Aminopyrazole oligomers for β-sheet stabilization of peptides

Article abstract of DOI:10.1055/s-2003-41031

A general concept for the stabilization of β-sheets by designed artificial ligands is introduced. The ligands have two key features: they contain acylated 3-aminopyrazoles with a DAD hydrogen bond donor and acceptor pattern, and they were synthesized as o

Full text of DOI:10.1055/s-2003-41031

PAPER
1815
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
A
minopyrazol
.
e
O
ligomers
R
for
-
Sheet Stabi
z
lization of
ePeptides pecki, M. Wehner, O. Molt, R. Zadmard, K. Harms, T. Schrader*
Philipps-Universität Marburg, Department of Chemistry, Hans-Meerwein-Straße, 35032 Marburg, Germany
Fax +49(6421)2828917; E-mail: schradet@mailer.uni-marburg.de
Received 7 July 2003
Dedicated to Wolfgang Steglich on the occasion of his 70th birthday.
any hydrogen bond donors on their outer side. This is our
new concept to prevent protein aggregation, which is be-
ing tested in a cooperation with biophysicists on the Prion
and Alzheimer’s protein.5
Abstract: A general concept for the stabilization of -sheets by de-
signed artificial ligands is introduced. The ligands have two key fea-
tures: they contain acylated 3-aminopyrazoles with a DAD
hydrogen bond donor and acceptor pattern, and they were synthe-
sized as oligomers in order to multiply their hydrogen bond interac-
tions with peptides in the -sheet conformation. Dimeric
aminopyrazoles were accessible by reaction of the N1-Boc-protect-
ed aminopyrazole derivative 1 with several acid dichlorides fol-
lowed by a standard deprotection procedure with trifluoroacetic
acid. For the oligomers, N1-PMB protection of new pyrazole amino
acids followed by an iterative extension protocol with peptide cou-
pling using PyClop or Mukaiyama’s reagent led to the target com-
pounds. All protecting groups were subsequently removed in a final
deprotection step with warm trifluoroacetic acid. Two dimeric key
compounds 3b and 3f were examined by NMR at various tempera-
tures, in NOESY experiments as well as by X-ray crystallography
in order to elucidate their conformational preference in solution and
the solid state. The emerging picture was the same for all methods:
both ligands adopt a flat conformation with a high degree of pre-ori-
entation and the correct DAD pattern for optimal interaction with
peptides in their extended conformation. Aggregation assays with
the Prion protein and the Alzheimer’s peptide A (1–40) show high-
ly promising results for some of the dimeric and oligomeric ligands
at very low concentrations.
Key words: amino acids, peptides, molecular recognition, bioor-
ganic chemistry, drugs, protecting groups
Figure 1 2:1-Complex formation between an N/C-protected dipep-
tide and aminopyrazole ligands. Top: force-field calculation (Cerius2,
Molecular Simulations, Dreiding 2.21). Bottom: Lewis structure.
Introduction
Protein folding diseases like Alzheimer’s or the Mad Cow
Disease (BSE)/Creutzfeldt–Jakob (CJD) are character-
ized by a precipitation of protein plaques in the nerve cells
of the brain. The key step is the misfolding of a water sol-
uble protein monomer (e.g., PrPC), existing mainly in the
-helical state, into a structure containing mainly -sheets
(PrPSc).1 It could be shown that, in the beginning phase,
these -sheet arrays form oligomers that eventually clus-
ter into insoluble aggregates.2
Concept
In order to increase the binding strength between the -
sheet binders and their peptide substrates we have now de-
veloped the second and third generation of receptor mole-
cules. The second generation contains two aminopyrazole
moieties covalently linked with a dicarboxylic spacer.
These new templates can form up to six hydrogen bonds
with a peptide if it resides in an extended conformation.
We deliberately chose various spacers with different ge-
ometries in order to be able to compare their ability to in-
teract with small peptides. For the third generation, we
introduced an additional carboxylate into the aminopyra-
zole structure, leading to a new non-natural amino acid.
Repetitive peptide coupling produced a growing oli-
gopeptide with a hydrogen pattern complementary to the
top and bottom face of a peptide in its -sheet conforma-
tion (Figure 2).
We have recently demonstrated, that acylated aminopyra-
zoles form complexes with small peptides by recognizing
their backbone.3 The aminopyrazoles can even be lined up
along both faces of peptide strands and thereby stabilize
their -sheet conformation4 (Figure 1). However, after be-
ing capped by external ligands, the peptides should not be
sticky any more, because the aminopyrazoles do not carry
Synthesis 2003, No. 12, Print: 02 09 2003. Web: 13 08 2003.
Art Id.1437-210X,E;2003,0,12,1815,1826,ftx,en;T06403SS.pdf.
DOI: 10.1055/s-2003-41031
© Georg Thieme Verlag Stuttgart · New York

1816
P. Rzepecki et al.
PAPER
Table 1 Free Dimeric Aminopyrazole Derivatives from the Double
Acylation with Subsequent Acidic Boc Removal (Scheme 1)
Synthesis
a) Dimeric Ligands
n
0
m R
Yield Yield 3a–f
(%) of 2 (%) of 3
Reaction of the N1-Boc-protected aminopyrazole deriva-
tive 1 with several acid dichlorides followed by a standard
deprotection procedure with trifluoroacetic acid led di-
rectly to the diacyl-bridged dimeric aminopyrazoles
(Scheme 1). The free heterocycles were obtained in a
highly pure state, however, they are in many cases only
sparingly soluble in pure chloroform. We assume, that in
nonpolar solvents hydrogen-bonded networks of these
aminopyrazole dimers are formed, with a mutual overlap
between neighboring dimers, leading to a belt-like struc-
ture. The spacers have been chosen with care: They pro-
duce dimers with either an extended arrangement of both
heterocycles (3a, 3b and 3d), a twisted structure (3c) or
even a flexible or rigid kinked geometry (3e and 3f). In ad-
dition, the spacer between the two dipeptide binders var-
ies in length (compare, e.g., 3b and 3d) (Scheme 1,
Table 1).
0
–
2a: 42 3a: 45
O
HN
N
NH
N
N
N
H
H
3a
1
1
0
1
–
2b: 56 3b: 71
O
O
HN
NH
NH
HN
N
N
3b
C(CH3)2
2c: 27 3c: 31
O
O
N
NH HN
N
HN
NH
3c
1
1
1
1
p-C6H4
m-C6H4
2d: 29 3d: 34
2e: 22 3e: 75
O
O
HN
O
NH
NH
H
N
N
N
3d
O
NH
HN
HN
N
N
NH
HN
3e
1
1
2,6-C5NH3 2f: 19 3f: 60
O
N
O
N
N
NH
NH
HN
3f
b) Oligomeric Ligands
To our surprise, the free 3-aminopyrazole-5-carboxylic
acid or derivatives thereof without an N-alkyl substituent
on the pyrazole nitrogen atom are unknown to date.6 We
prepared its basic skeleton by reducing the respective ni-
tro compound with sodium dithionate7 or - milder - by hy-
drogenation over Pd/C. However, even after its selective
conversion into the methyl ester and the N-acetyl deriva-
tive, no amide coupling could be effected under any reac-
tion condition tested. In all cases, the basic ring NH group
is more reactive than the aromatic amino group leading to
ring acylation instead of peptide coupling.8 Several stan-
dard protecting groups were tried, but after intensive in-
vestigations only one fulfilled the requirements of an
effective regioselective and reversible pyrazole ring pro-
tection. Boc- and Z-groups prevented the peptide coupling
(numerous coupling reagents were tested), SEM (trimeth-
ylsilylethoxymethyl) could not be cleaved off with TBAF,
and aqueous HCl led to considerable degradation of the
dimer. Benzyl protection came as a surprise, because it re-
sisted even harsh cleavage conditions such as hydrogenol-
ysis with Pd/C, Pd(OH)2/C, with or without acid additives
or even in pure acetic acid. Obviously, the tertiary nitro-
gen is extremely stable on the pyrazole nucleus. We
Figure 2 a) Optimized complex structure between the dimeric
-
sheet ligand 3 and tetraglycine (MacroModel 7.0, Amber*, chloro-
form); b) optimized complex structure between the tetrameric -sheet
ligand and octaalanine.
O
O
NH2
Rm
Cl
Cl
N
n
N
2a-f
Boc
1
O
O
NH
Rm
N
N
n H
NH
TFA
HN
N
3a-f
with n,m = 0,1
Scheme 1 Synthesis of the new dimeric aminopyrazole derivatives.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

