Synthesis of substituted pyrimidine hydrazine acids (PHA) and their use in peptide recognition

Article abstract of DOI:10.3987/COM-05-S(T)6

Substituted pyrimidine-hydrazine-acids (PHA) were prepared from orotic acid in five synthetic steps and high yields. Their geometry of hydrogen bond acceptor and donor sites make them suitable for the molecular recognition of peptide β-sheets. In non-protic solvents the PHA unit emits at around 420 nm after irradiation at 281 nm. The emission intensity decreases upon peptide binding and signals the binding event. Peptides consisting of PHAs and natural amino acids or a turn structure motif were prepared. The investigation of the intramolecular binding pattern by NMR spectroscopy revealed the expected interaction of the PHA and peptide β-sheet.

Full text of DOI:10.3987/COM-05-S(T)6

HETEROCYCLES, Vol. 67, No. 1, 2006
135
HETEROCYCLES, Vol. 67, No. 1, 2006, pp. 135 - 160. © The Japan Institute of Heterocyclic Chemistry
Received, 2nd March, 2005, Accepted, 15th April, 2005, Published online, 19th April, 2005. COM-05-S(T)6
SYNTHESIS OF SUBSTITUTED PYRIMIDINE HYDRAZINE
ACIDS
(PHA)
AND THEIR USE IN PEPTIDE
RECOGNITION
Stefan Miltschitzky, Veronika Michlova, Stefan Stadlbauer, and Burkhard
Koenig*
Department of Organic Chemistry, University of Regensburg,
Universitaetsstrasse 31, 93051 Regensburg, Germany
Dedicated to Prof. Barry Trost on the occasion of his 65th birthday
Abstract - Substituted pyrimidine-hydrazine-acids (PHA) were prepared from
orotic acid in five synthetic steps and high yields. Their geometry of hydrogen
bond acceptor and donor sites make them suitable for the molecular recognition
of peptide β-sheets. In non-protic solvents the PHA unit emits at around 420 nm
after irradiation at 281 nm. The emission intensity decreases upon peptide
binding and signals the binding event. Peptides consisting of PHAs and natural
amino acids or a turn structure motif were prepared. The investigation of the
intramolecular binding pattern by NMR spectroscopy revealed the expected
interaction of the PHA and peptide β-sheet.
Introduction
Interactions between peptide β-sheets are of importance for protein quaternary structure formation,
protein-protein interaction, and protein aggregation. They are involved in important biological processes
and the development of diseases - ranging from cancer and AIDS, to anthrax and Alzheimer´s.1,2 Small
synthetic molecules that mimic surface epitopes on proteins are a potential source of novel ligands for use
in drug and vaccine design.3 Over the last years peptidomimetics have attracted interest from both organic
and medicinal chemists. In the biological, chemical, and pharmaceutical areas they offer advantages over
physiologically active peptides, which as active substances are crucial for the organism and may lead to
severe side effects.4 There is much current interest in the preparation of peptides and peptidomimetics

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HETEROCYCLES, Vol. 67, No. 1, 2006
with a well-defined conformation.5 These conformationally constrained peptides are useful to probe
protein folding and to mimic peptide structures.
Since 1992, Nowick et al. have reported the development of peptidomimetic templates, which have been
termed molecular scaffolds.6 These templates have oligourea structure and reassemble, in some ways, β-
turns. Early in 1996, Nowick described an artificial antiparallel β-sheet (1), in which a 5-amino-2-
methoxybenzamide β-strand mimic (2) and a diurea molecular scaffold stabilize a β-sheet structure in an
attached peptide strand (Scheme 1).7
Me
NC
O
O
Me
O
H
N
N
N
H
Me
O
H
O
N
N
CH2
H
Bn
O
O
H
N
H2C
Me
N
N
N
β-strand mimic 2
H
H
iBu
Ph
O
artificial sheet 1
Scheme 1: Nowick´s artificial antiparallel β-sheet (1) and β-strand mimic (2)
Results and Discussions
We report in this paper the synthesis of substituted pyrimidine-hydrazine-acids (PHA), and their inter-
and intramolecular peptide binding properties as determined by NMR spectra and emission changes.
We envisioned 6-hydrazinopyrimidine-4-carboxylic acids as a building block for extended β-strand
mimics. Molecular modelling calculations on force field level (MMFF, program package Spartan)8 were
carried out to explore the conformational preferences of PHA. The calculations show that oligomers of
PHA adopt a linear conformation required to be complementary to peptide β-sheets.
The hydrazine provides the right pattern of hydrogen bond donor and acceptor geometry complementary
to extended peptide β-sheets and allows the formation of an intramolecular hydrogen bond. This should
keep the pyrimidine ring of PHA oligoamides in a planar arrangement.

HETEROCYCLES, Vol. 67, No. 1, 2006
137
Scheme 2: Low energy conformation of a PHA dimmer as calculated by force field method
Starting material for PHA derivatives is the commercial available orotic acid (3). The acid is converted
9
into the ester with methanol according to a literature procedure,9 followed by a halogenation with POCl3
or POBr3, respectively. The substitution of a halogen atom by Boc- or Cbz-monoprotected hydrazine
takes place selectively in 6-position. The yields of the substitution reaction are typically between 60 and
95 %.
O
O
POX3
1. SOCl2/DMF
2. MeOH
X = Cl, Br
HN
NH
HN
NH
HO
MeO
95 %
68 - 72 %
O
O
O
O
4
2
3
X
X
H2N-NH-PG
NEt3
N
N
H
N
N
N
MeO
MeO
PG
N
H
X
60 - 78%
O
O
5a X = Cl
5b X = Br
6a X = Cl, PG = Boc
6b X = Cl, PG = Cbz
6c X = Br, PG = Boc
6d X = Br, PG = Cbz
Scheme 3: Synthesis of the PHA parent structure
The selective substitution of the chlorine and bromine was investigated by Yamanaka et al. in detail.10
Computational and experimental studies both showed a dependency on the substituent in 4-position.
Acceptors, such as the methyl ester guide substitution in 6-position, while a methoxy groups yields
selective substitution in 2-position. The X-Ray structure analysis of 6a and 6b and the HMBC spectrum
of 6a confirm the expected substitution pattern.

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HETEROCYCLES, Vol. 67, No. 1, 2006
Scheme 4: Structures of 6a (left) and 6b (right) in the crystal
The HMBC spectra of an 8 mM solution in deuterated DMSO at room temperature of 6a shows a sharp
cross peak between the protons on hydrazine nitrogen NX and on carbon CH1, supporting the structure of
the expected compound. The double set of signals arises from the cis- and trans-isomers of the Boc
protecting group.11
CH1
NH3
NH2
CH1
100
110
120
130
140
150
160
170
Cl
N
N
H3
MeO
N
Boc
N
H2
O
H1
10.0
9.0
8.0
7.0
6.0
Scheme 5: HMBC-spectrum of 6a in deuterated DMSO.
However, the X-Ray structure of 6a does not show the assumed intramolecular hydrogen bond of the
1
proton on hydrazine nitrogen and the pyrimidin nitrogen. The temperature dependence of the H NMR
chemical shift of the NH2 proton (-5.15 ppb/K) in 10 mmol solution CDCl3 reflects its state of hydrogen
bonding.
The course of the subsequent substitution in 2-position with N-nucleophiles depends on the halogen atom.
Chlorine substitution occurs only when activated with DMAP in situ.

HETEROCYCLES, Vol. 67, No. 1, 2006
139
NMe2
R
N
R
N
Cl
N+
Cl
N
HNR1R2
base
N
N
N
N
DMAP
H
H
H
MeO
N
MeO
N
MeO
N
PG
Pg
PG
N
N
N
H
H
H
O
O
O
6a or 6d
7
8
Scheme 6: Proposed mechanism of the substitution of 2-chlorine atoms by N-nucleophiles
The DMAP displaces the chlorine atom as a nucleophile forming the charged intermediate (7). The amine
attacks in the subsequent step the more electrophilic carbon C2 under basic conditions. The amine is
nucleophile and base, but an excess of 10 equivalents is used. In case of NEt3 as an auxiliary base, still
5 equivalents of the nucleophile are required. Although the second step of the reaction releases the
DMAP, stoichiometric amounts are required to achieve good yields. The highest yields were obtained in
the presence of 1.2 equivalents of DMAP, 2 equivalents of NEt3 and 8 equivalents of amine (see
EXPERIMENTAL). The bromine derivative is, as expected, more reactive than the chlorine substituted
compound. No activation or addition of base is required for the substitution. In the presence of DMAP
and NEt3 however, complete conversion requires a 5-fold excess of amine. Best yields were obtained by
using 2 equivalents of NEt3 and a 10-fold excess of amine (see EXPERIMENTAL). Table 1 summarizes the
different compounds either derived from 6a (method A) or 6c (method B). In general, the more
nucleophilic secondary amines give higher yields than primary amines. The hydrazine protecting group
(PG) does not influence the yield of the substitution reaction.
*
PHA Amine
PG
Method
Yield (corrected ) [%]
8a
8a
8b
8c
HNEt2
HNEt2
HNEt2
H2NBu
Boc
Boc
Cbz
Boc
A
B
B
B
42
74
80
13 (26)
*
Yield of the reaction adjust to conversion in brackets
Table 1: Synthesized derivatives (8)
To avoid a side reaction by aminolysis of the ester, 4 was converted into the butylamide (9) according to a
literature procedure12 followed by bromination and subsequent substitution in 6- and 2-position with Boc-

