Synthesis of 2-(nitromethyl)ornithine from ornithine mediated by cobalt(III)

Article abstract of DOI:10.1016/S0020-1693(02)00691-6

Rac.-p-(tris(2-aminoethyl)amine-2-(nitromethyl)ornithine)cobalt(III) trichloride (2d) was obtained by a simple three-step procedure from ornithine using cobalt template chemistry. p-(Tris(2-aminoethyl)amine-ornithine)cobalt(III) trichloride (2a) was obtained from tris(2-aminoethyl)amine (tren) and (S)-ornithine in the presence of cobalt(II), which was oxidised to cobalt(III) during the reaction. Complex 2a was selectively oxidised with thionyl chloride-dimethyl formamide to p-(tris(2-aminoethyl)amine-dehydro-ornithine)cobalt(III) trichloride 2b. Complex 2c, in which reaction of thionyl chloride-dimethyl formamide has also occurred at the δ-amine of ornithine, was obtained at longer reaction times. Complex 2b reacted with nitromethane anion to give rac.-p-(tris(2-aminoethyl)amino-2-(nitromethyl)ornithine)cobalt(III) trichloride (2d). The amino acid rac.-2-(nitromethyl)ornithine (1b) was released by reducing complex 2d with aqueous ammonium sulfide. Complex 2d was expected to release 2-(nitromethyl)ornithine (1b) in hypoxic cells, where the amino acid could act as an inhibitor of ornithine decarboxylase. Preliminary data indicated that complex 2d was weakly cytotoxic in one cell type studied.

Full text of DOI:10.1016/S0020-1693(02)00691-6

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Inorganica Chimica Acta 331 (2002) 318–321
Synthesis of 2-(nitromethyl)ornithine from ornithine mediated by
cobalt(III)
Philip A. Butler a, Christopher G. Crane b, Bernard T. Golding a,*,
Anders Hammershøi c, David C. Hockless b, Tue B. Petersen c, Alan M. Sargeson b,
David C. Ware d
a Department of Chemistry, Bedson Building, Uni6ersity of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK
b Research School of Chemistry, Australian National Uni6ersity, Canberra, ACT 0200, Australia
c Department of Chemistry, Uni6ersity of Copenhagen, Uni6ersitetsparken 5, DK-2100, Copenhagen Ø, Denmark
d Department of Chemistry, Uni6ersity of Auckland, Pri6ate Bag 92019, Auckland, New Zealand
Received 21 August 2001; accepted 24 October 2001
Dedicated to Professor A.G. Sykes FRS in recognition of his major research contribution to the Department of Chemistry, University of
Newcastle upon Tyne
Abstract
Rac.-p-(tris(2-aminoethyl)amine-2-(nitromethyl)ornithine)cobalt(III) trichloride (2d) was obtained by a simple three-step proce-
dure from ornithine using cobalt template chemistry. p-(Tris(2-aminoethyl)amine-ornithine)cobalt(III) trichloride (2a) was
obtained from tris(2-aminoethyl)amine (tren) and (S)-ornithine in the presence of cobalt(II), which was oxidised to cobalt(III)
during the reaction. Complex 2a was selectively oxidised with thionyl chloride–dimethyl formamide to p-(tris(2-aminoethyl)amine-
dehydro-ornithine)cobalt(III) trichloride 2b. Complex 2c, in which reaction of thionyl chloride–dimethyl formamide has also
occurred at the d-amine of ornithine, was obtained at longer reaction times. Complex 2b reacted with nitromethane anion to give
rac.-p-(tris(2-aminoethyl)amino-2-(nitromethyl)ornithine)cobalt(III) trichloride (2d). The amino acid rac.-2-(nitromethyl)ornithine
(1b) was released by reducing complex 2d with aqueous ammonium sulfide. Complex 2d was expected to release 2-(ni-
tromethyl)ornithine (1b) in hypoxic cells, where the amino acid could act as an inhibitor of ornithine decarboxylase. Preliminary
data indicated that complex 2d was weakly cytotoxic in one cell type studied. © 2002 Published by Elsevier Science B.V.
