Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
%~20~3
Metal-complexing compounds find numerous uses in analy-
sis, research~ medicine and envixonmental technology.
Some examples are the recovery, removal and selective
separation of metal ions, the therapeutic removal of
toxic or radioactive metal ions from the body and the use
as ion carriers. Bonding, preferably covalent bonding,
between the complexing compound and a macromolecule or
other structures allowing the metal ions to be brought to
a particular action site is the aLm in many cases.
Particularly interesting fields of use are to be found in
medicine and diagnostics. Thus, cancer therapy with
monoclonal antibody~metal complex conjugates is described
(see Kozak et al., Trend in Biotechnology 4, 259-264
(1985) and Proc. Natl. Acad. Sci. USA 83, 474 tl986)).
The use of metal complexes in so-called magnetic reson-
ance imaging (= MRI) is also described (see, for example,
Magerstadt et al., Magnetic Resonance Imad. 3, B08-812
(1986)).
Lanthanide complexes are described, inter alia, as
"Reporter Groups" for diagnostic purposes (see, for
example, Clinical Biochem. 21, 173-178 tl988) and
EPA 298,939).
Further uses for m tal-complexing compounds and the
corresponding metal complexes can be found, for example,
Le A 28 223 - 1 -
:,
2~2~23
in WO 90/09379, WO 90/08138, WO 90/08134, WO 89/01476,
WP 89/11868, WO 89/11475, WO 89/05802 and WO 90/11282.
For use in medicine, diagnostics~ analysis and
environmental technology, the complexing agents should
have above all a high complexing constant (Rs). In
addition, the complexing agents, complexes and complex
6alts and their conjugates should meet the following
requirements:
good water-solubility, high selectivity under the condi-
tions of use, no toxicity, specificity for the si~e of
action, good metal selectivi~y and linkable structure.
These requirement~ can be influenced by the choice of
suitable structural components.
The 4-isothiocyanatobenzyl derivatives of diethylene~ri-
1~ aminepentaacetic acid and of ethylenediaminetetraacetic
acid are described as examples of metal-complexing
compounds (F. Keana et al., J. Org. Chem. 55, 2868-2871
(1990)). Linking to macromolecules and/or biomolecules is
effected here via a reaction with the isothiocyanate
group. The poor metal selec~ivity and th~ low stability
of the metal complex is a disadvantage of these com-
pounds.
Another complexing compound, N,N'~bis-(2-hydroxybenæyl)-
1-(4-bromoacetamidobenzyl)-1,2-ethylenediamine-N,N~-
diacetic acid, can be linked to antibodie~ by the 4-
bromoacetamidobenzyl group (see C.J. Mathias et al.,
Biocon~ugate Chem. 1, 204-211 (1990)). The metal
Le A 28 223 - 2 -
., . ~' ~
--" 2~2~2~
selectivity is adequate, but the stability of correspond-
ing complexes is low.
The present invention is therefore based on the ob~ect of
providing improved complexing compounds, in particular
compounds having an improved complexing affinity and at
the same time variable linkage possibilities, and the
corresponding complexes and/or complex salts.
The present invention relates to diethylenetriamine
derivatives of the formula (I)
~C~
ROOC-CH2~ ¦ ,CH2-COOR
,N- ( CH2 ) 2--N- ( CH2 ) 2-N ( I )
~OR [~OR
(~?' )n (R' )
in which
X represents Cg to Cl4-a~ylene which optionally con-
tains he~ero atom groupings, or Cl- to C20-alkylene
which contains hetero atom groupings (N, O, S (1 x,
several times)),
Y and, if appropriate, X + Y
Le A 28 223 - 3 -
:`
2~g2~23
represents NH ~ -S-S ~ ,
, ~ ~
-CH-CH21 -N ~
o
-N-oxysuccinimido, -N-maleimido, -NH2, -OH,
-COCH2-halogen, halogen, -NCO, -NCS, -CHO, -COOH,
o O O O O
Il 11 11 1~ 11 11
SH, C-halogen, -C o-c-~l, -C-ORl, -C-N~, -C-N
or CH=CH-CO2Rl,
wherein
Rl and R2 independently of one another each represent
hydrogen, a saturated or unsatllrated Cl- to C20-alkyl
radical which is optionally substituted by a phenyl
group, or a phenyl group,
X~ and X" independently of one another in each case
represent hydrogen, C1- to C10-alXyl or CB_ to C10-
aryl, it being po~sible for such alXyl or aryl
groups to b~ optionally substituted with a fuxther
substituent of the type Y,
R independently in each case represents hydrog~n,
ammonium or one equivalent of an alk~li metal or 1/2
Le A 28 223 - 4 -
.
