Synthesis of a peptidomimetic HCMV protease inhibitor library

Article abstract of DOI:10.1055/s-2002-31948

The human cytomegalovirus (HCMV) protease catalyzes the maturational process of the herpes virus assembly protein and plays a key role during the manufacture of viral capsid, and so is an attractive target for potential anti-herpes-virus agents with novel structures and new mechanisms. In this work, a peptidomimetic skeleton was designed and a chemical library containing 32 compounds with different substitutions on the skeleton was prepared by the oxidation of a precursor library, which was constructed from four types of building blocks: 4 carboxylic acids, 2 amines, 2 aldehydes and 2 isocyanides, based on multicomponent condensation following liquid phase strategies. The syntheses of the key building block isocyanides are presented.

Full text of DOI:10.1055/s-2002-31948

PAPER
1017
Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library
Synthesis of
a
i
Peptid
n
H
C
g
M
V
Protease Inhib
X
itor
L
ibrary u,* Wenwei Lin, Xiaomin Zou
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083,
P. R. China
Fax +86(010)62015584; E-mail: pingxuzh@public.bta.net.cn ; E-mail: pingxu@bjmu.edu.cn
Received 31 January 2002
Abstract: The human cytomegalovirus (HCMV) protease cata-
lyzes the maturational process of the herpes virus assembly protein
and plays a key role during the manufacture of viral capsid, and so
is an attractive target for potential anti-herpes-virus agents with
novel structures and new mechanisms. In this work, a peptidomi-
metic skeleton was designed and a chemical library containing 32
compounds with different substitutions on the skeleton was pre-
pared by the oxidation of a precursor library, which was constructed
from four types of building blocks: 4 carboxylic acids, 2 amines, 2
aldehydes and 2 isocyanides, based on multicomponent condensa-
tion following liquid phase strategies. The syntheses of the key
building block isocyanides are presented.
Key words: combinatorial chemistry, condensation, antiviral
agents, isocyanides, peptides
Figure 1 Chemical structure of library molecules
The human cytomegalovirus (HCMV) is a member of the
herpes virus family infecting 40–80% of the general pop-
ulation.1 HCMV, like all other herpes viruses, encodes a
binatorial synthetic method for peptidomimetic protease
inhibitors.
protease that is essential for viral replication. Inhibiting
this enzyme would show antiviral activity, and the en-
zyme has become a potential target for therapeutic
agents.2 Peptidomimetic compounds are very promising
molecules in developing new drugs, especially as protease
inhibitors, and there are many successful precedents.
Finding general synthetic methods for this kind of com-
pound, especially combinatorial synthetic methods that
can greatly improve the efficiency of finding new drugs,
is very useful. In this paper, a peptidomimetic HCMV
protease inhibitor library was designed and synthesized
with a liquid-phase, multicomponent condensation reac-
tion.
The library was synthesized by liquid-phase combinatori-
al synthesis, using a Ugi four-component condensation re-
action, following a two-step strategy (Scheme 1).The first
step was the Ugi condensation of four kinds of building
blocks: carboxylic acids II, amines III, aldehydes IV, and
isocyanides V (Figure 2), to produce the precursor library
VI. This was followed by oxidation of VI to form the ac-
tivated carbonyl group in compound I. Nakamura et al
have also reported similar work.6
Several crystallographic reports have shown that HCMV
protease represents a novel structure of serine protease
and in fact possesses a unique catalytic triad.3 A series of
peptidomimetic inhibitors of the HCMV protease has
been developed,4 and a crystal structure of HCMV pro-
tease in complex with a peptidomimetic inhibitor (cover-
ing the P4 to P1 positions) has been determined,5 which
revealed that the inhibitor was bound in an extended con-
formation, forming an anti-parallel -sheet with the pro-
tease, and that there were five hydrogen bonds and a
covalent bond between the inhibitor and the protease. On
the basis of this interaction mode, a peptidomimetic li-
brary I (Figure 1) was designed for building up a com-
Synthesis 2002, No. 8, 04 06 2002. Article Identifier:
1437-210X,E;2002,0,08,1017,1026,ftx,en;F01202SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881
Scheme 1

1018
P. Xu et al.
PAPER
Scheme 3
Figure 2 Chemical structures of the four kinds of building blocks
In the four building blocks, IIa, IIb, III and IV were com-
mercially available. Compounds IIc and IId can be pre-
pared conveniently (Scheme 2).
age of tartarate amides IX to form glyoxylate amides X.
Because of the poor solubility of IX in diethyl ether, sev-
eral other solvents were tried, such as THF, methanol and
ethanol. THF gave less improvement in solubility. Meth-
anol and ethanol can solve all reactants well, but the reac-
tions gave much more by-product. The mixture of diethyl
ether and ethanol (95:5) was found to be the best solvent
for the reaction both in solubility and good reaction. The
cleavage of IX in this mixed solvent can be carried out
smoothly and rapidly to produce glyoxylate amides X,
and no by-product was detected. The colorless oils X were
reacted immediately after preparation by addition of nitro-
ethane under the catalization of potassium fluoride on alu-
mina (KF/alumina) to generate 2-hydroxy-3-nitro-butyric
amides XI. The yields of these two steps were 61.1%
(XIa) and 41.3% (XIb), respectively. Alumina coated
with potassium fluoride proved to be a versatile solid base
for olefin- and acetylene- forming elimination, the Micha-
el addition, aldol condensation, and the Darzens conden-
sation.9 KF/alumina was tried as the solid base in
nitroethane addition to X and the procedure was improved
by packing the reaction mixture with black paper and
standing at r.t. for 48 h. This decreased the formation of
by-products and gave the product XI with higher purity
and yield. There are two chiral centers formed in the 2-hy-
droxy-3-nitrobutyric amide XI molecule when nitroet-
hane is added to glyoxylate amide X, and this theoretically
produces four optical isomers. The crystalline products
XIa and XIb were examined by TLC, which showed two
spots with XIa and one spot with XIb. The two samples
corresponding to the two spots with XIa were obtained by
silica gel column chromatography. They have different
melting points (98–100°C and 118–120°C, respectively),
Isocyanides V have to be produced from their precursors
XIII just before use owing to their instability. The syn-
thetic route to V is shown in Scheme 3. By following this
method, two isocyanides were synthesized with R group
equals benzyl (Va) and cyclohexyl (Vb), respectively.