Keywords: Cobalt complexes; Template chemistry; Ornithine
cobalt(III) complexes of ornithine, in which the metal
ion acts as both a protecting and activating group [5,6],
1. Introduction
to synthesise a cobalt chelate of 2-(nitromethyl)-
Derivatives of ornithine 1a that contain an a-
ornithine. This approach differs from that convention-
ally used by achieving an Umpolung at the a-centre of
the amino acid.
halogenomethyl substituent inhibit ornithine decar-
boxylase (ODC) irreversibly through interaction with
the pyridoxal cofactor (see Scheme 1) [1]. Such com-
pounds were synthesised from ornithine using classical
amino acid protection-activation chemistry [2,3], often
under quite vigorous conditions. The standard ap-
proach to the synthesis of a-substituted amino acids is
to alkylate a suitably protected/activated anion of the
parent a-amino acid [4]. We now report the use of
Scheme 1. Steps in the mechanism-based inhibition of ornithine
decarboxylase by an amino acid containing a suitably placed leaving
group (e.g. X=F) and involving a nucleophilic group (NuH) on
ornithine decarboxylase (PCHO=pyridoxal phosphate).
* Corresponding author.
0020-1693/02/$ - see front matter © 2002 Published by Elsevier Science B.V.
PII: S0020-1693(02)00691-6

P.A. Butler et al. / Inorganica Chimica Acta 331 (2002) 318–321
319
Cobalt(III) complexes of the type described herein
are potential mechanism-based inhibitors of ornithine
decarboxylase provided the cobalt(III) can be reduced
to the labile cobalt(II) state, which releases the active
agent. This may be feasible in solid tumours because
they usually contain a sub-population of hypoxic cells,
which can effect the reduction [7,8]. Such a strategy has
been used for liberating cytotoxic nitrogen mustards
complexed to cobalt(III) [9]. The released ornithine
derivative described herein may inhibit ODC as indi-
cated by Scheme 1 (X=NO2), provided NO−2 acts as
an efficient leaving group from an intermediate formed
with pyridoxal phosphate. The required nitrite elimina-
tion is analogous to that which occurs in the oxidation
of nitroethane by flavin adenine dinucleotide-dependent
C, 26.92; H, 7.50; N, 16.80. Calc. for
CoC11H30Cl3N6O2·3H2O: C, 26.54; H, 7.30; N, 16.89%.
2.2. p-(Tris(2-aminoethyl)amine-dehydro-ornithine)-
cobalt(III) trichloride (2b)
A solution of complex 2a (6.00 g, 12.05 mmol) in
trifluoromethanesulfonic acid (25.0 ml) was stirred un-
der nitrogen for 2 h. Dilution with diethyl ether (500
ml) gave a precipitate that was washed with ether and
dried under a nitrogen flow. The resulting solid was
taken up in dimethylformamide (120 ml), treated with
thionyl chloride (25 ml) drop wise and then stirred for
10 min at room temperature (r.t.). The mixture was
then carefully poured into water (250 cm3), stirred for
30 min and filtered (Celite filter aid). The filtrate,
diluted with water (2-l), was loaded onto a SP Sephadex
C25 Na+ column (22×6 cm) and eluted with water
(500 ml) followed by aqueous sodium acetate (0.4 M) to
give two minor bands followed by a major yellow band.
This last band was collected and desalted on AG
50W-X2 with hydrochloric acid (3 M). Concentration
to dryness gave a crude product that was dissolved in
hot 3 M HCl followed by the addition of ethanol until
the point of turbidity and the mixture was left at 5 °C
for crystallisation. The product (yellow solid) was col-
lected, washed with ethanol and diethyl ether. A further
crop of crystals was isolated from the mother liquor
resulting in a combined yield of 4.25 g (77%); 13C NMR
(D2O): l 189.9 (COO), 174.2 (CꢀN), 64.5, 61.7
(N(CH2)3), 47.9, 47.4 (CH2NH2), 41.2, 34.7, 25.0 (Orn-
CH2); UV–Vis spec: umax, nm (mmax, cm−1 mol−1): 461
(120); UV spec: umax, nm (mmax, cm−1 mol−1): 223
(22 710); IR (KBr, cm−1) 3393, 3195, 3045, 2856, 1667,
1612, 1283; Anal. Found: C, 28.98; H, 6.50; N, 18.12.
