USE OF COMPLEXES OF RADIOACTIVE METAL IONS WITH ALL-CIS-1,3,5-TRIAMINO-2,4, 6-CYCLOHEXANETRIOL AND ITS DERIVATIVES FOR RADIODIAGNOSTIC PURPOSES AND IN TUMOR THERAPY AND FOR THE PREPARATION OF AGENTS FOR RADIODIAGNOST C PURPOSES AND FOR TUMOR THERAPY

EMPLOI DE COMPLEXES D'IONS DE METAUX RADIO-ACTIFS ET DE L'ISOMERE UNIQUE CIS-1,3,5-TRIAMINO-2,4,6-CYCLOHEXANETRIOL ET DE SES DERIVES POUR LE RADIODIAGNOSTIC ET LES THERAPIES ANTITUMORALES ET POUR LA PREPARATION D'AGENTS EN VUE DU RADIODIAGNOSTIC ET DES THERAPIES ANTITUMORALES

English Abstract

- 13 -
Abstract:
Use of complexes of radioactive metal ions with
all-cis-1,3,5-triamino-2,4,6-trihydroxycyclohexane, its
derivatives, salts or quaternary ammonium salts for
radiodiagnostic purposes and in tumour therapy, and for
the preparation of agents for radiodiagnostic purposes
and for tumour therapy.

Claims

Laboratorien Hausmann AG
Use of complexes of radioactive metal ions with all-cis-
1,3,5-triamino-2,4,6-cyclohexanetriol and its derivatives
for radiodiagnostic purposes and in tumour therapy and
for the preparation of agents for radiodiagnostic pur-
poses and for tumour therapy.
Patent Claims
1. Use of complexes of radioactive metal ions with
all-cis-1,3,5-triamino-2,4,6-trihydroxycyclohexane or its
derivatives of the general formula (I)
<IMG> (I)
as ligands, in which the symbols R1, R2, R3, R4, R5 or R6
are identical or different and represent hydrogen atoms,
alkyl groups or -CO-alkyl groups, where the alkyl in the
alkyl or -CO-alkyl groups has 1 to 18 carbon atoms, and
where the alkyl and -CO-alkyl groups can each, indepen-
dently of one another, contain one or more, identical or
different functional groups, and their salts with
inorganic or organic pharmacologically customary acids or
their quaternary ammonium salts of the general formula
(II), (IIa) or (IIb)
<IMG> <IMG> <IMG>
II IIa IIb
with pharmaceutically utilisable anions, in which R1 to R6
and R7, R8 and R9 denote, independently of one another,
the unsubstituted or substituted alkyl groups or -CO-
alkyl groups defined above, or with all-cis-1,3,5-tri-
amino-2,4,6-trihydroxycyclohexane derivatives of the
general formula

- 2 -
<IMG> III <IMG> IV
or
n=4-8 n=4-8
in which the substituents on the nitrogen atoms are,
independently of one another, H or CH3, as ligands, for
radiodiagnostic purposes and in tumour therapy and for
the preparation of agents for radiodiagnostic purposes
and for tumour therapy.
2. Use according to Claim 1, characterised in that
the radioactive metal ions are .alpha., .beta. or ? emitters.
3. Use according to Claim 1 or 2, characterised in
that the radioactive metal ions are ions of radioactive
isotopes of iron, copper, gallium, yttrium, ruthenium,
technetium, rhodium, indium, dysprosium, samarium,
thulium, ytterbium or rhenium.
4. Use according to any of Claims 1 to 3, charac-
terised in that positively charged complexes are
employed, in particular for the radiodiagnostic demon-
stration of the heart and for preparing agents therefor.
5. Use according to ClaLm 4, characterised in that
complexes with the ligands of the formula (II), (IIa),
(IIb) or (IV) according to Claim 1 are employed.
6. Use according to Claim 5, characterised in that
the radicals R1 to R9 in the formulae (II), (IIa) and
(IIb) are each methyl groups and that the substituents on
the nitrogen atoms on the formula (IV) are methyl groups.
7. Use according to Claim 5, characterised in that
the radicals R1 to R6 in the formulae (II), (IIa) and
(IIb) are methyl groups and the radicals R7, R8 and R9 are
each H.