The amidation of diethyl tartarate with corresponding
amines is a good way to N,N -disubstituted tartaric acid
diamides IX. By following literature procedure,7 IXa was
produced in good yield (95.3%), and IXb in moderate
yield (76.7%). Kelly and co-workers reported an applica-
ble convenient, high-yielding method for the preparation
of esters of glyoxylic acid.8 The method consists of the
cleavage of the corresponding tartrate esters by ethereal
periodic acid. This method was applied here in the cleav-
Scheme 2
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

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Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library
1019
1
but exactly the same H NMR spectum. These two sam-
ples were believed to be a pair of enantiomers. The abso-
lute configurations of the two samples have not yet been
determined. The reported structure-activity relationships4
showed that the absolute configuration of the carbon atom
did not affect the bioactivity, so the pairs of enantiomers
of XI were retained unseparated.
The transformation of XI to XII was achieved by reduc-
tion of nitro group in XI to the amino group in XII. Sev-
eral methods were tried, e.g. Fe/HCl, Fe/HOAc,
HCOONH4/10% Pd-C, NaBH4/NiCl2, Sn/HCl, Zn/
NH4Cl, and H2/10% Pd-C. It was found that the
HCOONH4/10% Pd-C, Zn/NH4Cl, H2/10% Pd-C method
was better than the others, but the yield and purity of the
product were not satisfisfactory. Optimised results were
obtained with P–2 Ni10 (Scheme 4) and 4 atm H2/10% Pd-
C for 12 h, and then, after filtration, with Zn/NH4Cl for a
further 12 h. The products XII were adequate and could
be used in the next reaction without purification.
Scheme 5
Typically equal moles of the four components reacted
with each other in a Ugi reaction. But in the preparation of
compound XIV, the amount of benzylamine was doubled.
The extra one mole of the amine was used to transfer the
formylate group in V to the hydroxy group in XIV. Ac-
cording to the mechanism of the Ugi reaction, on route to
producing the condensation product XIV, the amino
group of benzylamine first reacts with the carbonyl group
of benzaldehyde to form a iminium intermediate state 1,
which is attacked by the nucleophilic isocyanide carbon to
form the intermediate state 2, this would produce a new
chiral center C* (Scheme 6). Because the attachment of
the isocyanide was equal from the two sides of the sp2
plane in transition state 1, the product XIV might be
formed equally as a pair of diastereomers. But in our ex-
periment XIV was yielded as one compound, as were all
the precursor library compounds VI. No structural data
were obtained to show the stereostructures.
Scheme 4
The formylation of XII was carried out by using HCOO-
Et, HCOOOCMe, DCC/HCOOH, HCOOCH2CN (imida-
zole),11 and DCC/HCOOH/NEt3/HCOOCH2CH2CN
(imidazole), respectively. Only DCC/HCOOH/NEt3/
HCOOCH2CH2CN (imidazole) gave good results. A cat-
alytic amount of cyanoethyl formate was needed. The
amino and hydroxyl groups in XII were formylated at
same time to give the diformylated products XIII. The
yields of the two steps (reduction and formylation) were
63.6% (XIIIa) and 52.0% (XIIIb), respectively.
The preparations of isocyanides has been reviewed.12 The
POCl3–NEt3 method was used in this work, and the two
isocyanides V were obtained from their precursors XIII.
The formation of the isocyanides was detected by the
characteristic 13C NMR peaks of the isocyanide groups at
= 160.654 (Va) and 158.642 (Vb), as well as the IR ab-
sorbance at 2200 cm–1. When we tried to purify the prod-
ucts by silica chromatography, the isocyanides were
found to be very unstable. They were therefore difficult to
purify, so the procedure was improved to finish the reac-
tion in shorter time (treated with POCl3 for 30 min instead
of 1 h, then with Na2CO3 for 5 min instead of 30 min),
then worked up and used immediately. The determina-
tions of the Ugi-4CC products confirm the correct struc-
tures of isocyanides V.
Scheme 6
Four carboxylic acids, 2 amines, 2 aldehydes and 2 isocy-
anides were chose to build up the precursor library VI,
which contained 32 molecules (compounds VI01–VI32,
Table 1), which were then oxidized to the target library I
(compounds I01–I32, Table 2). The oxidation of a hy-
droxy to a carbonyl group can be carried out by many
methods. In this work, Swern oxidation and Oppenauer
oxidation were tried, as well as other oxidants, e.g. CrO3
derivatives and DMP (Dess–Martin Periodinane).13 DMP
was found to be the best reagent to transform VI to I. The
reaction was done under mild conditions, and gave neat
product with high yield. DMP can be prepared from IBX
(2-iodoxybenzoic acid, Scheme 7).14
Before the precursor library VI was synthesized
(Scheme 1), a model reaction (Scheme 5) was undertaken
to test the method, and the products XIV and XV were de-
signed to be representative compounds of the precursor
and target libraries, respectively. The structures of XIV
and XV were confirmed by their EI–MS, elemental anal-
ysis, 1H NMR and IR spectra (see experimental section).
