New charge-deficient agmatine analogues

Article abstract of DOI:10.1007/s11171-005-0080-y

N,N′-Di-Boc-N″-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously unknown isosteric and charge-deficient agmatine analogues, hav

Full text of DOI:10.1007/s11171-005-0080-y

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 6, 2005, pp. 583–587. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 6, 2005, pp. 645–650.
Original Russian Text Copyright © 2005 by Simonyan, Grigorenko, Vepsalainen, Khomutov.
New Charge-Deficient Agmatine Analogues
,
1
A. R. Simonyan*, N. A. Grigorenko*, J. Vepsalainen**, and A. R. Khomutov*
*
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
*
* Department of Chemistry, University of Kuopio, P.O.Box 1627, FIN-70211, Kuopio, Finland
Received March 18, 2005; in final form, April 11, 2005
Abstract—N,N'-Di-Boc-N''-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-
substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously
unknown isosteric and charge-deficient agmatine analogues, have been synthesized. The possibilities of using
these compounds in studying polyamine metabolism are discussed.
Key words: agmatine, polyamines, O-substituted hydroxylamines
1
INTRODUCTION
Agmatine is one of key arginine metabolites
RESULTS AND DISCUSSION
AO-Agm is an isosteric charge-deficient (pK of
H NO group is ~5.0) analogue of Agm. Like other
a
2
(Scheme 1). It is widely distributed in bacteria, plants,
2
and Invertebrata [1]. In mid-1990s, Agm has also been O-susbstituted hydroxylamines, it forms stable oximes
found in eukaryotes; however, its content is low (from with aldehydes and ketones, including pyridoxal 5'-
a few to tens of mg per kg of tissue [2]). Agm is an phosphate (Fig. 1); the latter oxime is a coenzyme of
effector of NO synthase; it is bound by imidazoline ArgDC. The aminooxy analogues of the pyruvate- and
receptors and fulfills functions of neurotransmitter in pyridoxal 5'-phosphate-dependent enzymes of the
brain (see review [1] and references therein). Agm is amino acid metabolism are the efficient inhibitors of
actively transported into cells by the system of Put these enzyme (see review [5] and references therein),
transport and affects the polyamine homeostasis. In this and, therefore, one can expect a high activity of AO-
case, the exhaustion of the intracellular pool of sper- Agm (I) toward the ArgDC.
mine and spermidine sharply increases the efficiency of
Agm transport into cells [3].
Some guanidine-containing diamine derivatives,
including Agm and 1-guanidino-7-aminoheptane (an
The Agm conversion into Put is an initial stage of
polyamine biosynthesis in bacteria and plants [1]. In
A
animal cells, Put originates from ornithine. At the same
1
.5
time, there are data on the possibility of Put biosynthe-
sis from Agm [3]; however, the existence of this meta-
bolic pathway in eukaryotes is not generally admitted
1
.2
2
1
[4].
A productive approach to the clarification of role
0.9
and functions ofAgm in cell is the study of biochemical
effects of its analogues. We describe here the synthesis
of previously unknown isosteric charge-deficient Agm
analogues N-(3-aminooxypropyl)- and N-(3-aminopro-
poxy)guanidines on the basis of 3-aminooxy-1-amino-
propane. The possibility of using these compounds for
the chemical regulation of the activity of polyamine
metabolism enzymes is discussed.
0
0
.6
.3
0
3
00
330
360
390
420
450
nm
1
Corresponding author; phone: +7 (095) 135-6065; fax: +7 (095)
1
35-1405; e-mail: alexkhom@genome.eimb.relarn.ru
2
Abbreviations: Agm, agmatine (1-guanidino-4-aminobutane);
AO-Agm, N-(3-aminooxypropyl)guanidine; ArgDC, arginine
decarboxylase; GAPA, N-(3-aminopropoxy)guanidine; ODC,
ornithine decarboxylase; Put, putrescine (1,4-diaminobutane);
and Tf, triflate (trifluoromethanesulfonate).
Fig. 1. UV spectra of (curve 1) pyridoxal 5'-phosphate and
(curve 2) its oxime with AO-Agm. 1, pyridoxal 5'-phos-
–
4
phate (2.28 × 10 M) in 0.1 M sodium acetate buffer, pH
–
4
5.0; 2, the same but in the presence of 5 × 10 M AO-Agm
(1 min after mixing).
1
068-1620/05/3106-0583 © 2005 Pleiades Publishing, Inc.

