Decomposition of 1-(ω-aminoalkanoyl)guanidines under alkaline conditions

Article abstract of DOI:10.1016/j.tetlet.2007.07.147

The decomposition of some N^G-(ω-aminoalkanoyl)argininamides, which are key intermediates for the preparation of radiolabeled and fluorescent neuropeptide Y receptor ligands, prompted us to synthesize a small series of simple 1-(ω-aminoalkanoyl)guanidines, and to investigate these model compounds for stability in alkaline buffers. The degradation of acylguanidines was monitored by time resolved UV spectroscopy. The most labile compound, 1-(5-aminopentanoyl)guanidine, decomposed with a half life of 19 s to yield piperidin-2-one (pH 10.4 at 25 °C). In contrast the half life of 1-(6-aminohexanoyl)guanidine is 7.7 h, which is comparable to the hydrolysis of acetylguanidine (t_1/2 = 9.6 h) in alkaline solution.

Full text of DOI:10.1016/j.tetlet.2007.07.147

Tetrahedron Letters 48 (2007) 6996–6999
Decomposition of 1-(x-aminoalkanoyl)guanidines
under alkaline conditions
Albert Brennauer, Max Keller, Matthias Freund,
Gunther Bernhardt and Armin Buschauer*
¨
University of Regensburg, Institute of Pharmacy, Universita¨tsstr. 31, D-93040 Regensburg, Germany
Received 12 June 2007; revised 18 July 2007; accepted 24 July 2007
Available online 27 July 2007
Abstract—The decomposition of some NG-(x-aminoalkanoyl)argininamides, which are key intermediates for the preparation of
radiolabeled and fluorescent neuropeptide Y receptor ligands, prompted us to synthesize a small series of simple 1-(x-aminoalkano-
yl)guanidines, and to investigate these model compounds for stability in alkaline buffers. The degradation of acylguanidines was
monitored by time resolved UV spectroscopy. The most labile compound, 1-(5-aminopentanoyl)guanidine, decomposed with a half
life of 19 s to yield piperidin-2-one (pH 10.4 at 25 °C). In contrast the half life of 1-(6-aminohexanoyl)guanidine is 7.7 h, which is
comparable to the hydrolysis of acetylguanidine (t1/2 = 9.6 h) in alkaline solution.
Ó 2007 Elsevier Ltd. All rights reserved.
Guanidines are among the strongest non-anionic
organic bases. The corresponding guanidinium ions
are featuring the ability to form tight complexes with
carboxylates, phosphonates, and sulfonates;1 hence,
the guanidine substructure plays an important role for
molecular recognition processes in numerous biological
and artificial systems. Numerous natural products and
synthetic drugs comprising the guanidine motif are
known.2 As a natural amino acid arginine with its gua-
nidino side chain is frequently found in the interaction
sites of proteins.3
NG-acyl substituents do not necessarily impair the
carboxylate binding properties of guanidines:9,10 In
our lab we discovered that the neuropeptide Y (NPY)
Y1 receptor antagonistic potency of the argininamide
derivative BIBP 322611 can be enhanced by introduction
of acyl substituents at the guanidine nitrogen.8,12
Based on this concept, we tried to synthesize NG-(x-
aminoalkanoyl) substituted argininamides as precursors
for radio- or fluorescence labeled NPY receptor ligands.
However, it turned out that the x-amino group can dra-
matically accelerate the decomposition of these com-
pounds, depending on length and nature of the spacer.
In this work we report on the degradation and the half
lives of different x-aminoalkanoylguanidines in alkaline
buffer.
Due to their strong basicity (pKa ꢀ 12 of the conjugate
acids) guanidines are positively charged at physiological
pH. This is a major disadvantage with respect to the
pharmacokinetic profiles of guanidine derived drug can-
didates. Therefore, many efforts have been undertaken
to find less polar bioisosteric replacements for the guani-
dine motif.4–6
In an effort to label NG-(5-aminopentanoyl)-substituted
argininamides by acylation of the primary amino group
in the presence of a base, almost exclusively the NG-
unsubstituted arginine derivative could be isolated. This
supported the idea that the acyl-NG bond was cleaved by
intramolecular attack of the terminal amino function at
the carbonyl C-atom. In order to test this hypothesis we
synthesized 1-(5-aminopentanoyl)-guanidine (1d) as a
model compound for investigations on stability.
By introduction of an electron withdrawing acyl substi-
tuent at the guanidino nitrogen the pKa value can be
reduced by 4–5 orders of magnitude.7,8 Interestingly,
Keywords: Acylguanidines; Lactam formation; Degradation rate; UV
spectroscopy.
*
Opportunely, acylguanidines in general exhibit strong
UV absorbance around 230 nm.13 This rendered
Corresponding author. Tel.: +49 941 943 4827; fax: +49 941 943
4820; e-mail: armin.buschauer@chemie.uni-regensburg.de
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.07.147