PAPER
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
1817
moved to the PMB (p-methoxybenzyl) protecting group, the free oligomers. The dimeric fully protected intermedi-
but failed again, when we tried hydrogenolysis or the al- ates can also be converted at will into reagents with a free
ternative approach of oxidative cleavage with DDQ or amine group (H2-Pd/C) or a free carboxylic acid (LiOH/
CAN.9 Finally, we utilized the improved stabilization of MeOH).12 Their combination in a standard peptide cou-
the benzylic carbenium ion in the PMB group and em- pling protocol with PyClop produced the trimeric and tet-
ployed more drastic acidic conditions. After merely ef- rameric structures in one-step which were deprotected
fecting peptide cleavage with anisole/TFA, we used pure with TFA at elevated temperatures. The ring NH groups
anhydrous trifluoroacetic acid at elevated temperatures of of the free pyrazole oligomers turned out to be relatively
70 °C. Under these optimized conditions, a reproducible acidic, so that care has to be taken, when their TFA salts
quantitative conversion could be obtained for the PMB re- are brought back into the neutral form. It is therefore crit-
moval (Scheme 2).
ical to choose for each aminopyrazole derivative the ap-
propriate base with optimal pKa in order to ensure
complete neutralization. On the other hand, the amphoter-
ic character of the oligomers represents a convenient ac-
cess to water-soluble derivatives. The cationic form is
often much more soluble in aqueous mixtures, so that the
potential drugs become much easier to administer to pa-
tients.
O2N
O2N
HCl, MeOH
PMB-Br, K2CO3
DMF, 88%
N
N
COOMe
COOH
quant.
N
H
N
H
5
4
O2N
H2N
Pd/C, H2,
COOMe
N
N
COOMe
N
N
MeOH, 75%
PMB
PMB
8
O
6
method E,F
RI
H2N RII
LiOH, MeOH,
+
9a-e
Ra,b-COX,
53-68%
THF, H2O, 92%
OH
O
RVI
O2N
N
TFA, ∆
Ra,b
RIII
N
H
N
COOH
O
COOMe
N
PMB
7
N
PMB
14a-e
E: PyClop, DIEA
F: Mukaiyama's reagent, DIEA
12a, 12b
8, Mukaiyama's
reagent, DIEA
LiOH, MeOH,
THF, H2O,
78-95%
Scheme 3 Synthetic protocol for the key coupling step leading from
monomeric building blocks to dimeric, trimeric and tetrameric pyra-
zole amino acids.
CH2Cl2, 88%
O2N
O
Ra,b
O
N
N
N
OCH3
PMB
O
HN
PMB
COOH
N
N
Unfortunately, especially the higher free aminopyrazole
carboxylic acid oligomers turned out to be only sparingly
soluble in organic solvents. Large amounts of (in some
cases even boiling) DMSO were necessary for complete
dissolution. We therefore chose three different paths to in-
crease their solubility. Introduction of short ethylene gly-
col chains at the N-terminus could be brought about by
peptide coupling with glycolic acid derivatives
(Scheme 2, 13a). The methyl ester at the C-terminus
could be converted in high yields to alkyl amides by ami-
N
PMB
13a, 13b
Ra= triethylenglycole
9a
LiOH, MeOH,
THF, H2O,
88%
Pd/C, H2,
MeOH, 58%
R
b = methyl
O2N
H2N
O
O
O
O
N
N
N
PMB
OH
PMB
N
PMB
OCH3
HN
HN
N
N
N
N
PMB
10
11
Scheme 2 Synthetic pathway to dimeric pyrazole amino acids as fi-
nal products and synthetic intermediates
nolysis with n-alkylamines (Scheme 4, 16 and 18).13
A
double lipophilization was attempted with an early intro-
duction of an N-hexanoyl acyl protection, followed by
aminolysis of the methyl ester with n-butylamine
(Scheme 4, 17). To our disappointment, this derivative
was the least soluble of all – it even resisted boiling
DMSO. The two antiparallel tails might cause an unfavor-
able form of self-aggregation with complete desolvation
of the building blocks. In spite of the presence of two
strongly electron-withdrawing substituents on the pyra-
zole ring, the nitro ester 4 can be cleanly iodinated in the
vacant 4-position with iodine/CAN.14 Although Suzuki
and Heck couplings failed, we were successful with the at-
tachment of ethynyl groups by way of the Sonogashira
method (Pd-II, Cu-I, base, alkyne).15 This modification
may be the method of choice to introduce additional sub-
stituents at the back of the aminopyrazole ligands, where
Several coupling reagents were tried for the attachment of
the electron-poor aromatic amine onto an aromatic car-
boxylate. The best yields (75–94%) were obtained with
PyClop [tris(pyyrolidino)chlorophosphonium hexafluo-
rophosphate]10 and Mukaiyama’s reagent (2-chloro-N-
methylpyridinium chloride)11 (Scheme 3, Table 2). For a
repetitive protocol the nitro group was used as a transient
protecting group on the monomeric building block. Di-
rectly after amide coupling, it was reduced to the free
amine, which was subjected in the next cycle to peptide
coupling with another building block (Scheme 2). This
two-step procedure was repeated until the desired oligo-
mer size was reached. Finally, in the last step, all PMB
groups were cleaved off in one-step. Neutralization of the
acidic TFA solution with mild base (NaHCO3) furnished
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

1818
P. Rzepecki et al.
PAPER
Table 2 Final Oligomeric Pyrazole Amino Acid Products from the Coupling Reaction with Subsequent Acidic PMB Removal (Scheme 3)
H2N RII
O
Method
Yield (%) for 9 Yield (%) for 14
14a–e
RI
OH
7
8
F
9a: 88
9b: 80
14a: 75
14b: 50
O2N
N
O
O
N
H
OCH3
HN
NH
N
O2N
N
7
11
E
E
O
N
O
N
O
HN
OMe
H
N
NH
NH
N
H
10
11
9c: 51
9d: 66
14c: 35
14d: 63
O2N
N
COOMe
NH
N
O
O
N
H
O
HN
O
N
H
N
N
NH
NH
N
H
13a
8
F
O
O
NH
N
O
H
N
COOMe
2 CF3COOH
NH
N
NH
O
13b
8
F
9e: 46
14e: 90
AcHN
O
O
N
OCH3
N
HN
H
NH
N
2 CF3COOH
they cannot interfere with their hydrogen bonding to the these syntheses is always the distinction between the three
peptide.
basic nitrogen atoms in the aminopyrazole nucleus. A
careful choice of protecting groups solves this problem;
thus, selective Boc protection of the aminopyrazole
monomers allows their dimerization with diacid dichlo-
rides, while for the oligomers PMB protection of the new
In summary, we have outlined synthetic pathways to
dimeric and oligomeric aminopyrazole derivatives with a
hydrogen bond donor and acceptor pattern fully comple-
mentary to that of -sheets. The most critical aspect in
Scheme 4 Attachment of solubility enhancing groups to the periphery of oligomeric aminopyrazoles: aminolysis and Sonogashira-coupling.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

PAPER
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
1819
pyrazole amino acids followed by an iterative extension spective NH protons in only 6% DMSO–CDCl3. NOE ex-
protocol with PyClop coupling and catalytic reduction periments were also hampered by the low ligand
leads to the target compounds. All protecting groups are solubility. Nevertheless, a quite remarkable intramolecu-
subsequently removed in a final deprotection step with lar NOE pattern was found for 3b and 3f. In 3b a marked
warm trifluoroacetic acid. The two new classes of ligands cross-peak was found for the interaction of the amidic NH
have some common promising features: they are easily with the aromatic pyrazole CH proton, indicating a 180°
synthesized, contain nontoxic components,16 have low flip of the pyrazole nucleus. No intermolecular NOE
molecular weights around 300, and are neutral, stable peaks to the solvent (100% DMSO) could be found. By
molecules in their biologically active form.
contrast, strong cross peaks between every NH proton and
the solvent were evident for 3f, although it contained only
6% of DMSO. In this case, an additional intramolecular
NOE between the amidic NH and the aromatic pyrazole
CH indicated the expected torsional angle (0°).
Conformational Studies
Various spectroscopic experiments were carried out on
the most promising new ligands (vide infra), in order to
elucidate their conformational preference in solution as
well as their tautomer equilibrium. Since the oxalyl- and
the pyridinedicarboxyl-bridged dimers 3b, 3f displayed
the highest biological acitivity in a preliminary aggrega-
tion screening, these compounds were examined by NMR
at various temperatures, in NOESY experiments as well
as by X-ray crystallography. The very low solubility of 3b
suggests a massive self-association via hydrogen bonds.
Accordingly, even in 60% DMSO–CDCl3 only a very
small temperature dependence of 1.6 and 4.0 ppb/K was
found for its pyrazole NH and amidic protons.17 By con-
trast, the pyridine dicarboxamide, which is much better
soluble, produced a slope of 5.0 and 6.3 ppb/K for the re-
Interesting enough, the picture emerging from the VT and
NOE experiments, was fully confirmed by X-ray crystal-
lographic analyses (Figure 3).18 The oxalyl-bridged dimer
3b adopts a flat conformation, with both carbonyls point-
ing in opposite directions and a 180° rotation around the
pyrazole C–N bond. This conformation, however, is dif-
ferent to the above-postulated arrangement in peptide
complexes of 3b. We assume, that due to the positive
charge of the pyrazolium nucleus the dimer conformation
is stabilized by two additional intramolecular hydrogen
bonds with the bridging amide carbonyls. The flat pyri-
dine dicarboxamide dimer exhibits the expected intramo-
lecular preorganization by two hydrogen bonds between
the pyridine nitrogen and both amide NH groups. Howev-
er, here the pyrazole ring lines up all of its hydrogen bond
Figure 3 Crystal structures for two selected -sheet binders. Left: Oxalyl-bridged 3b: note the horizontal hydrogen bond formation to the
counterions [bis(trifluoroacetate) salt].20 Right: Pyridine dicarboxamide-bridged 3f: note the expected DAD pattern as well as the predicted pre-
organization by way of two intramolecular NH···N hydrogen bonds.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