5
84
SIMONYAN et al.
NH2
NH NH2
HOOC
NH
1
5
Arginine
NH2
NH2
3
NH2
NH NH2
O
HOOC
HOOC
Ornithine
Citruline
NH NH2
NH
H N
2
2
6
4
Putrescine
H N
2
HOOC
NH NH2
NH
NH2
Putrescine
γ-Guanidinebutyric acid
H N
2
NH
NH2
Spermidine
Scheme 1. Metabolism of agmatin [1]. 1, Arginase (EC 3.5.3.1); 2, ODC (ornithine decarboxylase, EC 4.1.1.17);
3
6
, ArgDC (arginine decarboxylase, EC 4.1.1.19); 4, agmatinase (EC 3.5.3.11); 5, NO synthase (EC 1.14.13.39); and
, diaminooxydase (EC 1.4.3.6).
efficient inhibitor of deoxyhypuzine synthase [6]), are are feeding with the seeds of this plant can cleave cana-
actively transferred into cells [3]. Consequently, one vanine to canavaline (γ-aminooxy-α-aminopropionic
can expect that GAPA (II), which, at physiological pH acid) [7]. Accordingly, the possibility of intracellular
values, is a doubly protonated structural analogue of degradation of GAPA to 3-aminooxy-1-aminopropane,
Agm (pK of the H N(NH)CNHO group of GAPA is
a
2
one of the most efficient ODC inhibitors [8–10], cannot
~
7.0–7.5, whereas the pK of NH group is ~10.0), be excluded. Therefore, (II) is interesting for the study
a
2
would be actively transported into cells exploiting the of special features of the Put active transport into cells;
Put transport system. There are known the examples of it can be regarded as an actively transportable proinhib-
catabolism of alkoxyguanidines. In particular, amino
acid canavanine (α-amino-γ-guanidinooxybutyric acid)
is widely distributed in the plants of family Legumino-
itor of ODC.
The guanidination of amino- and aminooxy groups
sae. This amino acid contains up to 13% of the whole was the key stage in the synthesis of AO-Agm and
bound nitrogen in the seeds of Dioclea megacarpa [7]. GAPA. A great number of guanidinating reagents are
The larvae of the corn beetle Caryedes brasiliensis that known from the chemistry of amino acids. Methyl
ethers of urea and thiourea, their N-protected deriva-
tives, and the corresponding pyrazoles are the most fre-
NH
quently used reagents among them [11–13]. However,
the reaction rates are low in a number of cases, and the
yields of target products are far from quantitative [6,
14]. Triflates of di-Boc, di-Cbz [15], and di-Fmoc [16]
derivatives of guanidine have recently been suggested
for the guanidinaion of amines. These reagents convert
amines into the corresponding guanidines for several
hours at room temperature in yields close to quantita-
tive. Therefore, in this work, we guanidinated amino
groups using the readily available di-Boc-guanidine tri-
flate.
NH2
H N
2
N
O
H
AO-Agm (I)
NH
NH2
O
N
NH2
H
GAPA (II)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

NEW CHARGE-DEFICIENT AGMATINE ANALOGUES
Me
585
Me
i
ii, iii
Et
Et
O
O
NH2
Et
O
NH NHBoc
O
NH NH · 2TosOH
2
O
O
N
O
N
H N
2
(III)
(IV)
NBoc
NH
AO-Agm (I)
iv
NBoc
Me
O
NHCbz
v
vi
NHCbz
O
NHCbz
BocNH NH
N
H2N
(V)
(VI)
(VII)
vii
NH
O
NH · 2HBr
2
NH2 NH
GAPA (II)
i
–
Reagents: i—(BocNH) C=NTf/Et N/CH Cl ; ii—HCl/Pr OH/H O; iii—AG 1-X8 TosO -form;
2
3
2
2
2
iv—Cbz-Cl/Et N/THF; v—HCl/EtOH/H O; vi—(BocNH) C=NTf/Et N/CH Cl /37°C;
3
2
2
3
2
2
vii—HBr/AcOH,
Scheme 2. Synthesis of hydroxylamine-containing agmatine analogues.
NH2
N
NHBoc
NBoc
O
O
(
BocNH) C=NTf
2
(VIII)
(IX)
1
2
3
Scheme 3. Synthesis of N -benzyloxy-N ,N -di-tert-butyloxycarbonylguanidine (IX).
AO-Agm (I) was obtained starting from 3-(1'-ethoxy- value of rate constant of the reaction of the second order
ethylidene)aminooxy-1-aminopropane (III) (Scheme 2). (molar ratios of O-benzylhydroxylamine and di-Boc-
The guanidination of free amino group in (III) with di- guanidine triflate) determined by the NMR method
Boc-guanidine triflate was achieved by a standard pro- according to the integral intensity of protons of CH2
cedure [15]. It led to di-Boc derivative (IV) in yield group of (VIII) (δ 4.63 ppm) and protons of CH group
2
close to quantitative. The removal of protective groups
of the di-Boc protected benzyloxyguanidine (IX) (δ
by HCl/EtOH resulted in theAO-Agm dihydrochloride,
which was slowly deliquesced in air and converted to a
well crystallized tosylate (I).
Yield, %
The synthesis of GAPA (II) was a more complicated
1
00
task, since, practically, the methods of guanidination of
aminooxy group were not developed. The conversion of
protected canaline into canavanine, described in [17],
proceeds as a result of treatment with S-methylisothio-
urea sulfate (yield 10%) or a boiling with cyanamide in
alcohol (yield 70%). We have used here di-Boc-guani-
dine triflate to obtain guanidinooxyalkanes for the first
time. Its interaction with O-benzylhydroxylamine
5
0
0
4
8
12 16 20 24
Time, h
(
VIII) (Scheme 3) showed that the corresponding
Fig. 2. The kinetics of guanidination of O-benzylhydroxyl-
guanidine derivative (IX) is formed in a high yield.
However, the reaction proceeds slower (Fig. 2) than in
the case of more nucleophilic aliphatic amines. The
amine (0.3 M) with di-Boc-guanidine triflate (0.3 M) in the
presence of triethylamine (0.3 M) in CDCl (0.5 ml) at
3
37°ë.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