A. Brennauer et al. / Tetrahedron Letters 48 (2007) 6996–6999
6997
Scheme 1. Degradation of 1-(5-aminopentanoyl)guanidine (1d) yield-
ing d-lactam and unsubstituted guanidine.
Figure 1. Time-dependent UV spectra of (5-aminopentanoyl)guani-
dine (1d) at pH 10.4.
possible to monitor the kinetics of the degradation of 1d
in alkaline buffers by time-resolved UV spectroscopy (cf.
Fig. 1).
Remarkably, the absorption of the acylguanidine moiety
was extinguished after only a few minutes. As acylguani-
dines in general are stable and isolable compounds,
obviously, the presence of the terminal amino group
dramatically enhanced the rate of decomposition by
intramolecular nucleophilic attack (6-exo-trig ring clo-
sure). In fact piperidin-2-one (d-lactam) was identified
as the degradation product of 1-(5-aminopentanoyl)gua-
nidine (1d) in alkaline solution by means of TLC and
RP-HPLC (cf. Scheme 1).
Scheme 2. Preparation of 1-(x-aminoacyl)guanidines by acylation of
1-(tert-butoxycarbonyl)guanidine with activated N-Boc-protected x-
amino acid derivatives and subsequent removal of Boc-protecting
groups with TFA-CH2Cl2 1:1.
In addition, x-aminoalkoxycarbonyl-guanidines, pre-
pared from N-Boc protected amino alcohols, were
probed as spacer variant. The alcohols were treated with
disuccinimidyl carbonate (DSC) yielding the corre-
sponding activated mixed carbonates, which were
allowed to react with 1-Boc-guanidine (cf. Scheme 2).
The exponential decay of the acylguanidine absorbance
at 230 nm allows for the calculation of (pseudo-)first
order rate constants (kobs) and the respective half-lives
(t1/2) by logarithmic plotting and linear regression of
the data points. For the degradation of 1-(5-amino-
pentanoyl)guanidine (1d) in alkaline aqueous buffers
we observed half-lives of 18.4 min, 42 s, and 19 s at
pH 8, 9, and 10.4, respectively.
The degradation rate in alkaline borate buffer (pH 10.4)
for all x-aminoacylguanidines (1–3) was determined
using time-resolved UV spectroscopy. Results are shown
in Table 1. The slow reaction of acetylguanidine (entry
4) and 4-(aminomethyl)cyclohexanecarbonylguanidine
(2), which are unable to form lactams, reveals that there
must be an alternative degradation pathway for acyl-
guanidines—most probably hydrolytic cleavage.13,15
Though, the isolation and characterization of the reac-
tion products was beyond the scope of this study, some
information about the potential degradation pathways
can be derived from comparison of the reaction rates
and the UV spectra.
In quest of alternative, more stable spacers we tested
various x-aminoalkanoylguanidines of various chain
lengths or with a cyclic scaffold for their stability in alka-
line buffers. The compounds were prepared by acylation
of 1-(tert-butoxycarbonyl)guanidine, which is accessible
by treating guanidine hydrochloride with a hypostoi-
chiometric amount of Boc2O in 4 M NaOH.14
The N-tert-butoxycarbonyl (Boc) protected amino acids
were either applied as succinimidyl esters, or activated
with 1,10-carbonyldiimidazole (CDI) and coupled with
1-Boc-guanidine. The resulting blocked aminoalkanoyl
guanidines were deprotected with 50% trifluoroacetic
acid (TFA) in CH2Cl2 (cf. Scheme 2). The obtained
di-trifluoroacetates are sufficiently stable in acidic
solutions.
In the case of glycylguanidine (1a) the formation of an
a-lactam is implausible. Nevertheless, 1a reacts much
faster than acetylguanidine, indicating that the a-amino
substituent participates in the degradation reaction.
Rink et al.16 found that Nx-(a-Fmoc-aminoacyl)argi-
nines form 2-amino-1H-imidazol-4(5H)-ones (5) when
the Fmoc group is cleaved off in the presence of an
excess of piperidine (cf. Scheme 3).
The UV spectrum of cyclic acylguanidine 5a shows an
For details cf. Supplementary data.
absorption maximum at 225 nm (in phosphate buffer

