mixture was stirred at -78 °C for 10 min. The Cbz-
L-tBuGly-OH/CDI solution prepared above was then added
dropwise to the lithium enolate at -78 °C. The resulting
mixture was stirred at -78 °C for 1 h, quenched with 1 M
HCl (100 mL), and extracted with EtOAc (2 × 100 mL). The
combined organic layers were washed with brine (150 mL),
dried over Na2SO4, and concentrated. The residue was purified
by flash chromatography on silica gel (10% EtOAc in hexanes)
to afford 35 (12.1 g, 44%, contaminated with ∼10% minor
impurities) as a pale yellow oil: IR (cm-1) 1717, 1508, 1265,
739; 1H NMR (CDCl3) δ 1.09 (s, 9H), 1.44 (s, 9H), 3.50 (s, 2H),
4.29 (d, 1H, J ) 9.3), 5.03 (s, 2H), 5.35-5.42 (m, 1H), 7.34 (s,
5H).
(R )-3-P h e n y l-2-t r iflu o r o m e t h a n e s u lfo n y lo x y p r o -
p ion ic Acid Meth yl Ester (36). 2,6-Lutidine (1.72 mL, 14.8
mmol, 3.3 equiv) and trifluoromethanesulfonic anhydride (2.50
mL, 14.8 mmol, 3.3 equiv) were added sequentially to a
solution of (R)-2-hydroxy-3-phenylpropionic acid methyl ester
(2.53 g, 14.06 mmol, 3.1 equiv) in CH2Cl2 (30 mL) at 0 °C. The
resulting pink mixture was stirred at 0 °C for 30 min and then
was partitioned between 0.5 M HCl (100 mL) and a 1:1 mixture
of EtOAc in hexanes (2 × 100 mL). The combined organic
layers were dried over Na2SO4 and were concentrated to afford
36. All of this material was utilized in the subsequent
transformation without further purification.
Cbz-L-tBu GlyΨ[COCH2]-L-P h e-OMe (38). A solution of
35 (1.64 g, 4.51 mmol, 1 equiv) in THF (100 mL) was added
dropwise to a stirred suspension of NaH (0.19 g of a 60%
dispersion in mineral oil, 4.74 mmol, 1.05 equiv) in THF (100
mL) at 0 °C. After the mixture was stirred for 10 min, a
solution of 36 (prepared above) in CH2Cl2 (10 mL) was added
dropwise. The resulting mixture was stirred at 23 °C for 24 h
and then was partitioned between 1.0 M HCl (50 mL) and
EtOAc (3 × 50 mL). The combined organic layers were washed
with brine (100 mL), dried over Na2SO4, and concentrated to
provide the crude coupling product (37) as a pale yellow oil.
Without further purification, the above oil was dissolved in
CH2Cl2 (10 mL), treated with trifluoroacetic acid (2 mL), and
Eth yl-3-{Boc-L-P h eΨ[COCH2]-L-P h e(4-F )-L-(Tr -Gln )}-E-
p r op en oa te (33). Lithium hydroxide (7.6 mL of a 1 M aqueous
solution, 7.6 mmol, 5.0 equiv) was added to a solution of 32
(0.630 g, 1.52 mmol, 1 equiv) in DME (8 mL) at 23 °C. The
resulting suspension was stirred at 23 °C for 20 min and then
was partitioned between 0.5 M HCl (100 mL) and EtOAc (2 ×
100 mL). The combined organic layers were dried over Na2-
SO4 and concentrated, and the residue was dissolved in a 1:1
mixture of CH2Cl2 and CH3CN (100 mL). 4-Methylmorpholine
