Susceptibility of methyl 3-Amino-1H-pyrazole-5-carboxylate to acylation

Article abstract of DOI:10.1080/00397910902898536

In the search for a new method of synthesis of hybrid peptides with aminopyrazole carboxylic acid, we tried to force selective acylation at the aromatic amino group instead of at the ring nitrogen atom with fairly gentle acylating agents. The acylating agents used were acid anhydrides: acetic anhydride, tert-butyl pyrocarbonate, and 2-(2-methoxyethoxy)ethoxyacetic acid/dicyclohexylcarbodiimide. We succceded in acylation at this amino group with almost none at the ring nitrogen atom. Sometimes, however, acylation in small quantities at the ring nitrogen atom was observed as a by-product. To remove this by-product, imidazole was used. Thus, we were able to obtain the hybrid peptides in question with no protection and subsequent removal required. We synthesized a few these free peptides with no protection of the pyrazole ring. This is a simpler method than that being used currently.

Full text of DOI:10.1080/00397910902898536

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Susceptibility of Methyl
3-Amino-1H-pyrazole-5-
carboxylate to Acylation
Anna Kusakiewicz-Dawid a , Łukasz Górecki a ,
Elżbieta Masiukiewicz a & Barbara Rzeszotarska a
a Institute of Chemistry, University of Opole, Opole,
Poland
Version of record first published: 16 Oct 2009
To cite this article: Anna Kusakiewicz-Dawid, Łukasz Górecki, Elżbieta Masiukiewicz
& Barbara Rzeszotarska (2009): Susceptibility of Methyl 3-Amino-1H-pyrazole-5-
carboxylate to Acylation, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 39:22, 4122-4132
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Synthetic Communications1, 39: 4122–4132, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910902898536
Susceptibility of Methyl 3-Amino-1H-
pyrazole-5-carboxylate to Acylation
ꢀ
Anna Kusakiewicz-Dawid, Łukasz Gorecki,
Elz˙bieta Masiukiewicz, and Barbara Rzeszotarska
Institute of Chemistry, University of Opole, Opole, Poland
Abstract: In the search for a new method of synthesis of hybrid peptides with
aminopyrazole carboxylic acid, we tried to force selective acylation at the
aromatic amino group instead of at the ring nitrogen atom with fairly gentle
acylating agents. The acylating agents used were acid anhydrides: acetic
anhydride, tert-butyl pyrocarbonate, and 2-(2-methoxyethoxy)ethoxyacetic acid=
dicyclohexylcarbodiimide. We succceded in acylation at this amino group with
almost none at the ring nitrogen atom. Sometimes, however, acylation in small
quantities at the ring nitrogen atom was observed as a by-product. To remove this
by-product, imidazole was used. Thus, we were able to obtain the hybrid peptides
in question with no protection and subsequent removal required. We synthesized
a few these free peptides with no protection of the pyrazole ring. This is a simpler
method than that being used currently.
Keywords: Acetylation, acylation, aromatic amine, 2-(2-methoxyethoxy)
ethoxyacetylation, methyl 3-amino-1H-pyrazole-5-carboxylate, tert-butyloxycar-
bonylation
A series of pathological processes is associated with the formation of
b-sheet structure and consecutive protein aggregation in the form of b-
amyloid deposition.[1] One of the most promising approaches aims at the
prevention of b-amyloid aggregation by using a specially designed b-sheet
peptide ligand containing methyl 3-amino-1H-pyrazole-5-carboxylate,[1]
a
Received June 30, 2008.
Address correspondence to Anna Kusakiewicz-Dawid, Institute of Chemistry,
University of Opole, 48 Oleska St., 45-052 Opole, Poland. E-mail: rzeszot@uni.
opole.pl
4122

