New heterocyclic β-sheet ligands with peptidic recognition elements

Article abstract of DOI:10.1021/jo0496603

A detailed and comprehensive overview is presented about the design, modeling, and synthesis, as well as spectroscopic characterization, of a new class of β-sheet ligands. The characteristic feature of these compounds is a peptidic chimeric structure formed from a specific combination of aminopyrazolecarboxylic acids with naturally occurring α-amino acids. These hybrid peptides are designed with the aid of molecular modeling to exist mainly in an extended conformation. All their hydrogen bond donors and acceptors can be aligned at the bottom face in such a way that a perfect complementarity toward β-sheets is obtained. Thus the aminopyrazoles impart rigidity and a highly efficient DAD sequence for the recognition of whole dipeptide fragments, whereas the natural α-amino acids are designed to mimick recognition sites in proteins, ultimately leading to sequence-selective protein recognition. The synthetic protocols either rely upon solution phase peptide coupling with a PMB protecting group strategy or solid-phase peptide coupling based on the Fmoc strategy, using the same protecting group. In solution, a key building block was prepared by catalytic reduction of a nitropyrazolecarboxylic acid precursor. Subsequently, it was (N-1)-protected with a PMB group, and elongated by HCTU- or T3P-assisted peptide coupling with dipeptide fragments, followed by PyClop-assisted coupling with another nitropyrazolecarboxylic acid building block. Final simultaneous deprotection of all PMB groups with hot TFA completed the high-yield protocol, which works racemization-free. After preparing a similar key building block with an Fmoc protection at N-3, we developed a strategy suitable for automated synthesis of larger hybrid ligands on a peptide synthesizer. Attachment of the first amino acid to a polystyrene resin over the Sieber amide linker is followed by an iterative sequence consisting of Fmoc deprotection with piperidine and subsequent coupling with natural α-amino acid via HATU/HOAt. High yields of free hybrid peptides are obtained after mild acidic cleavage from the resin, followed by deprotection of the PMB groups with hot TFA. The new aminopyrazole peptide hybrid compounds were characterized by various spectroscopic measurements including CD spectra, VT, and ROESY NMR experiments. All these accumulated data indicate the absence of any intramolecular hydrogen bonds and strongly support an extended conformation in solution, ideal for docking on to solvent-exposed β-sheets in proteins. Initial results from aggregation tests of pathological proteins with these and related ligands look extremely promising.

Full text of DOI:10.1021/jo0496603

New Heter ocyclic â-Sh eet Liga n d s w ith P ep tid ic Recogn ition
Elem en ts
P. Rzepecki,† H. Gallmeier,‡ N. Geib,† Katarina Cernovska,‡ B. Ko¨nig,*,‡ and T. Schrader*,†
Fachbereich Chemie, Universita¨t Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany, and
Institut fu¨r Organische Chemie, Universita¨t Regensburg, Universita¨tsstrasse 31,
93040 Regensburg, Germany
schradet@mailer.uni-marburg.de
Received March 1, 2004
A detailed and comprehensive overview is presented about the design, modeling, and synthesis, as
well as spectroscopic characterization, of a new class of â-sheet ligands. The characteristic feature
of these compounds is a peptidic chimeric structure formed from a specific combination of
aminopyrazolecarboxylic acids with naturally occurring R-amino acids. These hybrid peptides are
designed with the aid of molecular modeling to exist mainly in an extended conformation. All their
hydrogen bond donors and acceptors can be aligned at the bottom face in such a way that a perfect
complementarity toward â-sheets is obtained. Thus the aminopyrazoles impart rigidity and a highly
efficient DAD sequence for the recognition of whole dipeptide fragments, whereas the natural
R-amino acids are designed to mimick recognition sites in proteins, ultimately leading to sequence-
selective protein recognition. The synthetic protocols either rely upon solution phase peptide coupling
with a PMB protecting group strategy or solid-phase peptide coupling based on the Fmoc strategy,
using the same protecting group. In solution, a key building block was prepared by catalytic
reduction of a nitropyrazolecarboxylic acid precursor. Subsequently, it was (N-1)-protected with a
PMB group, and elongated by HCTU- or T3P-assisted peptide coupling with dipeptide fragments,
followed by PyClop-assisted coupling with another nitropyrazolecarboxylic acid building block. Final
simultaneous deprotection of all PMB groups with hot TFA completed the high-yield protocol, which
works racemization-free. After preparing a similar key building block with an Fmoc protection at
N-3, we developed a strategy suitable for automated synthesis of larger hybrid ligands on a peptide
synthesizer. Attachment of the first amino acid to a polystyrene resin over the Sieber amide linker
is followed by an iterative sequence consisting of Fmoc deprotection with piperidine and subsequent
coupling with natural R-amino acid via HATU/HOAt. High yields of free hybrid peptides are obtained
after mild acidic cleavage from the resin, followed by deprotection of the PMB groups with hot
TFA. The new aminopyrazole peptide hybrid compounds were characterized by various spectroscopic
measurements including CD spectra, VT, and ROESY NMR experiments. All these accumulated
data indicate the absence of any intramolecular hydrogen bonds and strongly support an extended
conformation in solution, ideal for docking on to solvent-exposed â-sheets in proteins. Initial results
from aggregation tests of pathological proteins with these and related ligands look extremely
promising.
In tr od u ction
fore a better understanding of the mechanism of ag-
gregation and the development of possible â-sheet binders
which can slow or even prevent the pathological process
is of high interest from both a mechanistic and a
therapeutic view.2 Despite their abundance and dramatic
progression there is virtually no therapy for protein
misfolding diseases such as BSE/Creutzfeldt-J akob or
Alzheimer’s. One of the most promising approaches aims
at the prevention of amyloid aggregation by designed
â-sheet ligands;3 this process is today considered the basic
A series of pathological processes is connected with the
formation of a â-sheet structure and consecutive protein
aggregation in the form of â-amyloid deposition. Those
amyloids are discussed as the primary cause for Alzhe-
imer’s disease; the conversion of R-helices to larger
â-sheet aggregates is also found with Creutzfeldt-J akob
disease, BSE, and other protein folding diseases.1 There-
† Universita¨t Marburg. Phone: +49-6421-2825544.
‡ Universita¨t Regensburg.
(1) (a) Wisniewski, T.; Aucouturier, P.; Soto, C.; Frangione, B.
Amyloid: Int. J . Exp. Clin. Invest. 1998, 5, 212. (b) Carrell, R. W.;
Lomas, D. A. Lancet 1997, 350, 134-138.
(2) (a) Lansbury, P. T., J r. Acc. Chem. Res. 1996, 29, 317-321. (b)
Riesner, D.; Kellings, K.; Post, K.; Wille, H.; Serban, M.; Baldwin, D.;
Groth, D. J . Virol. 1996, 70, 1714-1722. (c) Pitschke, M.; Prior, R.;
Haupt, M.; Riesner, D. Nat. Med. 1998, 4, 832-834.
10.1021/jo0496603 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/02/2004
5168
J . Org. Chem. 2004, 69, 5168-5178

New â-Sheet Ligands with Peptidic Recognition Elements
F IGURE 1. Capping the â-sheets of critical proteins with dimeric aminopyrazoles. Left: Geometrical constraints for â-sheet
ligands with a DAD pattern. Right: Schematic illustrating the interaction of the new ligands with solvent-exposed â-sheets on
the prion protein.8
F IGURE 2. Left: Attachment of a dipeptide at the C-terminus of a dimeric aminopyrazole leads to an enhanced hydrogen bond
network with a hexapeptide. Right: Monte Carlo simulation of a complex of a tetrameric ligand carrying two amino acids between
N- and C-terminal aminopyrazoles with a hexapeptide (VKLVFF) taken from the putative nucleation site of Aâ (MacroModel 7.2,
Schro¨dinger 2002, Amber*, water, 3000 steps).
pathological event in Alzheimer’s disease and is also
discussed as the major cause for related diseases.4
Several years ago, we introduced acylated 3-aminopy-
razoles as effective â-sheet ligands, which are able to
stabilize an extended conformation in small peptides.5
However, the monomeric ligands turned out to be biologi-
cally inactive toward recombinant prion protein in simple
aggregation assays. By a very straightforward synthetic
protocol, we obtained dimeric and oligomeric covalently
fused aminopyrazole ligands, which were fixed in a
planar arrangement and retained the essential DAD
hydrogen bond Donor and Acceptor pattern necessary for
complex formation with peptides in their â-sheet confor-
mation. These dimeric ligands gave very promising
preliminary results in biophysical experiments with the
prion protein and Aâ, where the aggregation kinetics
were monitored by Fluorescence Correlation Spectroscopy
(FCS) and density gradient centrifugation (Figure 1).6 A
very urgent question, however, remained unanswered,
as to how the pathologic proteins could be selectively
addressed, without harming other well-behaved native
proteins. This left us with the task of building into our
ligand structure a recognition element for a certain
protein. With Alzheimer’s disease, the general strategy
seemed obvious: in the future, we have to incorporate
in our ligands at least a truncated version of one of the
self-complementary pentapeptide sequences thought to
be the nucleation sites in Alzheimer’s aggregation: KLVFF
in the middle or VGGVV at the C-terminus.7 As an
additional advantage, the notorious solubility problem,
connected with the high tendency of aminopyrazoles to
undergo self-association, might be circumvented by in-
troducing amino acids into the ligand structure, which
cannot form π-stacks. To this end, we developed new
synthetic protocols for the regioselective incorporation of
proteinogenic (preferentially hydrophobic) amino acids
into the sequence of oligomeric aminopyrazoles. These
protocols rely either upon solution-phase peptide coupling
with a special protecting group strategy or solid-phase
peptide coupling based on the Fmoc strategy, using the
same protecting group. Herein we disclose the synthetic
strategies employed in a flexible general access to the
new class of peptide aminopyrazole hybrid compounds.
Results from biophysical and cell culture experiments will
be reported elsewhere.
(3) Another important approach is the immune-mediated Aâ-clear-
ance by active or passive immunization: Solomon, B. Curr. Med. Chem.
2002, 9, 1737-1750.
Mod elin g. Extensive modeling experiments including
(4) Selkoe, D. J . Science 1997, 275, 630-631.
Monte Carlo simulations in water9 suggest that a perfect
(5) (a) Schrader, T.; Kirsten, C. J . Chem. Soc., Chem. Commun.
1996, 2089. (b) Schrader, T.; Kirsten, C. N. J . Am. Chem. Soc. 1997,
119, 12061-12068.
(7) (a) Tjernberg, L. O.; Na¨slund, J .; Lindqvist, F.; J ohansson, J .;
Karlstro¨m, A. R.; Thyberg, J .; Terenius, L.; Nordstedt, C. J . Biol. Chem.
1996, 271, 8545. (b) Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar,
R. A.; Castano, E. M.; Frangione, B. Nat. Med. 1998, 4, 822-826.
(8) Scrapie protein model: Mestel, R. Science 1996, 273, 184-189.
(6) (a) Schrader, T.; Riesner, D.; Nagel-Steger, L.; Aschermann, K.;
Kirsten, C.; Rzepecki, P.; Molt, O.; Zadmard, R.; Wehner, M. Patent
application DE 102 21 052.7, 5/10/2002. (b) Rzepecki, P.; Wehner, M.;
Molt, O.; Zadmard, R.; Schrader, T. Synthesis 2003, 1815-1826.