N-oxide (0.357 g, 3.05 mmol, 2.0 equiv), powdered 4 Å
molecular sieves (0.70 g), and tetrapropylammonium perru-
thenate (0.054 g, 0.153 mmol, 0.10 equiv) were added sequen-
tially. The resulting dark reaction mixture was stirred for 3 h
at 23 °C and then was filtered through Celite. The filtrate was
concentrated under reduced pressure to provide a brown oil
which was dissolved in CH2Cl2 (40 mL). Crude ethyl-3-[H2N-
L-(Tr-Gln)]-E-propenoate‚HCl3 (26, 1.27 mmol, 1.2 equiv),
HOBt (0.268 g, 1.98 mmol, 1.3 equiv), 4-methylmorpholine
(0.670 mL, 6.09 mmol, 4.0 equiv), and EDC (0.380 g, 1.98
mmol, 1.3 equiv) were added sequentially. The reaction
mixture was stirred for 22 h at 23 °C 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 Na2SO4 and were concentrated. Purification of the residue
by flash column chromatography (40% EtOAc in hexanes)
provided 33 (0.558 g, 43%) as a white solid: mp ) 89-100 °C;
Rf ) 0.44 (50% EtOAc in hexanes); IR (cm-1) 3316, 2972, 1708,
1
1665; H NMR (CDCl3) δ 1.29 (t, 3H, J ) 7.2), 1.35 (s, 9H),
1.95-2.05 (m, 1H), 2.34-2.39 (m, 2H), 2.46 (d, 1H, J ) 16.8),
2.57-2.99 (m, 7H), 4.17 (q, 2H, J ) 7.2), 4.27-4.33 (m, 1H),
4.48 (s, br, 1H), 4.58 (d, 1H, J ) 6.9), 5.42 (d, 1H, J ) 15.3),
6.08 (d, 1H, J ) 8.4), 6.62 (dd, 1H, J ) 15.3, 4.8), 6.93-7.19
(m, 6H), 7.21-7.29 (m, 19H); Anal. (C52H56FN3O7) C, H, N.
Eth yl-3-{cyclop en tylSCO-L-P h eΨ[COCH2]-L-P h e(4-F )-
L-(Tr -Gln )}-E-p r op en oa te (34). A solution of HCl in 1,4-
dioxane (4.0 M, 8 mL) was added to a solution of 33 (0.302 g,
0.354 mmol, 1 equiv) in the same solvent (10 mL) at 23 °C.
The reaction mixture was stirred at 23 °C for 1.5 h and then
was concentrated. The resulting oil was dissolved in CH2Cl2
(15 mL) and cooled to 0 °C, and 4-methylmorpholine (0.117
mL, 1.06 mmol, 3.0 equiv) and cyclopentyl chlorothiolformate23
(0.087 mL, 0.528 mmol, 1.5 equiv) were added sequentially.
The reaction mixture was stirred for 30 min at 0 °C and then
was partitioned between water (100 mL) and a 1:1 mixture of
EtOAc and hexanes (2 × 100 mL). The combined organic layers
were dried over Na2SO4 and concentrated, and the residue was
chromatographed on silica gel (gradient elution, 30f40%
EtOAc in hexanes) to afford 34 (0.163 g, 52%) as a white
solid: mp ) 75-85 °C; Rf ) 0.48 (50% EtOAc in hexanes); IR
1
(cm-1) 3314, 1710, 1655; H NMR (CDCl3) δ 1.29 (t, 3H, J )
7.2), 1.48-1.67 (m, 6H), 1.97-2.03 (m, 2H), 2.29-2.42 (m, 2H),
2.54-2.97 (m, 8H), 3.54-3.63 (m, 1H), 4.18 (q, 2H, J ) 7.2),
4.51-4.58 (m, 2H), 5.44 (dd, 1H, J ) 15.6, 1.7), 5.59 (d, 1H, J
) 6.9), 5.90 (d, 1H, J ) 7.2), 6.16 (d, 1H, J ) 8.4), 6.64 (dd,
1H, J ) 15.6, 5.0), 6.91-7.08 (m, 5H), 7.14-7.29 (m, 20H).
Anal. (C53H56FN3O6S) C, H, N.