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HETEROCYCLES, Vol. 67, No. 1, 2006
protected hydrazine and amines. The amidic hydrogen atom allows the formation of another
intramolecular hydrogen bond to the pyrimidine nitrogen. Table 2 summarizes the results of the Br-
substitution in 2-position of compound (11) with several amines.
O
O
H2NBu
POBr3
72 %
HN
NH
HN
NH
H
MeO
N
Bu
O
O
O
O
9
4
Br
Br
H2N-NH-Boc
NEt3
N
N
N
N
H
H
H
N
N
N
Boc
N
82 %
Bu
Bu
Br
O
O
H
10
11
R1
N
R2
HNR1R2
NEt3
N
N
H
N
H
N
Bu
Boc
N
H
O
12
Scheme 7: Synthesis of compounds (12)
*
Entry
Amine
Yield (corrected ) [%]
12a
12b
12c
12d
12e
H2NBu
HNEt2
68
82
Morpholine
67
18-aza-crown-6
56
2-(2-Methoxy-ethoxy)-ethylamine
33 (68)
*
Reaction yield adjust to conversion given in brackets
Table 2: Synthesized derivatives (12)
A more extended peptide (15) was prepared to show the complementary structure and binding affinity of
PHA oligomers to peptide β-sheet structures. Cleaving off the ester and the Boc-group of 8a under basic

HETEROCYCLES, Vol. 67, No. 1, 2006
141
and acidic conditions gives the acid (13) and amine (14), respectively. A coupling of (13) and (14) under
standard peptide coupling conditions yields dipeptide (15) in moderate yield.13
NEt2
N
N
H
MeO
N
Boc
N
H
O
8a
HCl.Et2O
quant.
LiOH
94 %
NEt2
NEt2
N
N
N
N
H
Li+
.2HCl
MeO
NH2
O
N
+
Boc
N
N
H
H
O
O
HBTU
HOAt
DIEA
DMF
13
14
40 %
NEt2
NEt2
N
N
N
N
H
H
N
MeO
N
Boc
N
N
H
H
O
O
15
Scheme 8: Synthesis of dipeptide (15)
All synthesized PHA derivatives appear as mixtures of cis- and trans-isomers of the carbamate protecting
1
groups in the H NMR spectra. The pyrimidine ring and the hydrazine protons do not show
tautomerisation. Compounds (8, 12 and 15) emit around 420 nm when irradiated at 281 nm. The
halogenated compounds (5, 6, 10, and 11) have no luminescence. The emission intensity of solutions of
the compounds decreases from less polar (e.g. CHCl3) to polar aprotic solvents (e.g. MeCN). Polar protic
solvents, such as H2O, quench the emission completely. The intensity of emission changes with hydrogen
bonds to PHA. Therefore, fluorescence titration is a tool to monitor the binding process between PHA and
β-sheet peptides in aprotic solvents.
The mono PHA (8a) provides three binding sites as acceptor-donor-acceptor (ADA) motif, while bis
PHA (15) can form up to five (ADADA) intermolecular hydrogen bonds with a complementary binding
partner. Top and bottom face of an N- and C-terminal protected tripeptide differ in their hydrogen bond

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HETEROCYCLES, Vol. 67, No. 1, 2006
donor and acceptor pattern. While the top face can form four hydrogen bonds (DADA), the bottom face
has only three (ADA). An N- and C-terminally protected tetrapeptide offers five hydrogen binding sites
(ADADA) at the top face and four (DADA) on the bottom face.
The binding affinities of the PHA derivatives (8a) and (15) to small peptides of natural amino acids were
determined by 1H NMR spectra and fluorescence titrations in CDCl3 and CHCl3. Binding constants were
determined by non-linear fitting of chemical induced shifts (CIS) of several protons14 in the NMR
spectrum and of the decrease of emission intensity. Self-associations of the peptides and the PHA (8a
K11 = 17 L/mol, 15 K11 = 28 L/mol) were determined independently and were taken into account.
Emission intensities corrected for dilution during the titration are used. The stoichiometry of the
complexes was determined according to Job´s method of continuous variations.15 Table 3 summarizes the
results of our binding studies.
Entry
PHA
8a
Peptide
Stoichiometry Ka [L/mol]a
1
2
3
Ac-Phe-Val-Leu-OMe
Boc-Phe-Ala-Val-Leu-OMe
Boc-Phe-Gly-Val-Leu-OMe
1:1
1:1
2:1
101
78
15
15
2742
15381
aAll binding constants have errors of approx. ± 10 %.
Table 3: Ka values determined from NMR and fluorescence titrations in CDCl3 and CHCl3
The affinity of 8a to the tripeptide is 101 L/mol. Figure 3 shows the CIS of the acetyl singlet during
titration. By formation of three cooperative hydrogen bonds with PHA 8a the binding of the peptide´s top
face should be favoured (see Figure 1).
NEt2
N
NEt2
N
N
N
H
H
N
H
H
N
O
N
N
O
N
H
N
H
O
O
O
O
O
O
Bn
O
O
H
N
H
N
H
N
MeO
MeO
N
H
N
N
H
H
O
Bn
O
O
O
top face (favored)
bottom face (disfavored)
Figure 1: A 1:1 complex between 8a and Ac-Phe-Val-Leu-OMe

HETEROCYCLES, Vol. 67, No. 1, 2006
143
The binding affinity determined for 15 to the tetrapeptide Boc-Phe-Ala-Val-Leu-OMe is in the same
order of magnitude as the binding of 8a to a tripeptide, although an additional hydrogen bond formation
should be possible between 15 and the tetrapeptide. Figure 2 summarizes the possible 1:1 binding
motifs between 15 and Boc-Phe-Ala-Val-Leu-OMe.
NEt2
N
NEt2
N
NEt2
N
NEt2
N
N
N
N
N
H
H
H
H
MeO
N
O
N
MeO
N
N
O
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
Bn
O
O
O
O
Bn
H
N
H
N
H
N
H
N
top face
Boc
Boc
N
N
H
O
O
N
H
N
H
H
O
O
NEt2
N
NEt2
N
NEt2
N
NEt2
N
N
N
N
N
H
H
H
H
MeO
N
O
N
MeO
Boc
N
N
O
N
H
N
H
N
N
H
H
O
O
O
O
O
O
O
bottom face
O
O
O
O
H
H
H
H
O
N
N
N
N
N
H
N
H
Boc
N
N
H
H
O
O
Bn
Bn
O
O
Figure 2: Possible 1:1 complexes between 15 and Boc-Phe-Ala-Val-Leu-OMe
For steric reasons, the binding motifs containing Boc protecting group on different ends might be
favoured. The observed CIS and the calculated binding affinity result from an average value of the
experiment. The different aggregates are presumably in a fast dynamic equilibrium and NMR cannot
distinguish between them. All efforts to co-crystallize 8a and 15 with a peptide giving a picture of the
binding motif failed so far.
Figures 3 and 5 illustrate that a plateau in CIS or a minimum of emission is reached after the addition of 1
equivalent of the peptide to a solution of PHA. The 1H NMR Job´s plot of 8a and Ac-Phe-Val-Leu-OMe
(Figure 4) shows a maximum at x = 0.5, confirming a 1:1 binding motif.

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HETEROCYCLES, Vol. 67, No. 1, 2006
2.5
2
500
400
1.5
1
300
Emission PHA
calculated
Ac
calculated
200
100
0
0.5
0
0.00
2.00
4.00
6.00
8.00
0
5
10
15
20
25
Equivalents Ac-Phe-Val-Leu-OMe
Equivalents 8a
Figure 3: NMR titration curve (left) and fluorescence titration curve (right) of Ac-Phe-Val-Leu-OMe with
compound (8a) in CDCl3, c0 = 10 mmol/L, observing CIS of the acetyl hydrogen resonance and the
emission intensity at 423 nm (excitation at 280 nm)
80
60
40
20
0
0.00
0.20
0.40
0.60
0.80
1.00
x (8a)
Figure 4: 1H NMR job-plot of 8a and Ac-Phe-Val-Leu-OMe
50.00
40.00
30.00
calculated
pyrimidine H
20.00
10.00
0.00
0
0.5
1
1.5
2
Equivalents peptide
Figure 5: NMR spectral titration curve of Boc-Phe-Ala-Val-Leu-OMe with compound (15) in CDCl3,
c0 = 6.0 mmol/L, monitoring CIS of the pyrimidine hydrogen resonance.