1820
P. Rzepecki et al.
PAPER
MS (FD): m/z (%) = 448 (M+).
Anal. Calcd for C20H28N6O6: C, 53.56; H, 6.29; N, 18.74. Found: C,
donors and acceptors on the same side (torsional angle,
0°) in the correct DAD-pattern.19 Although some periph-
eral -stacking is observed, most hydrogen bond donors 53.30: H, 6.22; N, 18.79.
and acceptors available form intermolecular hydrogen
N-N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)-2,2-
dimethylmalonic Acid Diamide (2c)
bonds to the solvent (methanol).
The binding affinity of the new ligands towards model
peptides is currently under investigation with NMR tech-
niques. We are expecting even higher binding affinities
between model peptides and the higher oligomers. We
also started to test the new -sheet-binders on the prion
protein in cooperation with biophysicists.21 An initial pre-
cipitation experiment showed that 3b was able to keep
about one third of recombinant PrPC in solution. Under
these conditions, the same protein completely precipitated
in the absence of our ligand. In addition to the tests on the
recombinant prion protein, all new ligands are currently
screened in aggregation assays with the Alzheimer’s pep-
tide A (1-40); some of the dimeric and oligomeric
ligands show highly promising results at very low concen-
trations. These biophysical experiments will be reported
in due course.
Compound 1 (1.50 g, 7.60 mmol) was reacted with dimethylmalo-
nyl chloride in CHCl3 (30 mL) according to the general procedure
A. In this case, the CH2Cl2 was heated to reflux before the acid chlo-
ride was added. The residue was recrystallized from CH2Cl2–n-hex-
ane; yield: 1.0 g (27 %); colorless solid; mp 124 °C.
1H NMR (300 MHz, DMSO-d6): = 1.30 (s, 6 H), 1.43 (s, 18 H),
2.03 (s, 6 H), 6.43 (s, 2 H), 10.46 (s, 2 H).
13C NMR (50 MHz, CDCl3): = 14.4, 27.8, 27.9, 51.4, 86.5, 98.5,
150.7, 151.4, 153.2, 169.2.
MS (FD): m/z (%) = 490 (M+, 14), 390 (M+ – Boc, 75), 290 (M+ – 2
Boc, 10).
N-N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)tere-
phthalic Acid Diamide (2d)
Compound 1 (1.00 g, 5.1 mmol) was reacted with terephthaloyl
chloride in anhyd CH2Cl2 (30 mL) according to the general proce-
dure A. The residue was recrystallized from CHCl3–n-hexane;
yield: 780 mg (29%); colorless solid; mp 181 °C.
1H NMR (300 MHz, DMSO-d6): = 1.49 (s, 18 H), 2.19 (s, 6 H),
6.57 (s, 2 H), 8.02 (s, 4 H), 10.76 (s, 2 H).
Boc-Protected Dimers; General Procedure A
13C NMR (50 MHz, CDCl3): = 14.6, 28.4, 87.3, 98.6, 128.3,
136.6, 141.8, 151.8, 154.2, 161.4.
MS (FD): m/z (%) = 524 (M+, 36), 424 (M+ – Boc, 21), 325 (M+ – 2
N1-Boc-3-aminopyrazole 1 (2.00 equiv) and Et3N (3.00 equiv)
were dissolved in anhyd CH2Cl2 and treated with the diacid dichlo-
ride (1.00 equiv) at 0 °C and stirred overnight at r.t. The solvent was
evaporated to dryness and the resulting residue was extracted with
EtOAc. The organic layer was washed with aq 1 N HCl, sat. aq
NaHCO3, sat. aq NaCl and H2O, dried (MgSO4), and filtered. The
solvent was evaporated and the product was dried in vacuo. The
purification procedures are detailed together with the respective
compound characterization below.
Boc, 100).
Anal. Calcd for C26H32N6O6: C, 59.53; H, 6.15; N, 16.02. Found: C,
59.20; H, 6.55; N, 15.89.
N-N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)iso-
phthalic Acid Diamide (2e)
N,N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)urea
(2a)
Compound 1 (2.00 g, 10.2 mmol) was reacted with isophthaloyl
dichloride in anhyd CH2Cl2 (50 mL) according to general procedure
A; however, extraction was done with CHCl3. The residue was re-
crystallized from CHCl3–n-hexane; yield: 1.20 g (22%); colorless
solid; mp 171 °C.
1H NMR (300 MHz, DMSO-d6): = 1.33 (s, 18 H), 1.98 (s, 6 H),
6.40 (s, 2 H), 7.60 (t, J = 7.6 Hz, 1 H), 7.93 (d, J = 7.3 Hz, 1 H),
8.23 (s, 1 H), 10.62 (s, 2 H).
Compound 1 (1.22 g, 6.20 mmol) was coupled with phosgene in an-
hyd CH2Cl2 (40 mL) according to the general procedure A. For con-
venience, a 1.93 M solution of phosgene in toluene was used. The
product was recrystallized from CH2Cl2–n-hexane; yield: 1.09 g
(42%); colorless solid; mp 161 °C.
1H NMR (300 MHz, CDCl3): = 1.69 (s, 18 H), 2.28 (s, 6 H), 6.56
(s, 2 H), 9.73 (br s, 2 H).
13C NMR (50 MHz, CDCl3): = 14.4, 28.0, 86.5, 96.8, 141.8,
147.3, 151.0, 153.6.
MS (CI): m/z (%) = 421 (M + H+, 40), 318 (M+ – Boc, 6), 301 (100).
13C NMR (125 MHz, CDCl3): = 14.6, 28.2, 87.0, 98.7, 98.8,
126.9, 129.7, 130.8, 134.4, 141.6, 153.8, 162.3.
MS (FD): m/z (%) = 524 (M+ 14), 424 (M+ – Boc, 75), 324 (M+ – 2
,
Boc, 100).
Anal. Calcd for C19H28N6O5: C, 54.27; H, 6.71; N, 19.99. Found: C,
53.67; H, 6.87; N, 19.66.
N,N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)pyri-
dine-2,6-dicarboxylic Acid Diamide (2f)
Compound 1 (5.10 mmol) was reacted with pyridine-2,6-dicarbox-
ylic acid chloride in anhyd CH2Cl2 (50 mL) according to general
procedure A. The residue was recrystallized from CH2Cl2–n-hex-
ane; yield: 500 mg (19%); colorless solid; mp 165 °C.
1H NMR (500 MHz, CDCl3): = 1.57 (s, 18 H), 2.35 (s, 6 H), 6.82
(s, 2 H), 8.16 (t, J = 7.6 Hz, 1 H), 8.42 (d, J = 7.6 Hz, 1 H), 11.70,
(s, 2H), 12.02 (s, 2H).
N,N -Bis(1-tert-butyloxycarbonyl-5-methylpyrazol-3-yl)oxal-
diamide (2b)
Compound 1 (2.00 g, 10.0 mmol) was reacted with oxalyl chloride
in anhyd CH2Cl2 (40 mL) according to the general procedure A. The
product was recrystallized from CH2Cl2, the precipitate was finally
washed with cold n-hexane; yield: 1.10 g (56%); colorless solid; mp
170 °C.
1H NMR (300 MHz, CDCl3): = 1.71 (s, 18 H), 2.32 (s, 6 H), 6.81
(s, 2 H), 11.75 (br s, 2H).
13C NMR (75 MHz): = 10.8, 96.6, 125.1, 138.9, 139.6, 146.5,
148.9, 161.5.
13C NMR (75 MHz, CDCl3): = 14.6, 28.1, 87.4, 99.4, 139.4,
153.4, 155.4.
MS (FD): m/z (%) = 525 (M+, 100), 425 (M+ – Boc, 12), 325 (M+ –
2 Boc, 38).
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