- 3 -
8. Use according to any of Claims 1 to 4, charac-
terised in that the alkyl groups or -CO-alkyl groups
contain one or more functional groups which can co-
ordinate to metal ions.
9. Use according to Claim 4, characterised in that
the ligands of the general formula (I) are employed, in
which at least one of the radicals R1, R3 and R5 has the
general formula
- (CH2)n - N? (R)3
in which n = 1, 2 or 3, in particular 2, R is alkyl with
1, 2, 3 or 4 carbon atoms, and the radicals R2, R4 and R6
are hydrogen atoms or alkyl groups with 1 to 18 carbon
atoms.
10. Use according to any of Claims 1 to 3, charac-
terised in that ligands of the general formula (I), (II),
(IIa) or (IIb) are employed, in which the radicals R1, R3
and R5 are alkyl groups which have functional groups and
the general formula
- (CH2)n - COOH,
in which n = 1, 2 or 3 and, in particular 2 and the
radicals R2, R4 and R6 to R9 are each H.
11. Use according to Claim 10 for tumour therapy or
for the preparation of agents for tumour therapy.
12. Process for the preparation of complexes of
radioactive metal ions for radiodiagnostic purposes and
for tumour therapy, characterised in that an aqueous
solution or dispersion of a derivative of the general
formula (I), (II), (IIa), (IIb), (III) or (IV) as defined
in Claims 1, 7, 8, 9 or 10 is reacted with an aqueous
solution or dispersion of a salt, hydroxide or oxide of
a radioactive metal.
13. Process according to Claim 12, characterised in
that a salt, hydroxide or oxide of a metal which is an
.alpha., .beta. or ? emitter is used.

- 4 -
14. Process according to Claim 12 or 13, charac-
terised in that the salt, hydroxide or oxide of a metal
which comprises one or more radioactive isotopes of iron,
copper, gallium, yttrium, ruthenium, technetium, rhodium,
indium, dysprosium, samarium, thulium, ytterbium or
rhenium is used.
15. Process according to any of Claims 12 to 14,
characterised in that to prepare an agent suitable for
radiodiagnostic purposes or for tumour therapy, the
aqueous solution obtained according to any of Claims 12
to 14 is prepared, and the latter is mixed where appro-
priate with customary pharmaceutical additives.

Description

20~3~0
Use of complexes of radioactive metal ions with all-cis-
1,3,5-triamino-2,4,6-cyclohexane~riol and its derivatives
for radiodiagnostic purposes and in tumour therapy and
for the preparation of agents for radiodiagnostic pur-
5 poses and for tumour therapy.
The invention relates to the use of complexes of
all-cis-1,3,5-triamino-2,4,6-cyclohexanetriol and its
derivatives with ions of radioactive metal isotopes for
radiodiagnostic purposes and in tumour therapy and for
the preparation of agents for radiodiagnostic purposes
and fox tumour therapy.
The use of metal complexes for therapeutic or
diagnostic administration i5 described in Inorganic
Chemistry in Biology and Medicine, ACS Symposium Series
140, American Chemical Society, Washington, D.C. (1980),
pages 91 to 101, 103 to 119 and 121 to 140. Complexes of
radioactive isotopes have also been employed ~or pharma-
ceutical purposes, in particular for diagnostic purposes.
They can be used for the demonstration of various organs
of the body. A disadvantage of the demonstration methods
to date is the limited possibility of employment for
certain applications. For example, radiographic
demonstration of the human and animal heart has not been
possible.
The object of the invention is to provide novel
possibilities for radiodiagnostic demonstration and for
tumour treatment.
It has emerged that this object can be achieved
by using complexes of radioactive metals, in particular
radioactive metal cations with all-cis-1,3,5-triamino-
2,4,6-trihydroxycyclohexane and its derivatives for
,~ radiodiagnostic purposes and in tumour therapy or for the
preparation of agents for radiodiagnostic purposes and
for tumour therapy.
The complexes used according to the invention are
preferably based on radioactive metal i90topes which are
~, ~ or ~ emitters~ Examples of radioactive isotopes
- which can be used are those of technetium, iron, gallium,
ruthenium, indium, dysprosium, samarium, ~hulium,
- - - - .. .... .. . . ....
.
~' '