,
2~2~3
equivalent of an alkaline earth metal,
n independently in each case represents zero, 1, 2, 3
or 4 and
R~ independently in each case represents Cl- to C4-
alkyl, chlorine or bromine.
If X represents arylene containing hetero atoms, possible
hetero ~toms are, in particular, nitrogen atoms. Indivi-
dual example~ of arylene containing hetero atomi are~
pyridylene, pyrimidylene, indolylene and quinolylene.
If Y denote~ halogen or COCH2-halogen, possible halogen
atoms are, in par~icular, chlorine, bromine and iodine.
If R rapresents one equivalent of an alkali metal, sodium
or potassium are possible in particular, and if R repres-
ents 1/2 equivalent of an alkaline earth metal, magnesium
and calcium are possible in particular.
Praferably,
X is a C~- to C10 arylene or Cl- to C8-alkylene contain-
ing no hetero atom,
Y i~ -NH2 t -OH, -COCH2-halogen, halogen, -NCO, -NCS,
-N-oxy~uccinimido, -N-maleimido, -CHO, -COOH, SH or
Le A 28 223 - 5 -
~ . .
, . .. .
, ~ ,
- ?,~82~23
~s-s
X' and X" are identical and denote hydrogen, C1- to C6-
alkyl or phenyl,
the groupS R are identical and deno~e hydrogen, sodium,
potassium, magnesium or calcium,
the two n are identical and denote zero, 1 or 2 and
all the R' are identical and denote methyl, ethyl or
chlorine.
Particularly preferably,
0 X is phenylene, naphthylene, methylene, ethylene, n-
propylene, i-propylene, n butylene or i-butylene,
Y is hydroxyl, NH2 ~ SH, C02H or halogen,
X' and X" are identical and denote hydrogen, methyl,
ethyl or phenyl,
the R are identical and denote sodium or pota3sium,
the two n are identical and denote zexo or 1 and
the two R' are id~ntical and denota methyl or chlorine.
Le A 28 223 - 6 -
.
,
, :
: 2~82~23
The diethylenetriamine derivatives of the formula (I)
according to the invention can form complexes with the
most diverse metal ions. The present invention also
relates to such complexes. Possible central metal ions
for such complexes are, for example, ions of elements
having the atomic numbers 21 29, 31, 32, 38, 39, 42-46,
48, 49 and 57-83.
Certain central metal ions are preferred, depending on
the purpose for which the complexes are to be employed.
Di- and trivalent ions of elements having atomic numbPrs
21-29, 42, 44 and 57-70, in particular chromium(III),
manganese(II), iron(II), cobalt(II), nickel(II), copper-
(II), praseodymium(III), neodymium(III), samarium(III)
and ytterbium(III) ions, are preferred for use in MRI
diagnostics.
If a ~trong magnetic moment is desired, gadolinium(III),
terbium(III), dysprozium(III), holmium(III), erbium(III)
and iron(III) ions are preferred.
For use in nuclear medicine, all or some of the central
metal ions can be derived from radioactive elements.
Examples which may be mentioned are: radioisotopes of the
element~ copper, cobalt, gallium, germanium, yttrium,
strontium, technetium, indium, ytterbium, gatolinium,
samarium and iridium.
Those central metal ions which are derived from an
element of higher atomic number, for example from
Le A 28 223 - 7 -
:~ :
~- '.
2~82~s~
lanthanide element, are particularly suitable for use in
X-ray diagnostics.
The diethylenetriamine derivatives according to the
invention can be prepared in a manner which i5 known per
se starting from diethylenetriamine (see, for example,
Bioconjugate Chem. 1, 201-204 (1990) and Austr. J. Chem.
32, 519 (1979)).
The various central me~al ions can be introduced, for
example, by reacting the parent compound of the formula
(I) where all the R - hydrogen with any desired salt or
oxide of the metal.
