AU606146B2

AU606146B2 - Nitrogen-containing cyclic ligands, metallic complexes formed by these ligands, diagnostic compositions containing these complexes and process for the preparation of the ligands

Info

Publication number: AU606146B2
Application number: AU14611/88A
Authority: AU
Inventors: Bruno Bonnemain; Didier Doucet; Dominique Meyer; Michel Schaeffer
Original assignee: Guerbet SA
Current assignee: Guerbet SA
Priority date: 1987-04-14
Filing date: 1988-04-14
Publication date: 1991-01-31
Anticipated expiration: 2008-04-14
Also published as: DE3877799D1; FI93830B; FI93830C; NO881567D0; YU46691B; FR2614020B1; DK170946B1; NO176839B; IE62582B1; SI8810742A; KR880012572A; GR3007495T3; HRP920514A2; ATE85052T1; FI881708A0; PT87216B; IL86059A0; JPH01211573A; JP2675058B2; CN88102139A

Abstract

Ligands and complexes of the title and the applications of these complexes in magnetic resonance imaging, in X-ray radiology and as in-vivo chemical shift agents.

Description

COMMO NWE A'LT H OF A U S TR PATENT ACT 1952 COMPLETE SPECIFICATION vjoJ 1 (Original) FOR OFFICE USE Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: This document contain- t amendments made S :tion 49 and is correct tic printing.

Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: GUERBET S.A.

15, rue des Vanesses

Z.A.C.

Paris Nord II 93420 Villepinte,

FRANCE

Michel SCHAEFER Didier DOUCET Bruno BONNEMAIN Dominique MEYER DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: "NITROGEN-CONTAINING CYCLIC LIGANDS, METALLIC COMPLEXES FORMED BY THESE LIGANDS, DIAGNOSTIC COMPOSITIONS CONTAINING THESE COMPLEXES AND PROCESS FOR THE PAREPATION OF THE

LIGANDS"

The following statement is a full description of this invention, including the best method of performing it known to us -1-

JA

The present invention relates to new nitrogencontaining cyclic ligands and metallic complexes formed by the~se ligands, the uses of these complexes as magnetic resonance imaging agents, as X-ray-contrast agents and as chemical shift reagents in vivo.

The invention also relates to a process for the preparation of the ligands.

Thus, the invention relates to a ligand having the formula R HOOC -CHi CHi COOH N R-L 4

NJ

R R i 2 t" HOOC -CH in which R represents a radical of the formula

-(CH

2 CH CH 2 M

R

6

R

7 R R 6 being selected from the group consisting of C 1

-C

1 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl and a group of the formula 3. R CH-COON CH-COOH 'IN R I N -3 CH 1I (CH2 Z R 3 ALL~ CH- COON UU,

R

*1 2

R

1 'being selected from the. group consisting of the group A and the groups of the formula: -(CH 2) t- Y A Y (CH 2 t- A being selected from the group consisting of C 1

-C

8 alkylene, ClI-C 8 hydroxyalkylene and C 1 CC polyhydroxyalkylene, Y being selected from 0- and 0 -and t= 1 to 4, 0

R

7 being selected from the group consisting of hydrogen

C

1

C

4 lyl l- 4 hydroxyalkyl and C-C 4 polyhydroxyalkyl, m= 0 or I R 2 R 3 R 4 identical or different represent a radical of the formula (CH) CHI R P

R

8

R

9

R

8 and R 9 identical or dif ferent, being selectd frcm hydrogen c -c 1 alkyl, C -C 4 hydroxyalkyl and C -C 4 polyhydroxyalkyl p 1or 2 n 0, 1 or 2 and

R

5 is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, a C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl and Z is selected from the group consisting of oxygen and a group of the formula

N-

R 10 being selected from the group consisting of hydrogen C -C alkyl, C Chydroxyalkyl, C -C polyhydroxyalkyl, 1 14 1 4 a group of the formula CH COOH, R5having the meaning

R

given. previously, and a group of the formula 4 3

R

CM-COON CM-COON N R N 12 N 2-

'CH-COON

R

1 being selected from the group consisting of C Calkylene, C IC 1 8 hydroxyalkylene and. CCB polyhydroxyalik)'lene, as well as salts thereof, with the proviso that when Rio represents a macrocycle; R6 is selected from the group consisting Of Cl...

14 alkyl, Cl 1 4 hydroxyalky. and C 1 4 polyhydroxyalkyl.

