On the manner of cyclization of N-acylated aspartic and glutamic acid derivatives

Article abstract of DOI:10.1007/s10989-011-9246-z

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of Nacylated glutamic acid derivatives. During c

Full text of DOI:10.1007/s10989-011-9246-z

Int J Pept Res Ther (2011) 17:93–100
DOI 10.1007/s10989-011-9246-z
On the Manner of Cyclization of N-Acylated Aspartic
and Glutamic Acid Derivatives
_
•
•
•
Paweł Zajdel Christine Enjalbal Marek Zylewski
Gilles Subra
Accepted: 14 February 2011 / Published online: 17 March 2011
Ó Springer Science+Business Media, LLC 2011
Abstract When synthesizing arylpiperazine library
modified with N-acylated amino acid derivatives (e.g.,
cyclized aspartic acid, cyclized glutamic acid, proline) we
wished to rapidly determine the way of cyclization of N-
acylated glutamic acid derivatives. During concomitant
cleavage and cyclization two alternative routes were pos-
sible—either formation of six-member imide (glutarimide)
or five-member lactam. Application of MS/MS and 1H
NMR method allowed us to establish that cyclization of N-
acylated glutamic acid derivatives preceded to lactams—N-
acylated pyroglutamic acid derivatives.
field. Especially, amino acids-derived cyclic compounds, e.g.,
imides, lactones, lactams are interesting templates for com-
binatorial/parallel synthesis developments.
Although, solution-phase synthesis of imide derivatives
is quite well established, (Miller and Long 1951; Zajdel
et al. 2009; Chilmonczyk et al. 1995) few approaches for
solid-phase have been described. (Girdwood and Shute
1997; Barn and Morphy 1999). A common feature of
solid-supported approaches is the attachment of the a-
amino acid derivatives to the support through an amide or
ester linkage, thus resulting in a carboxylic acid or simple
amide residue on the final product upon cleavage. Alva-
rez-Gutierrez et al. (2000a) described the synthesis
of 1,3-disubstituted succinimide on p-methylbenzhydryl-
amine (MBHA) resin. Cyclization of aspartic acid was
accomplished at elevated temperature in the presence
of diphenylphosphorylazide (DPPA) and TEA in THF.
Interestingly, a similar strategy involving intramolecular
cyclization of glutamic acid derivative was applied for the
solid-phase synthesis of 1,5-disubstituted-2-pyrrolidinones
(Alvarez-Gutierrez et al. 2000b). One of the inconve-
niences of the latter approach was the use of strong acidic
conditions of HF to perform cleavage from the resin and
cyclization of glutamic acid derivatives requiring highly
basic conditions. Moreover, cyclization was performed
between the carboxylic function of the side chain and the
free or alkylated N-terminus of glutamic acid.
Keywords Solid-phase synthesis Á Aspartic acid
cyclization Á Glutamic acid cyclization Á Pyroglutamic
acid Á Mass spectrometry Á SynPhase Lanterns
Introduction
Synthesis of amino acid-derived small organic compounds for
biological studies and drug discovery is an attractive research
P. Zajdel (&)
Department of Medicinal Chemistry, Jagiellonian University
´
Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
e-mail: mfzajdel@cyf-kr.edu.pl
As an alternative, we have previously presented effi-
cient solid-phase strategy for the synthesis of N-acylated
amino acid derivatives, connected via an alkylen linker
with arylpiperazines (Zajdel et al. 2004, 2006), on BAL
linker functionalized SynPhase Lanterns (Jensen et al.
1998). The amino acids were N-acylated aspartic and
glutamic acids. In the last stage of the synthesis, com-
pounds bearing these dicarboxylic amino acid fragments
C. Enjalbal Á G. Subra
´
Equipe Aminoacides Peptides et Proteines, Institut des
´
Biomolecules Max Mousseron IBMM, UMR CNRS 5247,
´
Faculte de Pharmacie, Universite Montpellier I et II, 15 avenue
´
´
Charles Flahault, 34060 Montpellier, France
_
M. Zylewski
Department of Organic Chemistry, Jagiellonian University
´
Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
123

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Int J Pept Res Ther (2011) 17:93–100
underwent concomitant cleavage and cyclization in the
acidic conditions.