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

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P. Xu et al.
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peptidomimetic inhibitors. A chemical library containing
32 compounds with different substitutions on the skeleton
was prepared by the oxidation of a precursor library,
which was constructed from the four types of building
blocks: 4 carboxylic acids, 2 amines, 2 aldehydes and 2
isocyanides based on a multicomponent condensation fol-
lowing liquid phase strategies. The syntheses of the key
building block isocyanides are reported in detail. This liq-
uid phase method was not satisfactory enough to be ap-
plied to build up large scale libraries because of the
difficulty of purification and the low yields. It is necessary
to develop solid phase procedure to improve the method.
Scheme 7
Following the general procedure, all the library molecules
were synthesized, and large part of them were purified
from the reaction mixtures by chromatography and deter-
mined by MS spectrometry. The yields were 12.2–45.2%
for the precursor library VI preparation, 68.3–90.4% for
oxidation of library VI to target library I.
In conclusion a peptidomimetic skeleton was designed
based on the interactions between HCMV protease and its
Table 1 The Precursor Library VI Molecules
No.
R1
R2
R3
R4
R5
Yield
(%)
Formulae
(MW)
MSa,b (m/z)
Mp
(°C)
VI01
VI02
VI03
VI04
VI05
VI06
VI07
VI08
VI09
VI10
VI11
VI12
VI13
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
C6H11
19.7
32.7
28.6
36.4
45.2
C31H36N4O5
(544)
530a
(M + 1 –CH3)
55–57
85–87
67–69
69–71
66–68
H
C30H40N4O5
(536)
536a
(M)
H
(CH3)2CHC PhCH2
H2
C28H38N4O5
(510)
511b
(M +1)
H
(CH3)2CHC C6H11
H2
C27H42N4O5
(502)
503a
(M + 1)
H
(CH3)2CHC PhCH2
H2CH2
PhCH2
C29H40N4O5
(524)
524a
(M)
H
(CH3)2CHC PhCH2
H2CH2
C6H11
C28H44N4O5
(516)
H
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
33.4
C26H42N4O5
(490)
491b
(M + 1)
72–74
H
(CH3)2CHC (CH3)2CHC C6H11
H2CH2
C25H46N4O5
(482)
H2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
C6H11
36.2
24.6
41.1
40.2
44.5
C38H42N4O5
(634)
634a
(M)
65–67
72–74
73–75
67–69
69–71
PhCH2
PhCH2
PhCH2
PhCH2
C37H46N4O5
(626)
626a
(M)
(CH3)2CHC PhCH2
H2
C35H44N4O5
(600)
600a
(M)
(CH3)2CHC C6H11
H2
C34H48N4O5
(592)
592a
(M)
(CH3)2CHC PhCH2
H2CH2
PhCH2
C36H46N4O5
(614)
613a
(M – 1)
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

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Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library
1021
Table 1 The Precursor Library VI Molecules (continued)
No.
R1
R2
R3
R4
R5
Yield
(%)
Formulae
(MW)
MSa,b (m/z)
Mp
(°C)
VI14
VI15
VI16
VI17
VI18
VI19
VI20
VI21
VI22
VI23
VI24
VI25
VI26
VI27
CH3
CH3
CH3
PhCH2
PhCH2
PhCH2
H
(CH3)2CHC PhCH2
H2CH2
C6H11
C35H50N4O5
(606)
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
39
C33H48N4O5
(580)
580a
(M)
79–81
62–64
84–86
(CH3)2CHC (CH3)2CHC C6H11
19.0
36.9
C32H52N4O5
(572)
572a
(M )
H2CH2
H2
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
C6H11
C40H45N5O6
(691)
690b
(M – 1)
PhCH2NHCO
CH2CH2
H
PhCH2
PhCH2
PhCH2
PhCH2
C39H49N5O6
(683)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2
27.4
C37H47N5O6
(657)
658b
(M + 1)
92–94
PhCH2NHCO
CH2CH2
H
(CH3)2CHC C6H11
H2
C36H51N5O6
(649)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2CH2
PhCH2
C38H49N5O6
(671)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2CH2
C6H11
C37H53N5O6
(663)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
35
C35H51N5O6
(637)
638a
(M + 1)
96–98
76–78
83–85
PhCH2NHCO
CH2CH2
H
(CH3)2CHC (CH3)2CHC C6H11
C34H55N5O6
(629)
H2CH2
H2
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
C6H11
12.2
29.3
23.2
C47H51N5O6
(781)
781b
(M)
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
C46H55N5O6
(773)
774b
(M + 1)
PhCH2NHCO
CH2CH2
(CH3)2CHC PhCH2
H2
C44H53N5O6
(747)
807b
(M – 3 H +
Na + K)
VI28
VI29
VI30
VI31
VI32
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
(CH3)2CHC C6H11
H2
20.6
18.6
C43H57N5O6
(739)
56–88
87–89
PhCH2NHCO
CH2CH2
(CH3)2CHC PhCH2
H2CH2
PhCH2
C45H55N5O6
(761)
PhCH2NHCO
CH2CH2
(CH3)2CHC PhCH2
H2CH2
C6H11
C44H59N5O6
(753)
PhCH2NHCO
CH2CH2
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
34.4
C42H57N5O6
(727)
728b
(M + 1)
95–97
PhCH2NHCO
CH2CH2
(CH3)2CHC (CH3)2CHC C6H11
H2CH2 H2
C41H61N5O6
(719)
a EI–MS
b TOF–MS
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P. Xu et al.