Acylation of Methyl 3-Amino-1H-pyrazole-5-carboxylate
4123
so-called b-sheet breaker. However, the synthesis of these peptides is
difficult, because no amide coupling could be effected under any of the reac-
tion conditions tested.[2] In all cases, the ring NH group has been found to
be more reactive than the aromatic amine group, thus leading to ring acyla-
tion instead of peptide coupling.[2] Therefore, protection of the pyrazole
nitrogen is first required.[1–3] This process is not univocal, as two nitrogen
atoms are present in the pyrazole ring and two products are formed. It
was assumed that the susceptibility of the nitrogen atom to acylation is
dependent on the type and amount of acylating agent.
Therefore, methyl 3-amino-1H-pyrazole-5-carboxylate 1 acylation
was treated with 1.5 equivalent of Ac2O. In contrast to the previous
statement,[2] the monoacylated product 2 was always obtained at the
exoamino group (Scheme 1A). After 1 h of reaction with Ac2O in dioxane
at room temperature, there was 89% isolated product monoacetylated at
the amino group. No acetylation at the ring nitrogen atom was observed.
With 16 equivalents of acylation agent Ac2O at reflux, after 4 h 46%
isolated yield of diacylated product 3 was obtained (Scheme 1B).
For formation of the tert-butoxycarbonyl (Boc) derivative at the
exoamino group, action of tert-butyl pyrocarbonate at a higher tempera-
ture, preferably in the presence of a tertiary amine, is required. It can be
1
observed in the H NMR spectrum that the signal of NH is strongly
deshielded vs. tetramethylsilane (TMS, 12.3 ppm). This would suggest
that NH is strongly susceptible to the formation of an anion in the
presence of a tertiary amine. After 8 h of reaction with tert-butyl pyrocar-
bonate in dioxane, in the absence of a tertiary amine at reflux and with
consecutive treatment with imidazole to remove the tert-butoxycarbonyl
derivative at the ring nitrogen atom (Scheme 2C), 5 was obtained in
Scheme 1. Acetylation of 1.

4124
A. Kusakiewicz-Dawid et al.
Scheme 2. tert-Butyloxycarbonylation of 1.
analogy to the monoacetylated product in 72% isolated yield
(Scheme 2A). In the presence of a tertiary amine with tert-butyl pyrocar-
bonate, 4 is formed; thus the ring nitrogen atom is acylated (Scheme 2B).
2-(2-Methoxyethoxy)ethoxyacetylation, which is used for the pro-
nounced increase in solubility of product in water, follows the same
course. Using dicyclohexylcarbodiimide in dioxane as a condensing agent
at room temperature after 22 h, without ring nitrogen atom acylation, 6
was obtained in 75% yield (Scheme 3).
The obtained results show the possibility for the synthesis of the peptidic
ligands in question and additionally without the protection of nitrogen in
the pyrazole ring. Some peptide examples 7–9 were synthesized (Schemes 4
and 5). This method of synthesizing these compounds is simpler than that
Scheme 3. 2-(2-Methoxyethoxy)ethoxyacetylation of 1.

Acylation of Methyl 3-Amino-1H-pyrazole-5-carboxylate
4125
Scheme 4. Synthesis of methyl 3-(Na-tert-butoxycarbonyl-glycylamino)-1H-
pyrazole-5-carboxylate (7).
proposed by Schrader et al.[1–3] where protection of the pyrazole ring is
required. The synthesis of such peptides without protection of the pyrazole
ring is currently under way in this laboratory.
EXPERIMENTAL
General Procedure
The substrate methyl 3-amino-1H-pyrazole-5-carboxylate (1) of 97.9%
purity (determined by high-performance liquid chromatography, HPLC)
Scheme 5. Synthesis of methyl 3-(Na-benzyloxycarbonyl-Ne-tert-butoxycarbonyl-
L-lysylamino)-1H-pyrazole-5-carboxylate (8) and methyl 3-(Na-benzyloxycarbo-
nyl-Ne-tert-butoxycarbonyl-L-lysyl-Ne-tert-butoxycarbonyl-L-lysylamino)-1H-
pyrazole-5-carboxylate (9).