HETEROCYCLES, Vol. 67, No. 1, 2006
145
The titration of 15 with Boc-Phe-Gly-Val-Leu-OMe gives a different picture. A slight chance in the
peptide sequence from Ala to Gly in position 2 leads to a significant increase of the binding affinity and
change of the aggregate stoichiometry. The NMR spectra titration curve indicates two subsequent binding
processes.
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
80
60
40
20
0
OMe
calculated
0.00
0.20
0.40
0.60
0.80
1.00
0
0.5
1
1.5
2
2.5
3
3.5
4
x (15)
Equivalents pep
Figure 6: NMR spectral titration curve (left; CIS of the methyl ester group) and emission Job´s plot
analysis (right; emission intensity at 423 nm,excitation at 280 nm)) of Ac-Phe-Val-Leu-OMe with
compound (15) in CDCl3, c0 = 9.5 mmol/L.
The first part of the NMR titration curve is identical in shape to the titrations of 8a with the tripeptide
Boc-Phe-Val-Leu-OMe or 15 with the tetrapeptide Boc-Phe-Ala-Val-Leu-OMe, respectively. Addition of
one equivalent 15 to Boc-Phe-Gly-Val-Leu-OMe saturates the CIS. Addition of a second equivalent of
15 leads to a further CIS of the methoxy resonance signal. For the first binding process an affinity
constant of K11 = 2742 L/mol is derived, while the subsequent binding with K12 of 15381 L/mol indicates
a highly cooperative assembly process. We cannot provide a structure for the complex assembly, but the
exchange of alanine for glycine in the tripeptide sequence allows almost free rotation around the C-C and
the C-N bond in the glycine residue.16
To avoid higher aggregates and arrive at a more defined PHA – peptide interaction a short β-turn
fragment incorporating PHA was prepared. Scheme 9 shows the synthesis. Extension of Gellman´s β-turn
fragment Gly-D-Pro17 by H-Phe-Leu-Val-OMe required 1.1 equivalents of EDC and HOBt and 2
equivalents of Huenig´s Base. The product precipitates from DMF solution after the addition of water.
Lithium salt (14) and the deprotected pentapeptide couple to 16 in moderate yields.

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HETEROCYCLES, Vol. 67, No. 1, 2006
EDC/HOBT
DIEA
DMF, rt, 27 h
Boc-Phe-OH
HCl.Et2O
quant.
+
H-Phe-Val-Leu-OMe .HCl
H-Val-Leu-OMe.TFA
Boc-Phe-Val-Leu-OMe
95 %
Boc-D-Pro-Gly-OH
EDC/HOBT
DIEA
HCl.Et2O
quant.
DMF, rt, 27 h
H-D-Pro-Gly-Phe-Val-Leu-OMe .HCl
Boc-D-Pro-Gly-Phe-Val-Leu-OMe
78 %
NEt2
N
N
H
N
Li+
O
O
O
H
N
Boc
14
N
O
O
H
N
H
N
H
O
O
N
NH
EDC/HOBT
DIEA
O
O
O
H
N
DMF, rt, 28 h
52 %
N
O
N
H
N
N
16
Scheme 9: Synthesis of the β-turn fragment (16)
16
14
12
10
8
8a
16
6
4
2
0
350
400
450
500
550
600
λ [nm]
Figure 7: Emission spectra of (8a) and (16) in a 8 mM solution in acetonitrile
Figure 7 shows the emission intensity of equally concentrated solutions of 8a and 16 (8 mM) in
acetonitrile. The significant decreased emission intensity of 16 in comparison to 8a indicates an intra-
molecular binding process of PHA and peptide.
NMR spectra in 10 mM deuterated DMSO solution show only one conformer for (16) in solution.
Analysis of the observed intramolecular NOE contacts in deuterated DMSO clearly confirm a β-turn
structure as depicted in Figure 8.

HETEROCYCLES, Vol. 67, No. 1, 2006
147
H
H
O
O
H
N
O
H
N
H
N
H
H
H
O
O
H
N H
O
O
O
H
N
N
H
O
N
N
N
N
Figure 8: NOE contacts found in the analysis of compound (16)
ROESY experiments revealed numerous short-range and long-range NOEs. The NOE contact between
the pyrimidine proton and the Val-i-Pr-residue indicates a folded conformation. Due to signal overlap and
broad NH signals no further NOE contacts of PHA and peptide are observable.18
A more extended β-turn fragment was prepared incorporating dipeptide (15). The β-turn fragment Gly-D-
Pro was extended by H-Phe-Leu-Phe-Ala-NH2 and coupling of the deprotected hexapeptide with the
lithium carboxylate (17) gave 18 in good yield.
EDC/HOBt
.
H-Ala-NH2 HCl
DIEA
+
DMF
TFA
.
Boc-Phe-OH
H-Phe-Ala-NH2 TFA
Boc-Phe-Ala-NH2
74 %
quant.
Boc-Phe-Leu-OH
EDC/HOBt
DIEA
DMF
TFA
quant.
.
Boc-Phe-Leu-Phe-Ala-NH2
H-Phe-Leu-Phe-Ala-NH2 TFA
82 %
Boc-D-Pro-Gly-OH
EDC/HOBt
DIEA
TFA
DMF
Boc-D-Pro-Gly-Phe-Leu-Phe-Ala-NH2
quant.
61 %
NEt2
NEt2
N
N
N
N
17
Boc
H
H
+ O
Li
N
N
N
N
H
H
O
O
.
H-D-Pro-Gly-Phe-Leu-Phe-Ala-NH2 TFA
EDC/HOBt/DIEA
DMF, 75 %

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HETEROCYCLES, Vol. 67, No. 1, 2006
O
O
O
H
N
H
N
H
O
N
H
N
H
N
H
O
N
O
N
NH
O
O
O
O
H
H
N
N
N
N
H
N
H
N
N
N
N
18
Scheme 10: Synthesis of the β-turn fragment (18)
Figure 9 shows the analysis of the intramolecular NOE contacts of compound (18) in deuterated DMSO
solution. The NOE contacts between the N-terminal PHA unit and the C-terminal amino acid residue
indicate that the interaction of heterocycles and peptide chain propagates the turn. The proton on the
hydrazine of the terminal PHA unit shows a contact to the methyl group and the Cα proton of alanine to
the CH2 group of the phenylalanine. In both cases, signal overlap prohibits distinction of the diasterotopic
protons of the CH2 group in the phenylalanine and leucine side chain. The NOE contact of a PHA proton
and the glycine amide proton confirms the turn structure. Overall, eight NOE contacts support a solution
structure of 18 as shown in Figure 9.
Ph
Ph
O
O
O
H
N
H
N
H
O
N
H
N
H
N
H
O
N
O
H
NH
O
aH
N
O
cH
N
O
O
N
N
bH
N
dH
N
N
N
N
N
Figure 9: NOE contacts found in the analysis of compound (18)

HETEROCYCLES, Vol. 67, No. 1, 2006
149
Conclusion
We have reported the synthesis of substituted pyrimidine-hydrazine-acids (PHA) from orotic acid.
Standard peptide coupling procedures couple or incorporate the heterocyclic hydrazine acids into peptides
of natural amino acids. Their geometry of hydrogen bond acceptor and donor sites make them suitable for
complementary interaction with peptide β-sheets. An interesting feature is their luminescence in non-
polar solvents, which changes upon peptide binding. PHA´s are simple dipeptide mimics, with a build in
luminescence chemosensor monitoring the local polarity of the environment and interaction with adjacent
peptide chains.
EXPERIMENTAL
General Methods: Melting points were determined on a Tottoli micromelting point apparatus and are
1
uncorrected. H-NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer at 300 K.
Chemical shifts (δ) are reported in ppm downfield from internal TMS. Emission spectra were recorded on
a Cary Eclipse spectrophotometer. Thin layer chromatographic (TLC) analyses were performed on silica
gel 60 F-254 with a 0.2 mm layer thickness. Preparative chromatography columns were packed with silica
gel Geduran SI 60. Mass spectra were recorded on a Finnigan MAT TSQ 7000 (ESI) and on a Finnigan
MAT 95 (HRMS).
X-Ray Structures: Crystallographic data for the structures (6a) and (6b) have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication numbers CCDC 267816 &
267817. Copies of the data can be obtained, free of charge, on application to CDC, 12 Union Road,
Cambridge CB2 1EZ, UK [fax: +44(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].
1H-NMR and Emission Titrations: A solution of the PHA molecule (c = 15 mmol/L) was added in
portions via a microsyringe to a solution of a peptide (c = 1 mmol/L). Volume and concentration changes
were taken into account for the analysis of the binding event.14
Job´s Plot: Equimolar solutions (0.1 mM) of PHA molecule and peptide were prepared and mixed in
various amounts. Emission spectra of the mixtures were recorded, and the emission intensities were
analyzed by Job´s method modified for emission results.15
General procedure for the substitution of a halogen atom by a protected hydrazine (GP1): To a
solution of the halogenated pyrimidine (1 mmol) in DMF (1 mL/mmol) was added triethylamine
(1.1 mmol), followed by a solution of the protected hydrazine (1.1 mmol) in DMF (2 mL/mmol). The
solution was stirred at 60 °C until complete conversion of the pyrimidine (12-24 h). The reaction mixture
was allowed to cool to rt, diluted with water, and extracted with ethyl acetate. The organic phases were