Calc. for CoC11H28Cl3N6O2·H2O: C, 28.74; H, 6.53; N
18.28%.
D
-amino acid oxidase [10]. Alternatively, competitive
inhibition analogous to that reported [11] for a-methyl-
ornithine is a possibility.
2. Experimental
N.b. In all isolated complexes described below the
5-amine group of the ornithine fragment was
protonated.
2.1. p-(Tris(2-aminoethyl)amine-ornithine)cobalt(III)
trichloride (2a)
Tris(2-aminoethyl)amine (4.39 g, 30.0 mmol), NaOH
(1.20 g, 30.0 mmol) and activated charcoal (1.0 g) were
added to a solution of CoCl2·6H2O (7.14 g, 30.0 mmol)
and
L-(S)-ornithine monohydrochloride (5.06 g, 30.0
mmol) in water (100 ml). To the resulting mixture was
added 28% H2O2 (4.0 ml) in small portions over 1 h
and the solution was stirred for a further 1 h before 3.0
M HCl (10 ml) was added. The reaction mixture was
filtered (Celite filter aid) and the diluted filtrate (1-l)
absorbed on a column of AG 50W-X2 cation exchange
resin (22×6 cm). After washing with water, elution of
the cations with 0.5 M HCl revealed a minor purple
band, which was discarded. A major orange band was
eluted with 3 M HCl and the eluent evaporated to
dryness. The solid residue was dissolved in the mini-
mum amount of hot 3 M HCl followed by addition of
ethanol until the point of turbidity, the mixture was
then left at 5 °C for crystallisation. The product (or-
ange solid) was collected, washed with ethanol and
diethyl ether. Further product was isolated from the
mother liquor resulting in a combined yield of 9.56 g
(64%); 13C NMR (D2O): l 185.7 (COO), 63.2, 60.5
(N(CH2)3), 59.2 (CH), 47.2, 46.4, (CH2NH2), 40.5, 31.1,
25.3 (Orn-CH2); UV–Vis spec: umax, nm (mmax, cm−1
2.3. Rac.-p-(tris(2-aminoethyl)amine-2-(nitromethyl)-
ornithine)cobalt(III) trichloride (2d)
To complex 2b (3.30 g, 7.18 mmol) in 2:1 (v/v)
aqueous sodium hydroxide (0.1 M) and methanol (400
ml) was added nitromethane (23.7 ml). The mixture was
stirred for 2 h, quenched with hydrochloric acid (0.02
M) and diluted with water (500 ml). The resulting
solution was loaded onto a SP Sephadex C25 Na+
column (22×6 cm). Elution with water (500 ml), and
then aqueous sodium acetate (0.4 M) gave a minor
orange band followed by a major orange band. The
latter was collected and desalted on AG 50W-X2 with
hydrochloric acid (3 M). Concentration to dryness gave
a crude product that was dissolved in hot 3 M HCl
followed by the addition of ethanol until the point of
turbidity and the mixture was left at 5 °C for crystalli-
sation. The product was collected, washed with ethanol
mol−1): 342 (113), 473 (126); UV spec: umax, nm (mmax
,
cm−1 mol−1): 221 (20 407); IR (KBr, cm−1) 3432,
3185, 3078, 2947, 2891, 1644, 1593, 1381; Anal. Found:

320
P.A. Butler et al. / Inorganica Chimica Acta 331 (2002) 318–321
3. Results and discussion
The synthesis of a cobalt(III) complex of a 2-substi-
tuted ornithine was readily achieved in three steps as
shown in Scheme 2. Thus, the tris(2-aminoethyl)amine-
cobalt(III) complex of ornithine 2a was obtained di-
rectly from tris(2-aminoethyl)amine (tren) and
(S)-ornithine in the presence of cobalt(II), with in situ
oxidation to cobalt(III) by hydrogen peroxide. Com-
plex 2a was selectively oxidised with thionyl chloride–
dimethyl formamide [12] to the relatively stable imine
complex 2b. At longer reaction times complex 2c, in
which reaction of thionyl chloride–dimethyl formamide
has also occurred at the d-amine of ornithine, was
obtained in significant quantity. The imine function in
complexes such as 2b is known to be activated towards
nucleophiles such as the anion of nitromethane [13].