4126
A. Kusakiewicz-Dawid et al.
was obtained from commercial 3-nitro-1H-pyrazole-5-carboxylic acid of
99.6% purity (Aldrich) through the method highly recommended for
esterification in peptide chemistry wherein methanol and an excess of
thionyl chloride[4] (93% yield and 99.4% purity) are used. The methyl
ester was produced in its free form without hydrochloride (through ele-
mental analysis and mass spectroscopy). Consecutive reduction of the
nitro group with hydrogen in the presence of Pd=C[5] gave 81% yield of
amine of 99.7% purity.
The solvents from the reaction mixtures were removed in vacuo on
a rotary evaporator at a bath temperature not exceeding 40ꢀC.
Thin-layer chromatography (TLC) was performed on silica gel (DC
Alufolien Kieselgel Merck 1.05553) with detection by means of
chlorine–KI–tolidine in the solvent systems (A) CHCl3=MeOH=AcOH
(90:8:2), (B) CHCl3=MeOH=AcOH (95:5:3), (C) n-BuOH=AcOH=
AcOEt=H2O (1:1:1:1), (D) CHCl3=acetone (9:1), (E) n-BuOH=AcOH=
H2O (4:1:1), and (F) C6H6=MeOH=acetone=pyridine=AcOH (24:4:2:2:1).
Purities for the compounds were checked by HPLC by a Beckman
‘‘System Gold’’ with an Alltech Alltime C18 (RP 5 mm, 150 ꢁ 4.6 mm)
column and
a
loop (5 ml). The mobile phases were 0.1%
TFA=acetonitrile (A) 90:10, (B) 70:30, (C) 50:50, and (D) 40:60 at a
1 ml minꢂ1 rate of flow. Detection was made at 210 nm. Melting points
were recorded on a DSC-2010 calorimeter (Thermal Analysis Instru-
ments) under nitrogen in a closed copper vessel with a heating rate
of 2ꢀC minꢂ1. NMR spectra were measured on a Brucker Avance
400-MHz spectrometer in dimethyl sulfoxide (DMSO-d6) or CDCl3
in the presence of tetramethylsilane. Resonances were based on hetero-
nuclear multiple bond correlation (HMBC) and heteronuclear single
quantum correlation (HSQC) spectra. Fourier transform infrared
(FTIR) spectra were recorded on a PU 9800 Philips analytical spectro-
meter at 2 cmꢂ1 resolution in KBr or CHCl3.
Methyl 3-Nitro-1H-pyrazole-5-carboxylate
Absolute methanol (5.6 ml, 138 mmol) was cooled to ꢂ15ꢀC and thionyl
chloride (0.8 ml, 11 mmol) was added dropwise under stirring. After
15 min, the solution was followed by 3-nitro-1H-pyrazole-5-carboxylic
acid (1.26 g, 8 mmol). Stirring continued at ꢂ15ꢀC for 15 min and then
at room temperature for 24 h. The volatiles were evaporated, and the
resulting colorless crude precipitate was washed with acetone to neutra-
lize the pH and decay the sulfur compounds. It was then crystallized with
diethyl ether and hexane. A colorless solid resulted (1.28 g, 93% yield),
mp 145.5ꢀC, mp lit.[2] 138ꢀC; TLC: Rf (A) 0.74; HPLC (B): tR

Acylation of Methyl 3-Amino-1H-pyrazole-5-carboxylate
4127
5.29 min, 99.4% purity. Analysis calcd. for C5H5N3O4: C, 35.09%; H,
2.95%; N, 24.56%. Found: C, 35.18%; H,3.04%; N, 24.47%.
Methyl 3-Amino-1H-pyrazole-5-carboxylate (1)
Methyl 3-nitro-1H-pyrazole-5-carboxylate was suspended in methanol
(40 ml), 10% Pd=C (0.26 g) was added, and a Parr apparatus was filled
with hydrogen at five bars pressure. After 24 h, the catalyst was filtered
off and washed with methanol. The methanol was evaporated, and the
residue was crystallized with ethanol (10 ml). A colorless powder resulted
(1.14 g, 81% yield), mp 144.3ꢀC; TLC: Rf (A) 0.43; Rf (C) 0.83; HPLC
(A): tR 2.78 min, 99.7% purity. Analysis calcd. for C5H7N3O2; C,
42.55%; H, 4.96%; N, 29.79%. Found: C, 42.70%; H, 5.00%; N,
1
29.71%. H NMR (DMSO-d6) d (ppm): 3.742 (s, 3H, OCH3), 5.042 (s,
2H, NH2), 5.763 (s, 1H, C4H), 12.253 (s, 1H, NHring).
Methyl 3-(Acetylamino)-1H-pyrazole-5-carboxylate (2)
Ester 1 (0.16 g, 1.09 mmol) was suspended in dioxane (2 ml), and Ac2O
(0.15 ml, 1.5 mmol) was added. After several minutes, a precipitate
appeared, which after a further hour was filtered off to give 2
(0.177 g, 89% yield), mp 220ꢀC, TLC (A): Rf 0.57; HPLC (A): tR
1
6.99 min, 98.3% purity. H NMR (DMSO-d6) d (ppm): 2.017 (s, 3H,
CH3CO), 3.828 (s, 3H, OCH3), 6.989 (1H, C4H), 10.622 (s, 1H, NHamide),
13.556 (s, 1H, NHring); 13C NMR (DMSO-d6) d (ppm): 22.95 (CH3acetyl),
4
3
5
52.02 (CH3-ester), 99.36 (C ), 132.65 (C ), 148.00 (C ), 159.27 (C Oester),
=
167.84 (C Oamide). FTIR (KBr) (cmꢂ1) 3313, 3181 (nNH), 1726
(n C Oamide), 1694 (n C Oester). Analysis calcd. for C7H9N3O3:
C, 45.89%; H, 4.95%; N, 22.93%. Found: C, 46.05%; H, 5.07%; N
23.05.
=
=
=
Methyl 3-(Acetylamino)-1-acetyl-1H-pyrazole-5-carboxylate (3)
Ester (1) (0.14 g, 1.0 mmol) was suspended in Ac2O (1.52 ml, 16 mmol),
heated for 4 h, and left standing overnight. The formed precipitate was
filtered off (0.10 g, 46% yield), mp 153ꢀC, TLC (A): Rf 0.87; HPLC
1
(B): tR 3.7 min, 98.2% purity. H NMR (DMSO-d6) d (ppm): 2.192 (s,
3H, CH3acetyl amide), 2.679 (s, 3H, CH3acetyl ring), 3.861 (s, 3H, CH3ester),
7.019 (s, 1H, C4H), 10.342 (s, 1H NHamide); 13C NMR (DMSO-d6) d
(ppm): 23.40 (2 ꢁ CH3acetyl), 52.32 (CH3ester), 98.55 (C4), 141.11 (C3),