First, N-a-tert-butoxycarbonyl-L-glutamic acid c-benzyl ester
was directly coupled with 4-[4-(3-chlorophenyl)piperazin-1-
yl]butylamine, in the presence of 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). It
was then coupled with cyclohexane carboxylic acid to yield
compound 6. After removal of benzyl protection a corre-
sponding acid derivative of compound 6 was submitted to
cyclization in the presence of SOCl2 in chloroform. The final
pyroglytamyl derivative was purified by column chromatog-
raphy to yield pure 7 as white solid.
It is generally accepted that cyclization of sequences
including an aspartic acid regioselectively yields asparti-
mide moiety (3-amino-pyrrolidine-2,5-dione), while in the
case of glutamic acid possessing an additional methylene
group in the lateral chain, two alternative cyclization routes,
namely six-member imide (glutarimide) and five-member
lactam (pyrrolidine-5-on-2-carboxylic acid, pyroglutamic
acid), might occur. The possible ways of cyclization for
aspartic acid derivatives and glutamic acid are presented in
Schemes 1 and 2, respectively.
Mass Spectrometry
In this report, we will demonstrate application of MS/
MS and 1H NMR techniques for the elucidation of the type
of N-acylated glutamic acid derivatives cyclization.
The goal of this study was to determine the manner of
intramolecular cyclization of N-acylated glutamic acids
during cleavage from the BAL linker (Scheme 2). In the
first stage, mass spectrometry techniques were applied. Of
the available spectroscopic methods, mass spectrometry is
particularly well suited for probing combinatorial libraries,
since it provides rapid and sensitive measurements of the
cleaved mixtures of compounds. It is worth noting that this
technique has been successfully adapted for direct analysis
of the products attached to a solid support (MALDI,
S-SIMS) (Enjalbal et al. 2000).
Results and Discussion
Chemistry
Compound 7 was synthesized according to the standard
methods used in peptide chemistry and outlined in Scheme 3.
PS-SynPhase Lantern
O
Taking into account a structural similarity, four com-
pounds from the initial library were selected (Zajdel et al.
2004). Two of them were N-acylated aspartic acid deriv-
atives—compounds 1 and 2 (Scheme 4) and two were the
N
H
O
MeO
O
O
N
H
H
OMe
n
N
Cl
Cl
N
N
N
O
O
O
R
TFA, SOCl
2
O
N
N
N
n
O
H
O
H
O
R
n =1, 2
O
O
O
N
O
Cl
Structure
A
N
i
N
H
OH
N
N
Scheme 1 Cyclization of the aspartic acid derivatives to the 3-N-
acyl-amino-pyrrolidine-2,5-dione—Structure A
O
O
5
O
O
ii, iii
Boc-Glu(OBzl)-OH
PS-SynPhase Lantern
O
O
H
O
O
N
Cl
N
H
N
N
H
MeO
O
N
O
O
O
O
6
N
Cl
OMe
n
N
O
N
R
N
H
iv, v
n =1, 2
way 2
N
Cl
O
way 1
TFA/SOCl2
N
N
H
O
N
O
H
N
N
Cl
O
7
O
N
Cl
O
N
N
N
N
n
R
N
H
n
O
O
O
R
Scheme 3 Synthesis routes to the 1-N-cyclohexanoyl-{4-[4-(3-chlo-
rophenyl)piperazin-1-yl]-butyl}-pyrrolidine-5-one-2-carboxamide (7).