PAPER
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
1821
Boc Deprotection; General Procedure B
HRMS (EI): m/z calcd for C16H16N6O2: 324.1333; found: 324.1335.
The tert-butoxycarbonyl-protected compound (750 mol–
5.10 mmol) was dissolved in anhyd CH2Cl2 (30–50 mL) and stirred
at 0 °C with trifluoroacetic acid (TFA, 6 mL). The stirring was con-
tinued for 1 h after the starting material had disappeared in the TLC.
The solvent was evaporated and the resulting residue was extracted
with sat. aq NaHCO3 and boiling EtOAc (10 × 75 mL). The com-
bined organic layers were dried (MgSO4) and evaporated to dry-
ness.
N-N -Bis(5-methyl-1H-pyrazol-3-yl)isophthalic Acid Diamide
(3e)
Compound 2e (500 mg, 950 mol) was reacted with TFA in anhyd
CH2Cl2 (30 mL) according to general procedure B; yield: 230 mg
(75%); colorless solid; mp 172 °C.
1H NMR (300 MHz, DMSO-d6): = 2.30 (s, 6 H), 6.50 (s, 2 H),
7.67 (t, J = 7.6 Hz, 1 H), 8.16 (d, J = 8.0 Hz, 1 H), 8.61 (s, 2 H),
10.74 (s, 2 H), 12.21 (s, 2 H).
13C NMR (50 MHz, CD3OD): = 12.2, 97.5, 128.1, 130.0, 132.1,
135.6, 165.1.
N,N -Bis(5-methyl-1H-pyrazol-3-yl)urea (3a)
Compound 2a (500 mg, 1.20 mmol) was reacted with TFA in anhyd
CH2Cl2 (50 mL) according to the general procedure B; yield: 120
mg (45%); colorless solid; mp 202–206 °C.
1H NMR (300 MHz, CDCl3, 25 °C): = 1.69 (s, 18 H), 2.28 (s, 6
H), 6.56 (s, 2 H), 9.73 (br s, 2 H).
MS (EI): m/z (%) = 324 (M+, 53), 228 (M+ – aminopyrazole, 100),
206 (30), 97 (aminopyrazole+, 23).
HRMS (ESI): m/z calcd for C16H16N6O2: 324.1328; found:
13C NMR (50 MHz, CD3OD, 25 °C): = 10.9, 143.8, 147.2, 153.4.
324.1335.
MS (FAB + NBA): m/z (%) = 221 (M + H+, 60) 137 (M+ – pyrazole,
70), 51 (100).
N-N -Bis(5-methyl-1H-pyrazol-3-yl)pyridine-2,6-dicarboxylic
Acid Diamide (3f)
HRMS (ESI): m/z calcd for C9H12N6O: 220.1077; found: 220.1073.
Compound 2f (400 mg, 760 mol) was reacted with TFA in anhyd
CH2Cl2 (30 mL) according to general procedure B. After the solvent
was evaporated, the residue was suspended two times in toluene
and the solvent was again evaporated. The solid was washed with
10% aq NaOH (2 ×) and H2O and dried in vacuo; yield: 148 mg
(60%); colorless solid; mp 196 °C.
1H NMR (500 MHz, DMSO-d6, 25 °C): = 2.20 (s, 6 H), 6.43 (s, 2
H), 8.16 (t, J = 7.0 Hz, 1 H), 8.24 (d, J = 6.9 Hz, 1 H), 11.62 (s, 2
H).
N,N -Bis(5-methyl-1H-pyrazol-3-yl)oxaldiamide (3b)
Compound 2b (1.00 g, 5.10 mmol) was reacted with TFA in anhyd
CH2Cl2 (50 mL) according to the general procedure B. After the sol-
vent was evaporated, the residue was suspended twice in toluene
and the solvent was again evaporated. The solid was washed with
sat. aq NaHCO3 (2 ×) and H2O and dried in vacuo; yield: 885 mg
(71%); colorless solid; mp >230 °C.
1H NMR (300 MHz, DMSO-d6): = 2.37 (s, 6 H), 6.46 (s, 2 H),
10.70 (br s, 2 H), 12.39 (br s, 2 H).
13C NMR (125 MHz, DMSO-d6): = 10.8, 96.0, 139.6, 144.8,
157.5.
13C NMR (50 MHz, DMSO-d6, 25 °C): = 12.2, 97.9, 126.4, 140.0,
150.4, 162.8.
MS (CI, NH3, 200 °C): m/z (%) = 326 (M + H+, 100).
HRMS (ESI): m/z calcd for C15H15N7O2: 325.1271; found:
325.1287.
MS (EI): m/z (%) = 248 (M+), 124 (31), 97 (aminomethylpyrazole,
100).
HRMS (EI): m/z calcd for C10H12N6O2: 248.1017; found: 148.1022.
3-Nitropyrazole-5-carboxylic Acid Methyl Ester Hydrochloride
(5)
N-N -Bis(5-methyl-1H-pyrazol-3-yl)-2,2-dimethylmalonic Acid
Diamide (3c)
Compound 2c (530 mg, 1.10 mmol) was reacted with TFA in anhyd
CH2Cl2 (30 mL) according to the general procedure B; yield: 97 mg
(31%); colorless solid; mp 204 °C.
1H NMR (300 MHz, DMSO-d6): = 1.55 (s, 6 H), 2.25 (s, 6 H),
6.31 (s, 2 H), 9.85 (s, 2 H), 12.09 (s, 2 H).
In a three-necked flask with reflux condenser, compound 4 (10 g,
5.84 mmol) was dissolved in anhyd MeOH (250 mL). Gaseous HCl
was passed through this reaction mixture for 5 min; afterwards it
was stirred under reflux for 8 h. The solvent was evaporated and the
resulting solid was dried in vacuo; yield: 0.989 mg (99%); colorless
solid; mp 138 °C.
1H NMR (300 MHz, CDCl3): = 3.91 (s, 3 H), 7.52 (s, 1 H).
13C NMR (50 MHz, CD3OD): = 12.2, 24.6, 52.1, 97.2, 140.0,
148.2, 156.7, 172.1.
13C NMR (125 MHz, DMSO-d6): = 52.6, 104.7, 135.7, 155.8,
158.2.
MS (FD): m/z = 290 (M+).
MS (EI): m/z (%) = 171 (M+).
HRMS (ESI): m/z calcd for C13H18N6NaO2: 313.1351; found:
313.1389.
Anal. Calcd for C5H5N3O4: C, 35.10; H, 2.95; N, 24.56; found: C,
35.42; H, 3.33; N, 24.12.
N-N -Bis(5-methyl-1H-pyrazol-3-yl)terephthalic Acid Diamide
(3d)
2-(4-Methoxybenzyl)-5-nitro-2H-pyrazole-3-carboxylic Acid
Methyl Ester (6)
Compound 2d (500 mg, 0.95 mmol) was reacted with TFA in anhyd
CH2Cl2 (30 mL) according to the general procedure B; yield: 100
mg (33%); colorless solid; mp >230 °C.
1H NMR (500 MHz, DMSO-d6): = 2.35 (s, 6 H), 6.53 (s, 2 H),
8.18 (s, 4 H), 10.94 (s, 2 H), 12.24 (s, 2 H).
13C NMR (125 MHz, DMSO-d6): = 10.8, 96.3, 127.6, 136.6,
In an argon atmosphere, 3-nitropyrazole-5-carboxylic acid methyl
ester (5; 5.45 g, 20.2 mmol), 4-methoxybenzyl bromide (6.08 g,
30.2 mmol) and K2CO3 (5.57 g, 40.3 mmol) were reacted in anhyd
DMF (60 mL) for 18 h at 50 °C. After the reaction mixture was al-
lowed to cool to r.t., it was acidified with aq 1 M HCl and the aque-
ous solution was then extracted with MTBE (4 × 60 mL). The
combined organic layers were washed with H2O (30 mL) and sat. aq
NaCl (30 mL) and dried (Na2SO4). After filtration, the solvent was
evaporated and the yellow oil was purified by chromatography on a
silica gel column (eluent: n-pentane–EtOAc, 3:1); Rf 0.19; yield:
3.41 g (88%); pale yellow solid; mp 73 °C.
145.0, 163.6.
MS (EI): m/z (%) = 324 (M+, 13), 228 (M+ – aminopyrazole, 34),
206 (30).
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

1822
P. Rzepecki et al.
PAPER
1H NMR (300 MHz, CDCl3): = 3.85 (s, 3 H), 3.92 (s, 3 H), 5.76
(s, 2 H), 6.83–7.39 (m, 5 H), 3.82 (s, 3 H), 3.98 (s, 3 H), 5.80 (s, 2
H), 6.83–7.39 (m, 5 H).
13C NMR (75 MHz, CDCl3): = 52.8, 55.4, 56.2, 57.2, 107.8,
109.4, 114.3, 144.4, 127.1, 129.7, 129.9, 130.6, 158.7, 160.0.
Ms (EI): m/z (%) = 292 (M+ + H, 6), 291 (M+, 44), 121 (PMB+,
100).
18 h at r.t. The organic layer was washed with aq 1 M HCl, sat. aq
NaHCO3, sat. aq NaCl and H2O and dried (Na2SO4). After filtration,
the solvent was concentrated and the remaining residue was purified
by chromatography on silica gel column (eluent: n-pentane–EtOAc,
2:1); yellow solid; Rf 0.41; yield: 1.56 g (53%).
1H NMR (200 MHz, CDCl3): 3.30 (s, 3 H), 3.54–3.71 (m, 8 H)
3.73 (s, 3 H), 3.81 (s, 3 H), 4.09 (s, 2 H), 5.56 (s, 2 H), 6.76–7.26
(m, 5 H).
Anal. Calcd for C13H13N3O5: C, 53.61; H, 4.50; N, 14.43; found: C,
53.64; H, 4.69; N, 14.30.
13C NMR (125 MHz, CDCl3): = 52.0, 54.0, 55.3, 59.1, 70.3, 70.5,
70.8, 71.4, 72.0, 102.7, 114.0, 129.1, 131.2, 145.2, 159.2, 160.2,
167.9.
Reduction of 2-(4-Methoxybenzyl)-5-nitro-2H-pyrazoles; Gen-
eral Procedure C
MS (EI): m/z (%) = 421 (M+, 13), 121 (PMB+, 100).
To a solution of the nitro compound in THF (50 mL/10mmol) was
added MeOH (150 mL/10mmol) and Pd/C (10 mol%). The flask
was then evacuated and filled with H2. The reaction mixture was
stirred for 16–52 h, and then the catalyst was removed by filtration
over silica gel and the solution concentrated in vacuo. The residue
was purified by column chromatography.
Anal. Calcd for C20H27N3O7: C, 57.00; H, 6.46; N, 9.97. Found: C,
57.21; H, 6.46; N, 10.20.
5-Acetylamino-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic
Acid Methyl Ester (12b)
In an argon atmosphere, compound 8 (1.70 g, 6.50 mmol), acetyl
chloride (924 mg, 13.0 mmol), i-Pr2NEt (2.25 mL) and catalytic
amounts of DMAP in anhyd CH2Cl2 were heated to reflux for 3 h
and stirred for 20 h at r.t. The organic layer was washed with aq 1
M HCl, sat. aq NaHCO3, sat. aq NaCl and H2O and dried (Na2SO4).
After filtration, the solvent was concentrated and the remaining res-
idue was purified by chromatography on silica gel column (eluent:
n-pentane–EtOAc, 3:2); Rf 0.31; yield: 1.33 g (68%); mp 138 °C.
1H NMR (300 MHz, CDCl3): = 2.07 (s, 3 H), 3.70 (s, 3 H), 3.79 (s,
3 H), 5.51 (s, 2 H), 6.74–7.19 (m, 5 H), 7.74 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 24.0, 52.1, 54.0, 55.4, 102.6, 114.1,
129.2, 132.2, 145.8, 159.4, 167.5.
5-Amino-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic Acid
Methyl Ester (8)
Compound 6 (3.41 g, 11.7 mmol) was hydrogenated according to
the general procedure C. Purification was done by chromatography
on a silica gel column (eluent: n-pentane–EtOAc, 3:1); Rf 0.19;
yield: 2.28 g (75%); pale yellow solid; mp 93 °C; mixture of two re-
gioisomers.
1H NMR (300 MHz, CDCl3): (major isomer) = 3.79 (s, 3 H), 3.85
(s, 3 H), 5.52 (s, 2 H), 6.19 (s, 1 H) 6.82–7.29 (m, 4 H); (minor
isomer) = 3.70 (s, 3 H), 3.82 (s, 3 H), 5.15 (s, 2 H), 6.00 (s, 1 H)
6.76–7.21 (m, 4 H).
13C NMR (75 MHz, CDCl3): (major isomer) = 51.9, 53.6, 55.3,
98.3, 114.0, 129.0, 129.8, 132.5, 153.0, 159.2, 160.2; (minor iso-
mer) = 52.0, 52.4, 55.4, 94.3, 114.5, 127.4, 128.5, 141.8, 145.6,
159.6, 163.2.
MS (EI): m/z (%) = 303 (M+, 26), 121 (PMB+, 100).
Anal. Calcd for C15H17N3O4: C, 59.40; H, 5.65; N, 13.85. Found:
C, 59.06; H, 5.46; N, 13.77.
MS (EI): m/z (%) = 262 (M+ + H), 261 (M+), 121 (PMB+).
Saponification of N-(p-Methoxybenzyl)pyrazole Carboxylic
Acid Methyl Esters; General Procedure D
Anal. Calcd for C11H15N3O3: C, 59.76; H, 5.79; N, 16.08; found: C,
59.57; H, 5.53; N, 16.12.
In a round bottom flask, the N-(p-methoxybenzyl)pyrazole carbox-
ylic acid methyl ester was dissolved in a mixture of MeOH (50 mL/
15 mmol) and THF (50 mL/15 mmol). To this solution, was added
H2O (10 mL/15 mmol) and LiOH (1.10 equiv) and the mixture was
stirred until the starting material had disappeared on the TLC. The
solution was concentrated in vacuo; the residue was then dissolved
in H2O and acidified with aq 1 M HCl. The precipitate was filtered,
washed with aq 1 M HCl and dried in vacuo.
5-{[5-Amino-2-(4-methoxybenzyl)-2H-pyrazole-3-carbonyl]-
amino}-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic Acid
Methyl Ester (11)
Compound 9a (7.20 g, 13.8 mmol) was hydrogenated according to
the general procedure C. Purification was done by chromatography
on a silica gel column (eluent: CH2Cl2–MeOH, 30:1); Rf 0.19;
yield: 3.92 g (58%); yellow solid.
1H NMR (300 MHz, CDCl3): = 3.7 (s, 3 H), 3.75 (s, 3 H), 3.87 (s,
3 H), 5.50–5.61 (m, 4 H), 6.76–6.82 (m, 4 H), 7.18–7.33 (m, 6 H),
8.38 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 52.1, 53.2, 53.9, 55.1, 55.2, 94.4,
102.9, 113.8, 113.9, 128.8, 129.0, 129.1, 129.8, 132.1, 135.0, 145.4,
153.1, 157.0, 158.9, 159.2, 159.9.
2-(4-Methoxybenzyl)-5-nitro-2H-pyrazole-3-carboxylic Acid
(7)
Compound 6 (4.05 g, 16.2 mmol) and LiOH (421 mg, 17.8 mmol)
were reacted for 16 h according to the general procedure D; howev-
er, the reaction mixture was refluxed; yield: 4.14 g (92%); colorless
solid; mp 139 °C. Mixture of two regioisomers.
1H NMR (400 MHz, DMSO-d6): (major isomer) = 3.80 (s, 3 H),
5.53 (s, 2 H).
MS (EI): m/z (%) = 490 (M+, 19), 369 (M+ – PMB+, 100), 121
(PMB+, 61).
13C NMR (75 MHz, CDCl3): (major isomer) = 54.9, 55.8, 107.1,
114.1, 127.7, 129.3, 135.7, 153.6, 159.2, 161.4.
MS (EI): m/z (%) = 277 (M+, 29), 121 (PMB+, 121).
HRMS: m/z calcd for C25H26N6O5: 490.1965; found: 490.1975.
Anal. Calcd for C25H26N6O5: C, 61.22; H, 5.34; N, 17.13. Found: C,
61.19; H, 5.45; N, 16.57.
Anal. Calcd for C12H11N3O5: C, 51.99; H, 4.00; N, 15.16. Found: C,
51.45; H, 3.91; N, 14.78.
2-(4-Methoxybenzyl)-5-{2-[2-(2-methoxyethoxy)ethoxy]acetyl-
amino}-2H-pyrazole-3-carboxylic Methyl Ester (12a)
In an argon atmosphere, compound 8 (1.85 g, 7.08 mmol), 2-[2-(2-
methoxyethoxy)ethoxy]acetic acid (2.52 g, 7.08 mmol), i-Pr2NEt
(5.49 g, 42.5 mmol) and 2-chloro-1-methylpyridinium chloride
(15.6 mmol, 2.20 equiv) were reacted in anhyd CH2Cl2 (100 mL) for
2-(4-Methoxybenzyl)-5-{[2-(4-methoxybenzyl)-5-nitro-2H-
pyrazole-3-carbonyl]amino}-2H-pyrazole-3-carboxylic Acid
(10)
Compound 9a (5.20 g, 10 mmol) and LiOH (260 mg, 11.0 mmol)
were reacted according to the general procedure D, but the reaction
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