J . Org. Chem, Vol. 69, No. 16, 2004 5169

Rzepecki et al.
SCHEME 1. P r ep a r a tion of th e New P yr a zole 3-Am in o Acid by a Cycloa d d ition Rou te (left) a n d Cr ysta l
Str u ctu r e of 2 (r igh t)a
a
Reagents and conditions: (i) CHCl3/EAHCLsatd, rt, 39%; (ii) CH3CN, NEt3, rt, 98%; (iii) Fmoc-Gly-Cl, THF, 70 °C; (iv) 20% piperidine,
DMF; (v) Fmoc-Cl, THF, 70 °C, 96%; (vi) HCl, HOAc, EtOH; (vii) SOCl2, DMF.
SCHEME 2. Exa m p les for Syn th esized P yr a zole Bu ild in g Block sa
a
Reagents and conditions: (i) HCl, MeOH, ∆, quant.; (ii) PMB-Br, K2CO3, DMF, ∆.
â-sheet complementarity can be retained not only in
hybrid compounds with successive aminopyrazole and
peptide blocks but also in those with an even number of
amino acids between two aminopyrazole moieties (Figure
2).
nitrogen.11 Reaction of this building block with the acid
chloride of Fmoc-protected glycine led to peptide coupling
with concomitant urethane cleavage at the pyrazole
nucleus (3a ). Fmoc deprotection with piperidine yielded
3b; a second coupling step can subsequently be performed
with Fmoc-protected glycine acid chloride to give 4a ,
which affords, after final Fmoc removal, the diglycylpyra-
zole trimer 4b (Scheme 4). Unfortunately, its reaction
with the acid chloride of Fmoc-protected aminopyrazole
carboxylic acid 5 did not give the desired tetramer, but
rather transferred the Fmoc group on to the N-terminal
glycine of the trimer. Further attempts to effect C-
terminal peptide coupling of the new pyrazole-based
amino acid were hampered by competing acylation of the
softer ring nitrogen, so that a special protecting group
strategy became inevitable.
Syn th esis: (a ) Syn th esis of th e New Am in o Acid
by Cycloa d d ition of a Hyd r a zid e w ith a â-Cya n op y-
r u va te. Condensation of acetonitrile’s potassium salt
with diethyl oxalate in ethanol/diethyl ether leads in
quantitative yield to the potassium salt of ethyl cyan-
opyruvate (Scheme 1).10 This is reacted with hydrazi-
nomethylformate in a mixture of chloroform and ethyl
acetate, saturated with hydrogen chloride. In situ pro-
tonation of the potassium salt is followed by hydrazone
formation of the liberated R-keto group (1, 39%). Subse-
quent treatment with triethylamine in acetonitrile trig-
gers base-catalyzed cyclization of the intermediate and
furnishes the new pyrazole amino acid ester with a
carbamate protecting group on pyrazole’s N - 2 (2, 98%).
This key compound could be characterized by an X-ray
crystal structure confirming its 3,5-substitution pattern
as well as the location of the urethane at the ring
(b) Solu tion -P h a se Elon ga tion w ith P MB P r otec-
tion . In an alternative strategy, the new heterocyclic
amino acid was made accessible by way of catalytic
reduction of its nitro predecessor 6. The introduction of
the p-methoxybenzyl (PMB) protecting group at N-2
(11) For detailed crystallographic information, refer to the Cam-
bridge Crystallographic Database, where structure 2 has been depos-
ited with full data. Crystal structures of related aminopyrazole
derivatives: Saweczko, P.; Enright, G. D.; Kraatz, H. B. Inorg. Chem.
2001, 40, 4409-4419.
(9) MacroModel 7.2, Schro¨dinger 2002, Amber*, water, 3000 steps.
(10) Borsche, W.; Manteuffel, R. Liebigs Ann. Chem. 1934, 512, 102-
106.
5170 J . Org. Chem., Vol. 69, No. 16, 2004

New â-Sheet Ligands with Peptidic Recognition Elements
SCHEME 3. Step w ise Cou p lin g P r otocol for th e
Com bin a tion of P yr a zole-Ba sed a n d P r otein ogen ic
Am in o Acid s in Hybr id Liga n d s
functionality (14b). In addition the trimeric ligand 16 was
assembled by using the same strategy as described for
14a ,b. Finally, the aminopyrazole monomers or dimers
can also be placed at the N-terminus: coupling is most
conveniently accomplished by way of their free carboxylic
acid with peptide esters, using PyClop or Mukaiyama’s
reagent (15, 18). All the tetramers carry eight hydrogen
bond donors and acceptors capable in principle of stabi-
lizing a hexapeptide strand in its â-sheet conformation
by a double attack from the top and bottom face.
Although the five-membered pyrazole ring confers to the
ligand an overall shape similar to a halfmoon, self-
association seems to increase proportional to the number
of incorporated aminopyrazoles. Its solubility is, however,
markedly enhanced by the presence of two or more amino
acids, and especially with an oligomeric ethylene glycole
elongation.
(c) P r ep a r a tion of th e F m oc Bu ild in g Block . The
facile and highly variable access to â-sheet hybrid ligands
described above prompted us to develop a strategy
suitable for automated synthesis of larger hybrid ligands
on a peptide synthesizer. Starting from the PMB-
protected 3-nitropyrazole-5-carboxylic acid 9a , key com-
pound 19 was assembled through a two-step route:
reduction of the nitro group with H2/Pd-C and following
treatment with Fmoc-Cl yields in the corresponding Fmoc
protected amino acid.
allowed subsequent peptide coupling. Starting with
compound 6 several pyrazol intermediates could be
synthesized (7-10, for synthetic details see ref 6b).
With these building blocks (Scheme 2) subsequent
coupling with various amino acids and peptide esters
(Scheme 4) could be carried out. However, to avoid
racemization, higher oligomers were always prepared
from C- to N-terminus (Scheme 3). To this end, PMB-
protected ester 7 was coupled with N-Z-protected amino
acids or peptides with benztriazoles (HCTU or HATU),
phosphonate anhydrides (T3P), or EDC/HOBt.12 After
hydrogenolytic Z-removal, the PMB-protected nitropyra-
zol carboxylic acid (9a ) or an N-acylated aminopyrazole
carboxylic acid (9b,c) could be attached either with
PyClop or Mukaiyama’s reagent, since no racemization
can occur at the heterocylic amino acid.13 In principle
coupling with T3P is also possible; however, the received
yields were much lower. The final coupling step was
followed by complete removal of all PMB protecting
groups with hot trifluoroacetic acid, which leads to the
desired ligands (14a ,b). This procedure is racemization-
free despite its rather harsh acidic conditions.
The synthetic route outlined above proved to be
extremely flexible with respect to the number and order
of all coupling partners. In conclusion dimeric amino-
pyrazoles could be coupled at their free N-terminus with
N-acylated dipeptides by HCTU or T3P (17). If two amino
acids are to be inserted between flanking aminopyrazoles,
Z-protected dipeptides are the building blocks of choice:
The series of model compounds includes tetramers with
diglycyl bridges, either carrying an N-terminal acetyl-
amino (14a ) or an ethyleneglycol-elongated acetylamino
(d) Au tom a ted Iter a tive Solid -P h a se P ep tid e Syn -
th esis w ith th e New Bu ild in g Block . Two representa-
tive tetramers (20, 21) were prepared on an ACT Model
90 automated peptide synthesizer.14
To introduce an additional C-terminal amide group as
hydrogen bond donor, the solid-phase protocol starts from
a polystyrene resin, carrying the Sieber amide linker.15
After Fmoc deprotection, the first pyrazole amino acid is
attached by HOBt and diisopropylcarbodiimide (DIPCDI)
in N-methylpyrrolidone (NMP).16 All coupling steps were
carried out twice, to ensure complete conversion. Each
step was monitored by analytic HPLC or HPLC-MS, since
the low nucleophilicity of the pyrazole free amine group
does not guarantee positive color tests with NF-31.17
A
loading of ∼0.2 mmol/g of resin is achieved (gravimetric
determination; due to the low amount of free amino
groups a quantitative Kaiser test was not sufficiently
sensitive). An iterative sequence consisting of Fmoc
deprotection (piperidine) and coupling (HATU/HOAt,
collidine) follows.18 Aliphatic amino acids can also be
coupled by TBTU, HOBt, and diisopropylethylamine
(DIEA). Final Fmoc deprotection and acetylation of the
free N-terminus with acetic acid anhydride completes the
solid-phase synthesis. The PMB-protected tetramer is
cleaved off the Sieber amide resin by 2% TFA in dichlo-
romethane, whereas the following PMB deprotection
requires short heating to 70 °C. Semipreparative puri-
fication affords the analytically pure ligands as triflate
salts, which can eventually be converted to the free
(12) (a) HCTU is the “cheap” modification of HATU or HBTU; recent
findings distinguish between the guanidinium and the much more
potent uronium form: Carpino, L.; Imazumi, H.; El-Faham, A.; Ferrer,
F. J .; Zhang, C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.;
Mu¨gge, C.; Wenschuh, H.; Klose, J .; Beyermann, M.; Bienert, M.
Angew. Chem. 2002, 114, 457-461. (b) T3P: Wissmann, H.; Kleiner,
H.-J . Angew. Chem. 1980, 92, 129-130. Klose, J .; Bienert, M.;
Mollenkopf, C.; Wehle, D.; Zhang, C.; Carpino, L.; Henklein, P. Chem.
Commun. 1999, 1847-1848. EDC: Sheehan, J . C.; Cruickshank, P. A.;
Boshart, G. L. J . Org. Chem. 1961, 26, 2525.
(14) Automated Synthesizer, Model 90, Advanced ChemTech.
(15) Sieber, P. Tetrahedron Lett. 1987, 28, 2107-2110.
(16) HOBt, DIPCDI: Tartar, A.; Gesquiere, J . C. J . Org. Chem.
1979, 44, 5000.
(17) Madder, A.; Farcy, N.; Hosten, N. G. C.; DeMuynck, H.;
DeClercq, P. J .; Barry, J .; Davis, A. P. Eur. J . Org. Chem. 1946, 11,
2787-2791.
(18) HATU, HOAt: Carpino, L. A.; El-Faham, A. J . Org. Chem.
1995, 60, 3561-3564.
(13) PyClop: Coste, J .; Frerot, E.; J ouin, P. J . Org. Chem. 1994,
59, 2437-2446. Mukaiyama’s reagent: Mukaiyama, T. Angew. Chem.
1979, 91, 798-812.
J . Org. Chem, Vol. 69, No. 16, 2004 5171

Rzepecki et al.