(i) 4-[4-(3-chlorophenyl)piperazin-1-yl]butylamine, HBTU, TEA,
CH2Cl2, rt, 8 h; (ii) TFA; (iii) cyclohexane carboxylic acid, HBTU,
TEA, CH2Cl2, rt, 8 h; (iv) 30% HBr/AcOH; (v) SOCl2, CHCl3
Structure B
Structure C
Scheme 2 Possible routes of cyclization of the glutamic acid
derivatives: glutarimide—Structure B, and pyroglutamic acid—
Structure C
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Int J Pept Res Ther (2011) 17:93–100
95
studied N-acylated glutamic acid derivatives—compounds
3 and 4 (Scheme 5).
glutarimide ring and a five-membered pyroglutamyl moiety
(Scheme 5, Structures B and C, respectively).
The model compounds were submitted to ESI ? MS. In
MS spectra of compounds 1 and 2 apart from molecular
ions, isotopic ions (M ? 2)? were observed. Their inten-
sity equated to 33% of the molecular ion peak which
proved the presence of a chlorine ion. During spectra
investigation it was found that two routes of fragmentation
of Structure A occur simultaneously (Scheme 4). The first
one is the result of a loss of an acyl-amino moiety in
compound 2, forming a succinimide carbocation—348.2/
350.2 Da (way 1).
Even though, the five-member ring is favored due to the
less significant tensive interaction, six-membered glutari-
mides were also described in literature (Xiao et al. 2002).
In the mass spectra analysis of cyclized glutamic acid
derivatives three types of ions were observed. The cleavage
of an amide bond between the acyl residue and an amine
group at position 3 of a glutarimide led to formation of two
types of ions: ammonium ions—379.2/381.2 (way 1) and
iminium ions—377.2/379.2 (way 2). Furthermore, fission
between an imide nitrogen atom of Structure B and the
alkyl chain, or between a nitrogen atom of an amide bond
linking the pyroglutamyl residue with alkyl spacer in the
case of Structure C led to characteristic isotopic carboca-
tions for the halogen derivative—251.2/253.2 (way 3).
Determination of the manner of cyclization of glutamic
acid was problematic because the same fragmentation ions
were observed in two cases. Thus, at the next stage, com-
pound 4 was subjected to a Collisionally Activated
Decomposition (CAD) experiment. The spectrum analysis
proved the existence of protonated molecular ions (489.4/
491.4 Da), and the fragmentation ions presented in Table 1.
It could be noticed that fragment ions characterized by
masses higher than 200 Da contained chlorine atoms,
Further fragmentation caused by piperazine ring dis-
turbance resulted in the creation of succinimides carboca-
tions (compound 1: 181.1, compound 2: 195.2), and a
characteristic iminium cation containing a m-chlorophenyl
moiety of 152.1 Da observed for compound 2. The second
way of fragmentation involved selective cleavage between
an imide nitrogen atom and the tetramethylene aliphatic
spacer, resulting in a 251.2/253.2 Da ion for compound 2.
During cyclative cleavage from the solid-support, an
activated lateral carboxylic group of the glutamic acid
carboxamide derivative may attack two amide bond
localized in its proximity. This may result in creation of
two kinds of cyclic derivatives: a six-membered 3-amino-
Structure A
Scheme 4 Fragmentation
routes of the compounds 1 and 2
bearing aspartic acid residue
H
O
H
H
N
Cl
N
N
N
O
n
O
R
way 2
way 1
Compound 1, R=C6H11, n=1 (Structure A) : 461.3 / 463.3
Compound 2, R =C6H5, n=2 (Structure A) : 469.2 / 471.2
way 1
way 2
O
H
O
N
NH
R
N
NH
2
O
O
R
O
+
N
Cl
N
Cl
N
Cl
N
N
N
n
n
O
n
1: not observed
2: 251.2 / 253.2
1: 334.2 / 336.2
2: 348.2 / 350.2
O
O
+
+
1: 110.0
2: 110.0
N
N
N
N
+
Cl
n
O
- CO or -CH =CH
O
2
2
1: not observed
2: 152.1
1: 138.1
2: not observed
1: 181.1
2: 195.2