6998
A. Brennauer et al. / Tetrahedron Letters 48 (2007) 6996–6999
Table 1. Degradation of various aminoacylguanidines in alkaline buffer (pH 10.4) at 25 °C; (pseudo-) first-order rate constants and half-lives
Compound
Structure
k
max/nm
kobs/minꢁ1
t1/2
1a
1b
1c
1d
1e
n = 1
n = 2
n = 3
n = 4
n = 5
227
230
230
230
230
0.97
0.028
0.44
2.25
0.0015
43 s
4.1 h
96 s
19 s
7.7 h
2
231
0.0009
13 h
3a
3b
4
215
215
228
0.0043
0
2.7 h
1
0.0012
9.6 h
The huge difference in the reaction rates of 1d, the most
labile compound, and 1e, the most resistant compound
is surprising since these aminoalkanoylguanidines differ
only by one methylene group. Obviously, there is a
substantial difference in the tendency to form six-
membered and seven-membered lactams from 1-(x-
aminoalkanoyl)guanidines.
Scheme 3. Intramolecular reaction of 1-(a-aminoacyl)guanidines
according to Rink et al.16
x-Aminoalkoxycarbonyl substituted guanidines, which
were taken into account as alternative spacer groups,
are considerably more stable than the corresponding
x-aminoalkanoylguanidines. 1-Boc-guanidine and 1-[2-
(2-aminoethoxy)ethyloxycarbonyl]guanidine (3b) were
completely stable under the reaction conditions. The
absorption maxima of the alkoxycarbonylguanidines
are at shorter wavelengths (typically 215–225 nm) than
those of alkanoylguanidines.
at pH = 12)—but with a slightly lower molar extinction
compared to acyclic acylguanidines.15 These data are in
good agreement with the slight hypsochromic shift (227
vs 224 nm) and the minor hypochromism we observed
for the reaction of 1a in alkaline buffer. Thus, the forma-
tion of 5a is the most probable mechanism for the deg-
radation of 1a.
Within the series of linear 1-(x-aminoalkanoyl) guani-
dines b-alanyl (1b) and 6-aminohexanoyl (1e) substi-
tuted guanidines exhibit the lowest degradation rates
(cf. Table 1). The half lives for 1b and 1e are approxi-
mately 4 and 8 h, respectively. Obviously, the formation
of corresponding b- or e-lactams is significantly less
favored than the formation of the d-lactam, which
proceeds within minutes.
In contrast 1-(3-aminopropyloxycarbonyl)guanidine (3a),
an oxa analog of the highly reactive aminoalkanoyl-
guanidine 1d, reacts with a half-life of 2.7 h. In
principle the decomposition of both 3a and 1d can be
facilitated in a similar way by intramolecular nucleo-
philic attack (‘6-exo-trig’) of the terminal amino group.
However, the time-dependent absorption spectrum of 3a
in alkaline buffer shows the disappearance of the initial
maximum at 215 nm and simultaneously the appearance
of a new maximum at 224 nm. Since carbamates exhibit
no absorbance in this region, the formation of a cyclic
six-membered carbamate is implausible, whereas the
preservation of the chromophoric acylguanidine sub-
structure is more obvious. This can (only) be explained
by a rearrangement of 1-(3-aminopropyloxycarbonyl)-
guanidine (3a) to 1-(3-hydroxypropylaminocarbonyl)-
guanidine (6) by initial nucleophilic attack of the
terminal amino group at the acyl carbon atom and
subsequent cleavage of the ester bond (cf. Scheme 4).
Amidinourea shows a strong absorption maximum at
approximately 220–225 nm,17 which is in good agree-
For 1-(b-aminopropanoyl)guanidine (1b) the formation
of a cyclic acylguanidine is less likely than in the case
of 1a. The expected absorption maximum at 233 nm,
corresponding to 2-amino-5,6-dihydropyrimidin-4(1H)-
one, could not be observed; moreover, the hypothetical
product is known to be unstable in alkaline solution
(t1/2 = 4 h at pH 12).15 1-(4-Aminobutanoyl)guanidine
(1c), which can form a five-membered c-lactam, decom-
poses at a comparably rapid rate as 1a and 1d. The most
stable linear x-aminoalkanoyl substituted guanidine in
this series is 1e, the decomposition of which proceeds
only slightly faster than the alkaline hydrolysis of acet-
ylguanidine (4).