SCHEME 4. Over view of All New Am in op yr a zole P ep tid e Hybr id Liga n d s Syn th esized in Solu tion
SCHEME 5. Syn th esis of th e F m oc Bu ild in g Block
This might be due to a folded conformation in the
flexible peptide unit, acting as a â-turn. A ROESY
spectrum in connection with COSY and HMQC spectra
revealed the true conformation along the central peptide
chain: numerous cross-peaks were observed, which are
all consistent with an extended conformation of the
peptide hybrid molecule. Even at highest resolution no
cross-peak appeared, which would indicate a folded
conformation. Considering valine’s high propensity to
induce a â-sheet conformation in peptide strands, this
result is not surprising.20
Final proof came from variable temperature (VT)
experiments. If one conformation dominates in solution,
intramolecular hydrogen bonds only produce a small
temperature gradient for the corresponding NH signals
of ∆δ/∆T < 2-3 ppb/K in DMSO. Temperature gradients
of > 4 ppb/K, on the other hand, indicate intermolecular
strong hydrogen bonds with other peptide molecules.21
In the temperature range between 302 and 343 K the
NH protons 2 and 16 correspond to a gradient of 5.6 ppb/
K, those of NH-4 to 6.7 ppb/K, those of NH-18 to 6.1 ppb/
K, and those of NH-11 to 7.8 ppb/K. Thus, no intramo-
lecular hydrogen bonds are found in this ligand, which
must adopt an extended conformation in solution, ideal
for docking on to solvent-exposed â-sheets in proteins.
pyrazoles by neutralization with saturated NaHCO3
solution. No fluorine signals are found in the 19F NMR
spectrum.
Con for m a tion . The new aminopyrazole peptide hy-
brid compounds were characterized by various spectro-
scopic measurements including CD spectra, VT, and
ROESY NMR experiments. Contrary to oligopeptides
from proteinogenic amino acids, the hybrid compounds
displayed pronounced CD couplets in their CD spectra
(e.g., for 21: Cotton effect at 242 nm, ∆ꢀ ) + 17.1 mol-1
cm-1 and at 209 nm, ∆ꢀ ) -16.9 mol-1 cm-1).19 Obviously,
the flanking pyrazole chromophores interact with the
stereogenic environment of the dipeptide core unit.
Ou tlook
Although the new generation of â-sheet ligands dis-
plays a pronounced increase in solubility in organic
solvents, it is still only sparingly soluble in water.
Further optimization may be effected by releasing ionic
groups at the carboxy or amino terminus of the peptide
(19) Note that none of the molar ellipticity maximums corresponds
to formation of a â-sheet-like structure by self-association (218 nm).
(20) Kim, C. A.; Berg, J . M. Nature 1993, 362, 267. Nowick, J . S.;
Insaf, S. J . Am. Chem. Soc. 1997, 119, 10903.
(21) (a) Ribeiro, A. A.; Goodman, M.; Naider, F. Int. J . Peptide
Protein Res. 1979, 14, 414. (b) Kessler, H. Angew. Chem. 1982, 94.
509-520.
F IGURE 3. CD spectrum of tetramer 21 in acetonitrile
(dotted lines) and the CD curve (solid line) for a hexapeptide
derived from the putative nucleation site of Aâ (VKLVFF).
5172 J . Org. Chem., Vol. 69, No. 16, 2004

New â-Sheet Ligands with Peptidic Recognition Elements
SCHEME 6. Th e New Am in op yr a zole P ep tid e Hybr id Liga n d s Syn th esized w ith F m oc-SP P S
SCHEME 7. Solid -P h a se P r otocol for th e Au tom a ted Syn th esis of a Tetr a m er ic Am in op yr a zole P ep tid e
Hybr id Ba sed on th e F m oc Str a tegy
2-(Meth oxyca r bon ylh yd r a zon o)cya n op yr u va te (1). Po-
tassium (50.0 g, 1.28 mol) was dissolved in a mixture of dry
EtOH (320 mL) and dry Et2O (225 mL) under nitrogen. The
mixture was cooled to 0 °C in ice, diethyl oxalate (93.5 g, 0.64
mol), disolved in Et2O (30 mL), was added dropwise, and the
reaction mixture was stirred for 30 min. A solution of CH3CN
(26.3 g, 0.64 mol) in Et2O (30 mL) was added, and the mixture
was allowed to warm to room temperature and stirred for 1
h. The precipitated solid was collected by filtration to give 115
g (quantitative yield). The product was used without further
purification. Mp 105 °C dec; IR (KBr) νj [cm-1] 3393, 2170,
hybrids, or by introducing ionic amino acid residues.
Alternatively, solubility-conferring/enhancing protecting
groups may be attached at either terminus. Aggregation
tests of pathological proteins with these and related
ligands are underway in vitro in biophysical aggregation
experiments as well as in cell culture assays.22 Screening
is carried out for efficiency and selectivity toward Aâ
aggregation as well as toxicity for the cells. The prelimi-
nary results look extremely promising and will be
published in due course.
1
1626, 1558, 1363, 1310, 1112, 594, 526; H NMR (250 MHz,
3
3
D2O) δ [ppm] 1.19 (t, 3 H, J ) 7.1 Hz, CH3), 4.13 (q, 2 H, J
) 7.1 Hz, CH2), 4.80 (s, 1H, CH); MS (ESI, H2O) m/z (%) 140
(100) [M]-.
Exp er im en ta l Section
X-r a y Str u ctu r e of N-Meth oxyca r bon yl-3-a m in op yr a -
zole-5-ca r boxylic Acid Eth yl Ester (2). C8H11N3O4; M )
213.20 g/mol, triclinic, space group P1, a ) 6.3241(7) Å, R )
93.687(14)°, b ) 6.4956(8) Å, â ) 96.857(13)°, c ) 12.4132(15)
Å, γ ) 95.235(14)°, V ) 0.5027 nm3, Z ) 2, Dx ) 1.408 Mg/m3,
λ(MoKR) ) 71.073 pm, µ ) 0.114 mm-1, F(000) ) 224, T )
173 (1) K. A colorless needle-shaped crystal (0.70 × 0.12 ×
0.06 mm3) was used to collect on a STOE-IPDS diffractometer
4238 reflections (1567 independent signals), Rint ) 0.0380 from
3.16° to 25.73°. The structure was solved by direct methods
(SIR97)23 and the F2-value was optimized with the program
SHELXL-9724 for all non-hydrogen atoms. The postion of
hydrogen atoms was calculated. The final ωR(F2)-Wert for all
reflections was 0.0754; R(F) ) 0.0560, S ) 0.905, max ∆F )
To a suspension of the potassium cyano pyruvate (895 mg,
5 mmol) in chloroform (50 mL) was added HCl saturated ethyl
ester (3 mL). Methyl hydrazino formate (450 mg, 5 mmol) was
added, the mixture was stirred for 24 h at room temperature,
any precipitated solid was removed by filtration, and the
filtrate was concentrated to give an oil. The crude product was
purified by CC (ethyl acetate, Rf ) 0.72) to yield 1 (1.95 mmol,
39%) as a colorless solid. Mp 131 °C; IR (KBr) νj [cm-1] 3250 ,
2250, 1700; 1H NMR (250 MHz, CDCl3, TMS) δ [ppm] 1.40 (t,
3
3
3 H, J ) 7.1 Hz), 3.61 (s, 2 H), 3.89 (s, 3 H), 4.38 (q, J ) 7.1
Hz, 2 H), 11.93 (br s, 1 H, NH); MS (CI, CH2Cl2, NH3) m/z (%)
214 (34) [MH]+, 231 (100) [M + NH4]+. Anal. Calcd for
C8H11N3O4: C 45.07; H 5.20; N 19.71. Found: C 44.91; H 5.10;
N 19.50.
158 e‚nm-3
.
N-Meth oxycar bon yl-3-am in opyr azole-5-car boxylic Acid
Eth yl Ester (2). To a solution of 1 (107 mg, 0.5 mmol) in CH3-
CN (5 mL) was added triethylamine (0.14 mL, 1 mmol) and
the mixture was stirred for 10 min at room temperature. The
solvent was removed in vacuo and the solid residue was
recrystallized from ethyl acetate to give 2 (104 mg, 98%; Rf
(22) Cooperation with biophysicist Detlev Riesner at the University
of Du¨sseldorf, Germany, and J SW research, Graz, Austria.
(23) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A. J .
Appl. Crystallogr. 1993, 26, 343-350.
(24) Sheldrick, G. M. Programm fu¨r Kristallstrukturverfeinerung,
Universita¨t Go¨ttingen, 1997.
J . Org. Chem, Vol. 69, No. 16, 2004 5173

Rzepecki et al.
F IGURE 4. Phase-sensitive ROESY spectrum of tetramer 21 in DMSO-d6 (positive cross-peaks, exchange-effects; negative cross-
peaks, NOE effects); the signals of both pyrazole ring NH groups at 12.97 and 13.01 are not shown; the methyl groups H-8, H-9,
H-13, and H-14 are abbreviated by CH3 for clarity; smaller signals without assignment (arrows): minor diastereomer.
0.52) as colorless crystals. Mp 163 °C; IR (KBr) νj [cm-1] 3490,
3287, 3157, 1744, 1729, 1614, 1457, 1243; UV/vis (CH3CN) λmax
[nm] (log ꢀ) 216 (4.37), 292 (3.58); 1H NMR (250 MHz, CDCl3,
[nm] (log ꢀ) 208 (4.73), 220 (sh, 4.47), 266 (4.27), 276 (sh, 4.12),
1
289 (3.73), 300 (3.74); H NMR (250 MHz, DMSO-d6, TMS) δ
[ppm] 1.30 (t, 3J ) 7.1 Hz, 3 H, CH3), 3.81 (d, 2 H, 3J ) 5.7
Hz, CH2-glycine), 4.15-4.41 (m, 5 H, CH2-ester, CH2-Fmoc,
CH-Fmoc), 6.96 (br s, 1 H, CH-pyrazole), 7.29-7.47 (m, 4H,
3
TMS) δ [ppm] 1.38 (t, J ) 7.1 Hz, 3 H), 4.06 (s, 3 H), 4.39 (q,
3J ) 7.1 Hz, 2 H), 5.50 (br s, 2 H), 5.90 (s, 1 H); 13C NMR (62
MHz, CDCl3, TMS) δ [ppm] 14.3 (+), 54.8 (+), 61.5 (-), 89.8
(+), 147.3 (Cquat), 151.3 (Cquat), 152.2 (Cquat), 161.9 (Cquat); MS
(EI, CH2Cl2) m/z (%) 213 (36) [M+•], 169 (24) [M+• - C2H4O],
141 (100) [M+• - C3H4O2]; HRMS (C8H11N3O4) calcd 213.0750,
found 213.0752 ( 0.56 ppm. Anal. Calcd for C8H11N3O4: C
45.07; H 5.20, N 19.72. Found: C 44.87; H 5.04, N 19.44.
F m oc-Gly-P z(H)-OEt (3a ). Compound 2 (3.80 g, 17.82
mmol) was dissolved in anhydrous THF (120 mL) and the
solution was treated with 6.19 g (19.61 mmol) of N-(9-
fluorenylmethoxycarbonyl)glycine chloride and stirred for 24
h at 70 °C. After evaporation of the solvent the yellowish crude
product was absorbed onto silica gel and chromatographed over
silica gel eluting with ethyl acetate/petrol ether (4:1). Com-
pound 3a (7.50 g, 17.26 mmol, 97%) was isolated as a colorless
3
CH-arom Fmoc), 7.62 (t, 1 H, J ) 5.7 Hz, NH), 7.73 (d, 2 H,
3
3J ) 5.9 Hz, CH-arom Fmoc), 7.90 (d, 2 H, J ) 7.1 Hz, CH-
arom Fmoc), 10.67 (br s, 1 H, NH), 13.55 (br s, 1 H, NH-
pyrazole); 13C NMR (62 MHz, DMSO-d6) δ [ppm] 14.1 (+), 43.4
(-), 46.6 (+), 60.7 (-), 65.7 (-), 99.2 (+), 120.1 (+), 125.2 (+),
127.0 (+), 127.6 (+), 133.0 (Cquart), 140.7 (Cquart), 143.8 (Cquart),
147.5 (Cquart), 156.5 (Cquart), 158.8 (Cquart), 167.5 (Cquart); MS
(FAB, CH2Cl2, glycerin) m/z (%) 435 (100) [MH]+; HRMS
(C23H22N4O5) calcd 435.1668, found 435.1662.