Eth yl-3-{cyclop en tylSCO-L-P h eΨ[COCH2]-L-P h e(4-F )-
L-Gln }-E-p r op en oa te (12). Triisopropylsilane (0.10 mL, 0.488
mmol, 2.7 equiv) and trifluoroacetic acid (6 mL) were added
sequentially to a solution of 34 (0.160 g, 0.181 mmol, 1 equiv)
in CH2Cl2 (10 mL), producing a bright yellow solution. The
reaction mixture was stirred for 30 min at 23 °C and then
carbon tetrachloride (6 mL) was added, and the mixture was
concentrated under reduced pressure. The residue was purified
by flash column chromatography (5% CH3OH in CH2Cl2) to
afford 12 (0.082 g, 71%) as a white solid: mp ) 210-212 °C;
Rf ) 0.10 (10% CH3OH in CH2Cl2); IR (cm-1) 3284, 1717, 1637;

SAR of Ketomethylene-Containing Peptidomimetics
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1211
then maintained at 23 °C for 24 h. After dilution with CH2Cl2
(50 mL), the resulting solution was washed with saturated
NaHCO3 (50 mL) and brine (50 mL). The organic layer was
dried over Na2SO4 and concentrated. The residue was purified
by flash column chromatography on silica gel (10% EtOAc in
hexanes) to afford 38 (1.01 g, 54%) as a pale yellow oil: Rf )
0.41 (25% EtOAc in hexanes); IR (cm-1) 1711, 1514, 1233; 1H
NMR (CDCl3) δ 0.97 (s, 9H), 2.58-2.76 (m, 2H), 2.96-3.17 (m,
3H), 3.62 (s, 3H), 4.17 (d, 1H, J ) 8.1), 5.06-5.10 (s, 2H), 5.32
(d, 1H, J ) 8.6), 7.12-7.36 (m, 10H). Anal. (C25H31NO5‚
0.25H2O) C, H, N.
Boc-L-tBu GlyΨ[COCH2]-L-P h e-OMe (39). A sample of
10% Pd on C (0.110 g) was added to a solution of 38 (0.513 g,
1.33 mmol, 1 equiv) and di-tert-butyl dicarbonate (0.378 g, 1.73
mmol, 1.3 equiv) in CH3OH (10 mL) at 23 °C. The reaction
mixture was stirred at 23 °C under an H2 atmosphere (balloon)
overnight and then was filtered through Celite. The filtrate
was concentrated under reduced pressure, and the residue was
purified by flash column chromatography (10% EtOAc in
hexanes) to afford 39 (0.366 g, 70%) as white solid: mp ) 98-
99 °C; Rf ) 0.54 (25% EtOAc in hexanes); IR (cm-1) 1707, 1497,
1367; 1H NMR (CDCl3) δ 0.97 (s, 9H), 1.40 (s, 9H), 2.60-2.78
(m, 2H), 2.95-3.19 (m, 3H), 3.63 (s, 3H), 4.07-4.11 (m, 2H),
5.07 (d, 1H, J ) 9.3), 7.13-7.32 (m 5H). Anal. (C25H31NO5) C,
H, N.
Boc-L-tBu GlyΨ[COCH2]-L-P h e-OH (40). A sample of 2.0
M NaOH (3.35 mL, 6.7 mmol, 8.0 equiv) was added to a
solution of 39 (0.328 g, 0.84 mmol, 1 equiv) in CH3OH (6 mL)
at 0 °C over 10 min. The reaction mixture was stirred at 0 °C
for 2 h and then was partitioned between 10% KHSO4 (80 mL)
and CH2Cl2 (2 × 100 mL). The organic layers were dried over
Na2SO4 and were concentrated to give 40 (0.315 g, 99%) as a
white solid which was used without further purification: IR
(cm-1) 2960, 1710, 1498; 1H NMR (CDCl3) δ 0.94 (s, 9H), 1.39
(s, 9H), 2.60-2.80 (m, 2H), 2.95-3.16 (m, 3H), 4.08 (d, 2H, J
) 9.3), 5.09 (d, 1H, J ) 9.6), 7.12-7.31 (m, 5H).
