Template synthesis of peptidomimetics composed of aspartic acid moiety by ugi four-component condensation reaction

Article abstract of DOI:10.1081/SCC-200064913

Caspase-3 inhibitors were supposed to be of benefit to both acute and chronic neurodegenerative conditions. In this work, a CPP32-peptidomimetic- inhibitor library was designed. The necessary aspartate moiety was reserved at the C terminal. A facile and v

Full text of DOI:10.1081/SCC-200064913

Synthetic Communicationsw, 35: 1881–1888, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1081/SCC-200064913
Template Synthesis of Peptidomimetics
Composed of Aspartic Acid Moiety by Ugi
Four-Component Condensation Reaction
Xin Zhang, Xiaomin Zou, and Ping Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences,
Peking University Health Science Center, Beijing, China
Abstract: Caspase-3 inhibitors were supposed to be of benefit to both acute and
chronic neurodegenerative conditions. In this work, a CPP32-peptidomimetic-
inhibitor library was designed. The necessary aspartate moiety was reserved at the C
terminal. A facile and versatile method based on Ugi-4CR was developed to build
up the library. The aspartic acid–derived isocyanide 3, the most important
component of the four, was effectively synthesized from protected aspartic acid.
This Ugi procedure was tested by synthesizing a small library.
Keywords: Aspartic acid, isocyanide, peptidomimetics, Ugi four-component reaction
(Ugi-4CR)
INTRODUCTION
The concept of apoptosis was introduced in 1972 by Keer et al.[1] as a physio-
logic type of cell death with an important function, complementary to mitosis,
in tissue homeostasis. Abnormal apoptosis is related to a lot of diseases such as
tumors, AIDS, self-immuno diseases, and degenerate diseases.[2] Apoptotic
cell death plays an important role in neuronal cell death. Both in vitro and
in vivo evidence implicates ICE (Interlukin-1b Converting Enzyme) as an
important factor in neuronal apoptosis, especially under pathological con-
ditions.[3] Activation of the cysteine protease caspase-3 (CPP32) appears to
Received in Japan January 17, 2005
Address correspondence to Ping Xu, Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083,
China. Tel.: þ8610-82801505; Fax: þ8610-62015584; E-mail: pingxu@bjmu.edu.cn
1881

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X. Zhang, X. Zou, and P. Xu
Figure 1. CPP32 inhibitor library template.
be a key event in the execution of apoptosis in the central nervous system.
CPP32 inhibitors may become part of a practical therapeutic approach for
both acute and chronic neurodegenerative conditions.[4] Based on one of the
most effective CPP32 peptide aldehyde inhibitors, Ac-DEVD-CHO,[5] and
the three-dimensional structure of human CPP32 in complex with the irrevers-
ible tetrapeptide inhibitor Ac-DVAD fluoromethyl ketone,[6] a CPP32 pepti-
domimetic inhibitor library was designed in our project. The necessary
aspartate moiety was reserved at the C terminal. The structure of one of the
library templates is shown in Figure 1. The purpose of this work is to build
up an applicable synthetic method for this library.
RESULTS AND DISCUSSION
The multicomponent condensation reactions, especially Ugi four-component
condensation reaction (Ugi-4CR), have been used as a powerful tool in com-
binatorial synthesis.[7] In this article, a facile and versatile method was intro-
duced to build up one template of the library. This method was based on
Ugi-4CR. The required components of aldehydes, acids, and amines were
commercially available. Hence, the important works were the preparation of
the isocyanide component and development of the Ugi-4CR procedure.
The aspartic acid–derived isocyanide 3 was designed to be synthesized
by the method described in Scheme 1. The first step converted the starting
material Boc-Asp(OBzl)-OH to its Weinreb amide 1. According to Ref. 8,
this reaction should be carried out under nitrogen protection. However, we
found that the same result was produced without nitrogen. Then, the Boc pro-
tection of Weinreb amide 1 needed to be deprotected before the a-amino
group was formulated. Several deprotection procedures have been tried, and
it was found that the Boc protecting group in compound 1 was removed
thoroughly with a solution of 50% TFA (trifluoroacetic acid) and CH2Cl2.
The produced TFA salt of deprotected Weinreb amide 1 was then neutralized
with NMM (N-methylmorpholine). The following formulation reaction was
performed with anhydrous formic acid in the presence of EDC (N-ethyl-N0-
[3-dimethylaminopropyl]carbodiimide). The formulation failed if DCC (dicyclo-
hexylcarbodiimide) was used instead of EDC. The formamide 2 was