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Int J Pept Res Ther (2011) 17:93–100
Structure C
Structure B
Scheme 5 Fragmentation
routes of the compounds 3
(R = phenyl) and 4
(R = cyclohexyl), bearing
cyclized glutamic acid residue
H
O
H
O
N
Cl
way 2
H
way 1
O
H
N
N
Cl
N
N
N
R
N
H
OR
N
2H
2
2
O
O
O
R
way 3
way 3
+
2H
way 1
+
H
Compound 3, R= phenyl (structure B or C) : 483.3 / 485.3
Compound 4, R= cyclohexyl (structure B or C) : 489.3 / 491.3
way 2
way 3
way 1
way 2
3: 251.2 / 253.2
4: 251.2 / 253.2
3: 379.2 / 381.2 (not stable)
4: 379.2 / 381.2
O
H
N
N
Cl
N
N
Cl
Cl
N
Cl
O
N
N
N
N
N
+
H3N
H2
2
2
2
+
OR
O
O
N
2
3: 377.2 / 379.2
4: 377.2 / 379.2
O
N
Cl
H
N
Cl
+
H2N
O
N
N
N
N
N
+
2
2
H
OR
O
O
Table 1 Fragmentation ions
obtained for cyclized N-acylated
glutamic acid derivative
compound 3
Fragmentations ions for 3 (Da)
In the LC/MS/MS it appeared that the pyroglutamyl
cycle (lactone) was the most favored isomer. The most
abundant fragment ion—ammonium 379/381 Da, if of
glutarimide origin, would very easily lose an NH3 moiety,
resulting in an unobserved carbocation 362/364 Da
(Fig. 1). The above illustrated fragmentation was, however,
observed in the case of succinimide derivatives (Structure
A, Scheme 1).
489.4/491.4
379/381
278/280
251/253
183
111
83
1H NMR Structure Analysis
contrary to fragment ions with masses lower or equal to
200 Da. Additional information concerning the genealogy
of the fragment ions, namely of 379/381 and 251/253 Da,
was obtained. A proposed fragmentation is thus presented
in Scheme 6. The lines mark the fragment ions observed in
the CAD spectra of the parent molecule.
To evidence a creation of lactam derivative, a structure of
model pyroglutamic acid derivative was investigated by 1H
NMR technique. For this purpose, compound 4 was
re-synthesized on solid support in larger quantity (6 Lan-
terns) according to the previously described strategy (Zaj-
del et al. 2004), and purified by RP-HPLC. It was also
synthesized in liquid phase according to the standard Boc/
Bzl peptide (compound 7, Scheme 3).
It was difficult to determine the mechanism of creation
of the fragment ion possessing a chlorine atom with the
mass of 278/280 Da. Its parent fragment ion was
379/381 Da. When we compared those two ions (parent
and fragment), the second one turned out to contain an odd
number of nitrogen atoms. This observation may suggest
possible loss of the HCN nitrile in the fragment ion
379/381, but the mechanism remains unclear.
1
As it was anticipated, the classic one-dimensional H
NMR spectra for compounds synthesized on solid sup-
port (compound 4) and in a solution phase (compound 7)
were similar. Thus, in the next stage two-dimensional
spectra were prepared. Standard Varian pulse sequence
was used for the COSY 2D experiment (Fig. 2). The
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Int J Pept Res Ther (2011) 17:93–100
97
N
m/z 98 : iminium ion
CAD
N
Cl
m/z 251 / 253 : carbocation
(iminium ion)
N
O
+
N
N
Cl
Cl
N
N
N
H
NH
O
O
CAD
O
O
N
Cl
NH
N
N
H
H
NH2
m/z 379 / 381 : ammonium ion
N
O
H
m/z 183 : carbocation
(iminium ion)
CAD
CAD
O
N
Cl
N
N
H
N
2 H
m/z 278 / 280 :
O
obtained by the loss of the neutre nitrogen
m/z 379 / 381 - 101 ?
or
O
m/z 251 / 253 + HCN ?
mechanism unnknown
m/z 111 : acylium ion
- CO
m/z 83 : carbocation
Scheme 6 Fragmentation routes of the compound 4
Fig. 1 Proposed fragmentation
of glutarimide derivatives
m/z 379 / 381 : ion ammonium
O
N
Cl
+
+
N
N
2H
NH3
O
O
N
Cl
O
N
N
- NH3
R
N
H
O
m/z 362 / 364 : not observed
m/z 111 : ion acylium
spectrum was measured with 16 repetitions each 256
increments each. For data processing purpose the spec-
trum was zerofilled up to 2 K and sine bell apodization
was used.