The diethylenetriamine ~erivatives of the formula (I) can
be used for the most diverse diagnostic and therapeutic
purposes, for example for fluorescence lahelling and
radiolabelling of biomolecules (for example proteins,
nucleic acids and antibodies) and active compounds, in
nuclear medicine, magnetic resonance Lmaging
and in therapy with metal salts.
In the labelling proces~es, linking (chemical bonding) to
the particular biomolecule or active compound can be
effected via the Y group.
The diethylenetriamine derivatives according to the
invention have the advan~age that, because of their
molecular geometry, they are stable metal complexes,
great flexibility ex~sting in respect of the choica of
Le A 28 223 - 8 -
~ , ,
2~ 2 ~
the linking group Y.
Example 1
1,7-Diphthaloyldiethylenetriamine: (1)
192 g (1.3 mol) of phthalic anhydride are dissolved
completely in 2 1 of boiling chloroform. 62 g (O.6 mol)
o diethylenetriamine are added at 50C in the course of
80 minutes, while stirring vigorou~ly. The reaction
mixture become~ yellow and a light-coloured precipitate
forms. The chlorofoxm is distilled off and the residue is
heated at 135C for 2 hours. ~he crude product is pow-
dered, dissolved in minLmum amount~ of chloroform and
filtered hot. The product is preoipi~ated by addition of
three times the amount of ethanol, filtered off, dried
and recrystallised from chloroform-ethanol. After drying
at 120C, 135 g (61% yield) of a white solid of
melting point 175-176~C (literature = 180~C) are
obtained~
Example 2a)
4-(p-Nitrobenzyl)-1,7-diphthaloyldiethylenetriamine (2a)
9.6 g of ROH (168 mmol) are dissolved complet~ly in
100 ml of hot ethanol. 60 g (168 mmol) of the compound 1
obtained in Example 1 are introduced at room temperature
and the mixture is heated under reflux for 2.5 hours. A
~urbid mixture fgrms. 37.5 g (168 mmol3 of p-nitrobenzyl
Le A 28 ?23 - 9 -
2~82~23
bromide are added, while stirring vigorously, and the
mixture is heated under reflux for a further 16 hours.
The turbid mixture is filtered hot. After the filtrate
has cooled, the residue formed is separated off and dried
in ~acu~ 66 g (80% yield) of a white solid of
melting point 130-131C (literature = 129~130C) are
obtained.
Examples 2b) to 2c)
The compounds in the following Examples 2b and 2c are
prepared analogously to Example 2a) by N-alkylation of
1,7-diphthaloyldiethylenetriamine (1) with the corres-
ponding alkyl halide.
Example 2b)
4-(p-Bromobenzyl)-1,7-diphthaloyldiethylenetriamine (2b)
p-Bromobenzyl bromide is employ~d. ~ white solid of
melting point 149-150C is obtained in a yield of 65%.
H-NMR. (in DMSO-d6): 7.85 m 4H, 7.7 m 4H, 6.95 s 4H, 3.6
t (~ z 5~z) 4H, 3.53 s 2H, 2.67 t
(; = SHz) 4H.
MS : M~ = 531
IR s 3457 bw, 2945 w, 2834 w, 1770 w, 1710 ws, 1376 ~,
1058 m.
Le A 28 223 - 10
2 ~ 3
Exam~le 2c)
4-~3-Hydroxypropyl)-1,7-diphthaloyldiethylenetriamine
(2c)
3-Iodopropanol is employed. A white solid of melting
point 111-112~C is obtained.
H-NMR: (in CDCl3). 7.7 m 8H, 3.8 t (; = 6Hz), 4H, 3.5 t
(j = SHz) 2H, 2.83 t (; = 6Hz) 4H,
2.62 t (~ = SHz) 2H, 1.1 n-2H.
MS (CI): M+ = 421.
Example 3a!
4-(p-Nitrobenzyl~-diethylenetriamine (3a)
46 g (92 mmol) of the bisphthalimide described in Example
2a) are suspended in 0.8 1 of 6N HCl and the suspension
i8 heated at the boiling point. After 12 hours under
reflux, the clear solution is cooled, whereupon white
crystals separate out. The mixture is then filtered and
the fil~rate i~ concentrated in vacuo. The white residue
is taken up in 50 ml of 2N NaOH and extracted 3 times
with 200 ml of chloroform each time. After the organic
phase had been dried over sodium sulphate, it is concen-
trated in vacuum.