Synthesis of Peptidomimetics
1883
Scheme 1. Synthetic route of isocyanide 3.
dehydrated with POCl3 to yield the isocyanide 3, which is a key component for
the succedent Ugi-4CR. In this dehydration reaction NEt3 and KOH were used
to capture the released HCl because the resulting isocyano group was not
stable in acid conditions. The isocynaide has an active methine, which
might be susceptible to base-mediated racemization, theoretically. In our
experiment only one product was separated, which showed no rotation. The
1
structure of isocyanide 3 was characterized by H NMR, IR, and MS.
After obtaining the isocyanide 3, an Ugi-4CR model reaction was carried
out to produce compound 6 as a template synthesis (Scheme 2). b-Phenyl
propionic acid (4a), 3-methylbutyl amine (5a), phenylacetaldehyde, and iso-
cyanide 3 were used first. The ratio of the four components was 1:1:1:1 for
acid/amine/aldehyde/isocyanide. The reaction was done fluently in MeOH
solution at room temperature, which was the ordinary condition of Ugi-
4CR. According to the mechanism of Ugi reaction, to produce the conden-
sation product 6, the amino group of 3-methylbutyl amine first reacted with
Scheme 2. Ugi-4CR for template synthesis.

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X. Zhang, X. Zou, and P. Xu
the carbonyl group of phenylacetaldehyde to form a iminium intermediate
state, which was attached nucleophile by isocyanide carbon to produce a
new chiral center. Because the attachment of the isocyanide was equal from
the two sides of the sp2 plane in the transition state, the product 6 might be
formed equally in a pair of diastereomers. However, in our experiment, 6
was yielded as one compound and all the condensed products 7–12 did so.
No structural data were obtained to show the stereostructures. The structure
1
of the Ugi reaction product 6 was confirmed by H NMR and MS. This suc-
cessful synthesis of template compound laid a foundation for building up
our library. This procedure was tested more by synthesizing a small library
(compounds 6–12, Table 1). Because all these pure products were obtained
Table 1. Structures of template compounds
Formula
(Mw.)
Yield
(%)
Compd.
R1
R2
6
(CH3)2CHCH2CH2–
C36H45N3O6
615.77
20
7
8
C46H58N4O10
826.97
8.6
(CH3)2CHCH2CH2–
C43H56N4O10
788.93
12
30
16
9
C56H60N4O10
949.10
10
(CH3)2CHCH2CH2–
C53H58N4O10
911.05
11
12
C41H47N5O6
705.84
12
10
CH3CH2CH2CH2–
C40H41N5O6
574.67