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HETEROCYCLES, Vol. 67, No. 1, 2006
dried over Na2SO4, evaporated, and then concentrated under reduced pressure. If necessary, the residue
was purified by column chromatography (mixtures of hexanes and ethyl acetate) to afford the products.
General procedure for the substitution of a halogen atom by an amine (GP2): To a solution of the
brominated pyrimidine (1 mmol) in DMF (2 mL/mmol) was added the amine (10 mmol). The solution
was stirred at 60 °C until complete conversion of the pyrimidine was observed (18-36 h). The reaction
mixture was allowed to cool to rt, diluted with ethyl acetate, and washed with brine. The organic phases
were dried over Na2SO4, evaporated, concentrated under reduced pressure and purified by column
chromatography (mixtures of hexanes and ethyl acetate) to afford the products.
2,6-Dibromopyrimidine-4-carboxylic acid methyl ester (5b): Orotic acid methyl ester (4) (0.4 g,
2.35 mmol) was dissolved in acetonitrile (5 mL) and POBr3 (3.37 g, 11.8 mmol) was added. The reaction
mixture was stirred for 3 h under reflux. The resulting yellow solution was allowed to cool to rt, poured
into 20 mL of crushed ice water, and extracted with ethyl acetate. The combined extracts were dried over
Mg2SO4, and evaporated off. The residue was recrystallized from ethyl acetate/hexanes (3:1) to give 5b as
brown crystalline needles (541 mg, 78 %).
mp: > 185 °C (decomp). IR (KBr disk): 3116 cm-1, 3069, 1729, 1549, 1517, 1439, 1314, 1233, 1103, 957,
1
13
784, 730. H-NMR (DMSO-d6): δ: 3.94 ppm (s, 3 H, OCH3), 8.33 (s, 1 H, PHA-H). C-NMR (DMSO-
d6): δ: 53.4 ppm (+; HMBC: OCH3), 124.8 (+; HMBC: PHA-H), 150.8 (Cquat), 155.0 (Cquat), 157.0 (Cquat),
161.8 (Cquat; HMBC: -CO-OCH3). UV (MeCN): λmax (log ε): 283 nm (4.664). MS m/z (%): 313.9
+
[M+NH4 ] (100), 296.9 [MH+] (15).
Methyl 6-(N'-tert-butoxycarbonylhydrazino)-2-chloropyrimidine-4-carboxylate (6a): Compound
(5a) (1.32 g, 6.38 mmol) was allowed to react with triethylamine (1.06 mL, 0.78 g, 7.73 mmol) and Boc-
hydrazine (1.02 g, 7.73 mmol) following GP1 (hexanes/ethyl acetate 2:1, Rf = 0.30) to yield 6a (1.17 g,
60 %) as a colorless powder.
mp: 151-152 °C. IR (KBr disk): 3264 cm-1, 3230, 3109, 2982, 1735, 1609, 1446, 1263, 1159, 993, 754.
1H-NMR (CDCl3): δ: 1.39-1.41 ppm (m, 9 H, Boc-CH3), 3.85 (s, 3 H, O-CH3), 6.61 (s, 1 H, NH), 7.33 (s,
1 H, PHA-H), 7.77 (br, 1 H, NH). 13C-NMR (CDCl3): δ: 28.1 ppm (+; HMQC: Boc-CH3), 53.4 (+;
HMQC: OCH3), 82.8 (Cquat; HMBC: Boc), 102.1 (+; HMQC: PHA-H); 155.1 (Cquat), 156.6 (Cquat), 160.5
(Cquat), 163.8 (Cquat; HMBC: C=O ester), 168.0 (Cquat). UV/VIS (MeCN): λmax (log ε): 244 nm (4.860),
+
301 (4.517). MS m/z (%): 622.1 [2M+NH4 ] (1), 605.2 [2M+H+] (2), 303.2 [MH+] (100), 247.1 [MH+-
C4H8] (8), 203.1 [MH+-Boc] (2). Anal. Calcd for C11H15N4O4Cl: C: 43.6; H: 5.0; N: 18.5; Found: C: 43.9;
H: 5.0; N: 17.9.
Methyl 6-(N'-benzyloxycarbonylhydrazino)-2-chloropyrimidine-4-carboxylate (6b): Compound
(5a) (228 mg, 1.10 mmol) was allowed to react with triethylamine (128 µL, 122 mg, 1.21 mmol) and

HETEROCYCLES, Vol. 67, No. 1, 2006
151
Cbz-hydrazine (201 mg, 1.21 mmol) following GP1 (hexanes/ethyl acetate 3:2, Rf = 0.25) to yield 6a
(280 mg, 75 %) as a colorless powder.
mp: 94-96 °C. IR (KBr disk): 3264 cm-1, 3116, 2956, 1729, 1597, 1561, 1443, 1267, 1203, 978, 754. 1H-
NMR (CDCl3): δ: 3.98 ppm (s, 3 H, O-CH3), 5.21 (s, 2 H, CH2-Ar), 6.78 (br s, 1 H, N-H), 7.31-7.60 (m,
7 H, 5 Ar-H + PHA-H + N-H). 13C-NMR (CDCl3): δ: 53.3 ppm(+; HMQC: OCH3), 68.4 (-; HMQC: CH2
Bzl), 102.2 (+; HMQC: PHA-H), 128.3 (+; HMQC: Ar-H), 128.7 (+; HMQC: Ar-H), 135.0 (Cquat, Ar),
156.1 (Cquat), 156.7 (Cquat), 160.6 (Cquat), 163.6 (Cquat; HMBC: C=O ester), 168.2 (Cquat).
UV/VIS (MeCN): λmax (log ε): 234 nm (4.689), 300 (4.360). MS m/z (%): 337.1 [MH+] (100), 228.9
[MH+-PhCH2OH] (7), 202.9 [MH+-Cbz] (22), 188.1 [MH+-Cbz-NH] (29). Anal. Calcd for C14H13N4O4Cl:
C: 49.9; H: 3.9; N: 16.6; Found: C: 49.8; H: 4.2; N: 16.5.
Methyl 2-bromo-6-(N'-tert-butoxycarbonylhydrazino)pyrimidine-4-carboxylate (6c): Compound
(5b) (164 mg, 0.55 mmol) was allowed to react with triethylamine (91 µL, 87 mg, 0.66 mmol) and Boc-
hydrazine (87 mg, 0.66 mmol) following GP1 (hexanes/ethyl acetate 1:1, Rf = 0.54) to yield 6a (123 mg,
64 %) as a colorless powder.
1
mp: 227-228 °C. IR (KBr disk): 3228 cm-1, 3105, 2981, 1734, 1603, 1445, 1259, 1159, 752. H-NMR
(DMSO-d6, 363 K): δ: 1.44 ppm (s, 9 H, Boc-CH3), 3.89 (s, 3 H, OCH3), 7.36 (s, 1 H), 8.92 (bs, 1 H),
9.71 (s, 1 H). 13C-NMR (DMSO-d6): δ = 28.2 ppm (+; HMQC: Boc-CH3), 53.5 (+; HMQC: OCH3), 83.0
(Cquat; HMBC: Boc), 102.7 (+; HMQC: PHA-H), 152.1 (Cquat), 155.0 (Cquat; HMBC: C=O Boc), 156.3
(Cquat), 163.7 (Cquat; HMBC: C=O ester), 167.1 (Cquat). UV (MeCN): λmax (log ε): 235 nm (4.973), 303
(4.654). MS m/z (%): 340.0 [MH+] (100). HRMS Calcd for C11H15N4O4Br: 346.0277; Found:
346.0275±0.0002.
Methyl 6-(N'-benzyloxycarbonylhydrazino)-2-bromopyrimidine-4-carboxylate (6d): Compound
(5b) (836 mg, 2.83 mmol) was allowed to react with triethylamine (470 µL, 343 mg, 3.39 mmol) and
Cbz-hydrazine (563 mg, 3.39 mmol) following GP1 (ethyl acetate/hexanes 2:3, Rf = 0.22) to yield 6a
(839 mg, 78 %) as a colorless powder.
mp: 94-95 °C. IR (KBr disk): 3354 cm-1, 3270, 2958, 2930, 1721, 1691, 1591, 1512, 1376, 1287, 1211,
1
1121, 995, 968, 773, 743, 695. H-NMR (CDCl3): δ: 3.92 ppm (s, 3 H, OCH3), 5.16 (s, 2 H, CH2), 7.05-
7.58 (m, 7 H, Ar-H, PHA-H, NH), 8.60 (br s, 1 H, NH). 13C-NMR (CDCl3): δ: 53.5 ppm (+, OCH3), 68.4
(-, CH2), 102.4 (+, PHA-H), 127.3 (+, Ar-H), 127.7 (+, Ar-H), 134.1 (Cquat, Ar), 150.8 (Cquat), 155.2
(Cquat, C=O Cbz), 162.5 (Cquat), 162.6 (Cquat), 165.8 (Cquat). UV (MeCN): λmax (log ε): 301 nm (4.644). MS
m/z (%): 380.9 [MH+] (100). Anal. Calcd for C14H13N4O4Br: C: 44.1; H: 3.4; Br: 21.0; N: 14.7; O: 16.8;
Found: C: 44.6; H: 3.4; N: 14.9.
Methyl 6-(N'-tert-butoxycarbonylhydrazino)-2-diethylaminopyrimidine-4-carboxylate (8a): Method
A: A solution of 6a (117 mg, 0.39 mmol), DMAP (59 mg, 0.48 mmol) and diethylamine (80 µL, 57 mg,