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Table 2 The Target Library I Molecules
No.
I01
I02
I03
I04
I05
I06
I07
I08
I09
I10
I11
I12
I13
I14
I15
I16
I17
I18
I19
I20
I21
R1
R2
R3
R4
R5
Yield
(%)
Formula
(MW)
MSa–c (m/z)
Mp
(°C)
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
C6H11
71.3
82.3
68.3
77.8
78.6
C31H34N4O5
(542)
542a
(M)
58–60
87–89
54–56
68–70
54–56
H
C30H38N4O5
(534)
534a
(M)
H
(CH3)2CHC PhCH2
H2
C28H36N4O5
(508)
508a
(M)
H
(CH3)2CHC C6H11
H2
C27H40N4O5
(500)
500a
(M)
H
(CH3)2CHC PhCH2
H2CH2
PhCH2
C29H38N4O5
(522)
522a
(M)
H
(CH3)2CHC PhCH2
H2CH2
C6H11
C28H42N4O5
(514)
H
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
C26H40N4O5
(488)
H
(CH3)2CHC (CH3)2CHC C6H11
H2CH2
C25H44N4O5
(480)
H2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
H
PhCH2
PhCH2
PhCH2
C6H11
87
C38H40N4O5
(632)
632a
(M)
85–87
PhCH2
PhCH2
PhCH2
PhCH2
C37H44N4O5
(624)
(CH3)2CHC PhCH2
H2
79
C35H42N4O5
(598)
598a
(M)
(CH3)2CHC C6H11
H2
80.6
76.7
C34H46N4O5
(590)
590a
(M)
67–69
69–71
(CH3)2CHC PhCH2
H2CH2
PhCH2
C36H44N4O5
(612)
611a
(M – 1)
(CH3)2CHC PhCH2
H2CH2
C6H11
C35H48N4O5
(604)
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
83.5
73.8
C33H46N4O5
(578)
579a
(M + 1)
72–74
(CH3)2CHC (CH3)2CHC C6H11
H2CH2
C32H50N4O5
(570)
570a
(M)
H2
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
C6H11
C40H43N5O6
(689)
PhCH2NHCO
CH2CH2
H
PhCH2
PhCH2
PhCH2
PhCH2
C39H47N5O6
(681)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2
82.9
C37H45N5O6
(655)
654c
(M – 1)
91–93
PhCH2NHCO
CH2CH2
H
(CH3)2CHC C6H11
H2
C36H49N5O6
(647)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2CH2
PhCH2
C38H47N5O6
(669)
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Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library
1023
Table 2 The Target Library I Molecules (continued)
No.
I22
I23
I24
I25
I26
I27
I28
I29
I30
I31
I32
R1
R2
R3
R4
R5
Yield
(%)
Formula
(MW)
MSa–c (m/z)
Mp
(°C)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC PhCH2
H2CH2
C6H11
C37H51N5O6
(661)
PhCH2NHCO
CH2CH2
H
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
86.4
90.4
C35H49N5O6
(635)
635b
(M)
96–98
90–92
PhCH2NHCO
CH2CH2
H
(CH3)2CHC (CH3)2CHC C6H11
H2CH2
C34H53N5O6
(627)
H2
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
C6H11
C47H49N5O6
(779)
780c
(M + 1)
PhCH2NHCO
CH2CH2
PhCH2
PhCH2
PhCH2
PhCH2
C46H53N5O6
(771)
PhCH2NHCO
CH2CH2
(CH3)2CHC PhCH2
H2
69.1
76.4
82.5
C44H51N5O6
(745)
745c
(M)
94–96
55–57
87–89
PhCH2NHCO
CH2CH2
(CH3)2CHC C6H11
H2
C43H55N5O6
(737)
738b
(M + 1)
PhCH2NHCO
CH2CH2
(CH3)2CHC PhCH2
H2CH2
PhCH2
C45H53N5O6
(759)
760b
(M + 1)
PhCH2NHCO
CH2CH2-
(CH3)2CHC PhCH2
H2CH2
C6H11
C44H57N5O6
(751)
PhCH2NHCO
CH2CH2
(CH3)2CHC (CH3)2CHC PhCH2
H2CH2 H2
89
C42H55N5O6
(725)
725c
(M +1)
97–99
PhCH2NHCO
CH2CH2
(CH3)2CHC (CH3)2CHC C6H11
H2CH2 H2
C41H59N5O6
(717)
a EI–MS
b TOF–MS
c ES–MS
Unless otherwise mentioned, reagents were obtained commercially
and used without further purification. Melting points (uncorrected)
were taken with an X-4 apparatus and were uncorrected. IR (KBr),
1H NMR, 13C NMR and element data were taken with PE-983,
VXR-300S, Carloerba-1106 instruments, respectively. Petroleum
ether used had a boiling point range 60–90 °C.
evaporated under vacuum and the residual solid was washed with
Et2O to yield 3.33 g (97.8%) of white solid benzyl glycinate p-tolu-
enesulfonate; mp 127–129 °C. The solid was dispersed in CHCl3
(50 mL), then extracted with 10% aq Na2CO3/sat. aq NaCl (3 20
mL). The organic layer was dried (Na2SO4) for 30 min and filtered
to give the solution of benzyl glycinate in CHCl3.