14.6 g (67~ yield) of a yellow oil are obtained.
Le A 28 223
.: :
' ~ :
2~82~2~
H-NMR ~in CDCl3): 8.2 d (j = lOHz) 2H, 7.5 d (j = lOHz)
2H, 3.72 s 2H, 2.8 (t, j = 6.5Hz) 4H,
2.55 t (j = 6.5Hz~ 4H, 1.8 bs 4H.
Example 3b)
4-(p-Bromobenzyl)-diethylenetriamine (3b)
10 g (18.8 mmol) of ~he bisphthalimide described in
Example 2b) are reacted analogously to the procedure
descri~for Example 3a) to give 2.6 g (51% yield3 of
the title compound.
lH-NMR: 7.95 d (j = 9.5~z) 2H, 7.2 d (j = 9.5) 2H, 3.6 s
2H, 2.78 t (; = 6Hz) 4H, 2.64 t (j = 6Hz) 4H,
1.65 bs 4H
MS (CI): M~ = 272
Example 4
1,7-Bis-(salicylidene)-4-(p-nitrobenzyl)-diethylene-
triamine (4)
14 y (48 mmol) of the diamine deRcribed in Example 3a)
are dissolved in 500 ml of ab olu~e ethanol, and 14.4 g
(118 mmol) of ~alicylaldehyde are added. After the
mixture has been stirred at room temperature for
20 hours, the clear yellow solution i~ concentrated. The
residue is di~solved in 25Q ml of warm ethanol and kept
L A 28 223 - 12 -
,
at -4C overnight.
12.5 g (58% yield) of a yellow solid of melting point
91-93C (literature 86-88C) are obtained.
1H-NMR (in CDC13): 13.3 bs 2H, 8.25 s 2H, 7.95 dt (j = 9
S and lHz) 2H, 7.25-7.45 m 4H, 6.8-7.1
m 6H, 3.8 s 2H, 3.7 t 4H, 2.8 t 4H.
MS (CI): M+ = 446
Example_5
1,7-Bis-(2-hydro~yphenyl~-4-(p-nitrobenzyl)-diethylene-
triamine (5)
6 g (13.5 mmol3 of the Schiff~s base described in Example
4) are dissolved in 200 ml of absolute ethanol, and 1 g
(27 mmol) of sodium boro hydride is added. The reaction is
slightly exothermic. After the mix~ure has been stirred
at room temperature for 2 hours, the turbi d solution is
filtered and the residue is washed with ethanol. 4.5 g
(75~ yield) of a white solid are obtained. After
recrystallisation from warm ethanol, 2.5 g of a solid of
melting point 108-109C (literature 108-110C) are
formed.
Le A 28 223 - 13 -
.
, ., ~: ,, - ;. -
, . ,:
:
~20~
Example 6
Diethyl rl,7-bis-(2-hydroxyphenyl)-4-~p-nitrobenzyl)-
diethylenetriamine3diacetate (6)
1 g (2.24 mmol) of the diamine described in Example 5) is
dissolved in 60 ml of dry tetrahydrofuran, and 1.2 g
(7.32 mmol) of ethyl bromoacetate and 0.9 g (9 mmol) of
triethylamine are added. The clear solution is stirred at
40C for 48 hours. After cooling to 0C and subsequent
filtration, the solution is concentrated in vacu~ and the
residue is chromatographed over silica gel. (Mobile
phase: chloroform . methanol : acetonitrile = 98:1:1). -
856 mg (63% yield) of a yellow oil are obtained.
H-NMR (in CDC13): 9.7-9.8 bs 2H, 8~1 d (j = 8.5Hz) 2H,
7.38 d ~j = 8.5Hz) 2H, 7.2 dt (j = 8
and lHz) 2H, 6.8-7 m 6H, 4.17 q
(j = 7Hz) 4H, 3.72 s 4H, 3.52 s 2H,
3.25 s 4H, 2.5-2.8 m 8H, 1.22 t
(; = 7Hz) 6H.