2073V~0
ytterbium and y-t-trium. Specific examples are ions of the
f~llowing isotopes 59Fe, aZcu, b7G~, 63Ga 72Ga g5Ru 97Ru
Tc, ~h, LllIn, 1l3~n, 157D~ 153Sm l67Tm 163Yb 1~ZR
1~6Re
The ligands used for the complexes according to
the invention are all-cis-1,3,5-txiamino-2,4,6-
cyclohexanetriol and its derivatives as described, for
- example, in European Patent 0 190 676 (uS Patent
4 794 197) and in Helv. Chim. Acta. Vol 73, 1990, pages
97 to 105. They are prepared by the methods indicated
therein.
Particularly suitable ligands are all-cis-
1,3,5-triamino-2/4,6-trihydroxycyclohexane or its deriva-
tives of the general formula (I)
H0 R, a~
R ~ R
: in which the symbols Rl, R2, R3, R4, Rs or R6 are identical
or different and represent hydrogen atoms, alkyl groups
or -CO alkyl groups, where the alkyl in the alkyl or -CO-
alkyl groups has 1 to 18 carbon atoms, and where the
alkyl and -CO-alkyl groups can each, independently of one
: another, contain one or more, identical or.different
functional groups, and their salts with inorganic or
organic pharmacologically customary acids or their
quaternary ammonium salts of the general formula (II),
(IIa) or (IIb)
`f ~4'~
" 11
.. .. ... ...... ... .. ... .. . .. . . ....... ... ..... .... ... .. . . ...... ,, . . .. . , .~.. .. ~ ..

2~73~0
-- 3
with pharmaceuticall~I utilisable anions, in which R1 to R~
and R7, R8 and R~ denote, independently of one another,
the unsubstituted or substltuted alkyl groups or -CO-
alkyl groups defined above, or all-cis-1,3,5-triamino-
2,4,6-trihydroxycyclohexane deri~atives of the general
formula
~ Q~ n H~ ~ ~
in which the substituents on the nitrogen atoms are,
independently o~ one another, H or CH3 and are preferably
all CH3.
Examples for the radicals Rl to R~ in the ligands
are alkyl or -CO-alkyl groups which can contain one or
more, identical ox different functional groups. In each
:`case a maximum of one symbol of the symbols Rl to Rg on
:~15 each nitrogen atom is preferably a secondary or tertiary
alkyl substituent or a -CO-alkyl group. Moreover, the
:other symbols preferably represent hydrogen or methyl.
A preferred group of ligands are those in which
all the radicals R1 to R6 and Rl to R9 are methyl or -COCH3
groups.
:The alkyl groups or -CO-alkyl groups o~ the sub~
:~.stituents R1 to R6 and R1 to R~ preferably have one or more
functional groups which can coordinate to metal cations.
Examples of functional groups on the alkyl groups or -CO-
alkyl groups are -OH, -COO~ or salts thereof, -CONH2,
-CON(OH)R, where R represent~ an alkyl group with 1 to 6,
in particular with 1 to 4 carbon atoms, -OPO3H2 and salts
thereof, -PO3H2 or salts thereo, -SR (in which R has the
above meaning), and the ester~ of the above acids, -CN,
.. ., .. ., .... , .. ~... .... .... ~
'
-

- 2~73~
-- 4
Ho
1 =1~
~ and/or
J~ ~
and/or their salts.
:`Preferred examples are those of the ligands of
the formula (I), (II), (IIa) or (IIb) in which one or
~:more or all of the symbols Rl to Rg have, independently of
one another, the following meaning:
-i a) -H f) -(CH2)nCO~(OH)R
R=alkyl with C1-12
; b) -alkyl, with 1-12 C preferably C14
atoms or -CO-alkyl with
1-12 C atom~ in the g) -( CH2 ) nPO3~
alkyl
h) -(CH2)nPO32
c) - (CH2)nOH
i ) - ( CH2 ) nSR
d) -(CH2)nC02 R=alkyl with C1-l2,
preferably C14
e) -( CH2 ) nCONH2
~; . ' .
.`'~' ' . ' .
. .. . . . . .. . .. .. . . . ... . ... . . , .. .......... . .. . . . ~ . - .
`' ~ ' .
, ~ ,