PAPER
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
1823
mixture was refluxed for 16 h; yield: 4.45 g (88%); colorless solid;
mp 214 °C.
1H NMR (400 MHz, DMSO-d6): = 3.69 (s, 3 H), 3.70 (s, 3 H), 5.61
(s, 2 H), 5.78 (s, 2 H), 6.86–6.92, 7.13–7.27 (m, 9 H), 7.96 (s, 1 H),
11.56 (s, 1 H).
13C NMR (125 MHz, CDCl3): = 52.2, 54.1, 54.2, 55.4, 56.1, 98.8,
102.8, 103.4, 114.1, 114.2, 114.3, 127.2, 128.7, 128.8, 129.3, 129.4,
129.6, 130.1, 132.5, 134.9, 136.3, 144.6, 145.0, 154.2, 155.1, 156.3,
159.5, 159.6, 159.9, 160.0.
MS (ESI): m/z (%) = 749 (M+, 100).
13C NMR (75 MHz, DMSO-d6): = 52.9, 54.8, 54.9, 55.6, 102.6,
104.7, 106.7, 113.8, 126.7, 128.4, 128.5, 128,8, 132.7, 136.6, 143.5,
153.3, 155.4, 158.6, 158.8, 159.9.
HRMS: m/z calcd for C37H35KN9O9: 788.2195; found: 788.2190.
2-(4-Methoxybenzyl)-5-({2-(4-methoxybenzyl)-5-[(2-(4-meth-
oxybenzyl)-5-{[2-(4-methoxybenzyl)-5-nitro-2H-pyrazole-3-
carbonyl]amino}-2H-pyrazole-3-carbonyl)amino]-2H-pyra-
zole-3-carbonyl}amino)-2H-pyrazole-3-carboxylic Acid Methyl
Ester (9c)
MS (FD): m/z (%) = 506 (M+, 100).
Anal. Calcd for C24H22N6O7: C, 56.92; H, 4.38; N, 16.59. Found: C,
57.08; H, 4.45; N, 16.10.
Compounds 10 (418 mg, 826 mol) and 11 (405 mg, 826 mol)
were coupled according to the general procedure E. Purification by
chromatography on silica gel column (eluent: EtOAc–n-pentane,
2:1); Rf 0.89; yield: 412 mg (51%); pale yellow solid. Mixture of
several regioisomers.
1H NMR (400 MHz, CDCl3): (major isomer) = 3.58–3.68 (4 s, 12
H), 3.78 (s, 3 H), 5.49–5.66 (4 s, 8 H), 6.72–7.70 (m, 20 H), 8.41–
8.57 (3 s, 3 H).
13C NMR (75 MHz, CDCl3): = 52.2, 54.0, 55.3, 55.9, 56.0, 56.8,
61.5, 62.2, 99.1, 102.8, 103.7, 107.6, 126.1–160.0 (arom. carbons).
MS (MALDI): m/z (%) = 1002 (M+ + Na).
2-(4-Methoxybenzyl)-5-{2-[2-(2-methoxyethoxy)ethoxy]acetyl-
amino}-2H-pyrazole-3-carboxylic Acid (13a)
Compound 12a (1.52 g, 3.61 mmol) and LiOH (95.0 mg,
3.97 mmol) were reacted according to the general procedure D for
16 h; yield: 1.15 g (78%); colorless solid; mp 96 °C.
1H NMR (300 MHz, CDCl3): = 3.27 (s, 3 H), 3.51–3.62 (m, 8 H)
3.69 (s, 3 H), 4.08 (s, 2 H), 5.53 (s, 2 H), 6.73–7.12 (m, 4 H), 7.33
(s, 1 H), 9.23 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 54.2, 55.4, 59.1, 70.4, 70.6, 70.8,
71.5, 72.0, 104.0, 114.1, 129.1, 131.6, 145.3, 159.4, 162.1, 168.4.
MS (EI): m/z (%) = 408 (M+ + H, 4), 407 (M+, 16), 121 (PMB+,
100).
HRMS: m/z calcd for C49H46N12NaO11: 1001.3307; found:
1001.3322.
HRMS: m/z calcd for C19H25N3O7: 490.1965; found: 490.1975.
Coupling of Two Pyrazole Building Blocks; General Procedure
F
Anal. Calcd for C19H25N3O7: C, 56.01; H, 6.18; N, 10.31. Found: C,
55.71; H, 5.97; N, 10.29.
In an argon atmosphere, the pyrazole amino compound (1.00
equiv), the pyrazole carboxylic acid compound (1.10 equiv), 2-
chloro-1-methylpyridinium iodide (1.50 equiv) and i-Pr2NEt (3.00
equiv) were stirred at r.t. for 3 d. After washing the organic layers
with aq 1 M HCl, sat. aq NaHCO3, H2O and sat. aq NaCl (30 mL)
and drying (Na2SO4), the solvent was evaporated to dryness.
5-Acetylamino-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic
Acid (13b)
Compound 12b (1.21 g, 4.00 mmol) and excess of LiOH (155 mg,
6.00 mmol) were reacted according to the general procedure D. Re-
action time: 3 d; yield: 1.09 g (95%); colorless solid; mp 197 °C.
1H NMR (300 MHz, DMSO-d6): = 1.98 (s, 3 H), 3.70 (s, 3 H), 5.53
(s, 2 H), 6.82–6.88 (m, 2 H), 7.05 (s, 1 H), 7.19–7.37 (m, 2 H), 10.77
(s, 1 H), 13.37 (br s, 1 H).
13C NMR (75 MHz, DMSO-d6): = 23.0, 52.8, 55.0, 101.7, 113.8,
128.7, 129.5, 132.3, 146.2, 158.6, 160.4, 167.5.
2-(4-Methoxybenzyl)-5-{[2-(4-methoxybenzyl)-5-nitro-2H-
pyrazole-3-carbonyl]amino}-2H-pyrazole-3-carboxylic Acid
Methyl Ester (9a)
Compounds 7 (4.67 g, 16.8 mmol) and 8 (4.00 g, 15.3 mmol) were
coupled according to the general procedure F; yield: 7.90 g (88%);
colorless solid; mp 69 °C.
1H NMR (300 MHz, CDCl3): = 3.63 (s, 3 H), 3.66 (s, 3 H), 3.78
(s, 3 H), 5.33 (s, 2 H), 5.69 (s, 2 H), 6.64–6.79 (m, 4 H), 7.01–7.26
(m, 6 H), 9.18 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 52.1, 53.9, 55.1, 55.2, 55.7, 103.0,
103.7, 106.4, 107.6, 113.8, 113.9, 114.0, 114.3, 126.3, 127.3, 128.6,
129.0, 129.5, 130.0, 132.2, 134.7, 144.7, 146.2, 153.8, 155.2, 158.6,
159.2, 160.5, 171.4.
MS (EI): m/z (%) = 289 (M+, 25), 121 (PMB+, 100).
HRMS: m/z calcd for C14H15N3O4: 289.1063; found: 289.1055.
Coupling of Two Pyrazole Building Blocks; General Procedure
E
In an argon atmosphere, the pyrazole amino compound (1.00
equiv), the pyrazole carboxylic acid (1.00 equiv), PyClop (1.30
equiv) and (i-Pr)2NEt (4.00 equiv) were refluxed in anhyd CHCl3
for 45 h. Then the solvent was evaporated and the residue was puri-
fied by chromatography on a silica gel column (eluent: n-pentane–
EtOAc).
MS (EI): m/z (%) = 520 (M+, 27), 399 (M+ – PMB, 36), 121 (PMB+,
100).
Anal. Calcd for C25H24N6O7: C, 57.69; H, 4.65; N, 16.15. Found: C,
57.78; H, 4.78; N, 16.17.
1-(4-Methoxybenzyl)-5-{2-[(4-methoxybenzyl)-5-{[1-(4-meth-
oxybenzyl)-5-nitro-2H-pyrazole-3-carbonyl]pyrazol-3-carbon-
yl]amino}-2H-pyrazole-3-carbonylamino]-2H-pyrazole-3-
carboxylic Acid Methyl Ester (9b)
Compounds 7 (280 mg, 1.00 mmol) and 11 (490 mg, 1.00 mmol)
were coupled according to the general procedure E and purified by
chromatography on a silica gel column (eluent: EtOAc–n-pentane,
2:1); Rf 0.90; yield: 632 mg (83%); pale yellow solid.
1H NMR (400 MHz, CDCl3): = 3.77 (s, 6 H), 3.80 (s, 3 H), 3.91
(s, 3 H), 5.64 (s, 2 H), 5.65 (s, 2 H), 5.80 (s, 2 H), 6.80–6.87 (m, 6
H), 7.81 (s, 1 H), 7.24–7.40 (m, 8 H), 8.54 (s, 1 H), 8.56 (s, 1 H).
2-(4-Methoxybenzyl)-5-[(2-(4-methoxybenzyl)-5-{2-[2-(2-meth-
oxyethoxy)ethoxy]acetylamino}-2H-pyrazole-3-carbonyl)ami-
no]-2H-pyrazole-3-carboxylic Acid Methyl Ester (9d)
Compounds 13a (1.30 g, 3.21 mmol) and 8 (760 mg, 2.92 mmol)
were coupled according to the general procedure F and purified by
chromatography on a silica gel column (eluent: EtOAc); Rf 0.23;
yield: 1.25 g (66%); colorless solid; mp 102 °C.
1H NMR (300 MHz, CDCl3): = 3.27 (s, 3 H), 3.79–3.87 (m, 8 H)
3.95 (s, 3 H), 3.96 (s, 3 H), 4.06 (s, 3 H), 4.33 (s, 2 H), 5.79 (s, 2 H),
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