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HETEROCYCLES, Vol. 67, No. 1, 2006
0.78 mmol) in DMF (6 mL) is stirred for 1 d at 40 °C. The red solution is diluted with ethyl acetate
(8 mL) and washed with brine (2x15 mL). The organic extract is dried over MgSO4, evaporated under
reduced pressure and the residue purified by column chromatography (hexanes/ethyl acetate 3:1,
Rf = 0.22) to afford 8a (55 mg, 42 %) as a colourless powder.
Method B: A solution of 6c (200 mg, 0.58 mmol) and diethylamine (299 µL, 212 mg, 2.90 mmol) in
DMF (4 mL) is stirred for 8 h at 80 °C. The solvent is evaporated and the crude product purified by
column chromatography (hexanes/ethyl acetate 3:1, Rf = 0.22) to afford 8a (146 mg, 74 %).
1
mp: 176-177 °C. IR (KBr disk): 3237 cm-1, 2967, 2933, 1732, 1668, 1601, 1531, 1368, 1253, 1161. H-
3
NMR (DMSO-d6): δ: 1.09 ppm (t, J = 6.9 Hz, 6 H, CH3), 1.25-152 (m, 9 H, Boc-CH3), 3.54 (q,
13
3J = 6.9 Hz, 4 H, CH2), 3.81 (s, 3 H, OCH3), 6.32 (s, 1 H, PHA-H), 8.48-9.17 (m, 2 H, N-H). C-NMR
(DMSO-d6): δ: 13.1 ppm (+, CH3), 27.9 (+, Boc-CH3), 40.7 (-, CH2), 52.2 (+, OCH3), 78.8 (Cquat, Boc),
93.5 (+, PHA-H), 155.2 (Cquat), 155.6 (Cquat), 160.6 (Cquat), 164.3 (Cquat), 165.5 (Cquat). UV (MeCN):
λmax (log ε): 243 (5.316) nm, 329 (4.660). MS m/z (%): 701.5 [2MNa+] (6), 340.0 [MH+] (100)
Methyl 6-(N'-benzyloxycarbonylhydrazino)-2-diethylaminopyrimidine-4-carboxylate (8b): To a
solution of 6d (270 mg, 0.71 mmol) in DMF (5 mL) was added diethylamine (366 µL, 260 mg,
3.55 mmol). The reaction mixture was stirred for 16 h at 70 °C. The solution was diluted with ethyl
acetate (7 mL) and washed with brine (2x10 mL). The organic phase washed dried over MgSO4,
evaporated under reduced pressure and purified by column chromatography (hexanes/ethyl acetate 3:2,
Rf = 0.40) affording 8b (212 mg, 80 %).
1
mp: 174-177 °C. IR (KBr disk): 3262 cm-1, 2955, 1783, 1596, 1499, 1443, 1263, 1196, 976, 748. H-
3
3
NMR (CDCl3): δ: 1.12 ppm (t, J = 7.0 Hz, 6 H, NCH2CH3), 3.58 (q, J = 6.6 Hz, 4 H, NCH2CH3), 3.89
(s, 3 H, OCH3), 5.18 (s, 2 H, CH2 Cbz), 6.51-6.72 (m, 3 H, PHA-H, NH), 7.22-7.38 (m, 5 H, Ar-H). 13C-
NMR (CDCl3): δ: 13.1 ppm (+, NCH2CH3), 41.6 (-, NCH2CH3), 52.7 (+, OCH3), 67.9 (-, CH2 Cbz), 92.1
(+, PHA-H), 128.3 (+, Ar-H), 128.5 (+, Ar-H), 128.6 (+, Ar-H), 135.5 (Cquat, Ar), 156.0 (Cquat, C=O Cbz),
156.8 (Cquat), 161.1 (Cquat), 165.6 (Cquat), 166.0 (Cquat). UV (MeCN): λmax (log ε): 246 nm (5.289), 341
(4.579). MS m/z (%): 374.0 [MH+] (100).
Methyl 6-(N'-tert-butoxycarbonylhydrazino)-2-butylaminopyrimidine-4-carboxylate (8c): To a
stirred solution of 6c (100 mg, 0.29 mmol) in DMF (3 mL) was added butyl amine (71 µL, 53 mg,
0.72 mmol) and the reaction mixture was stirred for 18 h at 65 °C. The solution was diluted with ethyl
acetate (5 mL) and washed with brine (3x8 mL). The organic extract was dried over Na2SO4, evaporated
off and purified by column chromatography (hexanes/ethyl acetate 3:2, Rf = 0.14) to afford 8c (13 mg,
13 %) as colorless powder and unreacted 6c (49 mg, 0.14 mmol).
mp: 110-112 °C. IR (KBr disk): 3337 cm-1, 2959, 2873, 1735, 1591, 1542, 1439, 1368, 1249, 1160, 1102,
1
3
778. H-NMR (CDCl3): δ: 0.92 ppm (t, J = 7.2 Hz, CH3), 1.24-1.58 (13 H, Boc-CH3, CH3CH2,

HETEROCYCLES, Vol. 67, No. 1, 2006
153
CH3CH2CH2), 3.36 (q, 3J = 6.4 Hz, 2 H, CH2NH), 3.93 (s, 3 H, OCH3), 5.34 (br s, 1 H, NH), 6.55 (s, 1 H,
13
NH), 6.65 (s, 1 H, PHA-H), 6.76 (br s, 1 H, NH). C-NMR (DMSO-d6): δ: 13.8 ppm (+, CH3), 20.1 (-,
CH3CH2), 28.2 (+, Boc-CH3), 31.6 (-, CH3CH2), 36.9 (-, CH3CH2CH2), 53.0 (+, OCH3), 82.0 (Cquat, Boc),
93.2 (+, PHA-H), 155.6 (Cquat, 2 C), 162.4 (Cquat), 165.5 (Cquat), 166.7 (Cquat). UV (MeCN): λmax (log ε):
244 (5.213) nm, 323 (4.552). MS m/z (%): 340.1 [MH+] (100).
2,6-Dibromopyrimidine-4-carboxylic acid butylamide (10): Orotic acid butylamide (9) (1.65 g,
5.76 mmol) and POBr3 (7.74 g, 28.8 mmol) were suspended in acetonitrile (15 mL). The reaction mixture
was stirred for 3.5 h at 65 °C. The clear solution was allowed to cool to rt and poured on crushed ice
(20 mL) and water (10 mL) and extracted with ethyl acetate (3x45 mL). The organic extract was washed
with sat. NaHCO3 solution and dried over Na2SO4, evaporated off affording 10 (466 mg, 72 %) in
analytical pure form.mp: > 175 °C (decomp). IR (KBr disk): 3010 cm-1, 2987, 2979, 1650, 1636, 1225,
1
3
675. H-NMR (DMSO-d6): δ: 0.88 ppm (t, 3 H; J = 7.4 Hz, CH3), 1.18-1.36 (m, 2 H, CH3-CH2), 1.40-
3
1.57 (m, 2 H, CH2-CH2-NH), 3.27 (q, 2 H, J = 6.7 Hz, CH2-NH), 8.21 (s, 1 H, PHA-H), 9.02 (t, 1 H,
13
3J = 6.8 Hz, N-H). C-NMR (DMSO-d6): δ = 13.5 ppm (+, CH3), 19.4 (-, CH3-CH2), 30.8 (-, CH2-CH2-
NH), 38.8 (-, CH2-NH), 122.4 (+, PHA-H), 146.6 (Cquat), 150.1 (Cquat), 154.7 (Cquat), 160.1 (Cquat). UV
(MeCN): λmax (log ε): 276 nm (4.774). MS m/z (%): 333.7 [M+H+] (100).
2-Bromo-6-[N'-(tert-butoxyhydroxymethyl)hydrazino]pyrimidine-4-carboxylic acid butylamide
(11): 10 (350 mg, 1.04 mmol) was allowed to react with triethylamine (173 µL, 127 mg, 1.25 mmol)
and Boc-hydrazine (165 mg, 1.25 mmol) following GP1 (ethyl acetate/hexanes 3:5, Rf = 0.18) to yield 11
(331 mg, 82 %) as a colorless powder.
mp: 161-163 °C. IR (KBr disk): 3339 cm-1, 3227, 2974, 2932, 1725, 1651, 1593, 1526, 1392, 1272, 1254,
1
3
1165. H-NMR (CDCl3): δ: 0.95 ppm (t, 3 H, J = 7.3 Hz; HSQC: CH3), 1.36-1.45 (m, 2 H; ROESY:
3
1
CH3CH2), 1.47 (s, 9 3.43 (q, 2 H, J = 6.7 Hz, CH2-NH), 6.79 (s, 1 H; ROESY: NH), 7.46 (s, 1 H;
2
3
HSQC: PHA-H), 7.62 (bs, 1 H; ROESY: NH), 7.77 (t, J = 5.8 Hz, 1 H; HMBC: NHCO). 13C-NMR
(DMSO-d6): δ: 13.7 ppm (+; HSQC: CH3), 20.1 (-; HSQC: CH3CH2), 28.2 (+; HSQC: Boc-CH3), 31.5 (-;
HSQC: CH3CH2CH2), 39.4 (-; HSQC: CH2NH), 82.7 (Cquat; HMBC: Boc), 99.6 (+; HSQC: PHA-H),
150.8 (Cquat), 154.8 (Cquat; HMBC: C=O Boc), 161.7 (Cquat), 167.3 (Cquat). UV (MeCN): λmax (log ε):
240 nm (5.227), 295 (4.709). MS m/z (%): 388.0 [MH+] (88), 331.9 [MH+ - C4H8] (100). Anal. Calcd for
C14H22N5O3Br: C. 43.3; H: 5.7; N: 18.0; O: 12.4; Br: 20.6; Found: C: 43.6; H: 5.6; N: 17.9.
6-[N'-(tert-Butoxyhydroxymethyl)hydrazino]-2-butylaminopyrimidine-4-carboxylic acid butyl-
amide (12a): 11 (142 mg, 0.36 mmol) was allowed to react with butylamine (357 µL, 263 mg,
3.60 mmol) following GP2 (ethyl acetate/hexanes 1:1, Rf = 0.37) to give 12a (93 mg, 68 %) as a yellow
solid.