H-Gly-P z(H)-OEt (3b). Fmoc-protected 3a (200 mg, 0.46
mmol) was dissolved in 2 mL of 20% piperidine in DMF and
the solution was stirred for 15 min at ambient temperature.
The solvent was removed in vacuo and the resulting solid was
absorbed onto silica gel. After chromatographic purification
over silica gel eluting with CH2Cl2:MeOH:NH3 80:20:1, 91 mg
(0.43 mmol, 93%) of the free amine 3b was obtained as a
solid (Rf 0.32, EE:PE ) 2:1). Mp 132 °C; IR (KBr) νj [cm-1
]
3307, 3173, 1704, 1591, 1548, 1506, 1254; UV/vis (CH3CN) λmax
5174 J . Org. Chem., Vol. 69, No. 16, 2004

New â-Sheet Ligands with Peptidic Recognition Elements
colorless solid (Rf 0.54). Mp 188 °C dec; IR (KBr) νj [cm-1] 3374,
3236, 1722, 1654, 1528, 1472; UV/vis (CH3CN) λmax [nm] (log
ꢀ) 219 nm (3.79); 1H NMR (250 MHz, DMSO-d6, TMS) δ [ppm]
1.31 (t, 3 H, 3J ) 7.1 Hz, CH3), 3.32 (s, 2 H, CH2-glycine), 4.30
(q, 2 H, 3J ) 7.1 Hz CH2), 6.94 (s, 1 H, CH); 13C NMR (62
MHz, DMSO-d6, TMS) δ [ppm] 14.1 (+), 44.5 (-), 60.6 (-),
98.2 (+), 134.7 (Cquart), 145.8 (Cquart), 159.3 (Cquart), 170.9 (Cquart);
MS (EI, 70 eV) m/z (%) 212 (10) [M]•+, 155 (12) [M -
NHCOCH2NH2]•+; HRMS (C8H12N4O3) calcd 212.0909, found
212.0908.
δ [ppm] 3.67 (d, 3J ) 6.3 Hz, 2H, CH2-Gly), 3.71 (s, 3H, OCH3-
3
PMB), 3.83 (s, 3H, OCH3), 3.87 (d, J ) 5.7 Hz, 2H, CH2-Gly),
5.03 (s, 2H, CH2-Z), 5.54 (s, 2H, CH2-Bz), 6.87 (d, 3J ) 8.7 Hz,
2H, CH-arom), 7.06 (s, 1H, CH-pyrazole), 7.14 (d, 3J ) 8.7 Hz,
2H, CH-arom), 7.35 (m, 5H, CH-arom), 7.50 (t, 3J ) 6.2 Hz,
3
1H, NH-Gly), 8.18 (t, J ) 5.7 Hz, 1H, NH-Gly), 10.71 (s, 1H,
NH-pyrazole); 13C NMR (125 MHz, DMSO) δ [ppm] 42.2, 43.5,
52.2, 53.2, 55.0, 65.5, 101.7, 113.9, 127.7, 128.3, 128.7, 129.1,
131.2, 137.0, 145.9, 158.7, 159.3, 167.3, 169.5; MS (ESI) m/z
(%) 532 (M+ + Na, 100), 548 (M+ + K, 20), 510 (M+, 25); HRMS
calcd 532.181, found 532.180; Rf 0.10 in EtOAc/n-pentane 2:1;
mp 165-170 °C. Anal. Calcd for C25H27N5O7: C, 58.93; H, 5.34;
N, 13.75. Found: C, 58.93; H, 5.50; N, 13.48.
F m oc-Gly-Gly-P z(H)-OEt (4a ). Free amine 3b (100 mg,
0.23 mmol) and 145 mg (0.46 mmol) of N-(9-fluorenylmethoxy-
carbonyl)glycine chloride were dissolved in 4 mL of anhydrous
DMF. By addition of N,N-diisopropylethylamine the pH of the
solution was brought to 8-9. After the mixture was stirred
for 30 min at room temperature, the solvent was stripped off
in vacuo. The oily residue was suspended in CH2Cl2 and
sonicated for several minutes, and the solvent was subse-
quently removed again. The resulting solid was absorbed onto
silica gel and chromatographed over silica gel eluting with CH2-
Cl2:MeOH:NH3 100:10:1 (Rf 0.20). Trimer 4a (46 mg, 0.09
mmol, 41%) was isolated as a colorless solid. Mp 198 °C dec;
IR (KBr) νj [cm-1] 3325, 3140, 1701, 1640, 1571, 1211; UV/vis
(CH3CN) λmax [nm] (log ꢀ) 208 (4.73), 220 (sh, 4.49), 265 (4.28),
275 (sh, 4.12), 289 (3.69), 300 (3.70); 1H NMR (250 MHz,
H-Gly-Gly-P z(P MB)-OMe (12). Under inert atmosphere
700 mg of Z-Gly-Gly-Pz(PMB)-OMe (11) (1.37 mmol, 1.00
equiv) was dissolved in 2 mL of dichloromethan and the
solution was diluted with 25 mL of methanol. Next, 5 mol %
of Pd/C was added. The flask was then evacuated and filled
with H2. The reaction mixture was stirred for 16-52 h, then
the catalyst was removed by filtration over Celite and the
solution concentrated in vacuo. Yield 91% (470 µg, 1.25 µmol);
colorless solid. 1H NMR (200 MHz, DMSO-d6) δ [ppm] 3.61
(br s, 2H, CH2-Gly), 3.71 (s, 3H, OCH3-PMB), 3.83 (s, 3H,
3
OCH3), 3.97 (d, J ) 5.5 Hz, 2H, CH2-Gly), 5.54 (s, 2H, CH2-
Bz), 6.88 (d, 3J ) 8.7 Hz, 2H, CH-arom), 7.05 (s, 1H,
3
3
CH-pyrazole), 7.17 (d, J ) 8.7 Hz, 2H, CH-arom), 8.17 (br s,
DMSO-d6, TMS) δ [ppm] 1.30 (t, 3 H, J ) 7.1 Hz, CH3), 3.69
2H, NH2), 8.77 (t, 3J ) 5.6 Hz, 1H, NH), 10.86 (s, 1H, NH);
13C NMR (125 MHz, DMSO) δ [ppm] 42.2, 52.2, 52.9, 53.2, 55.1,
101.7, 113.9, 128.8, 129.1, 131.2, 145.8, 158.7, 159.3, 166.4,
166.9; MS (ESI) m/z (%) 398 (M+ + Na), 121 (PMB+); HRMS
calcd 398.144, found 398.143; Rf 0 in EtOAc/n-pentane 2:1; mp
219 °C.
3
3
(d, 2 H, J ) 6.0 Hz, CH2-glycine), 3.91 (d, 2 H, J ) 5.6 Hz
CH2-glycine), 4.19-4.36 (m, 5 H, 2 CH2-ester, CH2-Fmoc, CH-
Fmoc), 6.92 (br s, 1 H, CH-pyrazole), 7.29-7.47 (m, 4 H, CH-
3
arom Fmoc), 7.60 (t, 1 H, J ) 6.0 Hz, 1 H, NH), 7.72 (d, 2 H,
3J ) 7.1 Hz, CH-arom Fmoc), 7.89 (d, 2 H, J ) 7.1 Hz, CH-
3
arom Fmoc), 8.19 (t, 1 H, 3J ) 5.6 Hz, NH), 10.63 (s, 1 H, NH),
13.55 (br s, 1 H, NH-pyrazole); 13C NMR (100 MHz, DMSO-
d6, TMS) δ [ppm] 14.1 (+), 42.1 (-), 43.4 (-), 46.6 (+), 60.7
(-), 65.7 (-), 99.0 (+), 120.1 (+), 125.2 (+), 127.0 (+), 127.6
(+), 133.1 (Cquart), 140.7 (Cquart), 143.8 (Cquart), 147.3 (Cquart),
156.5 (Cquart), 159.0 (Cquart), 167.2 (Cquart), 169.5 (Cquart); MS
(FAB, DMSO/glycerin) m/z (%) 492 (100) [MH]+; HRMS
(C25H25N5O6) calcd 492.1883 [MH]+, found 492.1879 [MH]+.
Ac-P z(P MB)-Gly-Gly-P z(P MB)-OMe (13a ). In an argon
atmosphere a mixture of 158 mg of H-Gly-Gly-Pz(PMB)-OMe
(420 µmol, 1.00 equiv) (12), 120 mg (420 µmol, 1.00 equiv) of
5-Acetylamino-2-(4-methoxy-benzyl)-2H-pyrazole-3-carbon-
sa¨ure (9b),6b 260 µL of DIEA (1.52 mmol, 3.60 equiv), and 232
mg of chlortripyrolidinophosphoniumhexafluorphosphat (Py-
Clop) (550 µmol, 1.30 equiv) was stirred in dry dichlo-
romethane for 16 h. During the reaction
a yellow solid
H-Gly-Gly-P z(H)-OEt (4b). The Fmoc-protected 4a (360
mg, 0.73 mmol) was dissolved in 2 mL of 20% piperidine in
DMF and the solution was stirred for 15 min at room
temperature. The solvent was removed and the resulting solid
was suspended in CH2Cl2. After sonication the solution was
carefully removed by means of a pipet. This procedure was
repeated twice; the remaining solid was filtered off. After
purification over silica gel (Rf 0.16, CH2Cl2:MeOH:NH3 80:20:
1), free amine 4b was obtained in the form of a colorless solid
(193 mg, 0.72 mmol, 98%). Mp 190 °C; IR (KBr) νj [cm-1] 3363,
3249, 1723, 1663, 1547, 1473; UV/vis (CH3CN) λmax [nm] (log
ꢀ) 223 (3.60); 1H NMR (250 MHz, DMSO-d6, TMS) δ [ppm]
precipitated, which was filtrated, washed with n-pentane, and
dried in vacuo. Yield 61% (165 mg, 260 µmol), pale yellow solid.
1H NMR (300 MHz, DMSO) δ [ppm] 1.98 (s, 3H, CH3-acetyl),
3.68 (s, 2H, CH3-PMB), 3.70 (s, 3H, CH3-PMB), 3.82 (s, 3H,
CH3), 3.83-3.85 (m, 4H, CH2-glycine), 5.53 (s, 2H, CH2-Bz),
5.56 (m 2H, CH2-Bz), 6.82 (d, 3J ) 8.7 Hz, 2H, CH-arom), 6.85
3
(d, J ) 8.6 Hz, 2H, CH-arom), 7.06-7.22 (m, 6H, CH-arom,
pyrazole), 8.29 (br s, 1H, NH), 8.93 (br s, 1H, NH), 10.56 (s,
1H, NH), 10.64 (s, 1H, NH); MS (ESI) m/z (%) 669 (M+ + Na);
HRMS calcd 669.2397, found 669.2370; Rf 0.10 in CH2Cl2/
MeOH 10:1; mp 156 °C.