Eth yl-3-{Boc-L-tBu GlyΨ[COCH2]-L-P h e-L-(Tr -Gln )}-E-
p r op en oa te (41). HOBt (0.170 g, 1.26 mmol, 1.5 equiv),
4-methylmorpholine (0.77 mL, 2.52 mmol, 3 equiv), and EDC
(0.247 g, 1.26 mmol, 1.5 equiv) were added sequentially to a
solution of ethyl-3-[H2N-L-(Tr-Gln)]-E-propenoate‚HCl3 (26,
1.01 mmol, 1.2 equiv) and 40 (0.315 g, 0.84 mmol, 1 equiv) in
CH2Cl2 (10 mL) at 23 °C. The reaction mixture was stirred at
23 °C overnight and then was partitioned between water (100
mL) and CH2Cl2 (2 × 100 mL). The combined organic layers
were dried over Na2SO4 and concentrated, and the residue was
purified by flash column chromatography (35% EtOAc in
hexane) to afford 41 (0.474 g, 70%) as a white foam: Rf ) 0.58
(50% EtOAc in hexanes); IR (cm-1) 1702, 1669, 1494, 1169;
1H NMR (CDCl3) δ 0.85 (s, 9H), 1.30 (t, 3H, J ) 7.2), 1.41 (s,
9H), 1.56-1.65 (m, 1H), 1.95-2.02 (m, 1H), 2.22-2.42 (m, 2H),
2.62-2.88 (m, 4H), 3.09-3.18 (m, 1H), 4.00 (d, 1H, J ) 8.7),
4.17 (t, 2H, J ) 7.2), 4.46-4.51 (m, 1H), 4.93 (d, 1H, J ) 8.7),
5.37 (d, 1H, J ) 15.9), 5.69 (d, 1H, J ) 9.3), 6.54 (dd, 1H, J )
15.9, 4.8), 7.19-7.31 (m, 21H). Anal. (C49H59N3O7) C, H, N.
3.59-3.64 (m, 1H), 4.16 (q, 2H, J ) 7.2), 4.31 (d, 1H, J ) 8.4),
4.48 (m, 1H), 5.41 (dd, 1H, J ) 15.9, 1.8), 5.67 (d, 1H, J )
8.7), 5.82 (d, 1H, J ) 9.0), 6.56 (dd, 1H, J ) 15.6, 5.1), 7.19-
7.31 (m, 21H). Anal. (C50H59N3O6) C, H, N.
Eth yl-3-{cyclop en tylSCO-L-tBu Gly-Ψ[COCH2]-L-P h e-L-
Gln }-E-p r op en oa te (14). Triisopropylsilane (0.077 mL, 0.38
mmol, 1.0 equiv) and trifluoroacetic acid (4 mL) were added
sequentially to a solution of 42 (0.318 g, 0.38 mmol, 1 equiv)
in CH2Cl2 (4 mL) at 23 °C, producing a bright yellow solution.
After the mixture was stirred for 30 min, no yellow color
remained. The volatiles were removed under reduced pressure,
and the resulting white solid was triturated with Et2O (10 mL)
and filtered to give 14 (0.204 g, 91%): mp 65-68 °C; Rf ) 0.32
(10% CH3OH in CH2Cl2); IR (cm-1) 1715, 1652, 1520, 1193;
1H NMR (CDCl3) δ 0.96 (s, 9H), 1.31 (t, 3H, J ) 7.2), 1.45-
1.72 (m, 6H), 1.96-2.13 (m, 4H), 2.23 (t, 2H, J ) 7.5), 2.68-
2.79 (m, 2H), 2.84-2.95 (m, 2H), 3.11-3.21 (m, 1H), 3.59-
3.69 (m, 1H), 4.18 (q, 2H, J ) 7.2), 4.36 (d, 1H, J ) 8.1), 4.52-
4.59 (m, 1H), 5.37 (s, br, 1H), 5.42 (dd, 1H, J ) 15.9, 1.5), 5.80
(d, 1H, J ) 9.0), 5.90 (d, 1H, J ) 8.4), 6.46 (s, br, 1H), 6.61
(dd, 1H, J ) 15.9, 5.1), 7.18-7.30 (m, 5H). Anal. (C31H45N3O6S)
C, H, N.
Ack n ow led gm en t. We are grateful for many helpful
discussions throughout the course of this work with
Prof. Larry Overman and Dr. Steven Bender. We also
thank Dr. Kim Albizati, Dr. Srinivasan Babu, Terence
Moran, Ray Dagnino, and Miguel Pagan for the large-
scale preparation of several intermediates.
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Eth yl-3-{cyclop en tylSCO-L-tBu GlyΨ[COCH2]-L-P h e-L-
(Tr -Gln )}-E-p r op en oa te (42). A solution of HCl in 1,4-
dioxane (4.0 M, 4 mL) was added dropwise to a solution of 41
(0.441 g, 0.55 mmol, 1 equiv) in the same solvent at 23 °C.