To a solution of VII (2.07 g, 10 mmol) in anhyd CHCl3 (50 mL),
DCC (2.5 g, 12 mmol) was added with stirring. After 10 min, the so-
lution of benzyl glycinate in CHCl3 prepared above was added. The
mixture was stirred at r.t. for 2 h and filtered. The filtrate was
washed with 10% aq Na2CO3/sat. NaCl (2 20 mL), dried (Na2SO4)
for 30 min, filtered and evaporated to obtain crude VIIIc, which
was purified with silica chromatography (CHCl3–CH3OH 20:1).
Benzylaminocarbonylpropylic Acid (VII)
Anhydrous Et3N (6.5 g, 64 mmol) was added to a solution of suc-
cinic anhydride (3.1 g, 31 mmol) in anhyd CHCl3 (30 mL). After 5
min stirring, benzylamine (3.28 mL, 30mmol) was added and the
mixture was stirred at 60 °C for 12 h, and then mixed thoroughly
with l2 M aq NaOH (50 mL). The aq layer was extracted and
washed with CH2Cl2 (30 mL), and dried (Na2SO4). After filtration,
the pH was modulated to 2 with concd HCl. A large amount of white
solid was precipitated, filtered off, washed with water and dried un-
der reduced pressure to give 5.23 g (84.22%) of VII; mp 136 °C.
Yield: 2.62 g (73.9%); white crystals; mp 136–137°C.
IR: 3290, 3060, 1741, 1632, 1448, 1410, 891, 799 cm–1.
1H NMR (DMSO-d6): = 8.35 (m, 2 H, NHCO), 7.36–7.19 (m, 10
H, PhH), 5.11 (s, 2 H, OCH2Ph), 4.26 (d, 2 H, J = 6.0 Hz,
NCH2CO), 3.89 (d, 2 H, J = 6.0 Hz, PhCH2N), 2.41 (m, 4 H,
CH2CH2).
Benzyl N-(Benzylaminocarbonylpropyl)glycinate (VIIIc)
Glycine (0.75 g, 10 mmol) and p-toluenesulfonic acid monohydrate
(2.28 g, 12 mmol) were dissolved in benzyl alcohol (5 mL) and then
mixed with CCl4 (120 mL). The mixture was refluxed for 8 h and
the produced water was azeotropically distilled. The solvents were
MS (EI): m/z = 354 (M+).
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

1024
P. Xu et al.
PAPER
Anal. Calcd for C20H22N2O4 (354.41): C, 67.78; H, 6.26; N, 7.90.
Found C, 67.95; H, 6.58; N, 7.78.
Yield: 46.9 g (95.3%); white crystal; mp 201–203 °C.
IR: 3354, 3309, 1623, 1543, 1093 cm–1.
1H NMR (DMSO-d6): = 8.22 (t, 2 H, J = 6.2 Hz, CONHBn),
7.30–7.20 (m, 10 H, PhH), 5.70 (d, 2 H, J = 5.4 Hz, OH, disap-
peared with D2O), 4.42–4.28 (m, 6 H, OCH, PhCH2).
Benzyl N-(Benzylaminocarbonylpropyl)phenylalaninate (VII-
Id)
Following the same procedure with VIIIc, from phenylalanine
(1.65 g, 10 mmol) and p-toluenesulfonic acid monohydrate (2.28 g,
12 mmol), 4.12 g (96.5%) of white solid benzyl phenylalaninate p-
toluenesulfonate were obtained mp 186–188 °C.
MS (FAB): m/z = 329 (M + 1).
Anal. Calcd for C18H20N2O4 (328.37): C, 65.83; H, 6.15; N, 8.53.
Found C, 65.98; H, 6.13; N, 8.51.
Yield: 2.94 g (83%); white crystals; mp 144–146 °C.
IR: 3300, 3058, 1629, 1532, 1490, 1449, 844 cm–1.
1H NMR (DMSO-d6): = 8.42 (d, 1 H, J = 7.8 Hz, NHCO), 8.32 (t,
1 H, J = 5.7 Hz, BnNHCO), 7.37–7.18 (m, 15 H, PhH), 5.05 (m, 2
H, OCH2Ph), 4.50 (m, 1 H, NCHCO), 4.24 (d, 2 H, J = 5.7 Hz,
NCH2Ph), 3.04–2.86 (m, 2 H, CCH2Ph), 2.34 (m, 4 H, CH2CH2).
N,N -Dicyclohexyl-2,3-dihydroxysuccinic Diamide (IXb)
Following the same procedure with IXa, from cyclohexylamine
(35.7 g, 0.36 mol), IXb was obtained.
Yield: 35.9 g (76.7%); white crystal; mp 217–219 °C.
IR: 3344, 3297, 2929, 2848, 1645, 1619, 1548, 1118, 1102 cm–1.
MS (EI): m/z = 444 (M+).
1H NMR (DMSO-d6): = 7.32 (d, 2 H, J = 8.4 Hz, CONH), 5.49 (br
s, 2 H, OH, disappeared with D2O), 4.15 (s, 2 H, OCH), 1.67–1.52
(m, 10 H, CH, CH2), 1.25–1.08 (m, 12 H, CH2).
Anal. Calcd for C27H28N2O4 (444.53): C, 72.95; H, 6.35; N, 6.30.
Found C, 72.78; H, 6.48; N, 6.12.
MS (EI): m/z = 312 (M+).
N-(Benzylaminocarbonylpropyl)glycine (IIc)
Anal. Calcd for C16H28N2O4 (312.41): C, 61.50; H, 9.05; N, 8.97.
Found C, 61.76; H, 9.15; N, 8.95.
To a solution of VIIIc (3.54 g, 10 mmol) in CH3OH (50 mL), a cat-
alytic amount of 10% Pd-C and one drop of anhyd formic acid were
added. The mixture was stirred under 4 atm H2 at r.t. for 6 h. The
Pd/C was filtered off and the solvent evaporated to yield crude solid.