3
1.30 (t, 3 H, J ) 7.1 Hz CH3), 3.19 (s, 2 H, CH2-glycine), 3.93
Ac-P z(H)-Gly-Gly-P z(H)-OMe 2TF A (14a ). In an inert
atmosphere 64 mg (100 µmol, 1.00 equiv) of Ac-Pz(PMB)-Gly-
Gly-Pz(PMB)-OMe (13a ) was heated in 10 mL of anhydrous
TFA for 4 h to 70 °C. The solution was cooled to room
temperature and an excess of ice-cold diethyl ether was added.
The product precipitated as a colorless solid, which was filtered
off, washed several times with diethyl ether, and dried in
vacuo. Yield 47% (30 mg, 47 µmol); colorless solid. 1H NMR
(300 MHz, DMSO) δ [ppm] 2.02 (s, 3H, CH3-acetyl), 3.82 (s,
3H, CH3), 3.88-3.91 (m, 4H, CH2-glycine), 6.91 (s, 1H, CH-
pyrazole), 7.03 (s, 1H, CH-pyrazole), 8.25-8.28 (t, 3J ) 5.6 Hz,
1H, NH glycine), 8.73 (br s, 1H, NH glycine), 10.50 (s, 1H,
amide pyrazole), 10.63 (s, 1H, amide pyrazole); 13C NMR (100
MHz, DMSO-d6:) δ [ppm] 22.4, 52.18, 53.2, 55.1, 113.5, 113.8,
128.7, 128.8, 129.6, 145.6, 157.2, 159.7, 159.3, 168.5, 169.5;
mp >250 °C.
(s, 2 H, CH2-glycine), 4.29 (q, 2 H, 3J ) 7.1 Hz, CH2-ester),
6.90 (s, 1 H, CH), 8.24 (br s, 1 H, NH), 10.67 (br s, 1 H, NH);
13C NMR (62 MHz, DMSO-d6, TMS) δ [ppm] 14.1 (+), 41.9 (-),
44.2 (-), 60.6 (-), 98.8 (+), 134.1 (Cquart), 146.3 (Cquart), 159.2
(Cquart), 167.3 (Cquart), 172.8 (Cquart); MS (CI, NH3) m/z (%) 270
(58) [MH]+, 156 (100) [M - C4H5N2O2]+; HRMS (C10H15N5O4)
calcd 270.1202 [MH]+, found 270.1204 [MH]+.
Z-Gly-Gly-P z(P MB)-OMe (11). Under inert atmosphere a
mixture of 150 g of N-p-Methoxybenzyl-3-aminopyrazolee-5-
carbonylic acid methyl ester (7)6b (570 µmol, 1.00 equiv), 152
mg of N-benzyloxycarbonyl-glycinyl-glycin (570 µmol, 1.00
equiv), 136 mg of EDC (710 µmol, 1.25 equiv), 219 mg of
1-hydroxybenztriazol (HOBt) (1.43 mmol, 2.50 equiv), and 250
mL of DIEA (1.43 mmol, 2.50 equiv) were stirred for 1 h at 0
°C and for another 15 h at room temperature. The organic
layer was washed with satd aq NaHCO3, 1 M HCl, and satd
aq NaCl and dried over Na2SO4. After filtration the solution
was concentrated and crystallized from EtOAc. The colorless
solid was washed with EtOAc and dried in vacuo. Yield 42%
(120 mg, 240 µmol); colorless solid. 1H NMR (200 MHz, DMSO)
Glycol-P z(P MB)-Gly-Gly-P z(P MB)-OMe (13b). In an
argon atmosphere a mixture of 184 mg of Gly-Pz(PMB)-OH
(9c)6b (490 µmol, 1.00 equiv), 200 mg (490 µmol, 1.00 equiv )
of H-Gly-Gly-Pz(PMB)-OMe (14d ), 310 µL (1.77 mmol, 3.60
equiv) of DIEA, and 270 mg (640 µmol, 1.30 equiv) of PyClop
J . Org. Chem, Vol. 69, No. 16, 2004 5175

Rzepecki et al.
was stirred for 16 h at room temperature. The solvent was
evaporated and the residue was purified by chromatography
on silica gel column (gradient: CH2Cl2/CH3OH 30:1 and 10:
1). Finally the product was recrystallized from CHCl3 and
arom); 13C NMR (100 MHz, CDCl3) δ [ppm] 28.4, 37.6, 38.1,
38.4, 52.4, 53.4, 55.8, 127.1, 127.3, 128.6, 128.8, 129.3, 129.5,
170.9, 171.5; MS (ESI) m/z (%) 465 (M+ + K), 449 (M+ + Na);
HRMS calcd for C24H30N2O5Na 449.2052, found 449.2050; mp
132 °C; Rf 0.85 in CH2Cl2/MeOH 30:1; [R]Na +23.2 at T ) 21
°C, c 1.00 in CHCl3.
H-P h e-P h e-OMe‚TF A. Boc-L-Phe-L-Phe-OMe (2.50 g, 7.36
mmol, 1.00 equiv) was stirred in a mixture of dichloromethane
and trifluoroacidic acid (5:1) at 0 °C until complete conversion.
The dichloromethane was evaporated and ice-cold diethyl ether
was added to the remaining TFA solution. The precipitating
solid was filtered off, washed several times with diethyl ether,
and dried in vacuo. Yield 1.42 g (3.22 mmol, 44%); colorless
solid. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 2.87-3.13 (m,
4H, CH2-Phe), 3.61 (s, 3H, CH3), 4.02 (br s, 1H, R-CH), 4.54-
4.61 (m, 1H, R-CH), 7.21-7.35 (m, 10H, CH-arom), 8.10 (br s,
3H, NH3), 8.98 (d, J ) 7.63 Hz, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ [ppm] 36.6, 36.9, 52.0, 53.1, 53.8, 126.7, 127.2,
128,3, 128.4, 129.0, 129.5, 133.9, 136.6, 168.2, 171.1; MS (ESI)
m/z (%) 349 (M + Na)+, 327 (M+ + H)+; HRMS calcd for
1
diethyl ether. Yield 69 mg (98 µmol, 20%); colorless solid. H
NMR (300 MHz, CDCL3) δ [ppm] 3.31 (s, 3H, CH3-glycol),
3.52-3.73 (m, 10H, CH2-glycol), 3.67 (s, 3H, CH3-PMB), 3.73
3
(s, 3H, CH3-PMB), 3.81 (s, 3H, CH3), 4.05 (d, J ) 5.4 Hz, 2H,
CH2-Gly), 4.12-4.17 (m, 4H, CH2-Gly, OCH2), 5.50 (s, 2H, CH2-
Bz), 5.61 (s, 2H, CH2-Bz), 6.74-6.77 (m, 4H, CH2-arom), 7.19-
3
7.26 (m, 6H, H-CH2-arom, CH-pyrazole), 7.51 (t, J ) 5.4 Hz,
1H, NH), 8.16 (t, 3J ) 5.4 Hz, 1H, NH), 9.27, 9.80 (2s, 2H,
NH); 13C NMR (100 MHz, CDCl3) δ [ppm] 46.2, 46.3, 51.9, 53.8,
54.6, 55.2, 55.3, 59.0, 70.2, 70.4, 70.6, 71.4, 71.9, 98.9, 102.8,
113.8, 113.9, 128.9, 129.2, 129.3, 129.5, 131.8, 134.5, 144.7,
145.5, 159.1, 159.2, 160.0, 160.3, 167.0, 168.6, 170.2; MS (ESI)
m/z (%) 787 (M+ + Na); HRMS calcd 707.3027, found 787.3069;
mp 126 °C.
Glycol-P z(H )-Gly-Gly-P z(H )-OMe‚2TF A (14b ). In an
argon atmosphere 50 mg (71 µmol) of Glycol-Pz(PMB)-Gly-Gly-
Pz(PMB)-OMe (13b) was heated in 10 mL of anhydrous TFA
for 4 h to 70 °C. The solution was cooled to room temperature
and an excess of ice-cold diethyl ether was added. The product
precipitated as a colorless solid, which was filtrated, washed
several times with diethyl ether, and dried in vacuo. Yield 13
mg (25%,17.5 µmol); colorless solid. 1H NMR (300 MHz,
DMSO) δ [ppm] 3.23 (s, 3H, CH3-glycole), 3.44-3.47 (m, 2H,
CH2), 3.54-3.65 (m, 6H, CH2), 3.82 (s, 3H, CH3), 3.90-3.91
(m, 4H, CH2-glycine), 4.09 (s, 2H, CH2), 6.91 (s, 1H, CH-
pyrazole), 7.07 (s, 1H, CH-pyrazole), 8.27 (t, 3J ) 5.6 Hz, NH-
glycine), 8.75 (br s, 1H, NH-glycine), 10.11 (s, 1H, NH), 10.63
(s, 1H, NH); MS (ESI) m/z (%) 547 (M+ + Na); HRMS calcd
for C20H28N8NaO9 547.1877, found 547.1872.
C
19H23N2O: 327.1709, found 327.1706; m: 135 °C; [R]Na +4.9
at T ) 20 °C, c 1.00 in MeOH; Rf 0.13 in dichloromethane/
methanol 30:1.
Boc-L-Va l-P z(P MB)-OMe (16a ). In an inert atmosphere
a mixture of 820 mg (3.15 mmol, 1.00 equiv) of H-Pz(PMB)-
OMe (7), 685 mg (3.15 mmol, 1.00 equiv) of N-tert-butyloxy-
carbonyl-(S)-valine, 1.30 g (3.15 mmol, 1.00 equiv) of HCTU,
1.34 g (7.87 mmol, 2.50 equiv) of Cl-HOBT, and 1.10 mL (9.45
mmol, 3.00 equiv) of lutidin was stirred in a mixture of dry
CH2Cl2:dry DMF 3:1. After 2 h the organic layer was washed
with satd aq NaHCO3, 1 M HCl, and satd aq NaCl and dried
over Na2SO4. After filtration the solution was concentrated and
purified by chromatography over silica gel (eluent:EtOAc/n-
pentane 1:1). Yield 750 mg (1.63 mmol, 52%); colorless solid.
NO2-P z(P MB)-Gly-OMe (15a ). In an inert atmosphere 63
mg (500 µmol, 1.00 equiv) of glycin-methyl ester hydrochloride,
139 mg (500 µmol, 1.00 equiv) of 5-nitro-2-(4-methoxybenzyl)-
2H-pyrazole-3-carbonylic acid (9a ), 273 mg (650 µmol, 1.30
equiv) of PyClop, and 427 µL (2.45 mmol, 4.90 equiv) of
diisopropylethylamine were dissolved in dry dichloromethane
and the solution was stirred at room temperature for 16 h.