MS (Cl): M~ = 530
Example 7
di-t-Butyl C1,7-bis(2-hydroxybenzyl)-4-(p-nitrobenzyl)-
diethylenetriamin~ diacetate (7)
6 g (13.2 mmol) of the diamine described in Example 5)
Le A 28 223 - 14 ~
,
~82~3
are dissolved in 120 ml of dry tetrahydrofuran, and
5.16 g (26.4 mmol) of t-butyl bromoacetate and 3 g
(29.4 mmol) of triethylamine are added. The clear solu-
tion is stirred at 40C for 20 hours. Further t-butyl
bromoacetate and triethylamine are added until the
diamine has reacted completely. After cooling and subse-
quent filtration, the solution is concentrated in vac~
and the residue is chromatographed on silica gel.
6.6 g (73% yield) of a pale yellow solid of melting
point 105-107C are obtained.
H-NMR (in CDCl3): 9.9-9.7 bs 2H; 8.1 d (; = 8.5 Hz) 2H;
7.35 d (8.5 Hz); 7.2 dt (j = 8 and
1 Hz) 2H; 6.7-7 m 6H; 3.73 s 4H;
3.57 s 2H; 3.18 s 4H; 2.5-2.7 m 8H;
1.43 s 18 H
MS (CI): M+ = 678
Exam~le 8
1,7-Bis-(2-hydroxybenzyl)-4-(p-nitrobenzyl)-die~hylene-
triamine-1~7-diacetic acid (8)
i- from the triamine 5 by N-alkylation:
1 g (2.24 mmol) of the triamine described in Example 5)
is dissolved in 60 ml of dry tetrahydrofuran, and 1 g
(7.3 mmol3 of bromoacetic acid and 0.9 g t9 mmol~ of
triethylamine are added. After 6 hours at 40C, the clear
Le A 28 223 - 15 -
:.
'
~82~3
solution becomes turbid. After 30 hours at 40C, the
solution is concentrated in vacuo and the residue is
purified over an ion exchanger. 1.1 g (81% yield) of
a white solid are obtained.
The solution is concentrated and the residue is purified
over an ion exchangeresin. 350 mg (77% yield) of a white
solid are obtained.
ii - from the di-t-butyl ester 7 by ester cleavage-
O.5 g (O.8 mmol) of the diester described in Ex~mple 7)
was dissolved in 25 ml of benzene, and 4.8 ml (42 mmol)
of trifluoroacetic acid were added. The reaction was com-
plete after stirring for 2 hours at 60C as judged by TLC
(mobile phase chloroform : ethanol = 9 : 1).
lS Exam~le 9
Stability constant (Ks) of europium complexes with
complexing agent 8
1. Comparison substances:
A number of complexing agents were prepared in
order to compare their Ks with complexing agent 8.
2,6-Disubstituted pyridine derivatives
form particularly stable complexes with europium ions
~EPA 298 939). 4-Chloro-2,6-bis[N,N-bis(carboxy-
methyl)aminomethyl]pyridine (9~ was prPpared by the
Le A 28 223 - 16 -
. :,
~82~23
method of Yogtle and 9hm in Chem. Ber. 117, 948
(1984).
2. Europium complexes:
The europium complexes of 8 and 9 were prepared from
the corresponding sodium salts. To prepare the sodium salts,
4 molar equivalents of sodium hydroxide in aqueous
solution were added to complexing agents 8 and 9 and
the mixtures were then concentrated in vacu um.
3. Stability constant tKs)-
The equilibrium con~tan~ (R) for the formation of a
europium complex (EuL-) from the europium ion IEu3+)
and the ligands 8 and ~ (L4-) Is called the stability
constant Rs and is dete~mined from the law of mass
action as follows:
K = c(EuL-)
c(L4-) c(Eu3i)
Constant molar ratios of Europium (III) chloride
hexahydrate and the ligand in question were dissolved
in a defined volume of water. The concentration of non-
complexed europium wa~ determined at pH 4.65 by
titration with ~DTA ~andard ~olution against
xylenol or~nge a~ the indicatox. The EDTA complex is
weaker than the complexes under investigation.
Le A ~8 223 - 17 -
,...... .
- ~
: ,
. .
2~2~23
Therefor, only free Eu+3 are selectively detected.
Ks for 9 = 1.~7 x 102 moll-L
K9 for 8 = ~ (non-determinable)~
) Since no free europium ion was to be detected. The
detection limit of the method i6 approximately
3 10-5M
Le A 28 223 - 18 -
.