- 5
- (CH21 n~~ -OzC
C~2~n~ (~H2~n~
where n is 1, 2 or 3, and the salts thereof; and those in
which one or more or all of the symbols Rl to Rg have,
independently of one another, the following meaning:
a) -CO-(CH2)nOH h) ~3
~~~ n~~ Y
b) -CO-(cH2)ncO2
c) -CO~(CH2)nCONH2 ) ~~4 -(C ~-
; d) -CO-(cH2)ncON(OH)R
R=alkyl with Cl_l2 t
preferably C~
~, e) -CO-(cH2)nOPO3
_CO-(C~2)nPO32- k) ~ ~ ~n
'~ g ) --CO- ( CH2 ) nSR
R=alkyl with Cl-l2,
pref erablY C1-4
~` where n is 1, 2 or 3, and the salts thereof.
A particularly preferred group of ligands are
those of the formula (I), (II), (IIa) and (IIb) in which
at least one of the radicals R~ to Rs and, in particular,
in each case the radicals Rl, R3 and R5 represent the
group -(CH2)n-COOH in which n has the meaning o 1 to 3
and particularly preferably of 2. The remaining sub-
stituents R2, R4, R~ and R~ to Rg are preferably hydrogen
., ~ .. ..
'
.

2~3J)~
or CH3, and these rernaining substituents are preferably
identical. Compounds of this type of the formula (I) are
particularly well sulted for t~.mour therapy.
A particularly important group of ligands of the
complexes used according to the invention are those which
have positive charges so that the complexes prepared
therefrom are positively charged. Complexes of this type
are, for example, particularly suitable for
radiodiagnostic purposes, in particular for the
; 10 radiodiagnostic demonstration of the heart of warm-
blooded species (human and animal~. The positive charges
can derive either from the quaternary nitrogen atoms of
the compounds of the formula (II), (IIa), (IIb) and (IV)
or from substituents with-positively charged functional
groups which can be located on the ligands of the formula
(I) or (III) ~ut also on the ligands of the formulae
(II), (IIa), (IIb) and (IV). The positive charges can be
obtained, for example, by protonation or by
quaternisation of nitrogen atoms which are present.
Particularly favourable examples are compounds of the
formulae (II), (IIa), (IIb) and (IV) in which all the
substituents (in the formulae (II), (IIa), (IIb) the
substituent groups Rl to Rg) represent methyl groups.
Examples of substituents with positively charged
functional groups are alkyl groups with quaternary
ammonium groups, such as trimethylamino groups or
- triethylamino groups, for example those o the formula
-(CR2)D-NR3, in which n denotes 1 to 3 and, in particular,
2 and R represents alkyl with 1 to 4 carbon atoms, in
particular methyl.
Paxticularly preferred compounds of the formula
tI) are those in which Rl = R3 = R5 - -CH2CH2COOH and R2 =
R4 = R6 = ~; and those in which R1 = R3 = R5 ~ -CH2CH2N(CH3)3
and R2 = R4 = R~ - H.
The complexes used according to the invention are
easily prepared from aqueous solutions or suspensions of
salts, hydroxides or oxides of the radioactive metals o
interest in each case, and aqueous solutions or
',.
... .. . .. .. .
.

~73~5~
-- 7 --
suspensions of the ligands~ The aqueous solutions can,
for example, ~e acidic. The amounts generally used for
this purpose are such that 2 mole of ligands of the
formula (I), (II), (IIa), (IIb) or 1 mole o~ the formula
(III) or (IV) are present per 1 mole of metal ions. A
small excess of the ligand is preferably used.
To prepare technetium complex2s it i~ possible,
for example, to start from technetium hexafluoride or
technetium hexachloride solutions which can be initially
reduced so that technetium (IV) ions are present, after
which an aqueous solution with excess ligand is then
added.
A portion of the radioactive metals or metal
cations in the complexes employed according to the
invention can be replaced by corresponding non-radio-
active i50topes. For example, the complexes can be in the