Recrystallization from CH3OH–acetone–petroleum ether (5:1:1)
gave IIc.
N-Benzyl-2-hydroxy-3-nitrobutyric Amide (XIa)
To a solution of IXa (32.8 g, 0.1 mol) in dry 5% EtOH–Et2O (225
mL), paraperiodic acid (11.4 g, 0.05 mole) was added in portions
over 2 h under nitrogen with stirring. The reaction mixture was
stirred for 24 h at r.t., then petroleum ether (400 mL) was added with
stirring to separate out solid by-product completely. After filtration,
the filtrate solution was evaporated under vacuum to obtain the col-
orless oil of N-benzyl glyoxylic amide (Xa), which was then mixed
thoroughly with nitroethane (15 g, 0.2 mole), and absorbed homo-
geneously and completely with an appropriate amount of KF–Al2O3
(prepared as described9). The mixture was packed with black paper
and stood at r.t. for 48 h, then washed with acetone (ca. 500 mL).
Evaporation of the acetone solution gave crude product solid, and
recrystallization from petroleum ether–CHCl3 (10:1) yielded XIa.
Yield: 1.66 g (63%); white solid; mp 192–193 °C.
IR: 3308, 3066, 2923, 1702, 1636, 1547, 1415, 1332, 1237, 1034,
696 cm–1.
1H NMR (DMSO-d6): = 12.47 (s, 1 H, COOH, disappeared with
D2O), 8.34 (t, 1 H, J = 5.7 Hz, NHCO), 8.18 (t, 1 H, J = 5.7 Hz, NH-
CO), 7.30–7.21 (m, 5 H, PhH), 4.25 (d, 2 H, J = 5.7 Hz, NCH2CO),
3.73 (d, 2 H, J = 5.7 Hz, NCH2Ph), 2.38 (s, 4 H, CH2CH2).
MS (EI): m/z = 264 (M+).
Anal. Calcd for C13H16N2O4 (264.28): C, 58.08; H, 6.10; N, 10.60.
Found C, 58.47; H, 6.34; N, 10.34.
Yield: 29.0 g (61.1%); white plate crystal; mp 118–120 °C.
IR: 3328, 1630, 1547, 746 cm–1.
1H NMR (DMSO-d6): = 8.56 (t, 1 H, J = 6.0 Hz, CONH), 7.34–
7.20 (m, 5 H, PhH), 6.54 (d, 1 H, J = 6.3 Hz, OH, disappeared with
D2O), 5.00–4.91 (m, 1 H, CHNO2), 4.32 (d, 2 H, J = 6.0 Hz,
NCH2Ph), 4.27 (t, 1 H, J = 5.4 Hz, OCH), 1.46 (d, 3 H, J = 6.9 Hz,
CH3).
N-(Benzylaminocarbonylpropyl)phenylalanine (IId)
Following the same procedure with IIc, from VIIId (4.44 g, 10
mmol), IId was obtained.
Yield: 2.82 g (79.65%); white crystals; mp 166–168 °C.
IR: 3314, 3060, 3025, 2920, 1715, 1635, 1535, 1449, 1426, 892,
748 cm–1.
MS (EI): m/z = 238 (M+).
1H NMR (DMSO-d6): = 8.32 (t, 1 H, J = 6.0 Hz, BnNHCO), 8.10
(d, 1 H, J = 7.8 Hz, CONH), 7.31–7.17 (m, 10 H, PhH), 5.60 (d, 1
H, J = 8.1 Hz, COOH, disappeared with D2O), 4.35 (m, 1 H, NCH-
CO), 4.24 (d, 2 H, J = 6.0 Hz, PhCH2N), 3.06–2.79 (m, 2 H,
PhCH2C), 2.30 (t, 4 H, CH2CH2).
Anal. Calcd for C11H14N2O4 (238.24): C, 55.45; H, 5.93; N, 11.76.
Found C, 55.31; H, 5.93; N, 11.80.
N-Cyclohexyl-2-hydroxy-3-nitrobutyric Amide (XIb)
Following the same procedure with XIa, from IXb (31.2 g, 0.1
mol), XIb was obtained.
MS (EI): m/z = 354 (M+).
Anal. Calcd for C20H22N2O4 (354.41): C, 67.78; H, 6.26; N, 7.90.
Found C, 67.83; H, 6.25; N, 7.76.
Yield: 19.0 g (41.3%); white plate crystal; mp 130–132 °C.
IR: 3346, 2918, 2851, 1643, 1545, 1380 cm–1.
1H NMR (DMSO-d6): = 7.78 (d, 1 H, J = 8.1 Hz, CONH), 6.39 (d,
1 H, J = 6.6 Hz, OH, disappeared with D2O), 4.91–4.82 (m, 1 H,
CHNO2), 4.17 (t, 1 H, J = 5.9 Hz, OCH), 1.69 (m, 4 H, CH2), 1.58
(m, 1 H, CH), 1.43 (d, 3 H, J = 6.9 Hz, CH3), 1.29–1.12 (m, 6 H,
CH2).
N,N -Dibenzyl-2,3-dihydroxysuccinic Diamide (IXa)
A mixture of (+)-diethyl 2,3-dihydroxysuccinate (30.9 g, 0.15
mole), benzylamine (38.5 g, 0.36 mole), K2CO3 (0.5 g, 3.6 mmol)
and MeOH (150 mL) was refluxed for 7 h and then cooled. White
crystals separated. After filtration, a small amount of water was
added into the filtrate solution, and more crystals were obtained.