5
86
SIMONYAN et al.
–
2
–1
–1
5
.01 ppm) was shown to be 2.66 × 10 l mol min
75 min at 37°ë).
washed with 10% citric acid, water, 1 M NaHCO ,
3
(
τ
water, and saturated NaCl; and evaporated in a vacuum
1
/2
to dryness. The residue was dried in a vacuum over
Analogue (II) was synthesized from 3-(1'-ethoxy-
phosphorus pentoxide, and get (IV); a viscous oil; yield
ethylidene)aminooxy-1-aminopropane (III) (Scheme 2).
The free amino group of (III) was initially benzyloxy-
carbonylated, and the ethoxyethylidene protection was
then removed by HCl in aqueous alcohol. This resulted
1
0
.50 g (91%); R 0.76 (A); H NMR (CDCl ): 4.00–3.93
f
3
(
1
4 H, m, CH CH + NOCH ), 3.52 (2 H, m, CH NH),
.91 (3 H, s, CH C=NO), 1.89 (2 H, m, CH CH CH ),
3 2 2 2
3 2 2 2
in the corresponding hydrochloride, which was then 1.48 [9 H, s, C(CH ) ], 1.46 [9 H, s, C(CH ) ], 1.24 (3
converted into base (VI). The base was guanidinated by H, t, J 7.16, CH CH ).
3
3
3 3
3
2
di-Boc-guanidine triflate (24 h at 37°ë) to (VII)
obtained in a high yield. The simultaneous removal of
Boc and Cbz protective groups with HBr/AcOH at
room temperature led to the crystalline dihydrobromide
of (II).
Thus, we found that di-Boc-guanidine triflate effec-
tively guanidinates not only amines, but also the sub-
stantially less nucleophilic O-alkylhydroxylamines,
which allows the preparation of new isosteric charge-
deficient analogues of Agm: AO-Agm and GAPA.
N-(3-aminopropoxy)guanidine ditosylate (I). A
3
7% solution of HCl (0.5 ml) was added to a solution
of (IV) (0.34 g, 0.85 mmol) in isopropanol (5 ml). The
reaction mixture was kept for 5 h at room temperature,
evaporated in a vacuum to dryness; the oily residue was
several times coevaporated with absolute isopropanol
and dried in a vacuum over P O /KOH, to give crystal-
2
5
line slowly deliquescent N-(3-aminopropoxy)guanidine
1
dihydrochloride; yield 0.22 g; R 0.28 (B); H NMR
f
(
D O): 4.16 (2 H, t, J 6.0, H NOCH ), 3.33 (2 H, t, J 6.8,
2 2 2
13
CH NH), 1.99 (2 H, m, CH CH CH ); C NMR (D O):
2
2
2
2
2
EXPERIMENTAL
159.74 (s, NH CNH), 75.42 (t, H NOCH ), 40.63 (t,
2 2 2
CH NH), 29.43 (t, CH CH CH ). The dihydrochloride
2
2
2
2
The following reagents were used in this work: Cbz-
Cl and absolute ethanol from Fluka and ion-exchange
resin AG 1-X8 200–400 mesh from BioRad. 3-(1'-
was converted into ditosylate on a column packed with
–
ion-exchange resin AG 1-X8 in Tos form, which was
recrystallized from absolute ethanol and dried in a vac-
Ethoxyethylidene)aminooxy-1-aminopropane
was
uum over P O to give (I); yield 0.33 g (83%); mp 195–
synthesized as described in [18]; N,N'-di-Boc-guani-
dine triflate, as described in [15]; and O-benzylhydrox-
ylamine base, as described in [19]. TLC was carried out
2
5
1
96°ë. Found, %: C 45.62, H 6.03, N 11.87.
C H N O S . Calc., %: C 45.36, H 5.92, N 11.76.
18 28
4
7 2
on precoated Kieselgel 60 F2 plates (Merck) in the
54
3-Aminooxy-1-(N-benzyloxycarbonyl)aminopro-
pane (VI). A solution of (III) (1.33 g, 8.3 mmol) and
triethylamine (1.6 ml, 11 mmol) in THF (15 ml) was
cooled to 0°ë and treated at a vigorous stirring with
systems: (A) 4 : 1 chloroform–methanol; (B) 120 : 49 :
3
0 : 1 methanol–water–25% ammonia–trifluoroacet-
amide; (C) 95 : 5 CCl –MeOH; and (D) 7 : 3 dioxane–
4
2
5% ammonia. Substances were detected on the chro- Cbz-Cl (1.2 ml, 8.0 mmol) in three portions at 15-min