form of mixtures of complexes of radioactive metals or
metal cations with non-radioactive metals or metal
; cations. Moreover, the non-radioactive metals or metal
cations and the radioactive metals or metal cations can
be identical or different. For example, in the case wherP
radioactive iron isotopes are used they can be present
- together with non-radioactive iron. It is possi~le in the
preparation of products of this type, for example, to
form the complex from a solution of iron(III) ions in the
presence of radioactive iron~III) ions.
The complexes used according to the inv~ntion can
be prepared by the manufacturer or supplier; however,
they are often also prepared in situ in the x-ray
laboratory or treatment laboratory from the prepared
solutions.
The resulting aqueous solutions of the complexes
; can be employed for radiodiagnostic purposes and for
tumour therapy. Specific applications are x ray tomo~
graphy, positron emission tomography and, in particular,
demonstration of the human and animal heart, with posi-
tively charged complexes preferably being employed for
this purpose. It is paxtiaularly favourable for
.
.
:`
.

2~7~0
-- 8
diagnostic purpose~ to choose comylexes which are ~ or ~
emitters; ~ emitters are preferably employed for
controlling tumours, for ex~mple for controlling cancer.
The complexes used according to the invention are
employed in the form of aqueous solutions; they can be
administered in a variety of ways, in particular by
intravenous a~ministration.
Synthesis of all-cis-1,3,5-tris(2-carboxyethylamino)-
2,4,6-cyclohexanetriol
1 mmol (0.324 g) of all-cis-1,3,5-triammonio-
2,4,6-cyclohexanetriol sulphate was dissolved in a little
water and loaded onto an ion exchanger column (Dowex 1 x
4, 50/100 mesh, O~ form). It was eluted with water until
the eluate had a neutral reaction. The basic fractions
were concentrated in a rotaxy evaporator. This solution
of 1 mmol of triaminocyclohexanetriol was mixed with
3 mmol (3 ml of lM aqueous solution) of acrylonitrile and
left to stand in the dark under nitrogen for one week.
The intermediate product all-cis-1,3,5-tris(2-syanoethyl-
amino)-2,4,6-cyclohexanetriol was obtained by evaporation
in a rotary evaporator
H N~R (90 MHz, D2O, pD 2): 2.~7 ppm (t,6H); 3.49 ppm
(t,6H), 3.60 ppm (m,3H), 4.54 ppm (m,3H).
13C NMR (62.9 M~z, D2O, pD 3): 14.3 ppm, 40.6 ppm, 57.0
pm, 63.2 ppm, 117.4 ppm.
pK values (approximate): 3.0, 4.5, 6.3.
The nitrile was hydrolysed by boiling in con-
centrated hydrochloric acid under reflux for 6 hours. The
all-cis-1,3,5-tris~2-carboxyethylammonio)-2,4,6-cyclo-
hexanetriol trichloride was obtained by evaporation in a
rotary evaporator.
This was ta~en up in a little water and neutra-
lised to about p~ 4.5 with potassium hydroxide solution.
'.
.... ... . . . .. ...

2~730~0
.
g
The ~ cis-1,3,5~tris(2-carboxyethylamino)-2,4,6-cyclo-
hexanetriol precipitated during this. The product was
filtered off, dissolved in dilute acid (pH < 3) and again
precipitated by neutralisation, filtered off and finally
dried under high vacuum.
Element microanalysis:
Formula of the substance: C~5H27N309 MW: 393.39
C ~ N
calculated 45.80 % 6.92 %10.68 %
found 41.50 % 7.24 %9.31 %
These values are consistent with 2.8 mole of
water of crystallisation per mole of product.
H N~ (200 M~æ, D20, acid): 2.95 ppm (t,6H), 3.53 ppm
(t,6H), 3.72 ppm (m,3H), 4.72 ppm (m,3~).
lS 13~ NMR (200 MHz, D20, acid): 32.8 ppm, 43.5 ppm,
59.6 ppm, 66.1 ppm, 176.9 ppm.
pK values (approximate): 6.2, 8.2, 9.7.
Example 2:
Synthesis o~ t59Fe~ferric all-cis-1,3,5-tris(2-carboxy-
latoethylamino)-2,4,6-cyclohexanetriol
1 mmol of all-cis-1,3,5-tris(2-carboxyethyl-
amino)-2,4,6-cyclohexanetriol (from Example 1) was