1824
P. Rzepecki et al.
PAPER
5.86 (s, 2 H), 7.01–7.04 (m, 4 H), 7.41–7.46 (m, 4 H), 7.54 (s, 1 H),
7.56 (s, 1 H), 8.97 (s, 1 H), 9.23 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 52.1, 54.0, 54.1, 55.4, 59.1, 70.6,
70.4, 70.8, 71.5, 72.0, 98.9, 102.9, 114.1, 129.0, 129.4, 132.1,
134.6, 145.3, 145.3, 156.8, 159.3, 159.4, 160.1, 168.4.
13C NMR (75 MHz, CDCl3): = 14.1, 14.2, 20.4, 22.7, 25.5, 31.7,
31.9, 37.3, 39.7, 54.0, 55.5, 55.61, 98.4, 98.5, 99.41, 114.1, 114.2,
114.3, 129.3, 129.6, 129.71, 129.7, 135.0, 136.0, 145.3, 146.2,
157.3, 159.5, 159.9, 171.6.
MS (EI): m/z (%) = 629 (M+, 6), 121 (PMB+, 100).
MS (ESI): m/z (%) = 651 (M+ + H, 10), 650 (M+, 30), 529 (M+ –
PMB, 69).
Anal. Calcd for C34H43N7O5: C, 64.85; H, 6.88; N, 15.57. Found: C,
64.26; H, 6.88; N, 15.28.
HRMS: m/z calcd for C32H38N6O9: 650.2700; found: 650.2705.
4-Iodo-5-nitro-2H-pyrazole-3-carboxylic Acid Methyl Ester
(19)
5-{[5-Acetylamino-2-(4-methoxybenzyl)-2H-pyrazole-3-carbo-
nyl]amino}-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic
acid Methyl Ester (9e)
Compounds 13b (650 mg, 2.25 mg) and 8 (646 mg, 2.47 mmol)
were coupled according to the general procedure F and purified by
chromatography on a silica gel column (eluent: EtOAc–n-pentane,
1:1); Rf 0.10; yield: 553 mg (46%); colorless crystals; mp 98 °C.
1H NMR (300 MHz, CDCl3): = 2.15 (s, 3 H), 3.79 (s, 3 H), 3.81
(s, 3 H), 3.91 (s, 3 H), 5.64 (s, 2 H), 5.66 (s, 2 H), 6.82–6.88 (m, 4
H), 7.24–7.37 (m, 6 H), 7.93 (s, 1 H), 8.68 (s, 1 H).
13C NMR (75 MHz, CDCl3): = 24.0, 52.2, 52.4, 53.9, 54.1, 55.4,
102.9, 114.1, 114.3, 114.7, 128.7, 129.0, 129.2, 129.4, 132.3, 134.7,
145.1, 145.8, 156.7, 159.4, 159.5, 160.1, 167.8.
In an argon atmosphere, compound 5 (5.31 g, 30 mmol), iodine
(9.14 g, 36 mmol) and ceric ammonium nitrate (19.7 g, 36 mmol) in
anhyd MeCN were refluxed for 72 h. The solvent was evaporated
and the residue was dissolved in EtOAc. The organic layer was
washed with sat. aq NaHCO3 and sat. aq NaCl and dried (Na2SO4).
After filtration, the solution was concentrated and purified by chro-
matography on a silica gel column (eluent: EtOAc–n-pentane, 1:2);
yield: 7.72 g (87%); colorless solid; mp 165 °C.
1H NMR (200 MHz, DMSO-d6): = 3.90 (s, 3 H).
13C NMR (50 MHz, DMSO-d6): = 53.8, 63.2, 138.4, 158.0, 159.0.
MS (EI): m/z (%) = 296 (M+, 100).
4-Iodo-2-(4-methoxybenzyl)-5-nitro-2H-pyrazole-3-carboxylic
Acid Methyl Ester (20)
MS (EI): m/z (%) = 532 (M+), 411 (M+ – PMB).
HRMS (ESI): m/z calcd for C27H28N6NaO: 555.1968; found:
555.1958.
In an argon atmosphere, compound 19 (594 mg, 2.00 mmol), K2CO3
(297 mg, 3.00 mmol) and 4-methoxybenzyl bromide (483 mg, 2.40
mmol) were reacted in anhyd DMF for 3 h at 50 °C. The solvent was
evaporated and the resulting yellow oil was purified by chromatog-
raphy on a silica gel column (eluent: EtOAc–n-pentane, 1:5);
Rf 0.24; yield: 808 mg (97%); colorless solid; mp 110 °C.
5-{[5-Hexanoylamino-2-(4-methoxybenzyl)-2H-pyrazole-3-car-
bonyl]amino}-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic
Acid Methyl Ester (15)
In an argon atmosphere, compound 11 (120 mg, 245 mol) was dis-
solved in anhyd THF. Hexanoic acid chloride (33 L, 245 mol)
and (i-Pr)2NEt (65 L, 367 mol) were addded. The reaction mix-
ture was stirred at r.t. for 16 h. The solvent was concentrated and the
residue was purified by chromatography on a silica gel column (elu-
ent: EtOAc–n-pentane 1:1); Rf 0.72; yield: 141 mg (98%); colorless
solid.
1H NMR (300 MHz, CDCl3): = 1.03 (t, J = 6.8 Hz, 3 H), 1.42–
1.45 (m, 4 H), 1.77–1.82 (m, 2 H), 2.44 (t, J = 7.5 Hz, 2 H), 3.91 (s,
3 H), 3.94 (s, 3 H), 4.05 (s, 3 H), 5.77 (s, 2 H), 5.80 (s, 2 H), 6.94–
7.41 (m, 8 H), 7.54 (d, J = 6.1 Hz, 2 H), 8.52 (s, 1 H), 9.38 (s, 1 H).
1H NMR (300 MHz, CDCl3): = 3.94 (s, 3 H), 4.12 (s, 3 H), 5.92
(s, 2 H), 6.99 (d, J = 8.4 Hz, 2 H), 7.43 (d, J = 8.4 Hz, 2 H).
13C NMR (75 MHz, CDCl3): = 52.6, 55.2, 57.5, 58.1, 114.1,
129.3, 129.6, 136.8, 158.2, 159.9.
MS (EI): m/z (%) = 417 (M+, 30).
HRMS: m/z calcd for C13H12IKN3O5: 455.9459; found: 455.9442.
4-Hex-1-ynyl-1-(4-methoxybenzyl)-5-nitro-1H-pyrazole-3-car-
boxylic Acid Methyl Ester (21)
In an argon atmosphere, compound 20 (208 mg, 500 mol) and
Et3N were dissolved in 1,4-dioxane. The mixture was warmed gen-
tly until all components had dissolved. Subsequently bis(triphe-
nylphosphino)palladium dichloride (20 mg) and CuI (10 mg) were
added. The mixture was heated to 70 °C and a solution of hex-1-yne
(65 L, 550 mol) in 1,4-dioxane (1 mL) was added dropwise. The
reaction mixture was stirred for 22 h at 70 °C, and cooled to r.t.
CH2Cl2 was added and the organic layer was washed with aq 2%
HCl and H2O and dried (Na2SO4). The solvent was evaporated and
the residue was purified by chromatography on a silica gel column
(eluent: EtOAc–n-pentane, 1:6); Rf 0.11; yield: 36 mg (20%); col-
orless solid.
1H NMR (300 MHz, CDCl3): = 0.88 (t, J = 7.1 Hz, 3 H), 1.61–
1.34 (m, 4 H), 2.43 (t, J = 6.6 Hz, 2 H), 3.71 (s, 3 H), 3.86 (s, 3 H),
5.66 (s, 2 H), 6.77 (d, J = 8.7 Hz, 2 H), 7.22 (d, J = 8.7 Hz, 2 H).
13C NMR (75 MHz, CDCl3): = 13.7, 19.7, 21.9, 30.4, 52.7, 55.4,
55.9, 56.8, 68.5, 101.8, 107.8, 114.2, 126.9, 129.8, 135.1, 158.7,
159.9.
13C NMR (75 MHz, CDCl3): = 14.2, 22.7, 25.5, 31.7, 37.3, 52.3,
54.0, 54.3, 55.5, 55.6, 99.3, 103.4, 114.2, 114.3, 129.3, 129.5,
129.6, 129.7, 132.3, 1345.0, 145.6, 146.2, 157.2, 159.5, 159.6,
160.3, 171.6.
MS (EI): m/z (%) = 589 (M+ + H, 2), 588 (M+, 5), 467 (M+ – PMB+,
46), 121 (PMB+, 100).
Anal. Calcd for C31H36N6O6: C, 63.25; H, 6.16; N, 14.28. Found: C,
62.99; H, 6.29; N, 14.25.
5-{[5-Hexanoylamino-2-(4-methoxybenzyl)-2H-pyrazole-3-car-
bonyl]amino}-2-(4-methoxybenzyl)-2H-pyrazole-3-carboxylic
Acid Butylamide (16)
In an argon atmosphere, compound 15 (120 mg, 203 mol) was dis-
solved in n-butylamine (15 mL) and MeOH (15 mL) and refluxed
for 5 h. The solvent was evaporated and the residue was purified by
chromatography on a silica gel column (eluent: EtOAc–n-pentane,
1:1); Rf 0.58; yield: 126 mg (98%); colorless solid.
1H NMR (300 MHz, CDCl3): = 0.82 (t, J = 6.8 Hz, 3 H), 0.90 (t,
J = 7.3 Hz, 3 H), 1.21–1.61 (m, 12 H), 2.23 (t, J = 7.4 Hz, 2 H),
3.34 (q, J = 6.8 Hz, 2 H), 3.61 (s, 3 H), 3.63 (s, 3 H) 5.54 (s, 2 H),
5.55 (s, 2 H), 6.31 (t, J = 5.6 Hz, 1 H), 6.71–6.76 (m, 4 H), 7.04 (s,
1 H), 7.13 (d, J = 8.5 Hz, 4 H), 7.19 (d, J = 8.5 Hz, 4 H), 7.27 (s, 1
H), 8.23 (s, 1 H), 9.10 (s, 1 H).
PMB Deprotection; General Procedure G
In an argon atmosphere, the PMB-protected pyrazole compounds
were heated in anhyd TFA (10 mL/1.00 mmol) to 70 °C for 1–16 h.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