Conclusions
1
Summing up, application of mass spectrometry and 2D H
NMR has allowed determining the manner of cyclization of
the N-acylated glutamic acid derivatives. It was found that
both on solid support and in solution cyclization of N-acyl-
ated glutamic acid derivatives yielded a five-member lac-
tams—pyrrolidine-5-on-2-carboxylic acid (pyroglutamic
acid) derivatives.
In the 2D COSY spectrum of compound 4, the amide
proton observed at resonance 6.6 ppm exhibited a cross-
peak with the multiplet at resonance range 3.22–3.29 ppm.
The latter was attributed to 2 carbon protons from alkyl
spacer (C1-spacer) and 4 protons of the piperazine moiety.
Other observations indicated that the proton coming from
Ca, observed as a doublet of doublets at 4.61–4.64 ppm,
was coupled with two protons localized in the aliphatic
range of 2.17–2.24 ppm, which could be attributed to
carbon b of the pyroglutamyl moiety. Furthermore, the Ca
proton did not couple with the amide proton. This is of
particular importance, since such coupling should have
been observed for 6 member cycle—glutarimide (Structure
B, Scheme 2).
Experimental
Chemistry
All reagents and solvents were from Aldrich and Alfa
Aesar and were used without further purification. Protected
amino acids and HBTU were purchased from Iris.
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Int J Pept Res Ther (2011) 17:93–100
C
C1-spacer
out using a C18 Xterra MS, 21 9 3.0 mm column. A
flow rate of 500 ll/min and a gradient of (0–100)% B
over 5 min were used. Eluent A: water/0.1% TFA; eluent
B: acetonitrile/0.1% TFA. Positive ion electrospray mass
C
O
N
Cl
N
N
H
N
O
O
spectra were acquired at
a solvent flow rate of
100–500 ll/min. Nitrogen was used for both the nebu-
lizing gas and the drying gas. The data were obtained in
a scan mode ranging from 400 to 1400 m/z in 0.1 s
intervals; 10 scans were summed up to get the final
spectrum.
pyroglutamyl derivatives
compounds 4 (solid phase synthesis) and 7 (solution synthesis)
Collision activated dissociations (CAD) analyses were
carried out with the energy of 70 V, and all the fragmen-
tations were observed in the source.
1H NMR Experiments
NMR spectra were obtained using a Varian BB 200
(300 MHz) spectrometer at 28°C; chemical shifts (d) are
expressed in ppm downfield from the internal TMS as a
reference; J values are in hertz, and splitting patterns are
designated as follows: s (singlet), d (doublet), t (triplet), m
(multiplet). The samples were dissolved in CDCl3.
(S)-2-N-Tert-Butoxycarbonyl-4-{4-[4-(3-
Chlorophenyl)piperazin-1-yl]-Butyl}-Glutamyl Amide
5-Benzyl Ester (5)
1
Fig. 2 The COSY H NMR spectrum of the compounds 4 or 7. The
interaction between the amide protons and the protons of an aliphatic
spacer is shown in rectangle
O
O
O
N
Cl
N
N
H
N
H
Analytical HPLC were run on a Waters Alliance HPLC
instrument, equipped with a Chromolith SpeedROD col-
umn (4.6 9 50 mm). Standard conditions were eluent
system A (water/0.1% TFA), system B (acetonitrile/0.1%
TFA). A flow rate of 5 ml/min and a gradient of (0–100)%
B over 5 min were used, detection 214 nm. Retention
times (tR) are given in minutes. All melting points were
determined with Bu¨chi 353 capillary apparatus and remain
uncorrected. Elemental analyses were carried out using an
Elementar Vario EL III, and were within 0.4% of the
theoretical values. Silica gel 60 230–400 mesh, purchased
from Merck, was used for preparative chromatographic
purification.