4128
A. Kusakiewicz-Dawid et al.
=
=
=
145.10 (C5), 161.33 (C Oester), 167.75 (C Oamide), 173.72 (C Oring);
=
FTIR (cmꢂ1) (KBr): 3350 (nNH), 1741, 1725, 1709 (nC Oamide
nC Oester); (CHCl3): 3358 (nNH), 1730 (nC Oamide and C Oester).
Analysis calcd. for C9H11N3O4: C, 48.00%; H, 4.92%; N, 18.66%. Found:
C, 48.08%; H, 4.80%; N, 18.48%.
,
=
=
=
Methyl 3-(tert-Butoxycarbonylamino)-1H-pyrazole-5-carboxylate (5)
Ester (1) (0.282 g, 2 mmol) was dissolved in dioxane (8 ml), tert-butyl
pyrocarbonate (0.654 g, 3 mmol) was added, and the entire mixture was
then refluxed for 8 h. The solution was left standing overnight. Imidazole
(0.613 g, 9 mmol) was added and refluxed for 2 h, and dioxane was
evaporated. The residue was dissolved in ethyl acetate, extracted with
HCl (0.5 M, 3 ꢁ 5 ml), washed with water, and dried, and the ethyl acetate
was evaporated. A colorless powder resulted (0.349 g, 72% yield),
mp 179–182ꢀC; TLC: (B) Rf 0.70; HPLC: (C) tR 3.1 min, 99% purity;
1H NMR (DMSO-d6) d (ppm): 1.450 (s, 9H, CH3urethane), 3.827 (s, 3H
CH3ester), 6.765 (s, 1H, C4H), 9.822 (s, 1H, NHamide), 13.431 (s, 1H,
NHring); 13C NMR (DMSO-d6) d (ppm): 27.84 (CH3urethane), 51.87
(CH3-ester), 79.05 (Cquaternary), 98.46 (C4), 132.53 (C3), 148.16 (C5),
152.67 (C Oamide), 159.22 (C Oester); FTIR (cmꢂ1) (KBr): 3326, 3181
=
=
=
=
(nNH), 1736, 1720 (nC O); (CHCl3): 3441, 3329 (nNH), 1719 (nC O).
Analysis for C10H15N3O4 calcd.: C, 49.79%; H, 6.27%; N, 17.42%.
Found: C, 49.63%; H, 6.16; N, 17.53.
Methyl 1-tert-Butoxycarbonyl-3-amino-1H-pyrazole-5-carboxylate (4)
Ester (1) (0.141 g, 1 mmol) was dissolved in dioxane (6 ml), triethylamine
(0.104 ml, 1.5 mmol) and tert-butyl pyrocarbonate (0.327 g, 1.5 mmol)
were added, and the whole mixture was heated at reflux for 1 h. The
solvent was evaporated, and the residue was dissolved in ethyl acetate,
extracted with 5% citric acid (2 ꢁ 5 ml), washed with water, and dried.
Ethyl acetate was then evaporated. The precipitate was treated with
diethyl ether and filtered off. A colorless powder resulted (1.08 g, 45%
yield), mp 135–137ꢀC; TLC: (B) 0.65; HPLC: (C) tR 3.47 min, 98% purity;
1H NMR (DMSO-d6) d (ppm): 1.591 (s, 9H, CH3urethane), 3.803 (s, 3H,
CH3ester), 5.702 (s, 1H, C4H), 6.535 (s, 2H, NH2); 13C NMR (DMSO-d6)
d (ppm): 27.53 (CH3urethane), 51.88 (CH3ester), 81.47 (Cquaternary), 88.00
4
5
3
=
=
(C ), 145.14 (C ), 149.59 (C Oamide), 152.11 (C ), 162.17 (C Oester).
=
FTIR (cmꢂ1
=
) (KBr): 3487, 3353 (nNH2), 1739 (nC Ourethane),
1730 (nC Oester), 1609 (dNH2); (CHCl3): 3508, 3393 (nNH2), 1737