Accordingly, complex 2b readily reacted with ni-
tromethane anion to give rac.-p-(tris(2-aminoethyl)-
amino-2-(nitromethyl)ornithine)cobalt(III) trichloride
(2d). The amino acid rac.-2-(nitromethyl)ornithine (1b)
was released by reducing complex 2d with aqueous
ammonium sulfide. This important chemoselective re-
duction opens the way to the synthesis of a variety of
a-(nitroalkyl)-substituted amino acids from amino acids
and nitroalkanes.
Scheme 2. Synthesis of rac-[p-(tris(2-aminoethyl)amine-2-(ni-
tromethyl)ornithinato)cobalt(III)]3+ (2d) [reagents: (i) SOCl2–
Me2NCHO; (ii) MeNO2–NaOH (2b“2d)]; n.b. for clarity, hydrogen
atoms have been omitted from the tris(2-aminoethyl)amine ligand.
and diethyl ether to give an orange solid (2.25 g, 56%);
13C NMR (D2O): l 183.9 (COO), 81.2 (CH2NO2), 65.5
(quaternary C), 64.7, 64.5, 61.9 (N(CH2)3), 49.0, 48.4,
47.6 (CH2NH2), 41.3, 35.4, 23.9 (Orn-CH2); UV–Vis
spec: umax, nm (mmax, cm2 mol−1): 480 (149), 343 (162);
UV spec: umax, nm (mmax, cm2 mol−1): 221 (23 164); IR
(KBr, cm−1) 3451, 3323, 3204, 3121, 3059, 3008, 1651,
1608, 1539, 1388; Anal. Found: C, 25.47; H, 6.57; N,
17.01. Calc. for CoC12H31Cl3N7O4·4H2O: C, 25.07; H,
6.85; N, 17.06%.
2.4. 2-(Nitromethyl)ornithine
(2,5-diamino-2-nitromethyl-pentanoic acid) (1b)
Aqueous ammonium sulfide (20% w/v, 1 ml) was
The coordinated 2-(nitromethyl)ornithine in complex
2d can bind to the tren-cobalt(III) template in two
orientations (p and t, where the carboxylate of the
amino acid is trans to a primary amine of tren or to the
tertiary amine group, respectively). X-ray crystallo-
graphic analysis of the imine complex 2b defines the
orientation as p (see Fig. 1)1. The general kinetic inert-
added to
a
stirred solution of rac.-p-tris(2-
aminoethyl)amine-a-nitromethylornithinato-cobalt(III)
trichloride tetrahydrate (0.60 g, 1.05 mmol) in water (40
ml). After 30 min the mixture was acidified with HCl
(37% w/v, 2 ml), purged with N2 for 20 min and filtered
through Kieselguhr. The filtrate was diluted with water
(400 ml) and loaded onto a Bio-Rad AG 50W-X2
column (3×10 cm). The column was washed with
water (500 ml) and the subsequent eluent was passed
through a flow cell in a spectrophotometer monitoring
at 240 nm. Elution with 0.5 M HCl (500 ml) removed
Co2+ ions. Elution with 1 M HCl produced a single
band that was collected and the hydrochloric acid was
removed. To the residue was added ethanol to the point
of turbidity. Crystallisation was allowed to proceed at
5 °C to give fine white crystals (0.20 g, 78%) of the title
compound, m.p. 180–182 °C (dec.).