O
O
A solution of N-2-tert-butoxycarbonyl-L-glutamic acid
5-benzyl ester (0.67 g, 2 mmol), HBTU (0.83 g, 2.2 mmol)
in CH2Cl2 (25 ml) was stirred at room temperature for 2 min
and then 4-[4-(3-chlorophenyl)piperazin-1-yl]butylamine
(0.49 g, 1.8 mmol) in CH2Cl2 (2 ml) was added followed by
the addition of TEA (0.84 ml, 6 mmol). After the reaction
has been stirred for 8 h, the solvent was removed under
reduced pressure. The oily residue was dissolved in AcOEt
(50 ml) and washed with saturated NaHCO3 (3 9 30 ml),
water (3 9 30 ml), brine, dried over Na2SO4, and finally
concentrated in vacuum. Purification by column chroma-
tography on silica gel (CH2Cl2/MeOH: 9/1) gave 5 (0.94 g,
81% yield) as white solid.
LC/MS Analysis
Samples were prepared in acetonitrile/water (50:50 v/v),
containing a 0.1% TFA. The LC/MS system consisted of
a Waters Alliance 2690 HPLC, coupled to a Micromass
(Manchester, UK) Platform II spectrometer (electrospray
ionization mode, ESI?). All the analyses were carried
1
Melting point: 106–108°C. HPLC: tR = 2.73. H NMR
(CDCl3) d (ppm) 1.42 (s, 9H, C(CH3)3), 1.54–1.56 (m, 4H,
NCH2CH2CH2), 1.90–1.95 (m, 1H, Hb Glu), 2.09–2.13 (m,
1H, Hb Glu), 2.40–2.54 (m, 4H, CH2CH2N(CH2)2, Hc Glu),
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Int J Pept Res Ther (2011) 17:93–100
99
(S)-1-N-Cyclohexanoyl-{4-[4-(3-
Chlorophenyl)piperazin-1-yl]-Butyl}-Pyrrolidin-5-on-
2-Carboxamide (7)
2.56–2.60 (m, 4H, N(CH2)2), 3.18–3.28 (m, 6H, (CH2)2N,
NCH2), 4.11–4.20 (m, 1H, Ha Glu), 5.06–5.16 (q, 2H,
CH2Ph), 5.21–5.23 (m, 1H, NHCH), 6.45 (b, 1H, NHCH2)
6.76–6.81 (m, 2H, Ph), 6.86–6.85 (m, 1H, Ph), 7.12–7.18
(t, 1H, Ph, J = 8.1 Hz), 7.32–7.37 (m, 5H, Ph). ESI
(M ? H?) calcd for C31H43ClN4O5 (monoisotope): 586.3;
found 587.5. Elemental analysis—Found (%): C, 63.66; H,
7.33; N, 9.60. Calcd (%) for C31H43ClN4O5: C, 63.41; H,
7.38; N, 9.54.
N
Cl
O
N
N
H
N
O
O
(S)-2-N-Cyclohexanoyl-4-{4-[4-(3-
Chlorophenyl)piperazin-1-yl]-Butyl}-Glutamyl Amide
5-Benzyl Ester (6)
Compound 2 was dissolved in 5 ml of a 30% solution of HBr
in acetic acid in a round bottom flask. The mixture was stirred
at 40°C for 3 h. After complete deprotection (TLC control),
the solution was concentrated under reduced pressure, and
residue co-evaporated several times with CH2Cl2 and ether
to give a slightly yellow foam. The obtained acid was left to
dry in desiccator over P2O5 overnight. Carboxylic acid was
used without further analytical identification. It was (0.63 g,
1.25 mmol) dissolved in chloroform (4 ml) and thionyl
chloride (0.1 g, 1.25 mmol) was added at 0°C. The mixture
was allowed to warm in room temperature for 60 min, and
then the mixture was heated under reflux for 6 h while stir-
ring. Then, the mixture was concentrated under reduced
pressure and the residue was dissolved in AcOEt (40 ml).