Acylation of Methyl 3-Amino-1H-pyrazole-5-carboxylate
4129
=
=
(nC Ourethane), 1727 (nC Oester), 1613 (dNH2). Analysis for C10H15N3O4 ꢁ
0.1 tBuOH calcd.: C, 50.19%; H, 6.43%; N, 16.89%. Found: C, 49.79%; H,
6.37%; N, 16.55%.
Methyl 2-(2-Methoxyethoxy)ethoxyacetylamino-1H-
pyrazole-5-carboxylate (6)
Ester (1) (0.705 g, 5 mmol) was dissolved in warm dioxane (12.5 ml). After
cooling to room temperature, 2-(2-methoxyethoxy)ethoxyacetic acid
(0.77 ml, 5 mmol) and dicyclohexylcarbodiimide (1.03 g, 5 mmol) were
added, and after 40 h, dicyclohexylurea was filtered off and washed with
dioxane (2 ꢁ 12.5 ml). The dioxane was then evaporated. The residue was
dissolved in chloroform (5 ml), applied on a silica-gel column (60H
Merck 1.05553, / 4 cm, h 6 cm), and equilibrated with chloroform. The
column was eluted with chloroform (50 ml) and followed with solutions
with increased concentrations in acetone, from 2.5% to 32.5%. A color-
less powder resulted (1.125 g, 75% yield), mp 82.5–83.5ꢀC (83.86ꢀC
according to DSC); TLC: Rf (D) 0.49, (E) 0.57, (F) 0.66; HPLC: (B)
tR 2.71 min, 100% purity; 1H NMR (CDCl3) d (ppm): 3.398 (s, 3H,
CH3glycol), 3.617–3.639 (m, 2H, CH2glycol), 3.713–3.373 (m, 4H,
2 ꢁ CH2glycol), 3.786–3.793 (m, 2H, CH2glycol), 3.922 (s, 3H, OCH3),
4.190 (s, 2H, CH2glycol), 7.110 (s, 1H, C4H), 9.845 (s, 1H, NHamide),
11.263 (s, 1H, NHring); 13C NMR (CDCl3), d (ppm): 52.20 (OCH3),
58.88 (CH3glycol), 70.07 (CH2glycol), 70.30 (CH2glycol), 70.44 (CH2glycol),
71.21 (CH2glycol), 71.64 (CH2glycol), 98.54 (C4), 137.67 (C5), 143.87
3
(C ), 161.13 (C Oester), 168.48 (C Oamide). FTIR (cmꢂ1) (KBr):
=
=
3444, 3268, 3139 (nNHamide, nNHring), 2989, 2959, 2928, 2882, 2848
=
=
=
(nCH2), 1732 (nC Oester), 1653 (nC Oamide), 1594 (nC Nring),
(CH2Cl2): 3429, 3399, 3311 (nNHamide, nNHring) 2916 (nCH2) 1725
=
=
=
(nC Oester), 1696 (nC Oamide), 1604 (nC Nring). Analysis for
C12H19N3O6 calcd.: C, 47.84%; H, 6.36%; N, 13.95%. Found: C,
47.74%; H, 6.40%; N, 13.95%.
Methyl 3-(Na-tert-Butoxycarbonyl-glycylamino)-1H-
pyrazole-5-carboxylate (7)
Compound 1 (0.141 g, 1 mmol) was dissolved in DMF (2.3 ml), and
Boc-Gly-ONp (0.355 g, 1.2 mmol) and HOBt ꢁ H2O (0.153 g, 1 mmol)
were added. After 21 h at room temperature, the solvent was removed.
The residue was triturated with 5% NaHCO3 (1.5 ml). The precipitate
was filtered off and washed with 5% NaHCO3 (3 ꢁ 1.5 ml), with water