matograms by the UV-light absorption and the color intervals. The mixture was then stirred for 1 h at 0°ë
reaction with ninhydrin; aminooxy compounds, in the and for 3 h at room temperature. The triethylamine
form of fluorescing oximes of pyridoxal 5'-phosphate. hydrochloride was filtered off; the filtrate was evapo-
Silica gel of the trade mark Kieselgel (40–63 µm, rated in a vacuum to dryness; and the residue was dis-
Merck) was used for column chromatography; elution solved in chloroform (20 ml), cooled to 4°ë, and suc-
systems are given in the text.
cessively washed with cool water, 10% citric acid,
water, 1 M NaHCO , and water. The extract was dried
NMR spectra were registered on a Bruker Avance
3
with MgSO and evaporated in a vacuum. The residue
5
00 DRX instrument (Germany) at the working fre-
4
1
13
was dried in a vacuum over ê é to give (V) as a vis-
quency of 500.1 MHz for H and 125.8 MHz for C
2
5
nuclei. The substances were coevaporated with CD OD cous oil; yield 2.39 g (92%); R
0.58 (C).
3
f
and then dissolved in CDCl . Tetramethylsilane (for
3
A solution of (V) (2.39 g, 7.7 mmol) in absolute eth-
CDCl solutions) and sodium 3-trimethylsilyl-1-pro-
3
anol (13 ml) was mixed with 37% HCl (1.7 ml), kept
panesulfonate (for D O solutions) were used as internal for 10 min at room temperature, and evaporated in a
2
standards. Chemical shifts are given in ppm and spin– vacuum to dryness. The residue was recrystallized from
spin coupling constants in Hz.
EtOH–EtOAc and get (VI) hydrochloride; yield 1.47 g
1
2
(73%); R 0.62 (E). A solution of (VI) hydrochloride
f
N -[3-(1'-Ethoxyethylidene)aminooxypropyl]-N ,
3
(0.52 g, 2 mmol) in water (7 ml) was treated with 10 M
NaOH (0.2 ml), extracted with chloroform, and the
extract was dried with K CO . The solution was evapo-
N -di-tert-butyloxycarbonylguanidine (IV). A solu-
tion of di-Boc-guanidine triflate (0.53 g (1.35 mmol) in
dichloromethane (3 ml) added to a solution of (III)
0.24 g, 1.5 mmol) and triethylamine (0.21 ml,
.5 mmol) in dichloromethane (3 ml). The reaction
mixture was kept for 1.5 h at room temperature; diluted
with an equal volume of dichloromethane; successively m, C
2
3
rated in a vacuum to dryness, and the solid residue was
(
1
dried in a vacuum over P O to give (VI); yield 0.38 g
2
5
1
(
86%); R 0.51 (Ä); H NMR (CDCl ): 7.34–7.30 (5 H,
f
3
H
6
), 5.37 (2 H, s, PhCH ), 3.70 (2 H, t, J 5.92,
2
5
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

NEW CHARGE-DEFICIENT AGMATINE ANALOGUES
587
H NOCH ), 3.29–3.24 (2 H, m, CH NHZ), 1.79–1.73
ACKNOWLEDGMENTS
2
2
2
(
2 H, m, CH CH CH ).
2 2 2
This work was supported by the Russian Foundation
of Basis Research, project no. 03-04-49080, and the
Academy of Sciences of Finland.
1
2
3
N -(3-Benzyloxycarbonylaminopropoxy)-N ,N -
di-tert-butyloxycarbonylguanidine (VII). A solution
of di-Boc-guanidine triflate (0.49 g, 1.26 mmol) in
chloroform (3 ml) was added to a solution of (VI)
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9
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(
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1
1
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(
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f
3
1
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7
.31 (5 H, m, C H ), 5.01 (2 H, s, C H CH ), 1.48 [9 H,
6 5 6 5 2
s, C(CH ) ], 1.45 [9 H, s, C(CH ) ].
3
3
3 3
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005