PAPER
Aminopyrazole Oligomers for -Sheet Stabilization of Peptides
1825
5-[(5-Nitro-2H-pyrazole-3-carbonyl)amino]-2H-pyrazole-3-
HRMS (ESI): m/z calcd for C16H22N6NaO7: 433.1448; found:
carboxylic Acid Methyl Ester (14a)
433.1458.
Compound 9a (260 mg, 500 mol) was reacted for 16 h according
to the general deprotection procedure G. The solvent was evaporat-
ed, and the remaining brown solid was washed with n-pentane–
Et2O (1:1), sat. aq NaHCO3 and H2O, and dried in vacuo over P4O10;
yield: 105 mg (75%); colorless solid; mp 213 °C.
1H NMR (300 MHz, DMSO-d6): = 3.86 (s, 3 H), 7.11 (s, 1 H),
7.93 (s, 1 H), 11.54 (s, 1 H), 11.26 (s, 1 H), 13.85 (s, 1 H), 15.02 (s,
2 H).
5-[(5-Acetylamino-2H-pyrazole-3-carbonyl)amino]-2H-pyra-
zole-3-carboxylic Acid Methyl Ester Trifluoracetate Salt (14e)
Compound 9e (100 mg, 187 mol) was reacted according to the
general deprotection procedure G. After adding cold Et2O, the prod-
uct precipitated. The precipitate was filtered, washed with Et2O and
dried in vacuo; Yield: 49 mg (90%); colorless solid.
1H NMR (300 MHz, DMSO-d6): = 2.03 (s, 3 H), 3.83 (s, 3 H), 6.98
(s, 1 H), 7.28 (s, 1 H), 10.51 (s, 1 H), 11.04 (s, 1 H).
13C NMR (75 MHz, DMSO-d6): = 23.4, 52.2, 97.1, 99.8, 135.2,
137.6, 145.6, 146.7, 157.5, 160.3, 167.9.
13C NMR (75 MHz, DMSO-d6): = 51.93, 99.7, 102.4, 156.7.
MS (EI): m/z (%) = 280 (M+, 48), 281 (M+ + H, 4).
19F NMR (188 MHz, DMSO-d6): = –70.4.
MS (ESI): m/z (%) = 293 (M+ + H, 35).
HRMS (EI): m/z calcd for C9H8N6O5: 280.0556; found: 280.0549.
5-{[5-({5-[(Nitro-2H-pyrazole-3-carbonyl)amino]-2H-pyrazole-
3-carbonyl}amino]-2H-pyrazole-3-carboxylic Acid Methyl Es-
ter (14b)
HRMS (ESI): m/z calcd for C11H12N6O4 + H: 293.0998; found:
293.0994.
Compound 9b (150 mg, 200 mol) was reacted according to the
general deprotection procedure G for 4.5 h. The remaining solid
was washed with CH2Cl2, H2O and ammonia solution (25%); yield:
39 mg (50%).
1H NMR (300 MHz, DMSO-d6): = 4.02 (s, 3 H), 7.23 (s, 1 H),
7.74 (s, 1 H), 8.11, (s, 1 H), 11.41 (br s, 1 H), 11.61 (s, 1 H), 11.61
(br s, 1 H), 13.73 (br s, 2 H), 15.15 (br s, 1 H).
13C NMR (125 MHz, DMSO-d6): = 52.0, 98.3, 96.8, 102.3, 132.8,
136.1, 146.5, 147.2, 154.7, 155.4, 156.5, 157.3, 159.2.
MS (FD): m/z (%) = 389 (M+, 25) 388 (M+ – H, 100).
5-[(5-Hexanoylamino-2H-pyrazole-3-carbonyl)amino]-2H-
pyrazole-3-carboxylic Acid Butyl Amide (17)
Compound 16 (120 mg, 190 mol) was reacted according to the
general deprotection procedure G. The solvent was evaporated, and
the residue was washed with a mixture of Et2O–n-pentane; yield: 64
mg (86%); colorless solid.
1H NMR (300 MHz, DMSO-d6): = 0.82–0.89 (m, 6 H), 1.25–1.57
(m, 10 H), 2.29 (t, J = 7.4 Hz, 2 H), 3.19–3.31 (m, 2 H), 7.19 (s, 1
H), 7.38 (s, 1 H), 8.47 (br s, 1 H), 10.42 (s, 1 H), 10.93 (s, 1 H),
13.10 (br s, 2 H).
HRMS (ESI): m/z calcd for C13H11N9O6: 390.0911, found:
390.0956.
Due to its low solubility and the strong quadrupole moment of the
nitrogen nuclei no 13C NMR spectrum could be obtained.
MS (ESI): m/z (%) = 390 (M+).
5-{[5-({5-[(5-Nitro-2H-pyrazole-3-carbonyl)amino]-2H-pyra-
zole-3-carbonyl}amino)-2H-pyrazole-3-carbonyl]amino}-2H-
pyrazole-3-carboxylic Acid Methyl Ester (14c)
HRMS (ESI): m/z calcd for C18H27N7O3 + H: 390.2254; found:
390.2296.
Compound 9c (103 mg) was reacted according to the general depro-
tection procedure G for 16 h. The remaining solid was washed with
CH2Cl2, H2O and aq ammonia (25%); yield: 35%.
5-[(5-Nitro-2H-pyrazole-3-carbonyl)amino]-2H-pyrazole-3
carboxylic Acid Butylamide (18)
In an argon atmosphere, compound 14a (120 mg, 203 mol) was
dissolved in n-butylamine (15 mL) and MeOH (15 mL) and re-
fluxed for 5 h. The solvent was evaporated and the residue was pur-
ified by chromatography over silica gel (CH2Cl2–MeOH, 5:1);
Rf 0.07; yield: 60 mg (88%); colorless solid; mp 294 °C.
1H NMR (400 MHz, DMSO-d6): (T = 380 K) = 3.89 (s, 3 H), 6.95
(s, 1 H), 7.12 (s, 1 H), 7.17(s, 1 H), 7.55 (s, 1H); additional signals
at T = 300 K: 11.27 (br s, 3 H), 11.54 (s, 1 H), 13.9 (s, 1 H), 15.02
(s, 1 H).
Due to its low solubility and the strong quadrupole moment of the
nitrogen nuclei no 13C NMR spectrum could be obtained.
MS (ESI): m/z (%) = 497 (M+ – H).
H NMR (300 MHz, DMSO-d6): = 0.90 (t, J = 6.9 Hz, 3 H), 1.29–
1.52 (m, 4 H), 3.25 (m, 2 H), 7.26 (s, 1 H), 7.87 (s, 1 H), 8.55 (br s,
1 H), 11.26 (s, 1 H), 13.26 (s, 1 H).
13C NMR (75 MHz, DMSO-d6): = 14.32, 20.2, 31.7, 38.9, 97.4,
102.9, 137.8, 139.9, 147.0, 156.0, 156.6, 159.0.
HRMS (ESI): m/z calcd for C17H13N12O7: 497.1030; found:
497.0628.
MS (FD): m/z (%) = 322 (M+,100), 321 (M+ + H, 20).
5-[(5-{2-[2-(2-Methoxyethoxy)ethoxy]acetylamino}-2H-pyra-
zole-3-carbonyl)amino]-2H-pyrazole-3-carboxylic Acid Methyl
Ester Trifluoracetate Salt (14d)
HRMS (ESI): m/z calcd for C12H15N7O4: 360.0823; found:
360.0813.
Compound 9d (180 mg, 276 mol) was reacted according to the
general deprotection procedure G. The solvent was evaporated, and
the residue was washed with a mixture of Et2O–n-pentane (1:1);
yield: 111 mg (63%); colorless solid; mp 197 °C.
1H NMR (300 MHz, DMSO-d6): = 3.29 (s, 3 H), 3.34–3.67 (m, 8
H), 3.84 (s, 3 H), 4.10 (s, 2 H), 6.98 (s, 1 H), 7.32 (s, 1 H), 10.13, (s,
1 H), 11.06 (s, 1 H).
13C NMR (125 MHz, DMSO-d6): = 51.8, 58.0, 69.5, 69.6, 69.7,
70.4, 71.2, 160.1, 168.0.
19F NMR (188 MHz, DMSO-d6): = –70.8.
MS (ESI): m/z (%) = 433 (M+, 100).
Computational Methods
Force-field calculations were initially carried out as molecular me-
chanics calculations without solvent. To establish the minimum en-
ergy conformation of the free ligands well as their 1:1 complex with
respective model peptides Monte-Carlo simulations were carried
out in CHCl3 (MacroModel 7.0, Schrödinger Inc., 2000. Force-
field: Amber*). The three best structures for both the free ligands as
well as their 1:1 complexes varied in their total energy by only 10
kJ/mol. The -sheet conformation of model peptides was stabilized
with some of the dimeric (e.g, with 3b, but not with 3f), but with all
of the trimeric and tetrameric aminopyrazoles.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York