4130
A. Kusakiewicz-Dawid et al.
(2 ꢁ 2 ml), and with diethyl ether (2 ꢁ 2 ml). Compound 7 was obtained
(0.221 g, 74% yield), mp 201.23ꢀC; TLC (B): Rf 0.27; HPLC (C):
1
tR ¼ 2.16 min, 100% purity; H NMR (DMSO-d6) d (ppm): 1.392 (s,
9H, CH3-tBu), 3.718 and 3.733 (d, 2H, CH2-Gly), 3.834 (s, 3H,
OCH3), 6.920 (s, lH, CHring), 7.067 (s, lH, NHurethane), 10.645 (s,
1H, NHamide), 13.563 (s, lH, NHring); 13C NMR (DMSO-d6) d
(ppm): 28.23 (3C, CH3-tBu), 43.23 (CH2-Gly), 51.99 (OCH3), 78.11
4
5
3
=
(C-tBu), 98.79 (C ), 133.80 (C ), 147.00 (C ), 155.94 (C Ourethane),
=
=
159.71 (C Oester), 167.87 (C Oamide). Analysis calcd. for
C12H18N4O5: C, 48.32%; H, 6.08%; N, 18.78%. Found: C, 48.56%;
H, 6.19%; N, 18.98%.
Methyl 3-(Na-Benzyloxycarbonyl-Ne-tert-butoxycarbonyl-L-
lysylamino)-1H-pyrazole-5-carboxylate (8)
Compound 1 (0.282 g, 2 mmol) was dissolved in DMF (3 ml), and
Z-Lys(Boc)-ONp (1.103 g, 2.2 mmol) and HOBt ꢁ H2O (0.306 g, 2 mmol)
were added. After 24 h at room temperature, the solvent was removed.
The residue (oil) was dissolved in ethyl acetate (12 ml) and extracted with
5% NaHCO3 (4 ꢁ 3 ml), brine (3 ꢁ 3 ml), and water (2 ꢁ 3 ml). The ethyl
acetate layer was dried with MgSO4. The ethyl acetate was evaporated,
and the residue (oil) was dissolved in chloroform (2.5 ml), applied on a
column (60H Merck 1.05553, U 3 cm, h 6 cm), and equilibrated with
chloroform (100 ml) and 2.5%, 5.0%, 7.5%, 10.0%, 15.0%, and 20.0%
acetone in chloroform (each 50 ml) and 25% acetone in chloroform
(150 ml). The solutions was evaporated; the precipitate was dissolved in
ethyl acetate and extracted with 0.5 N HCl (2 ꢁ 2 ml) and brine (5 ꢁ 3 ml).
The ethyl acetate layer was dried with MgSO4. Compound 9 (0.532 g,
53% yield) was obtained, mp 59.96ꢀC; TLC (B): Rf 0.51; HPLC: (D),
1
tR 3.16 min, 100% purity; H NMR (DMSO-d6) d (ppm): 1.403 (s, 9H,
CH3-tBu), 1.492 (brs, 2H, CH2-Lys), 1.797 (brs, lH, CH2-Lys), 1.915 (brs,
lH, CH2-Lys), 3.076 (brs, 3H, CH2-Lys), 3.921 (s, 4H, OCH3), 4.527 and
4.541 (d, lH, a-CH), 4.730 (s, lH, NHurethane Boc), 5.078–5.162 (m, 3H,
CH2-Z), 6.022 and 6.038 (d, lH, NHurethane Z), 7.303 (s, lH, CHring),
7.326 (m, 5H, CHarom.), 10.728 (s, lH, NHamide); 13C NMR (DMSO-d6)
d (ppm): 22.62 (CH2-Lys), 28.39 (3C, CH3-tBu), 29.68 (CH2-Lys), 32.70
(CH2-Lys), 40.12 (CH2-Lys), 52.67 (OCH3), 55.18 (a-CH), 67.25 (CH2-Z),
79.27 (C-tBu), 100.29 (C4), 128.16–128.51 (5C, C2-C6arom:), 134.59 (C3),
3
1
=
136.05 (Carom:), 147.07 (C ), 156.19 (C Ourethane Boc), 156.47 (C Ourethane Z),
=
=
=
160.79 (C Oester), 170.16 (C Oamide). Analysis calcd. for C24H33N3O7:
C, 57.25%; H, 6.61%; N, 13.91%. Found: C, 57.12%; H, 6.37%; N,
13.88%.