Synthesis of Peptidomimetics
1885
by prepared TLC developed three times, the yields of these compounds
(10–30%) were not good. The structures of compounds 7–12 was character-
ized by 1H NMR and MS.
CONCLUSION
In conclusion, an Ugi reaction–based method was developed to build up one
template of the designed CPP32 peptidomimetic inhibitor library. The key
component of the reaction, aspartate-derived isocyanide, has to be synthesized
before use. The applicability of this method was limited by preparation of
diverse aspartate derived isocyanide.
EXPERIMENTAL
Unless otherwise mentioned, reagents were obtained commercially and used
without further purification. The petroleum ether used had a boiling point
range of 60–908C. Melting points were taken with an X-4 apparatus and
1
are uncorrected. IR (KBr), H NMR, MS, and element data were taken with
Perkin-Elmer983, JNM-AL300 FT NMR System, VG-ZAB-HS20–250, and
Flash EA 1112 instruments respectively.
N-(tert-Butoxycarbonyl)-L-aspartic Acid 4-Benzyl Ester
N0-Methoxy-N0-methylamide (1)
Boc-Asp(OBzl)-OH (3.23 g, 0.01 mol) was dissolved in CH2Cl2 (15 mL). The
solution was cooled to 2138C. Under a nitrogen atmosphere, N-methylmor-
pholine (2.2 mL, 0.02 mol) was added, followed by dropwise addition of
isobutyl chloroformate (1.3 mL, 0.01 mol). The reaction mixture was stirred
vigorously for 15 min. N,O-Dimethylhydroxylamine hydrochloride (0.97 g,
0.01 mol) was added, then the ice bath was removed, and the reaction
mixture was stirred for another 1 h. After filtration the solution was purified
by silica-gel column chromatography (EtOAc/petroleum ether ¼ 3/2) to
1
afford 1 as a colorless liquid in 94% yield (3.44 g). H NMR (CDCl3) d
7.344 (m, 5H, –Ph), 5.440 (s, 1H, –NH–), 5.113 (s, 2H, –OCH2Ph), 5.031
(m, 1H, –CH–), 3.747 (s, 3H, –OCH3), 3.177 (s, 3H, –NCH3), 2.817
(m, 2H, –CH2), 1.429 (s, 9H, Boc). MS (ESI): 366.8 (Mþ).
N-Formyl-L-aspartic Acid 4-Benzyl Ester N0-Methoxy-
N0-methylamide (2)
Compound 1 (3.66 g, 0.01 mol) was stirred with 50% TFA/CH2Cl2 (20 mL)
for 0.5 h to remove the Boc protection, and then the solution was evaporated
in vacuum. The residue was dissolved in a solution of N-methylmorpholine

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X. Zhang, X. Zou, and P. Xu
(2.2 mL, 0.02 mol) in CH2Cl2 (50 mL). N-Ethyl-N0-(3-dimethylaminopropyl)-
carbodiimide (3.82 g, 0.02 mol) was added to a solution of formic acid (2.1 g,
0.05 mol) in CH2Cl2 (50 mL) and the mixture was stirred for 15 min. The two
solutions were combined and stirred overnight at room temperature. The
reaction mixture was washed with 5% HCl, saturated NaHCO3, and NaCl in
sequence. The organic layer was dried over Na2SO4, and then evaporated to
1
give 2 as a yellow oil in 96% yield (2.85 g). H NMR (CDCl3) d 8.168
(s, 1H, –CHO), 7.354 (m, 5H, –Ph), 6.646 (bs, 1H, –CONH–), 5.373
(m, 1H, N–CH), 5.117 (s, 2H, –OCH2Ph), 3.756 (s, 3H, –OCH3), 3.190
(s, 3H, –NCH3), 2.850 (m, 2H, –CH2).
2-Isocyano-succinic acid 4-Benzyl Ester N0-Methoxy-
N0-methylamide (3)
Compound 2 (1 g, 3.4 mmol) was dissolved in CH2Cl2 (15 mL), and the
solution was cooled in an ice bath. Triethyl amine (0.7 mL, 5.6 mmol) was
dropped into the solution; then POCl3 (0.4 mL, 4 mmol) was added. After
the mixture had been stirred for 1 h, a solution of KOH (500 mg) in water
(5 mL) was added and stirred for another 1 h. The layers were separated and
the organic layer was washed with water and dried over Na2SO4. After eva-
poration, the residue was purified by silica-gel column chromatography
(EtOAc/P.ether ¼ 3/2) to obtain 3 as a yellow oil in 83% yield (780 mg).
1H NMR (CDCl3) d 7.360 (m, 5H, –Ph), 5.211 (q, 2H, –OCH2Ph), 5.056
(t, 1H, CN–CH–), 3.814 (s, 3H, –OCH3), 3.261 (s, 3H, –NCH3), 2.978
(m, 2H, –CH2). IR (KBr) cm21: 2923.89, 2141.26 (CN–), 1735.24,
1677.61. MS (ESI): 276.2 (Mþ).
General Procedure for Template Synthesis (6–12)
b-Phenyl propionic acid (4a, 150 mg, 1 mmol), 3-methylbutyl amine (5a,
40 mg, 1 mmol), and phenylacetaldehyde (120 mg, 1 mmol) were dissolved
in methanol (5 mL). Then, isocyanide 3 (276 mg, 1 mmol) was added to this
solution, and the mixture was stirred for 48 h at room temperature. The
reaction mixture was separated by prepared TLC (CH2Cl2/ethanol ¼ 3/1 as
eluent) three times to produce 6 as a soft solid in 20% yield (123 mg),
mp 308C. 1H NMR (CDCl3) d 8.589 (s, 1H, CO–NH–), 7.828–7.518
(m, 15H, 3Ph–), 5.299 (m, 2H, 2N–CHCO–), 5.161 (s, 2H, –OCH2Ph),
4.306 (t, 2H, PhCH2–), 4.217 (t, 2H, PhCH2–), 4.131 (t, 2H, NCH2–),
4.078 (s, 3H, –OCH3), 3.736 (s, 3H, –NCH3), 2.302 (m, 4H, 2COCH2–),
1.302 (m, 1H, –CH–), 1.253 (m, 2H, CH2–), 1.053 (d, 3H, –CH3), 0.996
(d, 3H, –CH3). MS (ESI): 615.6 (Mþ).
Following the same procedure as with 6, from Boc-Asp(Obzl)-OH (4b,
160 mg, 0.5 mmol), 2-(1-cyclohexenyl)ethylamine (5b, 65 mg, 0.5 mmol),