The organic phase was washed with saturated NaHCO3
(2 9 30 ml), water (2 9 30 ml), dried over MgSO4, and
finally concentrated in vacuum to give a crude product as a
yellow oil. It was purified by column chromatography over
silica gel (CH2Cl2/MeOH: 9/1) to give 7 as a white solid
(yield: 47%).
O
O
O
N
Cl
N
N
H
N
H
O
Compound 1 (0.9 g, 1.5 mmol) was stirred in a mixture
TFA/CH2Cl2 (9/1, v/v) at room temperature for 60 min.
The volatiles were removed under reduced pressure to
yield the corresponding TFA salt as orange oil (0.92 g,
100% yield). It was then dissolved in CH2Cl2 (20 ml) and
added to the mixture of cyclohexane carboxylic acid
(0.21 g, 1.65 mmol), HBTU (0.62 g, 1.65 mmol), and TEA
(0.69 ml, 4.95 mmol) in 20 ml of CH2Cl2. The reaction
was stirred at room temperature for 8 h. After concentra-
tion the resulting crude was dissolved in AcOEt (50 ml),
washed with saturated NaHCO3 (2 9 40 ml), water
(1 9 40 ml), brine, dried over MgSO4, and finally con-
centrated. Purification by column chromatography on silica
gel (CH2Cl2/MeOH: 9/1) gave 6 (0.69 g, 75% yield) as a
white powder.
1
Melting point: 131–133°C. HPLC: tR = 2.47. H NMR
(CDCl3) d (ppm) 1.14–2.00 (m, 14H, cluster-cHex,
NHCH2CH2CH2), 2.09–2.30 (m, 2H, H–Cb Pyr), 2.46–2.63
(m, 3H, CH2CH2N(CH2)2, H–Cc Pyr), 2.65–2.80 (m, 4H,
N(CH2)2), 2.97 (dt, 1H, H–Cc Pyr, J = 17.60, 10.18 Hz),
3.21–3.45 (m, 6H, (CH2)2N, NCH2), 3.47–3.61 (m, 1H,
cHex), 4.63 (dd, 1H, H–Ca Pyr, J = 7.84, 2.89 Hz), 6.60 (m,
1H, NHCH2), 6.78–6.88 (m, 2H, Ar), 6.88–6.94 (m, 1H, Ar),
7.20 (t, 1H, Ar). ESI (M ? H?) calcd for C26H37ClN4O3
(monoisotope): 488.3; found 489.4. Elemental analysis—
Found (%): C, 63.64; H, 7.96; N, 11.24. Calcd (%) for
C26H37ClN4O3: C, 63.85; H, 7.63; N, 11.46.
1
Melting point: 142–143°C. HPLC: tR = 2.81. H NMR
(CDCl3) d (ppm) 1.16–1.84 (m, 14H, cluster-cHex,
NCH2CH2CH2), 1.89–2.18 (m, 3H, Hb Glu, CH2CHCH2),
2.34–2.44 (m, 3H, CH2CH2N(CH2)2, Hc Glu), 2.53–2.63
(m, 5H, N(CH2)2, Hc Glu), 3.19–3.49 (m, 6H, (CH2)2N,
NCH2), 4.36–4.43 (m, 1H, Ha Glu), 5.06–5.12 (q, 2H,
CH2Ph), 6.33–6.37 (d, 1H, NHCH, J = 7.70 Hz), 6.65 (b,
1H, NHCH2), 6.76–6.81 (m, 2H, Ph), 6.86–6.87 (m, 1H,
Ph), 7.12–7.18 (t, 1H, Ph, J = 8.1 Hz), 7.30–7.39 (m, 5H,
Ph), ESI (M ? H?) calcd for C33H45ClN4O4 (monoiso-
tope): 596.3; found 597.3. Elemental analysis—Found (%):
C, 66.66; H, 7.54; N, 9.43. Calcd (%) for C33H45ClN4O4:
C, 66.37; H, 7.60; N, 9.38.
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