A. Brennauer et al. / Tetrahedron Letters 48 (2007) 6996–6999
6999
Acknowledgment
Support of this work by the Research Training Program
(Graduiertenkolleg GRK 760) of the Deutsche For-
schungsgemeinschaft is gratefully acknowledged.
Supplementary data
Supplementary data includes synthesis and analytical
data of the discussed compounds as well as details about
the kinetic measurements. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.tetlet.2007.07.147.
Scheme 4. Hypothetical mechanism for the formation of putative
isomerization product 6 from 1-(3-aminopropyloxycarbonyl)guanidine
3a, supported by HPLC–MS analysis: after an incubation period of
60 min two peaks of identical mass, 161 Da (MH+), with capacity
factors of 0.55 (3a) and 1.88 (6) were detected, whereas at 0 min only
one peak corresponding to 3a was identified.
References and notes
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In conclusion, acylguanidines are promising, less polar
bioisosteres of the strongly basic guanidino group.
However, acylguanidines tend to decompose when sub-
jected to alkaline conditions. The alkaline hydrolysis of
acetylguanidine proceeds with a half-life of 9.6 h.
Decomposition can be extremely accelerated if an
intramolecular nucleophilic attack is possible, as demon-
strated by the cleavage of 1-(5-aminopentanoyl)guani-
dine, which is completely converted to d-lactam within
minutes in alkaline solution. However, there are pro-
nounced differences in the reaction rates of linear 1-(x-
aminoalkanoyl)guanidines depending on the length of
the chain.
Compared to aminoalkanoylguanidines, the analogous
aminoalkoxycarbonyl-substituted guanidines are con-
siderably more stable toward alkaline hydrolysis. In gen-
eral the oxa analogs are inert in aqueous buffer at pH
10.4 and 25 °C. However, the reaction can be facilitated
by intramolecular nucleophilic groups in appropriate
distance to the electrophilic center.
On one hand, the degradation pathways described in
this study should be taken into account in the design
and synthesis of aminoalkanoylguanidines such as
NG-substituted argininamides, which are useful building
blocks for the preparation of fluorescent and radiola-
beled NPY receptor antagonists. On the other hand,
the 5-aminopentanoyl spacer could potentially serve as
an easily cleavable linker for the immobilization of gua-
nidines on solid support or as tunable protecting group
for the guanidino function based on the principle of
‘assisted cleavage’.
16. Rink, H.; Sieber, P.; Raschdorf, F. Tetrahedron Lett. 1984,
25, 621–624.
17. Takimoto, M. Nippon Kagaku Zasshi 1964, 85, 159–168.