1826
P. Rzepecki et al.
PAPER
Mass Spectrometric Measurements
(2) (a) Riesner, D.; Kellings, K.; Post, K.; Wille, H.; Serban, H.;
Baldwin, M.; Groth, D. J. Virol. 1996, 70, 1714.
(b) Pitschke, M.; Prior, R.; Haupt, M.; Riesner, D. Nat. Med.
(NY) 1998, 4, 832.
(3) Schrader, T.; Kirsten, C. Chem. Commun. 1996, 2089.
(4) Schrader, T.; Kirsten, C. N. J. Am. Chem. Soc. 1997, 119,
12061.
(5) Schrader, T.; Riesner, D.; Nagel-Steger, L.; Aschermann,
K.; Kirsten, C.; Rzepecki, P.; Molt, O.; Zadmard, R.;
Wehner, M. DE 102 21 052.7 application, 2002.
(6) Some of Dervan’s minor groove binders contain N-alkylated
pyrazole amino acids: Zhan, Z.-Y. J.; Dervan, P. B. Bioorg.
Med. Chem. 2000, 8, 2467.
EI mass spectra were recorded on a Finnigan MAT CH7A spec-
trometer. ESI mass spectra were recorded on a Finnigan MAT 95
spectrometer. Samples (20 L) were introduced as 10–5 M solutions
in MeOH at flow rates of 20 L min–1. Heated capillary tempera-
ture: 150 °C. Ion spray potential: 3.5 kV (positive ESI), 3.0 kV
(negative ESI). About 20–30 scans were averaged to improve the
signal-to-noise ratio. MALDI-TOF spectra were recorded on a
Bruker Flex III spectrometer, matrix: sinapinic acid.
Variable Temperature Experiments
The ligands 3b and 3f were both measured at millimolar concentra-
tions in solvent mixtures containing 60% (3b) or 6% (3f) of DMSO-
d6 at various temperatures increasing in 5 °C increments from 5 to
75 °C. The temperature dependent change of chemical shifts for the
pyrazole and amidic NH protons was plotted in a conventional V/T
diagram. From its slope the temperature dependence was inferred in
ppb/K.
(7) Robins, R. K.; Furcht, F. W.; Grauer, A. D.; Jones, J. W. J.
Am. Chem Soc. 1956, 78, 2418.
(8) This is a general problem often observed with pyrazole
derivatives: In agreement with the HSAB principle, hard
acids such as acid chlorides preferentially attack the hard
base NH2,while softer acids (such as anhydrides) perform
acylation of the softer ring nitrogen.
NOESY Experiments
Standard 1H NOESY experiments were performed with the host 3b
and 3f on the Bruker 500 MHz NMR spectrometer. Mixing times
varied between 100 and 200 ms. In spite of its high viscosity leading
to enforced spin-lattice relaxation we chose solvent mixtures of
CDCl3 with DMSO-d6, because only in these mixtures the strongly
self-associating ligands could be dissolved at concentrations higher
than 10–4 M. Only medium to strong reciprocal NOE peaks were
chosen for a structural interpretation.
(9) (a) Removal with TFA/anisole or TFA: Subramanyam, C.
Synth. Commun. 1995, 25, 761. (b) With DDQ: Singh, S. B.
Tetrahedron Lett. 1995, 35, 2009.
(10) Coste, J.; Frerot, E.; Jouin, P. J. Org. Chem. 1994, 59, 2437.
(11) Mukaiyama, T. Angew. Chem., Int. Ed. Engl. 1979, 18, 707;
Angew. Chem. 1979, 91, 798.
(12) Nakhle, B. M.; Trammell, S. A.; Sigel, K. M.; Meyer, T. J.;
Erickson, B. W. Tetrahedron 1999, 55, 2835.
(13) Wang, Y.; Inguaggiato, G.; Jasamai, M.; Shah, M.; Hughes,
D.; Slater, M.; Simons, C. Bioorg. Med. Chem. 1999, 7, 481.
(14) Rodriguz-Franco, M. I.; Dorronsoro, I.; Hernandez-
Higueras, A. I.; Antequera, G. Tetrahedron Lett. 2001, 42,
863.
X-Ray Crystallography22
Both crystals were measured using a STOE IPDS2 image plate de-
tector system at 190 K. Crystal structure solution and refinement
were performed using the SHELX97 program system.
(15) Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N.
Synthesis 1980, 627.
3b
C10H12N6O2, 4(CF3OH), M = 704.37, monoclinic, a = 18.180(2),
b = 11.2188(9), c = 14.7656(19) Å, = 112.037(9)°, space group
P21/n, Z = 4. No final refinement has been done due to the poor
crystal quality (weak diffraction, big mosaicity).
(16) The aminopyrazole nucleus is incorporated in Viagra; 3-
aminopyrazoles have been tested as antiinflammatory
agents: Gadad, A. K.; Kittur, B. S.; Kapsi, S. G.;
Mahajanshetti, C. S.; Rajur, S. B. Arzneim.-Forsch. 1996,
46, 1082.
3f
(17) Ribeiro, A. A.; Goodman, M.; Naider, F. Int. J. Peptide
Protein Res. 1979, 14, 414.
(18) For detailed crystallographic information, refer to the
Cambridge Crystallographic Database, where structure 3f
has been deposited with full data.
C15H15N7O2, 2(CH3OH), M = 389.42, monoclinic, a = 13.6837(13),
b = 8.2937(9), c = 16.2244(15) Å, = 94.960(7)°, space group C2/
c, Z = 4, wR2 = 0.1013, R1 = 0.0384.
(19) A similar DAD pattern has been observed in a crystal
structure for a related aminopyrazole derivative: Saweczko,
P.; Enright, G. D.; Kraatz, H. B. Inorg. Chem. 2001, 40,
4409.
(20) Due to the low crystal quality, the crystal structure of 3b has
not been fully refined.
Acknowledgments
This work was supported by the Volkswagen foundation (priority
area conformational control of biomolecular function). We thank
Gerhard Schäfer, Daniel Rübeling, Nina Geib, Christoph Christo-
phis, Radovan Bast, Christian Kleeberg and Nicola Springer for
preparative support.
(21) Professor Dr. D. Riesner, Institut für Physikalische Biologie,
Universität Düsseldorf.
(22) CCDC 214082 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44
1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
References
(1) (a) Wisniewski, T.; Aucouturier, P.; Soto, C.; Frangione, B.
Amyloid 1998, 5, 212. (b) Carrell, R. W.; Lomas, D. A.
Lancet 1997, 350, 134.
Synthesis 2003, No. 12, 1815–1826 © Thieme Stuttgart · New York