dissolved in about 20 ml of 0.1 M hydrochloric acid.
1 mmol o Fe(III) (1 ml of lM Fe~13 with 2 mCi of 59FeCl3
(Amersham) in HCl lM) was added. XOH ~about 1 M) was
slowly added to the clear yellowish solution, which
became darker yellow during this. The product began to
precipitate out at about pH 3. Alkali was then added
until the pH was ahout 4.5.
The precipitated [59Fe]ferric all-cis~1,3,5-tris-
(2-carboxylatoethylamino)-2,4,6-cyclohexanetriolwasfil-
.
'
;

2~7~
- 10
tered off on a G4 sintered disc and subsequently purified
by dissolving in 0.1 M HCl and precipitating at pH 4.5.
The product was finally dried under high vacuum. Struc-
tural identity with the non-radioactively labelled
S product was established on the basis o spectroscopic
-~ properties.
Analysis of the non-radioactively labelled
complex leads to the conclusion that the formula is
Cl5H24N309Fe .
Example 3:
Synthesisofall-cis-1,3,5-trihydroxy-2,4,6-tri-N-acetyl~
aminocyclohexanè
3.28 g (18.5 mmol) of the amine (from Example 1~
were dissolved in 100 ml of methanol, and 35 ml
(370 mmol) of acetic anhydride were added. ~he mixture
- was stirred at room temperature overnight. Methanol and
excess acetic anhydride were removed in vacuo. The
resulting product was recrystallised from ethanol and
dried over phosphorus pentoxide und~r high vacuum for
24 h. Yield 86 %, melting point 300C.
n Element microanalysiq:
Formula of the substance: C15H2lN3O6 MW: 303~3
C H N
calculated 47.52 % 6.98 % 13.85 %
found 46.21 % 6.82 % 13.61 %
H NMR (200 MHz; D20) 2.05 ppm (s;3H), 3.97-4.04 (q;2H)
13C NMR ~200 MHz, D20) 176.4 ppm; 72.8 ppm; 53.5 ppm,
i 24.7 ppm
Solubility: readily soluble in water

2~73~
- lL -
~a~
Synthesis of [ Fe]ferric all-cis-1,3,5-trihydroxy-
2,4,6-tri-N-acetylaminocyclohexane
1 mmol of all-cis-1,3,5-trihydroxy-2,4,6-tri-N-
acetylaminocyclohexane was dissolved in about 20 ml of
O.1 M hydrochloric acid. 0.5 mmol of Fe(III) (O.S ml of
lM FeCl3 in HCl lM) with 2mCi of 59FeCl3 (Amersham) was
added.
Then exactly the calculated amount of KOH (lM)
for the hydrochloric acid and a further 8 equivalents of
; alkali per Fe were added all at once (pH > 8). Back~
neutralisation with HCl(lM) until the pH was in the
region 5.0 i 0.2 was subsequen~ly carried out, checking
with a pH meter. The uncharged iron complex with the
composition Fe(H3L2) (L = ligand) precipitated. The
complex is soluble in a buffer with physiological pH and
above that.
Structural identity with the non-radioactively
labelled product was established on the basis af spectro-
scopic properties.
Analysis of the non-radioactively labelled
complex leads to the conclusion that the formula is
Fe(:~24~39N6Ol2 8H2-
Exam~le 5:
Synthesis of gadolinium all-cis-1,3,5-tris(2-carboxylato-
ethylamino)-2,4,6-cyclohexanetriol
1 mmol of all-cis-1,3,5-tris(2-carboxyethyl-
amino)-2,4,6-cyclohexanetriol (from Example 1) was
dissolved in about 8 ml of 1 mol~l hydrochloric acid.
1 mmol of GdC13 (100 mg of GdCl3 x 6H2O/ml of HCl 1 mol/l)
was added. The pH of the clear colourless solution was
~lowly adjusted to 4.5 with NaOH (about 2 mol/l). This
resulted in initial turbidity at pH 3 and precipitation
at pH 4.5. The precipitated gadolinium all-cis-1,3,5-
tris(2-carboxylatoethylamino)-2,4,6-cyclohexanetriol was
- filtered on a G4 sintered disc and sub~equently washed
with distilled water. ~he product waY finally dried under
~ . .. .. ..
'
,
:`

2~73~0
- 12 -
high vacuum at 40C.
Element microanalysis:
Formula of the substance: C1sH24N3OgGd MW: 547.62
C H N Gd
calculated 32.90% 4.42% 7.67% 28.72
found 22.13% 6.58% 5.17% 19.10%
calculated* 22.03% 6.65% 5.14% 19.23%
(* 15 ~2 )
These values are consistent with 15 mole of water
of crystallisation per mole of product.
.
~`
. ... ...... .....
: .