The solvent was evaporated and the residue was purified by
chromatography on silica gel column (eluent EtOAc/n-pentane
1:2). Yield 170 mg (495 µmol, 99%); colorless solid. 1H NMR
(300 MHz, DMSO-d6) δ [ppm] 3.67 (s, 3H, CH3-PMB), 3.72 (s,
3
1H NMR (500 MHz, CDCl3) δ [ppm] 0.92 (d, J ) 7.1 Hz, 3H,
CH3-ValR), 0.99 (d, 3J ) 6.9 Hz, 3H, CH3-Valâ), 1.34 (s, 9H,
t-Bu), 2.24-2.31 (m,1H, CH-i-Pr), 3.76 (s, 3H, CH3-PMB), 3.85
(s, 3H, CH3), 4.12 (br s, 1H, R-H), 5.03 (br s, 1H, NH-Boc),
5.57 (s, 2H, CH2-PMB), 6.82 (d, 3J ) 8.6 Hz, 2H, CH-arom),
7.21 (d, 3J ) 8.6 Hz, 2H, CH-arom), 7.27 (s, 1H, CH-pyrazole),
8.42 (s, 1H, NH); 13C NMR (125 MHz, CDCl3) δ [ppm] 17.7,
19.6, 28.5, 30.8, 52.2, 54.1, 55.4, 60.5, 102.8, 114.2, 129.2, 129.3,
132.2, 145.4, 156.1, 159.5 160.2, 169.6; MS (ESI) m/z (%) 483
(M+ + Na); MS (FD) m/z (%) 460 (M+); HRMS (ESI) calcd for
3
3H, CH3-glycine), 4.05 (d, J ) 5.3 Hz, 2H, CH2-glycine), 5.75
C
C
23H32N4O6Na 483.2220, found 483.2232. Anal. Calcd for
23H32N4O6: C, 59.99; H, 7.00; N, 12.17. Found: C, 59.93; H,
3
(s, 2H, CH2-Bz), 6.89 (d, J ) 8.3 Hz, 2H, CH2-arom), 7.25 (d,
3J ) 8.3 Hz, 2H, CH2-arom), 7.67 (s, 1H, CH2-pyrazole), 9.38
7.03; N, 12.10. Rf 0.28 in n-pentane/diethyl ether 1:1; [R]Na
-11.4, T ) 20 °C, c 0.50 in CHCl3; mp 130 °C.
3
(t, J ) 5.3 Hz, 1H, NH); MS (ESI) m/z 349 (M+ + H); Rf 0.19
in ETOAc/n-pentane 1:2.
H-L-Va l-P z(P MB)-OMe‚TF A (16b). Boc-L-Val-Pz(PMB)-
OMe (16a ) (700 mg, 1.52 mmol) was stirred at room temper-
ature in a mixture of CH2Cl2 and trifluoroacidic acid (3:1) until
the starting material disappeared on TLC. The solvent was
evaporated and the residue was dissolved two times in toluene
and again evaporated. Finally the residue was dissoved in
acetone, the solvent was again evaporated, and the solid was
dried in vacuo. Yield 725 mg (1.52 mmol, quant.); solid. 1H
NMR (300 MHz, CDCl3) δ [ppm] 0.82 (d, 3J ) 6.0 Hz, 6H, CH3),
1.95-2.07 (m, 1H, CH-i-Pr), 3.61 (s, 3H, CH3-PMB), 3.71 (s,
NO2-P z(H)-Gly-OMe (15). In an argon atmosphere 50 mg
(146 µmol, 1.00 equiv) of NO2-Pz(PMB)-Gly-OMe (15a ) were
heated in 10 mL of anhydrous TFA for 4 h to 70 °C. The solvent
was evaporated and the residue was purified by chromatog-
raphy on silica gel column (eluent CH2Cl2/CH3OH 10:1) Yield
20 mg (88 µmol, 60%); colorless solid. 1H NMR (300 MHz,
DMSO-d6) δ [ppm] 3.67 (s, 3H, CH3), 4.07 (d, 3J ) 5.9 Hz, 2H,
CH2), 7.62 (s, 1H, CH), 9.29 (t, 3J ) 5.4 Hz, 1H, NH), 14.87 (s,
1H, NH-pyrazole); MS (EI) m/z 228 (M+); Rf 0.55 in dichlo-
rmethan/methanol 10:1; mp 167 °C.
Boc-L-P h e-L-P h e-OMe. N-tert-Butyloxycarbonyl-(S)-phen-
ylalanine (1.22 g, 4.60 mmol, 1.00 equiv) and 1.00 g (4.60
mmol, 1.00 equiv) of (S)-Phenylalanine-methyl ester were
stirred together with 1.90 g (4.60 mmol, 1.00 equiv) of HCTU,
11.5 mmol (2.50 equiv) of Cl-HOBt, and 2.14 mL (17.6 mmol,
4.00 equiv) of 4,6-lutidin. The organic layer was washed with
satd aq NaHCO3, 1 M HCl, and satd aq NaCl and dried over
Na2SO4. After filtration the solution was evaporated and the
residue dried in vacuo. Yield 1.57 g (3.68 mmol, 80%); colorless
solid. 1H NMR (300 MHz, CDCl3) δ [ppm] 1.39 (s, 9H, CH3-
Boc), 2.95-3.04 (m, 4H, CH2-phe), 3.67 (s, 3H, CH3), 4.31-
4.33 (m, 1H, R-CH), 4.76-4.79 (m, 1H, R-CH), 4.91 (br s, 1H,
NH), 6.25 (d, J ) 6.6 Hz, 1H, NH), 6.95-7.29 (2m, 10H, CH-
2
3H, CH3), 3.96-3.98 (m, 1H, RH), 5.43 (d, J ) 14.9 Hz, 1H,
2
3
CH-benz.R), 5.54 (d, J ) 14.6 Hz, 1H, CH-benz.â), 6.65 (d, J
3
) 8.6 Hz, 2H, CH-arom), 7.00 (d, J ) 8.3 Hz, 2H, CH-arom),
7.03 (s, 1H, CH-pyrazole), 8.06 (br s, 3H, NH3), 10.17 (s, 1H,
NH); 13C NMR (75 MHz, CDCl3) δ [ppm] 17.3, 18.1, 30.4, 52.2,
54.0, 55.3, 59.2, 103.3, 114.1, 128.8, 129.0, 132.6, 144.7, 159.4,
159.7, 166.8; mp 75 °C; [R]Na +18.0, T ) 20 °C, c 1.00 in MeOH;
MS (ESI) m/z (%) 383 (M+ + Na).
NO2-P z(P MB)-L-Va l-P z(P MB)-OMe (16c). A mixture of
167 mg (604 µmol, 1.00 equiv) of 5-nitro-2-(4-methoxybenzyl)-
2H-pyrazole-3-carbonylic acid (9a ),6b H-L-Val-Pz(PMB)-OMe‚
TFA (16b) (549 µmol, 1.00 equiv), 699 mg (1.10 mmol, 1.10
equiv) of T3P, and 845 µL (3.84 mmol, 7.00 equiv) of N-
methylmorpholin was stirred under argon for 24 h in dry
5176 J . Org. Chem., Vol. 69, No. 16, 2004

New â-Sheet Ligands with Peptidic Recognition Elements
dichloromethane. The solvent was evaporated and the residue
was purified by chromatography on a silica gel column (eluent
EtOAc/n-pentane 1:2). Yield 50% (165 mg, 275 µmol); colorless
solid. 1H NMR (300 MHz, CDCl3) δ [ppm] 089 (d, 3J ) 6.6 Hz,
3H, CH3-ValR), 0.93 (d, 3J ) 5.6 Hz, 3H, CH3-Val â), 2.10-
2.17 (m, 1H, CH-i-Pr), 3.66 (s, 3H, OCH3-PMB), 3.71 (s, 3H,
OCH3-PMB), 3.79 (s, 3H, OCH3), 4.43 (dd, J ) 6.6 Hz, J )
8.3 Hz, 3H, R-H-Val), 5.50-5.68 (m, 4H, CH2-Bz), 6.65-6.80,
7.13-7.73 (2m, 7H, H-arom), 8.21 (s, 1H, H-NH-pyrazole); MS
(FD) m/z (%) 619 (M+); Rf 0.31 in n-pentane/EtOAc 2:1.
50:1). Yield 77 mg (95 µmol, 95%); colorless solid. 1H NMR (400
MHz, DMSO-d6) δ [ppm] 2.87-3.10 (m, 4H, CH2-Phe), 3.59
(s, 3H, CH3), 3.70 (s, 3H, CH3), 3.71 (s, 3H, CH3), 4.51-4.57
(m, 1H, RH), 4.74-4.79 (m, 1H, RH), 5.40 (d, 1H, 2J ) 14.4
Hz, CHR-PMB), 5.54 (d, 1H, 2J ) 14.4 Hz, CHâ-PMB), 5.82
(s, 2H, CH2), 6.79 (d, 3J ) 8.5 Hz, 2H, CH-PMB), 6.89 (d, 3J )
8.8 Hz, 2H, CH-PMB), 6.91 (d, 3J ) 8.5 Hz, 2H, CH-PMB),
7.15-7.32 (m, 12H, H-arom), 7.38 (s, 1H, CH-pyrazole), 7.91
3
3
3
(s, 1H, CH-pyrazole), 8.56 (d, J ) 7.5 Hz, 1H, NH), 8.65 (d,
3J ) 8.8 Hz, 1H, NH), 11.45 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ [ppm] 36.6, 36.7, 51.9, 52.7, 53.7, 53.9, 55.0, 55.1,
55.2, 99.9, 105.1, 133.7, 114.1, 126.3, 126.5, 126.6, 128.0, 128.1,
128.3, 129.1, 129.3, 129.5, 134.8, 136.8, 137.1, 138.1, 144.8,
153.6, 155.5, 158.6, 158.8, 159.1, 171.2, 171.8; MS (ESI) m/z
(%) 837 (M+ + Na); mp 215 °C.
NO2-P z(H )-L-Va l-P z(H )-OMe (16). Under inert atmo-
sphere 45 mg (73 µmol, 1.00 equiv) of NO2-Pz(PMB)-L-Val-Pz-
(PMB)-OMe (16c) was heated in 10 mL of anhydrous TFA for
4 h to 70 °C. The solvent was evaporated and the residue was
purified by chromatography on silica gel column (eluent CH2-
Cl2/CH3OH 30:2). Yield 90% (26 mg, 66 µmol); colorless solid.
NO2-P z-P z-L-P h e-L-P h e-OMe (18). In an argon atmo-
sphere 20 mg (24.5 µmol, equiv) of NO2-Pz(PMB)-Pz(PMB)-L-
Phe-L-Phe-OMe (18a ) was heated in 2 mL of anhydrous TFA
for 2 h to 70 °C. The solvent was cooled to room temperature
and an excess of an ice-cold mixture of diethyl ether and
n-pentane (1:1) was added. The product precipitated as a
colorless solid, which was filtered off, washed several times
with n-pentane, and dried in vacuo. Yield 10 mg (12.4 µmol,
3
1H NMR (300 MHz, DMSO-d6) δ [ppm] 1.03 (d, J ) 6.3 Hz,
3
3H, CH3-Val R), 1.05 (d, J ) 6.3 Hz, 3H, CH3-Val â), 2.25-
2.35 (m, 1H, CH-i-Pr), 3.92 (s, 3H, OCH3), 4.19 (m, 1H, R-H-
Val), 7.09 (s, 1H, CH-pyrazole), 8.00 (s, 1H, H-CH-pyrazole),
8.93 (d, 3J ) 8.6 Hz, 1H, NH-Val), 10.98 (s, 1H, amid NH-
pyrazole), 13.72, 14.86 (2s, 2H, H-NH-pyrazole; 13C NMR (75
MHz, DMSO-d6) δ [ppm] 18.6, 18.9, 30.1, 51.8, 59.7, 99.4,
102.2, 113.9, 129.5, 139.7, 129.5, 138.7, 155.7, 157.3, 169.1;
MS (FD) m/z 379 (M+); HRMS (ESI) calcd for C14H18N7O6
380.1319, found 380.1349; mp 182 °C.