Acylation of Methyl 3-Amino-1H-pyrazole-5-carboxylate
4131
Methyl 3-(Na-benzyloxycarbonyl-Ne-tert-butoxycarbonyl-L-lysyl-Ne-tert-
butoxycarbonyl-L-lysylamino)-1H-pyrazole-5-carboxylate (9)
Compound 8 (0.532 g, 1.05 mmol) was dissolved in MeOH (7 ml); 10%
Pd=C (0.053 g) and H2 were added. After 3 h, the catalyst was filtered
off and MeOH was evaporated. Lys(Boc)-Pz-OMe (0.306 g, 0.8 mmol)
was obtained and dissolved in DMF (3 ml). Z-Lys(Boc)-ONp (0.441 g,
0.88 mmol) and HOBt ꢁ H2O (0.135 g, 0.88 mmol) were added. After
20 h of stirring at room temperature, the DMF was evaporated. The resi-
due was dissolved in chloroform (20 ml), extracted with 5% NaHCO3
(4 ꢁ 2 ml), brine (4 ꢁ 4 ml), and water (2 ꢁ 4 ml), and dried with MgSO4.
Chloroform was evaporated, and ethyl ether was added to the residue
and left standing in the refrigerator. The precipitate was filtered off
(0.521 g), washed with ethyl ether (3 ꢁ 3 ml), and dissolved in warm
methanol (8 ml). After cooling to room temperature, ethyl ether (20 ml)
was added. Compound 9 (0.479 g, 62% yield, two crops) was obtained,
mp 163.39ꢀC; TLC (B): Rf 0.57; HPLC: (D) tR 4.56 min, first crop
1
100% purity, second crop 98.8%. H NMR (DMSO-d6) d (ppm): 1.365
(s, 22H, CH3-tBu ꢁ 2, CH2-Lys ꢁ 2), 1.516 (s, 4H, CH2-Lys ꢁ 2), 1,610 (s,
4H, CH2-Lys ꢁ 2), 2.890 (s, 4H, CH2-Lys ꢁ 2), 3.836 (s, 3H, OCH3),
4.014 (s, 1H, a-CH-Lys), 1.417 (s, 1H, a-CH-Lys), 5.041 (s, 2H, CH2-Z),
6.771 (s, 2H, NHurethane Boc ꢁ 2), 6.996 (s, 1H, C4), 7.324 (s, 1H,
NHurethane Z), 7.365 (m, 5H, CHarom.), 8.037 (s, lH, NHamide Lys-Lys),
10.745 (s, lH, NHamide Lys-pyrazole), 13.597 (s, 1H, NHring); 13C NMR
(DMSO-d6) d (ppm): 22.58 (CH2-Lys), 22.82 (CH2-Lys), 28.18 (6C,
CH3-tBu), 29.13 (2C, CH2-Lys), 31.51 (CH2-Lys), 31.69 (CH2-Lys), 40.04
(2C, CH2-Lys), 51.87 (OCH3), 52.62 (a-CH-Lys), 54.57 (a-CH-Lys), 65.32
(CH2-Z), 77.25 (C-tBu), 99,59 (C4), 127.63 and 128.26 (5C, C2-Ca6rom:),
5
3
1
=
132.99 (C ), 136.97 (Carom:), 144.50 (C ), 155.49 (2C, C Ourethane Boc),
=
155.91 (C Ourethane Z), 159.47 (C Oester), 170.14 (C Oamide Lys-Lys),
=
=
=
171.99 (C Oamide Lys-pyrazole). Analysis calcd. for C35H53N7O10: C,
57.44%; H, 7.30%; N, 13.40%. Found: C, 57.07%; H, 7.31%; N, 13.47%.
ACKNOWLEDGMENT
The authors gratefully acknowledge the University of Opole for grant
number 1=ICh=S-2007.
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¨