IXa was yielded by recrystallization of the crude material from
EtOH–H2O (1:1).
MS (EI): m/z = 230 (M+).
Anal. Calcd for C10H18N2O4 (230.26): C, 52.15; H, 7.89; N, 12.17.
Found C, 52.28; H, 7.97; N, 12.17.
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library
IR: 3500, 3412, 3330, 2936, 1659, 1526 cm–1.
1025
N-Benzyl-2-hydroxy-3-aminobutyric Amide (XIIa)
To a solution of XIa (2.38 g, 10 mmol) in CH3OH (120 mL), a cat-
alytic amount of 10% Pd-C, one drop of anhyd formic acid and a
catalytic amount of P-2 Ni (prepared as described10) were added.
The mixture was stirred under 4 atm H2 at r.t. for 12 h. After filtra-
tion, to the filtrate solution, NH4Cl (7.0 g, 120 mmol), zinc powder
(4.9 g, 75 mmol) and H2O (5 mL) were added. After further stirring
at r.t. for 12 h, the mixture was filtered and the filtrate was evapo-
rated under vacuum to dryness to yield crude solid XIIa. This crude
XIIa can be used directly in the next reaction without further puri-
fication. An analytical sample was obtained from the crude material
with silica gel column (CHCl3–MeOH, 6:1) and then recrystalliza-
tion from CHCl3–Et2O–petroleum ether (5:2:1).
1H NMR (DMSO-d6): = 7.99 (s, 1 H, OCHO), 7.93 (m, 1 H, NH-
CO), 7.51 (d, 1 H, J = 8.4 Hz, CONH), 5.78 (d, 1 H, J = 5.7 Hz,
NCHO, disappeared with D2O), 4.20 (m, 1 H, OCH), 3.92 (m, 1 H,
NCH), 3.57 (m, 1 H, CH), 1.68–0.99 (m, 10 H, CH2), 0.89 (d, 3 H,
J = 6.3 Hz, CH3).
MS (EI): m/z = 256 (M+).
Anal. calcd for C12H20N2O4 (256.30): C, 56.24; H, 7.87; N, 10.93.
Found C, 56.35; H, 7.93; N, 10.76.
N-Benzyl-2-hydroxy-3-[acetylglycyl-(N-benzyl-2-phenyl)gly-
cyl]aminobutyric Amide (XIV)
After cooling with ice bath, Et3N (0.9 mL, 0.65 g, 6.2 mmol) and
POCl3 (0.19 mL, 0.3 g, 2 mmol) were added into the solution of XI-
IIa (0.528 g, 2 mmol) in anhyd CH2Cl2 (20 mL), and stirred for
30min.Then a solution of Na2CO3 (0.24 g) in H2O (10 mL) was add-
ed to the mixture and stirred for 10 min more. The organic layer was
separated out, dried (Na2SO4) for 30 min, and filtered. This solution
of isocyanide Va in CH2Cl2 was used immediately in the next Ugi
reaction.
Crude yield: 1.96 g (94.3%); mp 142–144 °C.
IR: 3323, 3065, 2932, 1652, 1611, 1569, 1524, 1344 cm–1.
1H NMR (DMSO-d6): = 8.55 (d, 1 H, J = 5.7 Hz, OH, disappeared
with D2O), 8.37 (s, 1 H, CONH), 7.53 (br s, 2 H, NH2, disappeared
with D2O), 7.32–7.21 (m, 5 H, PhH), 4.28 (m, 2 H, CH2Ph), 4.18 (s,
1 H, NCH), 3.45 (m, 1 H, OCH), 0.98 (d, 3 H, J = 6.6 Hz, CH3).
MS (EI): m/z = 208 (M+).
A solution of acetylglycine (0.234 g, 2 mmol), benzylamine (0.428
g, 4 mmol) and benzaldehyde (0.212 g, 2 mmol) in MeOH (5 mL)
was prepared in advance. The above solution of isocyanide Va in
CH2Cl2 was added to the prepared mixture, and stirred at r.t. for 48
h. Condensed product XIV was obtained from the reaction mixture
with silica gel column chromatography (eluted with CH2Cl2–
CH3OH, 20:1).
Anal. Calcd for C11H16N2O2 (208.26): C, 63.44; H, 7.74; N, 13.45.
Found C, 63.50; H, 7.76; N, 13.41.
N-Cyclohexyl-2-hydroxy-3-aminobutyric Amide (XIIb)
Following the same procedure with XIIa, from XIb (2.30 g, 10
mmol), crude solid XIIb was obtained.
Crude yield: 1.65 g (82.4%); analytical sample gave mp 222–223
°C (sublimed).
IR: 3408, 3325, 3297, 3285, 1646 cm–1.
1H NMR (DMSO-d6): = 8.05 (s, 2 H, NH2, disappeared with
D2O), 7.77 (d, 1 H, J = 8.4 Hz, CONH), 6.36 (d, 1 H, J = 5.7 Hz,
OH, disappeared with D2O), 4.16 (m, 1 H, NCH), 3.49 (m, 1 H,
OCH), 1.68 (m, 4 H, CH2), 1.58 (m, 1 H, CH), 1.26 (m, 6 H, CH2),
1.03 (d, 3 H, J = 6.6 Hz, CH3).
Yield: 0.357 g (33.7% from XIIIa to XIV); white solid; mp 60–62
°C.
IR: 3302, 3057, 2969, 2868, 1948, 1644, 1526, 1447, 1263, 1078,
844, 698 cm–1.