1
51%); colorless solid. H NMR (300 MHz, DMSO-d6) δ [ppm]
2.92-3.10 (m, 4H, CH2-Phe), 3.59 (s, 3H, CH3), 4.49-4.56 (m,
1H, RCH), 4.71-4.75 (m, 1H, RCH), 7.15-7.37 (m, 11H, CH-
arom), 7.93 (s, 1H, CH-pyrazole), 8.61 (d, 3J ) 7.3 Hz, 1H, NH),
Ac-Gly-Gly-P z(P MB)-P z(P MB)-OMe (17a ). In an argon
atmosphere 130 mg (265 µmol, 1.00 equiv) of 3-{[5-Amino-2-
(4-methoxy-benzyl)-2H-pyrazole-3-carbonyl]amino}-2-(4-meth-
oxybenzyl)-2H-pyrazole-3-carbonylic acid methylester (8),6b 92
mg (530 µmol, 2.00 equiv) of N-Acetyl-glycinyl-glycine, 138 mL
(795 µmol, 3.00 equiv) of DIEA, and 75 mg (291 µmol, 1.10
equiv) of 2-chlor-1-methylpyridiniumiodid were stirred for 2
h. The organic layer was washed with satd aq NaHCO3, 1 M
HCl, and satd aq NaCl and dried over Na2SO4. After filtration
the solution was concentrated and purified by chromatography
over silica gel (eluent CH2Cl2/CH3OH 20:1). Yield 64% (110
3
8.80 (d, J ) 8.9 Hz, 1H, NH), 11.38 (s, 1H, NH), 13.2 (s, 1H,
NH), 14.9 (s, 1H, NH); mp 140 °C; MS (ESI) m/z (%) 613 (M+
+ Na).
N-4-Meth oxyben zyl-3-(9-flu or en ylm eth oxyca r bon yl)-
a m in op yr a zole-5-ca r boxylic Acid (F m oc-P z(P MB)-OH)
(19). In a Schlenck tube a solution of 910 mg (3.28 mmol) of
N-4-methoxybenzyl-3-nitropyrazole-5-carboxylic acid methyl
ester in 4 mL of anhydrous DMF was treated with 65 mg of
Pd/C (10%). The mixture was subsequently transferred into
an autoclave and stirred at a hydrogen pressure of 5 bar for 4
h at room temperature. After filtration over Celite at 0 °C,
1.26 mL (959 mg, 7.42 mmol) of N,N-diisopropylethylamine
and 891 mg (3.44 mmol) of 9-(fluorenylmethyl)chloroformiate
were added. The resulting solution was stirred for 5 h at room
temperature, diluted with 4 mL of water, acidified with 1 N
HCl (pH ∼1) and extracted with Et2O (3 × 5 mL). The
combined organic layers were washed with water and dried
over Na2SO4. The solvent was removed in vacuo and the crude
product was purified over silica gel (eluent CH2Cl2:MeOH:NH3
100:10:1, Rf 0.2), furnishing pure 5 as a colorless solid (784
mg, 1.67 mmol, 51%). Mp 236 °C; IR (KBr) νj [cm-1] 3424, 3263,
3100, 2930, 1733, 1586, 1513, 1249; UV/vis (CH3CN) λmax [nm]
(log ꢀ) 208 (4.51), 265 (4.03), 275 (sh, 3.88), 289 (3.45), 300
(3.44); 1H NMR (200 MHz, DMSO-d6, TMS) δ [ppm] 3.71 (s, 3
H, CH3), 4.20-4.36 (m, 3 H, CH2-Fmoc, CHFmoc), 5.63 (s, 2 H,
CH2Bzl), 6.62 (br s, 1 H, CHPyraz.), 6.82-6.89 (m, 2 H, CHarom),
7.14-7.22 (m, 2 H, CHarom), 7.28-7.47 (m, 4 H, CHFmoc), 7.78
(d, 2 H, 3J ) 7.1 Hz, CHarom), 7.90 (d, 2 H, 3J ) 7.1 Hz, CHarom),
10.19 (br s, 1 H, NH); 13C NMR (62 MHz, DMSO-d6, TMS) δ
[ppm] 46.4 (+), 52.0 (-), 55.0 (+), 66.0 (-), 99.4 (+), 113.6 (+),
120.0 (+), 125.4 (+), 127.0 (+), 127.6 (+), 128.8 (+), 130.6
(Cquart), 138.6 (Cquart), 140.6 (Cquart), 143.7 (Cquart), 145.4 (Cquart),
153.4 (Cquart), 158.4 (Cquart), 162.1 (Cquart); MS (FAB, glycerin/
MeOH) m/z (%) 470 (100) [MH]+; HRMS (C27H23N3O5) calcd
470.1716 [MH]+, found 470.1708 [MH]+ (1.32 ppm. Anal.
1
mg, 170 mmol); colorless solid. H NMR (300 MHz, CDCl3) δ
[ppm] 1.82 (s, 3H, CH3-acetyl), 3.69, 3.72 (2s, 6H, OCH3-PMB),
3.86 (br s, 5H, OCH3, CH2-Gly), 4.03 (s, 2H, CH2-Gly), 5.56,
5.59 (2s, 4H, CH2-Bz), 6.75 (d, 3J ) 8.6 Hz, 2H, CH-arom),
3
3
6.75 (d, J ) 8.6 Hz, 2H, CH-arom), 6.75 (d, J ) 7.9 Hz, 2H,
CH-arom), 7.22 (br s, 1H, NH), 7.34 (s, 1H, NH), 9.27, 9.58
(2s, 2H, NH); 13C NMR (100 MHz, CDCl3) δ [ppm] 22.1, 43.5,
43.6, 52.2, 54.1, 55.3, 103.6, 114.1, 114.2, 128.7, 129.2, 129.6,
129.7, 132.3, 145.2, 159.4, 159.5, 159.5, 159.6; MS (FD) m/z
(%) 669 (M+ + Na); mp 122 °C.
Ac-Gly-Gly-P z(H)-P z(H)-OMe (17). In an argon atmo-
sphere 100 mg (154 µmol, 1.00 equiv) of Ac-Gly-Gly-Pz(PMB)-
Pz(PMB)-OMe (17a ) was heated in 10 mL of anhydrous TFA
for 4 h to 70 °C. The solvent was evaporated and the residue
was washed several times with a mixture of n-pentane/diethyl
ether (1:1) and finally with satd aq NaHCO3. The product was
dried in vacuo. Yield 60% (50 mg, 92 µmol), pale yellow solid.
1H NMR (300 MHz, DMSO-d6) δ [ppm] 1.87 (s, 3H, CH3-acetyl),
3.73 (m, 2H, CH2-Gly), 3.84 (s, 3H, CH3), 3.89 (d, 2H, CH2-
Gly), 7.02 (s, 1H, CH-pyrazole), 7.13 (s, 1H, CH-pyrazole),
8.14-8.20 (m, 2H, NH-gly), 10.48, 11.12 (2brs, 2H, amidNH),
13.31, 13.77 (2 br s, 2H, NH-pyrazole); 13C NMR (100 MHz,
DMSO-d6) δ [ppm] 22.4, 52.18, 53.2, 55.1, 113.5, 113.8, 128.7,
128.8, 129.6, 145.6, 157.2, 159.7, 159.3, 168.5, 169.5; MS (FD)
m/z (%) 429 (M+ + Na); mp 200 °C dec.
NO2-P z(P MB)-P z(P MB)-L-P h e-L-P h e-OMe (18a ). NO2-
Pz(PMB)-Pz(PMB)-OH (10) (50.6 mg,100 µmol, 1.00 equiv), 48
mg (110 µmol, 1.10 equiv) of H-Phe-Phe-OMe‚TFA, 31 mg (120
µmol, 1.20 equiv) of 3-chloro-1-methylpyridinium iodide, and
55 µL (300 µmol, 3.00 equiv) of DIEA were stirred in dry
dichloromethane overnight. The organic layer was washed
with satd aq NaHCO3, 1 M HCl, and satd aq NaCl and dried
over Na2SO4. After filtration the solution was evaporated and
the residue dried in vacuo. The residue was purified by
chromatography on silica gel column (eluent CH2Cl2/2-propanol
Calcd for C27H23N3O5: C 69.07; H 4.94, N 8.95. Found:
68.93; H 4.88; N 8.64.
C
Gen er a l P r oced u r e for th e Solid -P h a se P ep tid e Syn -
th esis (F m oc Str a tegy). In the first step, the Fmoc-protected
amino acid was coupled to the Sieber amide resin. To this end,
the Fmoc group was cleaved off the resin by 20% piperidine
in NMP (30 min, 2 runs). The first coupling step was performed
twice with 5 equiv of Fmoc-protected amino acid, 5 equiv of
HOBt, and 5 equiv of DIPCDI in NMP (6 h, maximum loading
∼0.2 mmol/g of resin, determined by gravimetry). All reagents
J . Org. Chem, Vol. 69, No. 16, 2004 5177

Rzepecki et al.
were added in quantities of 8 mL/g of resin. Deprotection of
the resin-bound first amino acid was again effected with 20%
piperidine in NMP (20 min, 2 runs). The next Fmoc-protected
amino acid (3 equiv) was coupled twice with 3.3 equiv of
HATU, 3.3 equiv of HOAt, and 30 equiv of collidine in NMP
or alternatively 3.3 equiv of TBTU, 3.3 equiv of HOBt, and
8.4 equiv of DIEA in NMP (7 h each run), followed by a double
deprotection step with 20% piperidine in NMP (30 min, 2
runs).25 After the last amino acid was attached, the free
N-terminal amino group was acetylated by treating 1.00 g of
the resin (loading ∼0.2 mmol/g) twice with a solution of 190
µL (2.0 mmol, 10 equiv) of Ac2O and 68 µL (0.2 mmol, 1 equiv)
of DIEA in 3 mL of CH2Cl2 (30 min, 2 runs). Finally the solvent
was evaporated and the resin-bound peptide was triply washed
with CH2Cl2. The Sieber amide resin was subsequently
transferred into a PE syringe with integrated filterplate and
swollen for 10 min in CH2Cl2. Then TFA in CH2Cl2 (2% v/v, 8
mL/g of resin) was added and the solution shaken for 5 min.
The peptide cleavage procedure was repeated 8-10 times and
the combined filtrates were evaporated to dryness on a rotatory
evaporater. The resulting solid was dissolved in a little CH2-
Cl2, sonicated intensively, and precipitated again by addition
of Et2O. After filtration the crude product was dried in vacuo
and purified by column chromatography or semipreparative
HPLC. For the final PMB removal, the PMB-protected oligoa-
mide was dissolved in a small amount of dry TFA and heated
under argon within 20 min to 70-72 °C. The reaction mixture
was kept for 5 min at this temperature; then the solvent was
removed in vacuo and the remaining solid was dissolved in a
small amount of dichloromethane, sonicated, and precipitated
again by addition of ether. The crude product was collected
by filtration, dried in high vacuum, and purified by HPLC.