Synthesis of Peptidomimetics
1887
phenylacetaldehyde (60 mg, 0.5 mmol), and 3 (140 mg, 0.5 mmol), 7 was
1
obtained as a colorless oil in 8.6% yield (35 mg). H NMR (CDCl3) d 8.451
(s, 1H, CO–NH–), 7.768–7.547 (m, 15H, 3Ph–), 5.631 (m, 1H, –CH55),
5.541 (s, 1H, BocNH–), 5.487 (m, 1H, –CH55), 5.302 (m, 3H, 3N–CH–
CO), 5.147 (s, 4H, 2OCH2Ph), 4.128 (t, 2H, NCH2–), 4.104 (s, 3H,
–OCH3), 4.011 (t, 2H, PhCH2–), 3.678 (s, 3H, –NCH3), 2.324 (m, 4H,
2COCH2–), 1.950 (m, 1H, –CH–), 1.435 (s, 9H, Boc), 1.323 (m, 8H,
4CH2–). MS (ESI): 827.0 (Mþ).
Following the same procedure as with 6, from Boc-Asp(Obzl)-OH (4b,
160 mg, 0.5 mmol), 3-methylbutyl amine (5a, 20 mg, 0.5 mmol), phenylacetal-
dehyde (60 mg, 0.5 mmol), and 3 (140 mg, 0.5 mmol), 8 was obtained as a
1
colorless oil in 12% yield (47 mg). H NMR (CDCl3) d 8.497 (s, 1H, CO–
NH–), 7.813–7.585 (m, 15H, 3Ph–), 5.481 (s, 1H, BocNH–), 5.311
(m, 3H, 3N–CH–CO), 5.094 (s, 4H, 2OCH2Ph), 4.204 (t, 2H, NCH2–),
4.082 (s, 3H, –OCH3), 4.043 (t, 2H, PhCH2–), 3.757 (s, 3H, –NCH3),
2.895 (m, 4H, 2COCH2–), 1.435 (s, 9H, Boc), 1.388 (m, 1H, –CH–),
1.235 (m, 2H, –CH2–), 1.108 (d, 3H, –CH3), 1.002 (d, 3H, –CH3). MS
(ESI): 789.0 (Mþ).
Following the same procedure as with 6, from Fmoc-Asp(Obzl)-OH (4c,
205 mg, 0.5 mmol), 2-(1-cyclohexenyl)ethylamine (5b, 65 mg, 0.5 mmol),
phenylacetaldehyde (60 mg, 0.5 mmol), and 3 (140 mg, 0.5 mmol), 9 was
obtained as a colorless oil in 30% yield (142 mg). 1H NMR (CDCl3)
d 8.468 (s, 1H, CO–NH–), 7.852–7.523 (m, 23H, Ar–), 5.834 (s, 1H,
FmocNH–), 5.627 (m, 1H, –CH55), 5.573 (m, 1H, –CH55), 5.338 (m, 3H,
3N–CH–CO), 5.205 (s, 4H, 2OCH2Ph), 4.709 (d, 2H, OCH2–), 4.461
(m, 1H, ArCHAr), 4.102 (t, 2H, NCH2–), 4.092 (s, 3H, –OCH3), 3.996
(t, 2H, PhCH2–), 3.711 (s, 3H, –NCH3), 2.469 (m, 4H, 2COCH2–), 1.976
(m, 1H, –CH–), 1.550 (m, 8H, 4CH2–). MS (ESI): 949.7 (Mþ).
Following the same procedure as with 6, from Fmoc-Asp(Obzl)-OH (4c,
205 mg, 0.5 mmol), 3-methylbutyl amine (5a, 20 mg, 0.5 mmol), phenylacetal-
dehyde (60 mg, 0.5 mmol), and 3 (140 mg, 0.5 mmol), 10 was obtained as a
1
colorless oil in 16% yield (73 mg). H NMR (CDCl3) d 8.531 (s, 1H, CO–
NH–), 7.864–7.539 (m, 23H, Ar–), 5.893 (s, 1H, FmocNH–), 5.347
(m, 3H, 3N–CH–CO), 5.340 (s, 4H, 2OCH2Ph), 4.716 (d, 2H, OCH2–),
4.458 (m, 1H, ArCHAr), 4.097 (t, 2H, NCH2–), 4.052 (s, 3H, –OCH3),
3.988 (t, 2H, PhCH2–), 3.696 (s, 3H, –NCH3), 2.880 (m, 4H, 2COCH2–),
1.405 (m, 1H, –CH–), 1.261 (m, 2H, –CH2–), 1.079 (d, 3H, –CH3), 0.992
(d, 3H, –CH3). MS (ESI): 911.0 (Mþ).
Following the same procedure as with 6, from b-phenyl propionic acid
(4a, 75 mg, 0.5 mmol), 4-amino-1-phenylpiperazine (5c, 89 mg, 0.5 mmol),
phenylacetaldehyde (60 mg, 0.5 mmol), and 3 (140 mg, 0.5 mmol), 11 was
1
obtained as a colorless oil in 12% yield (42 mg). H NMR (CDCl3) d 8.564
(s, 1H, CO–NH–), 7.749–6.607 (m, 20H, 4Ph–), 5.286 (m, 2H, 2N
–CHCO–), 5.193 (s, 2H, –OCH2Ph), 4.313 (t, 2H, PhCH2–), 4.248 (t, 2H,
PhCH2–), 4.101 (s, 3H, –OCH3), 3.825 (s, 3H, –NCH3), 3.470 (t, 4H,