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HETEROCYCLES, Vol. 67, No. 1, 2006
mp: 184-186 °C. IR (KBr disk): 3372 cm-1, 3213, 2962, 1735, 1665, 1589, 1528, 1460, 1367, 1250, 1161,
1
1015, 771. H-NMR (CDCl3): δ: 0.73-0.97 ppm (m, 6 H, 2 CH3), 1.32-1.66 (m, 17 H, Boc-CH3, 4 CH2),
3.32-3.45 (m, 4 H, 2 CH2NH), 5.14 (br s, 1 H, NH), 6.73 (br s, 1 H, NH), 6.80 (s, 1 H, PHA-H), 7.09 (br
13
s, 1 H, NH), 7.89 (br s, 1 H, NHCO). C-NMR (CDCl3): δ: 13.8 ppm (+, CH3), 13.9 (+, CH3), 20.0 (-),
20.1 (-) 28.2 (+, Boc-CH3), 31.6 (-), 31.7 (-), 39.0 (-), 41.2 (-), 88.9 (Cquat, Boc), 91.0 (+, PHA-H), 155.7
(Cquat), 156.0 (Cquat), 161.3 (Cquat), 161.4 (Cquat), 163.8 (Cquat), 164.1 (Cquat). UV (MeOH): λmax (log ε):
236 nm (5.197), 328 (4.460). MS m/z (%): 381.1 [MH+] (100).
6-[N'-(tert-Butoxy-hydroxymethyl)hydrazino]-2-diethylaminopyrimidine-4-carboxylic
acid
butylamide (12b): To a solution of 11 (242 mg, 0.62 mmol) in DMF (3 mL) was added diethylamine
(312 µL, 228 mg, 3.12 mg) and the solution was stirred for 16 h at 70 °C. The solution was diluted with
ethyl acetate (10 mL) and washed with brine (2x20 mL). The organic extract was dried over MgSO4 and
evaporated off. The residue was purified by column chromatography (hexanes/ethyl acetate 1:2,
Rf = 0.33) to afford 12b (200 mg, 82 %).
1
mp: 165-167 °C. IR (KBr disk): 3226cm-1, 2967, 2933, 1732, 1668, 1601, 1531, 1368, 1253, 1161. H-
3
3
NMR (CDCl3): δ: 0.95 ppm (t, J = 7.3 Hz, 3 H, CH3 Bu), 1.17 (t, J = 7.0 Hz, 6 H, CH3 Et), 1.30-1.73
(m, 13 H, Boc-CH3, CH3CH2, CH3CH2CH2), 3.41 (q, 3J = 6.7 Hz, CH2NHCO), 3.57 (q, 3J = 7.0 Hz, CH2
Et), 6.68-6.77 (m, 2 H, NH), 6.91 (s, 1 H, PHA-H), 7.88 (br s, 1 H, NHCO). 13C-NMR (CDCl3): δ:
13.5 ppm (+, CH3 Bu), 13.6 (+, CH3 Et), 19.4 (-, CH3CH2), 27.9 (+, Boc-CH3), 31.0 (-, CH3CH2CH2),
38.2 (-, CH2 Et), 38.5 (-, CH2 Et), 40.7 (-, CH2NHCO), 80.2 (Cquat, Boc), 98.3 (+, PHA-H), 155.6 (Cquat
,
C=O Boc), 158.6 (Cquat), 161.3 (Cquat), 163.5 (Cquat), 167.2 (Cquat). UV (MeCN): λmax (log ε): 243 nm
(5.316), 330 (4.645). MS m/z (%): 783.6 [2M+Na+] (4), 761.7 [2M+H+], 381.0 [M+H+] (100), 324.9
[MH+-C4H8] (32), 280.8 [MH+-Boc] (48).
tert-Butyl N'-(6-butylcarbamoyl-2-morpholin-4-ylpyrimidin-4-yl)hydrazine carboxylate (12c):
Compound (11) (100 mg, 0.26 mmol) was allowed to react with 1,4,7,10,13-pentaoxa-16-aza-cyclo-octa-
decane (225 µL, 225 mg, 2.58 mmol) following GP2 (ethyl acetate/hexanes 1:1, Rf = 0.24) to give 12c
(68 mg, 0.17 mmol, 66 %) as a colourless, hygroscopic solid.
mp: 167-170 °C. IR (KBr disk): 3356 cm-1, 3290, 3197, 2691, 1724, 1659, 1558, 1523, 1447, 1361, 1254,
1
3
1161, 1104, 1002, 875. H-NMR (CDCl3): δ: 0.95 ppm (t, J = 7.3 Hz, 3 H, CH3), 1.23-1.64 (m, 13 H,
3
3
Boc-CH3, CH3CH2, CH3CH2CH2), 3.41 (dd, J = 7.1 Hz, J = 13.4 Hz, 2 H, CH2NHCO), 3.75 (br s, 8 H,
3
CH2 morpholine), 6.61-6.82 (m, 3 H, PHA-H, 2 NH), 7.79 (t, J = 5.6 Hz). 13C-NMR (CDCl3): δ:
13.8 ppm (+, CH3), 20.2 (-, CH3CH2), 28.2 (+, Boc-CH3), 31.7 (-, CH3CH2CH2), 39.1 (-, CH2NHCO),
44.3 (-, CH2 morpholine), 66.8 (-, CH2 morpholine), 81.8 (Cquat, Boc), 91.2 (+, PHA-H), 155.8 (Cquat
,
C=O Boc), 157.4 (Cquat), 160.7 (Cquat), 163.8 (Cquat), 166.7 (Cquat). UV (MeOH): λmax (log ε): 242 nm
(5.449), 328 (4.680). MS m/z (%): 395.1 [MH+] (100), 296.8 [MH+-Boc] (52). HRMS Calcd for

HETEROCYCLES, Vol. 67, No. 1, 2006
155
C18H30N6O4: 394.2329; Found: 394.2330±0.0004. Anal. Calcd for C18H30N6O4: C: 54.8; H: 7.7; N: 21.3;
O: 16.2; Found: C: 54.8; H: 7.5; N: 21.5.
tert-Butyl
N'-[6-butylcarbamoyl-2-(1,4,7,10,13-pentaoxa-16-azacyclooctadec-16-yl)pyrimidin-4-
yl]hydrazine carboxylate (12d): Compound (11) (145 mg, 0.37 mmol) and triethylamine (154 µL,
112 mg, 1.11 mmol) were dissolved in DMF (3 mL) and 1,4,7,10,13-pentaoxa-16-aza-cyclooctadecane
(292 mg, 1.11 mmol) was added. The resulting solution was stirred for 36 h at 65 °C, diluted with ethyl
acetate (5 mL) and washed with brine (2x10 mL). The organic extract was dried over MgSO4, evaporated
off and purified by column chromatography (ethyl acetate, Rf = 0.17) to afford 12d (118 mg, 56 %).
1
mp: > 180 °C (decomp). IR (CH2Cl2): 3110 cm-1, 2979, 1665, 1571, 1259, 902, 886. H-NMR (CDCl3):
3
3
δ: 0.87 ppm (t, J = 7.3 Hz, 3 H, CH3), 1.21-1.72 (m, 13 H, Boc-CH3, 2 CH2), 3.32 (q, J = 6.8 Hz, 2 H,
CH2NHCO), 3.41-3.94 (m, 24 H, CH2 crown ether), 6.39-6.99 (m, 3 H, PHA-H, 2 NH), 7.84 (t,
13
3J = 5.6 Hz, 1 H, NHCO). C-NMR (CDCl3): δ = 13.8 ppm (+, CH3), 20.1 (-, CH3CH2), 28.2 (+, Boc-
CH3), 39.0 (-, CH3CH2CH2), 48.8 (-, CH2NHCO), 69.5 (-, CH2 crown ether), 70.7 (-, CH2 crown ether),
70.8 (-, CH2 crown ether), 81.6 (Cquat, Boc), 90.1 (+, PHA-H), 155.8 (Cquat, Boc C=O), 157.4 (Cquat),
160.4 (Cquat), 164.1 (Cquat), 166.6 (Cquat). UV (MeCN): λmax (log ε): 244 nm (5.255), 331 (4.578). MS m/z
(%): 609.3 [M+K+] (12), 593.3 [M+Na+] (10), 571.4 [MH+] (100). HRMS Calcd for C26H46N6O8:
570.3377; Found: 570.3379±0.0004.
tert-Butyl
N'-{6-butylcarbamoyl-2-[2-(2-methoxyethoxy)ethylamino]pyrimidin-4-yl}hydrazine
carboxylate (12e): Compound (11) (110 mg, 0.28 mmol) was allowed to react with 2-(2-methoxy-
ethoxy)ethylamine (361 µL, 334 mg, 2.80 mmol) following GP2 (ethyl acetate, Rf = 0.16) to give 12e
(33 mg, 28 %) as a colourless powder and unreacted 11 (51 mg, 0.13 mmol).
mp: 88-89 °C. IR (KBr disk): 3436 cm-1, 3376, 3242, 2963, 1730, 1670, 1590, 1569, 1522, 1440, 1257,
1
3
1149, 1091, 802. H-NMR (CDCl3): δ: 0.95 ppm (t, J = 7.3 Hz, 3 H, CH3), 1.36-1.46 (m, 11 H, Boc-
CH3, 2 CH2), 1.53-1.63 (m, 2 H, CH2), 3.36-3.43 (m, 5 H, OCH3, CH2), 3.55-3.65 (m, 8 H, 4 CH2), 5.55
13
(br s, 1 H, NH), 6.74-6.81 (m, 2 H, PHA-H, NH), 7.00 (br s, 1 H, NH), 7.86 (br s, 1 H, NH). C-NMR
(CDCl3): δ: 12.8 ppm (+, CH3 Bu), 19.1 (-), 27.2 (+, Boc-CH3), 30.6 (-), 38.1 (-), 40.1 (-), 58.0 (+,
OCH3), 69.0 (-), 69.3 (-), 70.9 (-), 80.8 (Cquat, Boc), 90.3 (+, PHA-H), 154.6 (Cquat, C=O Boc), 158.6
(Cquat), 160.2 (Cquat), 162.7 (Cquat), 166.1 (Cquat). UV (MeOH): λmax (log ε): 242 nm (4.758). MS m/z (%):
427.1 [MH+] (100), 853.6 [2M+H+] (8).
Methyl 2-diethylamino-6-hydrazinopyrimidin-4-carboxylate bishydrochloride (13): Compound (8a)
(53 mg, 0.16 mmol) is dissolved in ether saturated with HCl (2 mL) at 0 °C and stirred for 15 min.
Removal of the solvent under reduced pressure gives 13 in quantitative yield (50 mg). The product is used
without further purification.