The combined fractions were evaporated to dryness and
lyophilized. To liberate the free pyrazole amines, the resulting
solid was suspended in saturated aqueous NaHCO3, sonicated
for 10 min, filtered, and dried. The colorless solids were usually
spectroscopically pure.
purified by column chromatography over silica gel (eluent CH2-
Cl2:MeOH 10:1, Rf 0.32). Yield 124 mg (0.17 mmol, 91%) of a
colorless solid. Mp 185 °C; [R]20 +19.4 (c 0.07 in DMSO); IR
D
(KBr) νj [cm-1] 3414, 3298, 2963, 2930, 1668, 1577, 1514, 1250;
UV/vis (CH3CN) λmax [nm] (log ꢀ) 222 (4.72), 271 (sh, 4.13); 1H
NMR (400 MHz, DMSO-d6, TMS) δ [ppm] 0.85-0.93 (m, 12
H, CH3-i-Pr), 2.00 (s, 3 H, CH3-acetyl), 2.02-2.20 (m, 2 H, CH-
i-Pr), 3.69 (s, 3 H, CH3), 3.71 (s, 3 H, CH3), 4.28 (dd, 1 H, 3J )
3
3
3
8.7 Hz, J ) 7.3 Hz, R-CH), 4.34 (dd, 1 H, J ) 8.5 Hz, J )
7.3 Hz, R-CH), 5.45-5.65 (m, 4 H, CH2-PMB), 6.79-6.89 (m,
4 H, CH-arom PMB), 7.12-7.18 (m, 5 H, CH-arom pMB, CH-
pyrazole), 7.26 (s, 1 H, CH-pyrazole), 7.51 (br s, 1 H, NH2),
3
7.97 (d, 1 H, J ) 8.5 Hz, NH), 8.06 (br s, 1 H, NH), 8.63 (d, 1
H, J ) 8.7 Hz, NH), 10.58 (s, 1 H, NH), 10.67 (s, 1 H, NH);
3
13C NMR (62 MHz, DMSO-d6, TMS) δ [ppm] 18.1 (+), 19.0 (+),
19.2 (+), 23.0 (+), 29.5 (+), 30.6 (+), 52.5 (-), 52.5 (-), 55.0
(+), 55.0 (+), 57.8 (+), 59.0 (+), 98.7 (+), 99.1 (+), 113.6 (+),
113.7 (+), 128.9 (+), 129.0 (+), 129.7 (Cquart), 129.9 (Cquart),
134.6 (Cquart), 134.7 (Cquart), 145.3 (Cquart), 145.9 (Cquart), 158.5
(Cquart), 158.6 (Cquart), 159.2 (Cquart), 160.8 (Cquart), 167.5 (Cquart),
169.2 (Cquart), 170.7 (Cquart); MS (FAB, glycerol/DMSO) m/z (%)
716 (100) [MH]+; HRMS (C36H45N9O7) calcd 716.3520 [MH]+,
found 716.3528 [MH]+ (1.23 ppm.
Ac-Val-P z(H)-P z(H)-Val-NH2 (21a). From Ac-Val-Pz(PMB)-
Pz(PMB)-Val-NH2 (13a ): 100 mg (0.14 mmol). The crude
product was purified by semipreparative HPLC. A colorless
solid (47 mg) was obtained (0.11 mmol, 71%). Mp:188 °C; [R]20
D
+38.8 (c 0.07 in DMSO); IR (KBr) νj [cm-1] 3402, 2967, 1664,
1600, 1541; UV/vis (CH3CN) λmax [nm] (log ꢀ) 222 (4.56); 1H
NMR (400 MHz, DMSO-d6, TMS) δ [ppm] 0.87-0.94 (m, 12
H, CH3-i-Pr), 1.90 (s, 3 H, CH3-acetyl), 1.96-2.12 (m, 2 H, CH-
i-Pr), 4.25-4.35 (m, 2 H, R-CH), 7.07-7.36 (br, 3 H, 2 NH,
CH-pyrazole), 7.52 (br s, 1 H, CH-pyrazole), 8.01 (d, 1 H,3J )
8.5 Hz, NH), 8.05-8.27 (br s, 1 H, NH), 10.60 (br s, 1 H, NH),
10.92 (s, 1 H, NH), 12.60-13.80 (br, 2 H, 2 NH-pyrazole); 13
C
NMR (100 MHz, DMSO-d6, TMS) δ [ppm] 18.3 (+), 19.1 (+),
19.3 (+), 22.4 (+), 30.3 (+), 57.6 (+), 58.0 (+), 97.0 (+), 137.0
(Cquart), 145.9 (Cquart), 156.9 (Cquart), 156.2 (Cquart), 169.4 (Cquart),
169.8 (Cquart), 172.7 (Cquart); MS (FAB, glycerin/DMSO) m/z (%)
476 (38) [MH]+; HRMS (C20H29N9O5) calcd 476.2370 [MH]+,
found 476.2366 [MH]+ (1.48 ppm.
Ac-Va l-P z(P MB)-P z(P MB)-Va l-NH2 (20a ). Fmoc-Sieber
amide resin: 1.00 g (0.20 mmol/g); first loading, Fmoc-Pz-
(PMB)-Sieber amide resin: 0.19 mmol/g. The product was
purified by column chromatography over silica gel (eluent CH2-
Cl2:MeOH 10:1, Rf 0.34). Yield 125 mg (0.18 mmol, 92%) of a
colorless solid. Mp 251 °C; [R]20 +26.7 (c 0.06 in DMSO); IR
D
(KBr) νj [cm-1] 3423, 2965, 1669, 1576, 1514, 1470, 1250; UV/
vis (CH3CN) λmax [nm] (log ꢀ) 227 (4.37), 271 (sh, 4.05); 1H NMR
(400 MHz, DMSO-d6, TMS) δ [ppm] 0.84-0.93 (m, 12 H, CH3-
i-Pr), 1.88 (s, 3 H, CH3-acetyl), 1.95-2.02 (m, 1 H, CH-i-Pr),
2.05-2.12 (m,1 H, CH-i-Pr), 3.70 (s, 3 H, CH3), 3.71 (s, 3 H,
CH3), 4.21 (dd, 1 H, 3J ) 8.6 Hz, 3J ) 7.0 Hz, R-CH), 4.29 (dd,
1 H, 3J ) 8.6 Hz, 3J ) 7.2 Hz, R-CH), 5.50-5.67 (m, 4 H, CH2-
PMB), 6.83-6.89 (m, 4 H, CH-arom PMB), 6.83-6.89 (m, 4
H, CH-arom PMB), 7.09 (br s, 1 H, NH), 7.13-7.20 (m, 4 H,
CH-arom PMB), 7.41 (s, 1 H, CH-pyrazole), 7.45 (br s, 1 H,
NH), 7.45 (br s, 1 H, NH), 7.46 (s, 1 H, CH-pyrazole), 7.96 (d,
1 H, J ) 8.6 Hz, NH), 8.47 (d, 1 H, J ) 8.6 Hz, NH), 10.59
(s, 1 H, NH), 11.18 (s, 1 H, NH); 13C NMR (62 MHz, DMSO-
d6, TMS) δ [ppm] 18.2 (+), 18.7 (+), 19.1 (+), 19.3 (+), 22.4
(+), 30.2 (+), 31.2 (+), 52.7 (-), 52.8 (-), 55.0 (+), 55.0 (+),
58.1 (+), 58.5 (+), 100.0 (+), 100.1 (+), 113.7 (+), 113.8 (+),
128.7 (+), 129.0 (+), 129.7 (Cquart), 129.7 (Cquart), 134.1 (Cquart),
134.8 (Cquart), 145.1 (Cquart), 145.5 (Cquart), 157.1 (Cquart), 158.6
(Cquart), 158.6 (Cquart), 159.1 (Cquart), 169.3 (Cquart), 169.8 (Cquart),
172.6 (Cquart); MS (FAB, glycerin/DMSO) m/z (%) 716 (100)
[MH]+; HRMS (C36H45N9O7) calcd 716.3520 [MH]+, found
716.3509 [MH]+ (1.21 ppm.
Ac-P z(H)-Va l-Va l-P z(H)-NH2 (21). From Ac-Pz(PMB)-Val-
Val-Pz(PMB)-NH2 (14a ): 100 mg (0.14 mmol). The oligoamide
14b was obtained as a colorless solid (54 mg, 0.17 mmol, 81%).
Mp 201 °C; [R]20 +34.3 (c 0.07 in DMSO); IR (KBr) νj [cm-1
]
D
3264, 2967, 1665, 1594, 1526; UV/vis (CH3CN) λmax [nm] (log
ꢀ) 222 (4.62); 1H NMR (400 MHz, DMSO-d6, TMS) δ [ppm]
0.86-0.94 (m, 12 H, CH3-Val), 1.98-2.13 (m, 5 H, 2H-i-Pr,
CH3), 4.29-4.41 (m, 2H, 2RH), 7.10 (s, 1 H, CH-pyrazole), 7.28
(s, 1 H, CH-pyrazol), 7.37 (s, NH), 7.90 (br, 2 H, 2NH), 8.44
(d, 1 H, J ) 8.1 Hz, NH), 10.43 (s, 1 H, NH), 10.49 (s, 1 H,
NH), 12.97 (s, 1 H, NH-pyrazole), 13.01 (s, 1 H, NH-pyrazole);
13C NMR (100 MHz, DMSO-d6, TMS) δ [ppm] 18.2 (+), 18.7
(+), 19.0 (+), 19.2 (+), 23.0 (+), 30.1 (+), 30.5 (+), 57.8 (+),
57.9 (+), 95.9 (+), 96.5 (+), 137.4 (Cquart), 146.3 (Cquart), 158.8
(Cquart), 160.4 (Cquart), 167.5 (Cquart), 169.1 (Cquart), 170.8 (Cquart);
MS (ESI, MeOH + 10 mmol of NH4Ac) m/z (%) 474 (100) [M]-;
HRMS (C20H29N9O5) calcd 476.2370 [MH]+, found 476.2362
[MH]+ (0.90 ppm.
3
3
Ack n ow led gm en t . We thank Clariant GmbH,
Frankfurt, Germany, for a generous donation of their
peptide coupling reagent T3P. This work was supported
by the Volkswagen-Stiftung. K.C. thanks the DAAD for
a stipend within the Internatiol Quality Network Me-
dicinal Chemistry (IQN-MC).
Ac-P z(P MB)-Va l-Va l-P z(P MB)-NH2 (20). Fmoc-Sieber
amide resin: 1.00 g (0.20 mmol/g); first loading, Fmoc-Pz-
(PMB)-Sieber amide resin: 0.19 mmol/g. The product was
(25) This method is used for the direct coupling of pyrazole-based
and proteinogenic amino acids. In the case of two aliphatic amino acids,
coupling times can be considerably shortened.
J O0496603
5178 J . Org. Chem., Vol. 69, No. 16, 2004