1H NMR (DMSO-d6): = 8.26–8.02 (m, 3 H, NH), 7.27–7.12 (m,
15 H, PhH), 5.89 (d, 1 H, J = 5.7 Hz, OH, disappeared with D2O),
4.30–3.78 (m, 9 H, CH, CH2), 1.24 (s, 3 H, CH3CO), 1.05 (d, 3 H,
J = 6.9 Hz, CH3C).
MS (EI): m/z = 201 (M + 1).
MS (EI): m/z = 531 (M + 1).
Anal. Calcd for C10H20N2O2 (200.28): C, 59.97; H, 10.06; N, 13.99.
Found C, 59.78; H, 10.09; N, 13.91.
Anal. Calcd for C30H34N4O5 (530.59): C, 67.91; H, 6.45; N, 10.56.
Found C, 67.87; H, 6.50; N, 10.61.
N-Benzyl-2-formyloxy-3-formamidobutyric Amide (XIIIa)
The crude product of XIIa (1.96 g, 9.4 mmol) was mixed with a cat-
alytic amount of imidazole and cyanomethyl formate (1.875 g, 25
mmol, prepared as described11) and stirred at r.t. for 48 h. After
evaporation in vacuum, the mixture was separated on a silica gel
column (CH2Cl2–MeOH, 20:1) to yield XIIIa.
N-Benzyl-2-oxo-3-[acetylglycyl-(N-benzyl-2-phenyl)glycyl]ami-
nobutyric Amide (XV)
The XIV white solid (0.530 g, 1 mmol) and DMAP (0.64 g, 1.5
mmol, prepared as described13,14) were placed with a solution of
CF3COOH in CH2Cl2 (0.05 mol/L, 20 mL), and stirred for 30 min
at r.t.. Then a solution of Na2S2O3 in sat. aq NaHCO3 (60%, 8 mL)
was added and stirred for a further10 min. The CH2Cl2 layer was
separated out, dried (Na2SO4), filtered and evaporated to remove
solvent. XV was obtained with silica gel column chromatography
(eluted with CH2Cl2–MeOH, 20:1).
Yield: 1.68 g (63.6% from XIa to XIIIa); white solid; mp 98–99 °C.
IR: 3376, 3183, 3025, 1932, 1643, 1531, 1447, 1339, 1294, 1124,
884, 690 cm–1.
1H NMR (DMSO-d6): = 8.27 (t, 1 H, J = 5.9 Hz, BnNHCO), 7.92
(s, 1 H, OCHO), 7.78 (m, 1 H, NHCO), 7.40–7.20 (m, 5 H, PhH),
5.90 (d, 1 H, J = 5.7 Hz, NCHO, disappeared with D2O), 4.30 (m, 2
H, CH2Ph), 4.22 (d, 1 H, J = 6.0 Hz, OCH), 3.92 (m, 1 H, NCH),
1.05 (d, 3 H, J = 6.6 Hz, CH3).
Yield: 0.453 g (85.8%); white solid; mp: 65–66.5 °C.
1H NMR (DMSO-d6): = 8.23–8.00 (m, 3 H, NH), 7.21–7.02 (m,
15 H, PhH), 4.25–3.70 (m, 8 H, CH, CH2), 1.80 (s, 3 H, CH3CO),
1.03 (d, 3 H, J = 6.7 Hz, CH3C).
MS (EI): m/z = 263 (M+ – 1).
MS (EI): m/z = 529 (M + 1).
Anal. Calcd for C13H16N2O4 (264.28): C, 59.08; H, 6.10; N, 10.60.
Found C, 59.21; H, 6.02; N, 10.68.
Anal. Calcd for C30H32N4O5 (528.61): C, 68.16; H, 6.10; N, 10.60.
Found C, 68.02; H, 6.33; N, 10.54.
N-Cyclohexyl-2-formyloxy-3-formamidobutyric Amide (XIIIb)
Following the same procedure with XIIIa, from crude XIIb (1.65
g, 8.2 mmol), XIIIb was obtained.
Preparation of Library VI and I; General procedure
Following the same procedures with XIV and XV, the precursor li-
brary VI and the target library I were prepared. Most products were
white to pale yellow solids, obtained by silica gel column chroma-
tography, eluted with CH2Cl2–MeOH, 20:1.
Yield: 1.33 g (52.0% from XIb to XIIIb); white solid; mp 147–
149 °C.
Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York

1026
P. Xu et al.
PAPER
Margosiak, S. A.; Pinco, C.; Matthews, D. A.; Kan, C.-C.
Cell 1996, 86, 835.
Acknowledgement
This work was supported by the Chinese National Natural Science
Foundation (39800186) and the National Major Program for Basic
Research Development (973 projects) from the Ministry of Science
and Technology of China (G1998051114).
(4) Ogilvie, W.; Bailey, M.; Poupart, M.-A.; Abraham, A.;
Bhavsar, A.; Bonneau, P.; Bordeleau, J.; Bousquet, Y.;
Chabot, C.; Guse, I.; Halmos, T.; Lavallee, P.; Leach, M.;
Malenfant, E.; O’Meara, J.; Plante, R.; Plouffe, C.; Poirier,
M.; Soucy, F.; Yoakim, C.; Deziel, R. J. Med. Chem. 1997,
40, 4113.
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(6) Nakamura, M.; Inoue, J.; Yamada, T. Bioorg. Med. Chem.
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(7) Tan, W.-H.; Zhang, S.-Q.; Zhang, S.-Y.; Feng, R. Youji
Huaxue 1994, 14, 181.
(8) Kelly, T. R.; Schmidt, T. E.; Haggerty, J. G. Synthesis 1972,
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Synthesis 2002, No. 8, 1017–1026 ISSN 0039-7881 © Thieme Stuttgart · New York