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X. Zhang, X. Zou, and P. Xu
2NCH2–), 2.778 (t, 4H, 2NCH2–), 2.630 (m, 4H, 2COCH2–). MS (ESI):
705.7 (Mþ).
Following the same procedure as with 6, from nicotinic acid (4e, 62 mg,
0.5 mmol), 1-butylamine (5d, 37 mg, 0.5 mmol), phenylacetaldehyde (60 mg,
0.5 mmol), and 3 (140 mg, 0.5 mmol), 12 was obtained as a colorless oil in
1
10% yield (29 mg). H NMR (CDCl3) d 9.171 (s, 1H, Py), 8.843 (d, 1H,
Py), 8.572 (s, 1H, CO–NH–), 8.324 (d, 1H, Py), 7.638–7.359 (m, 11H,
2Ph–, Py), 5.289 (m, 2H, 2N–CHCO–), 5.091 (s, 2H, –OCH2Ph), 3.972
(t, 2H, PhCH2–), 4.135 (t, 2H, NCH2–), 3.997 (s, 3H, –OCH3), 3.723
(s, 3H, –NCH3), 2.882 (m, 2H, COCH2–), 1.356 (m, 4H, –2CH2–), 0.969
(t, 3H, –CH3). MS (ESI): 574.5 (Mþ).
ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation of
China (20272004).
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