The reaction mixture was stirred for 2 h at 23 °C and then
was concentrated to provide the amine salt as a white foam.
This material was dissolved in CH2Cl2 (20 mL) and cooled to
0 °C, and 4-methylmorpholine (0.181 mL, 1.65 mmol, 3 equiv)
and cyclopentyl chlorothiolformate23 (0.135 mL, 0.82 mmol, 1.5
equiv) were added sequentially. The reaction mixture was
stirred at 0 °C for 30 min and then was partitioned between
H2O (50 mL) and a 1:1 mixture of EtOAc and hexanes (3 × 50
mL). The combined organic layers were dried over Na2SO4 and
were concentrated. The residue was purified by flash column
chromatography (35% EtOAc in hexane) to provide 42 (0.347
g, 76%) as a white foam: Rf ) 0.48 (50% EtOAc in hexanes);
1
IR (cm-1) 1718, 1656, 1493, 1186; H NMR (CDCl3) δ 0.86 (s,
9H), 1.27 (t, 3H, J ) 7.2), 1.56-1.78 (m, 6H), 1.95-2.16 (m,
4H), 2.22-2.39 (m, 2H), 2.60-2.90 (m, 4H), 3.05-3.14 (m, 1H),

1212 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
(7) A similar series of HRV 3CP inhibitors has also recently been
described. See: Kong, J .-s.; Venkatraman, S.; Furness, K.;
Nimkar, S.; Shepherd, T. A.; Wang, Q. M.; Aube´, J .; Hanzlik, R.
P. Synthesis and Evaluation of Peptidyl Michael Acceptors That
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Zhou, R.; Webber, S. E.; Marakovits, J . T.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Lee, C. A.; Ford, C. E.; Burke,
B. J .; Rejto, P. A.; Hendrickson, T. F.; Tuntland, T.; Brown, E.
L.; Meador, J . W., III.; Ferre, R. A.; Harr, J . E. V.; Kosa, M. B.;
Worland, S. T. Structure-Based Design, Synthesis, and Biological
Evaluation of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 4. Incorporation of P1 Lactam Moieties as L-
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Reaction. J . Am. Chem. Soc. 1996, 118, 11970-11971.
(26) Luly, J . R.; Dellaria, J . F.; Plattner, J . J .; Soderquist, J . L.; Yi,
N. A Synthesis of Protected Aminoalkyl Epoxides from R-Amino
Acids. J . Org. Chem. 1987, 52, 1487-1492.
(27) Hoffman, R. V.; Tao, J . A Simple, Stereoselective Synthesis of
Ketomethylene Dipeptide Isosteres. Tetrahedron 1997, 53, 7119-
7126.
(28) The basic hydrolysis utilized to prepare γ-keto-acid 40 did not
result in substantial epimerization as evidenced by the lack of
significant diastereomers of the subsequent coupling product
(41). However, considerable epimerization was noted when
similar hydrolyses were attempted on other less-hindered γ-keto-
ester substrates (e.g., the Val-Phe isostere corresponding to
γ-keto-ester 39).
(13) In several cases, the solubility of a given inhibitor was deter-
mined to be less than the reported CC50 value. However, the
most active antiviral agents were clearly soluble and nontoxic
to more than 10× the reported EC50 values.
(14) The antirhinoviral properties of certain 3CP inhibitors were
determined under similar conditions against several additional
HRV serotypes in cell culture. In general, the activity observed
for a given compound vs serotypes 1A, 2, and 10 was representa-
tive of its broad-spectrum antiviral properties. Serotype 14
nearly always proved to be the most susceptible to the antiviral
agents under study. Full details of these experiments will be
presented elsewhere (Patick, A. K. Manuscript in preparation).
(15) Dragovich, P. S.; Zhou, R.; Skalitzky, D. J .; Fuhrman, S. A.;
Patick, A. K.; Ford, C. E.; Meador, J . W., III; Worland, S. T. Solid-
Phase Synthesis of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 1. Optimization of Tripeptides Incorporating N-
Terminal Amides. Bioorg. Med. Chem., in press.
(29) Compound 15 was synthesized by a method analogous to that
reported previously for the preparation of tripeptide-derived,
irreversible 3CP inhibitors. See refs 3 and 4 above.
J M980537B