1 Data were collected at −6091 °C for an orange plate crystal
(C11H28Cl3CoN6O2, M=651.8) with dimensions approximately
0.30×0.24×0.10 mm using a Rigaku AFC6R diffractometer with
graphite monochromated Cu Ka radiation and a 12 kW rotating
anode generator. Primitive monoclinic cell dimensions were: a=
11.419(4), b=15.000(2), c=15.476(3) A, i=111.85(2)o, V=
,
3
−3
,
2460.4(10) A ; for Z=4 the calculated density was 1.76 g cm
;
space group P21/a (c14). The structure was solved by heavy-atom
Patterson methods and expanded using Fourier techniques. The
non-hydrogen atoms were refined anisotropically. Hydrogen atoms
were included in calculated positions but not refined. Water hydro-
gens were located from a difference map. The final cycle of full-ma-
trix least-squares refinement was based on 2333 observed reflections
(I\3|(I)) and 326 variable parameters and converged with un-
weighted and weighted agreement factors of: R=SꢀꢀFoꢀ−ꢀFcꢀꢀ/
SꢀFoꢀ=0.070; Rw=[I(Sw(ꢀFoꢀ−ꢀFcꢀ)2/(SwFo2)]1/2=0.080, good-
ness-of-fit=3.60. All calculations were performed using the TEXSAN
crystallographic software package [Crystal Structure Analysis Pack-
age, Molecular Structure Corporation (1985 and 1992)].
2
1H NMR (D2O): l 4.95 (dd, 2H, CH2NO2, JHH=16
Hz), 2.88 (t, 2H, CH2NH3), 1.59 (m, 4H, CCH2CH2);
13C NMR (D2O): l 171.8 (COO), 77.8 (CH2NO2), 62.8
(quaternary C), 40.8, 32.6, 22.9 (Orn-CH2); IR: 3393,
2987, 2514, 2000, 1644, 1566, 1514, 1395, 1288 cm−1
;
Anal. Found: C, 29.58; H, 6.06; N, 17.13. Calc. for
C6H13ClN3O4·H2O: C, 29.46; H, 6.18; N, 17.18%.

P.A. Butler et al. / Inorganica Chimica Acta 331 (2002) 318–321
321
use of tris(2-aminoethyl)amine as supporting ligand at
cobalt necessarily leads to racemic 1b. In principle,
monochiral [14] amino acids can be obtained by em-
ploying a chiral cobalt moiety, e.g. bis(ethane-1,2-
diamine)-cobalt.
Acknowledgements
We thank Dr. W.R. Wilson for the biological assays
and the EPSRC (CASE award with Phoenix Chemicals,
Brombrough to P.A.B.) and Australian National Uni-
versity for support.
Fig. 1. X-ray crystal structure of p-(tris(2-aminoethyl)amine-dehydro-
ornithine)cobalt(III) trichloride (2b).
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Complex 2d has been tested (4 h exposure to 2d in
air) with four cancer cell lines: Chinese hamster ovary
(CHO) AA8, CHO UV4 (a repair deficient mutant of
the AA8 cells), human ovary SKOV-6 and human
colon EMT6. For the CHO UV4 cells the IC50 was 89
mM, whilst for the other cell lines the IC50 was \1
mM. The modest activity of complex 2d towards the
UV4 cells should be enhanced by changing the support-
ing ligands at cobalt, to achieve a more positive redox
potential and hence readier access to the kinetically
labile cobalt(II) oxidation state. Experiments are there-
fore in progress to generate such cobalt(III) complexes
in order to enhance the release of a-substituted or-
nithine derivatives. The finding that the amino acid 1b
can be released from complex 2d by reduction with
aqueous ammonium sulfide (see above) shows that
chemoselective reduction of Co(III) to Co(II) can at
least be chemically achieved.
The strategy described for the synthesis of complex
2d and hence amino acid 1b should be applicable to the
synthesis of a variety of a-substituted amino acids from
imino acids and carbanions. In the present work, the