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mp: > 185 °C (decomp). IR (KBr disk): 3446 cm-1, 2887, 1744, 1643, 1591, 1521, 1449, 1337, 1254,
1182, 1058. 1H-NMR (DMSO-d6): δ: 1.14 ppm (t, 3J = 6.9 Hz, 6 H, CH3), 3.64 (q, 3J = 6.9 Hz, 4 H, CH2),
3.86 (s, 3 H, OCH3), 6.56 (s, 1 H, PHAt-H), 10.55 (br s, 5 H, NH). MS m/z (%): 240.0 [MH+] (100).
Lithium 6-(N'-tert-butoxycarbonylhydrazino)-2-diethylaminopyrimidine-4-carboxylate (14): To a
solution of 8a (108 mg, 0.32 mmol) in a 4:1 mixture of acetone/water (6 mL) was added LiOH·2H2O
(13.4 mg, 0.32 mmol). The reaction mixture was stirred for 6 h at rt. The solvents are removed under
reduced pressure to afford 14 (100 mg, 94 %) in almost quantitative yield. The salt is used without further
purification.
mp: > 185 °C (decomp). IR (KBr disk): 3298 cm-1, 2974, 1725, 1589, 1520, 1420, 1362, 1247, 1164,
1
3
1077, 859, 784. H-NMR (DMSO-d6): δ: 1.06 ppm (t, J = 6.9 Hz, 6 H, CH3), 1.42 (s, 9 H, Boc-CH3),
3.51 (q, 3J = 6.9 Hz, 4 H, CH2), 6.33 (s, 1 H, PHA-H), 8.68 (bs, 1 H, NH), 8.95 (bs, 1 H, NH). 13C-NMR
(DMSO-d6): δ: 13.8 ppm (+, CH3), 29.5 (+, Boc-CH3), 40.6 (-, CH2), 78.9 (Cquat, Boc), 90.4 (+, PHA-H),
155.7 (Cquat, C=O Boc), 160.4 (Cquat), 166.9 (Cquat), 167.7 (Cquat), 174.6 (Cquat, C=O carboxylate). MS m/z
(%): 326.0 [M-H+] (100).
Methyl
6-{N'-[6-(N'-tert-butoxycarbonylhydrazino)-2-diethylaminopyrimidine-4-carbonyl]-
hydrazino}-2-diethylaminopyrimidine-4-carboxylate (15): Lithium salt (14) (198 mg, 0.35 mmol),
hydrazine dihydrochloride (13) (109 mg, 0.35 mmol), HOAt (57 mg, 0.42 mmol), HATU (160 mg,
0.42 mmol), and Huenig´s Base (238 µL, 181 mg, 1.40 mmol) are dissolved in dichloromethane (5 mL).
The solution is stirred for 30 h at rt. The solvent is removed under reduced pressure and the yellow
residue purified by column chromatography (ethyl acetate/hexanes 3:1, Rf = 0.68) to afford 15 (78 mg,
40 %) as a colorless powder.
mp: 154-156 °C. IR (KBr disk): 3257 cm-1, 3388, 2974, 1720, 1604, 1583, 1548, 1474, 1435, 1130, 1259,
1
1165, 1122. H-NMR (DMSO-d6): δ: 0.81-1.22 ppm (m, 12 H, 4 CH3), 1.28-1.54 (m, 9 H, Boc-CH3),
3.34-3.78 (m, 8 H, 4 CH2), 3.80 (s, 3 H, OCH3), 6.15-6.70 (m, 4 H, 2 N-H, 2 PHA-H), 8.50-10.2 (m, br s,
2 H, N-H). 13C-NMR (DMSO-d6): δ: 12.7 ppm (+, CH3), 13.1 (+, CH3), 28.0 (+, Boc-CH3), 40.8 (-, CH2),
52.2 (+, OCH3), 85.8 (Cquat, Boc), 121.6 (+, PHA-H), 129.5 (+, PHA-H), 152.3 (Cquat, C=O Boc), 160.5
(Cquat, C=O), 165.4 (Cquat, C=O); further signals could not be labelled. UV (MeCN): λmax (log ε): 211 nm
(5.483), 245 (5.519), 346 (4.933). MS m/z (%): 547.4 [MH+] (100). HRMS Calcd for C24H38N10O5:
546.3027; Found: 546.3026±0.0004.
Methyl 2-(2-{2-[2-({1-[6-(N'-tert-butoxycarbonylhydrazino)-2-diethylaminopyrimidine-4-carbonyl]-
pyrrolidine-2-carbonyl}amino)acetylamino]-3-phenylpropionylamino}-4-methylpentanoylamino)-
3-methylbutyrate (16): Compound (14) (75 mg, 0.23 mmol), H-D-Pro-Gly-Phe-Val-Leu-OMe (131 mg,
0.23 mmol), EDC (45 µL, 39 mg, 0.25 mmol), HOBt (34 mg, 0.25 mmol), and Huenig´s Base (75 µL,

HETEROCYCLES, Vol. 67, No. 1, 2006
157
57 mg, 0.46 mmol) were dissolved in DMF (3 mL). The reaction mixture was stirred for 18 h at rt. At
0 °C, water (5 mL) was added to precipitate (16) (99 mg, 52 %) in analytical pure form.
1
mp: 121 °C. IR (KBr disk): 3349cm-1, 3024, 2995, 1754, 1670, 1591, 1364, 1275, 1161, 913, 898. H-
NMR (DMSO-d6): δ: 0.74-0.95 ppm (m, 12 H; HSQC: CH3 Leu, CH3 Val), 1.04-1.18 (m, 6 H; HSQC:
CH3 PHA), 1.34-1.45 (m, 9 H; HSQC: Boc-CH3), 1.46-1.53 (m, 1 H; COSY: CHs Val), 1.54-1.70 (m,
2 H; COSY: CH2 Pro), 1.75-1.85 (m, 2 H; COSY CH2 Pro), 1.86-2.01 (m, 2 H; COSY: CH2 Leu), 2.02-
2.16 (m, 1 H; COSY: CHs Leu), 2.70-2.89 (m, 1 H; HMBC: CH2 Phe), 2.94-3.08 (m, 1 H; HMBC: CH2
Phe), 3.41-3.81 (m, 11 H; COSY: CδH2 Pro, CH2 PHA; HSQC: OCH3, CH2 Gly), 4.12-4.22 (m, 1 H;
COSY: CH Pro), 4.24-4.33 (m, 1 H; COSY: CH Val), 4.38-4.47 (m, 1 H; COSY: CH Leu), 4.51-4.71 (m,
1 H; COSY: CH Phe), 5.99-6.24 (m, 1 H; HSQC: PHA-H), 7.10-7.29 (m, 5 H; HSQC: Ar-H), 7.79-7.91
(m, 1 H, NH), 7.92-8.00 (m, 1 H; NH), 8.04-8.11 (m, 1 H; NH), 8.14-8.24 (m, 1 H; NH), 8.30-8.38 (m,
13
1 H; NH), 8.45-8.55 (m, 1 H; NH). C-NMR (DMSO-d6): δ: 13.5 ppm (+; HMQC: CH3 PHA), 18.5 (+;
HMQC: CH3 Leu), 18.7 (+; HMQC: CH3 Leu), 19.5 (+; HMQC: CH3 Val), 21.7 (+; HMQC: CH3 Val),
23.2 (-; HSQC: CH2 Pro), 24.6 (+; HSQC: CHs Leu), 28.5 (+; HMQC: Boc-CH3), 28.6 (+; HMQC; Boc-
CH3), 29.6 (-; HSQC: CH2Pro), 31.1 (+; HSQC: CHs Val), 40.2 (-; HSQC: CH2 PHA), 40.7 (-; HSQC:
CH2 Leu), 41.8 (-; HSQC: CH2 Pro), 42.5 (-; HSQC: CH2 Gly), 50.7 (-; HSQC: CH2 Phe), 52.2 (+;
HSQC: OCH3), 54.4 (+; HSQC: CH Val), 58.2 (+; HMBC: CH Phe), 60.7 (+; HSQC: CH Pro), 62.5 (+;
HSQC: CH Leu), 79.4 (Cquart; HMBC: Boc), 116.7 (+; HSQC: PHA-H), 126.7 (+; HSQC: Ar-H), 128.5
(+; HSQC: Ar-H), 129.7 (+; HSQC: Ar-H), 138.2 (Cquart; HMBC: Ar), 156.8 (Cquart; HMBC: C=O Boc),
167.5 (Cquart), 168.4 (Cquart; HMBC: C=O Gly), 168.5 (Cquart; HMBC: CHet-NEt2), 169.3 (Cquart), 170.6
(Cquart; HMBC: C=O Phe), 170.7 (Cquart; HMBC: C=O Pro), 172.5 (Cquart; HMBC: C=O Leu), 172.7
(Cquart; HMBC: C=O Val). UV (MeOH): λmax (log ε): 330 nm (6.961). MS m/z (%): 853.6 [MH+] (100).
+
HRMS Calcd for C42H65N10O9 : 853.4936; Found: 853.4926±0.0006.
Lithium 6-{N'-[6-(N'-tert-butoxycarbonylhydrazino)-2-methylpyrimidine-4-carbonyl]hydrazino}-
2-methylpyrimidine-4-carboxylate (17): To a solution of 15 (216 mg, 0.50 mmol) in a 4:1 mixture of
acetone/water (8 mL) was added LiOH·2H2O (20.6 mg, 0.50 mmol). The reaction mixture was stirred for
14 h under reflux. The solvents are removed under reduced pressure to afford 17 (202 mg, 95 %) in
almost quantitative yield. The salt is used without further purification.
1
mp: 185 °C (decomp). IR (KBr disk): 3332 cm-1, 2977, 1725, 1588, 1524, 1360, 1247, 1167. H-NMR
(DMSO-d6): δ: 0.96-1.11 ppm (m, 12 H, CH3), 1.44 (s, 9 H, Boc-CH3), 3.38-3.55 (m, 8 H, CH2), 6.39 (s,
1 H, PHA-H), 6.89 (s, 1 H, PHA-H), 8.24 (br s, 1 H, NH), 8.68 (br s, 1 H, NH), 8.87 (br s, 1 H, NH), 8.95
(br s, 1 H, NH). 13C-NMR (DMSO-d6): δ: 13.6 ppm (+, CH3), 13.8 (+, CH3), 29.5 (+, Boc-CH3), 40.2 (-,
CH2), 40.6 (-, CH2), 78.5 (Cquat, Boc), 90.4 (+, PHA-H), 98.7 (+, PHA-H), 155.7 (Cquat, C=O Boc), 160.4

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HETEROCYCLES, Vol. 67, No. 1, 2006
(Cquat), 160.9 (Cquat), 164.7 (Cquat), 165.4 (Cquat), 166.9 (Cquat), 167.7 (Cquat), 169.6 (Cquat), 174.8 (Cquat
,
C=O carboxylate). MS m/z (%): 419.1 [M-H+] (100).
Turn structure (18): Compound (17) (43 mg, 0.10 mmol), D-Pro-Gly-Phe-Leu-Phe-Ala-NH2.TFA
(73 mg, 0.10 mmol), EDC (54 µL, 47 mg, 0.30 mmol), HOBt (41 mg, 0.30 mmol), and Huenig´s Base
(65 µL, 50 mg, 0.40 mmol) were dissolved in DMF (4 mL). The reaction mixture was stirred for 15 h at
rt. At 0 °C, water (5 mL) was added to precipitate (18) (87 mg, 75 %) in analytical pure form.
mp: 118 °C. IR (KBr disk): 3321 cm-1, 2961, 2297, 1728, 1662, 1576, 1531, 1456, 1373, 1272, 1161,
1
1125, 1074, 779, 703. H-NMR (DMSO-d6, 290 K): δ: 0.61-0.64 ppm (m, 3 H; COSY: CH3 Leu), 0.72-
3
0.76 (m, 3 H; COSY: CH3 Leu), 0.90-1.14 (m, 18 H; COSY: CH3 PHA), 1.21 (d, J = 7.05 Hz, 3 H;
COSY: CH3 Ala), 1.25-1.33 (m, 3 H; COSY: CH2 Leu, CHs Leu), 1.35-1.44 (m, 13 H; HMBC: Boc-CH3;
COSY: 2xCH2 Pro), 1.76-1.87 (m, 1 H; COSY: CH2 Pro), 2.06-2.13 (m, 1 H; COSY: CH2 Pro), 2.75-2.85
(m, 1 H; NOESY: CH2 Phe), 2.89-2.98 (m, 1 H; NOESY: CH2 Phe), 3.02-3.10 (m, 2 H; NOESY: CH2
Phe), 3.27-3.68 (m, 8 H; COSY: CH2 PHA), 3.70-3.83 (m, 2 H; COSY: CH2 Gly), 4.06-4.20 (m, 2 H;
COSY: CH Leu, CH Ala), 4.35-4.55 (m, 3 H; COSY: CH Pro; NOESY: CH Phe, CH Phe), 6.29 (s, 1 H:
3
HMBC: PHA-H), 6.99-7.37 (m, 13 H; HMBC: Ar-H, PHA-NH, Ala-NH2), 7.70 (d, J = 7.8 Hz, 1 H;
3
COSY: NH Phe), 7.74-7.82 (m, 1 H; COSY: NH Ala), 7.85 (d, J = 7.4 Hz, 1 H; NOESY: PHA-NH),
7.88-7.96 (m, 1 H; COSY: NH Phe), 7.97-8.00 (m, 1 H; COSY: NH Leu), 8.14-8.17 (m, 1 H; NOESY:
PHA-NH), 8.93 (bs, 1 H; NOESY: PHA-NHNH), 10.09 (s, 1 H; NOESY: PHA-NHNH). 13C-NMR
(DMSO-d6): δ: 15.7 ppm (+; HMQC: CH3 Ala) 17.4 (+; HMQC: CH3 Leu), 18.9 (+; HMQC: CH3 Leu),
22.0 (+; HMQC: CH3 PHA), 24.0 (-; HMQC: CH2 Pro), 27.8 (-; HMQC: CH2 Pro), 29.8 (+; HMBC: CHs
Leu), 31.2 (+; HMQC: Boc-CH3), 33.7 (-; HMQC: CH2 Pro), 36.7 (-; HMQC: CH2 Phe), 37.4 (-; HMQC:
CH2 Phe), 39.4 (-; HMBC: CH2 Leu), 47.4 (-; HMQC: CH2 Gly), 50.1 (+; HMQC: CH Pro), 51.3 (-;
HMQC: CH2 PHA) 53.5 (+; HMQC: CH Phe), 57.4 (+; HMBC: CH Leu), 57.7 (+; HMQC: CH Ala),
60.3 (+; HMQC: CH Phe), 79.7 (Cquat; HMBC: Boc), 126.0 (+; HMBC: Ar-H), 127.7 (+; HMBC: Ar-H),
128.1 (+; HMBC: Ar-H), 129.1 (+; HMBC: Ar-H), 130.7 (+; HMBC: Ar-H), 137.3 (Cquat; HMBC: Ar),
137.5 (Cquat; HMBC: Ar). Further signals are not detectable. UV (MeOH): λmax (log ε): 245 nm (3.275),
346 (1.730). UV (MeOH): λmax (lg ε): 245 nm (3.275), 346 nm (1.730). MS m/z (%): 1186.8 [M+Na+]
+
(30), 1164.7 [MH+] (80), 583.1 [M+2H+] (100). HRMS Calcd for C57H82N17O10 : 1164.6431; Found:
1164.6436±0.0005.
ACKNOWLEDGEMENTS
V. M. thanks the International Quality Network Medicinal Chemistry (IQN-MC; funded by the DAAD)
for a postdoctoral stipend. We acknowledge support by the Graduiertenkolleg (GRK 760) Medicinal
Chemistry and the Fonds der Chemischen Industrie.

HETEROCYCLES, Vol. 67, No. 1, 2006
159
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13.
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15.
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16.
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18.
Additional NOE contacts between the PHA unit and the peptide strand are expected, but not
observed due to broad NH signals of the protons of the hydrazine. Resonances of NH protons of
16 appear shifted downfield of the analogous protons of the controls Boc-D-Pro-Gly-Phe-Val-
Leu-OMe and 8a. The analysis of the NH-CαH coupling constants is not possible due to signal
overlap.