The ligands of formula I can be prepared by reaction of a compound of formula: X -CH -COOH (I fail inwichR 5 has the meaning given above and X represents a tit# labile group such as a chlorine, bromine or iodine atom or a tosyloxy group or a mesyloxy group, with a cyclic amine of the formula N N in whc I# 'R4adnhv h ennsgvnaoe N CH CH-- 22 R' R L6 being selected from the group consisting of C 1 1 akl

C

1

-C

4 hdoykyc- 4 polyhydroxyalkyl and a group of the korrnula 4 n R CH R R C H C H rn

H

R

2 R, R 4

R

7 Rl 1 m, n, having the meanings given above and Z' is chosen from among an oxygen atom and a group of formula N RI R' 10being selected from the group consisting of hydrogen,

C

1

-C

1 4 alkyl, Ci-C 4 hydroxyalkyl, c 1

-C

4 polyhydroxyalkyl, and a group of thefoiua H H R /R -R -2N-R

H

R~,R

2

,R

3

,R

4

,R

12 and n having the meanings given above with the proviso that when R 10 represents a racrocycle; R'is selected from the group consisting Of Cl- 14 alkyl, c..

4 hydroxyalkyl and Cl 1 4 polyhydroxyaikyl.

The ligands of formula I can also be prepared according to a Strecker reaction, by reaction of a cyclic amine of formula III with an aldehyde of formula: SCHO IlJa in which R has the meaning given previously, in the presence of hydrogen 5 cyanide or more usually cyanide ions (KCN, NaCN).

The compounds of formula III in which Z'is a group: I can be prepared.

a) by reaction of a polyamine of formula:

R?

R'HN NJ}~~R P NHR' IV n In which n, Rand R 4 have the meanings given previously and RI represonts a toayl, mesyl or benzene sulfonyl group, vitb a c in which represent a chlorini b) by rea -6 apound of formula: X -R Z N- 3 X k 2 R 3 anid R' have the meanings given previously and X aa labile group such as a tosyloxy or mesyloxy group or ior bromine or iodine atom, or :tion of a diamine of formula: P HN NH R in which R' 1 and RI have the meanings indicated previously, with a compound of formula: X F14 N N X n2 Ii

II

I

4? This cycliaation reaction is carried out advantageously In the presence-of a O*ase transfer catalyst.

The compounds of formula III in which Z' is an oxygen atom can be prepared by reaction of a polyamine of formula: R' HN-[R 4

N]-RI-NHR'IV

in which n, R, and R 4 have the meanings given previously and R' represents a tosyl, mesyl or benzene sulfonyl group, with a compound of formula:

X-R

2

R-R

3

-X

in which R 2

R

3 have the meanings given previously, RI is as defined above and X represents a labile group, Tht-pollamines of formula IV can be obtained f rom dihydroxylamlies according to the followinig scheac: HO -1 [f 4 -j rl OM n Q 'C IPyrjcid n* -7- I i NaN I/CH 3 CN/N 0O t3 J- 1 -N V III V1 Reduction H PdIC R Cl/pyridins RMN N1 ~.NHR IV -n As an alternative, pnthalimide is made to react with the Compounds of formula Vil and hydrazinolyais is carried out in order to convert the Compounds of formula VII into the compounds of formula IX.

The compounds of formula III containing 2 nitrogen-containin$ ringS can be prepared according to the procedures specified previously.

Thus, it is possible to react a polyamina of the formulai A x II CH CM 2 CM -CH R N H NH 4'R'M NM R' in whi~ch A and R' have the meanings given previously, with a compound of formula XI in order to obtain a compound of formula III in which RiIs a group A.

The polyamine of formula XII can be prepared from a tetrahalogenated derivative by nucleophilic substitution in the presence of sodium azide followed by a reduction in the presence of hydrogen and palladlum on charcoal.

As an alteencoraive, ring andpoind ohic fru aCgopoe A, ca2b A aogn ateative, theg compund ofic frulsa gropoe of 2a b prepared by condensation of a compound of formula: Is

IS

-8 Et OOC COO Et CH A -CH xi I I Et OOC COO Et With a polyanine of for ula< H H H H HN -RZ- N -R 3 N-R NJ-H t I *i 041? 41 *6 4 @404 4 0 4444 4 *44,4*

S

*0 S O 44 4 44 @4 0 4 @4 followed by reduction with diborane according to a procedure described by Tabushi et al. (Tetra Letters 12, 1049, 1977).

The compounds of formula I composed of 2 nitrogen-containing rings are then prepared from compounds of formula III with 2 rings as described previously.

As an alternative, compounds of formula I composed of 2 nitrogencontaining rings can be prepared by condensation of a compound of formula I in which 1 is a radical of formula: CH CH 2C M C IC S6 '7 in which R6 is a hydroalkyl group, with an actlvatable bifunctional compound of formula: X, A X, X, being a COOH group, a COC1 group or- an acid anhydride.

The compounds of formula I exhibiting 2 nitrogen-containing rings can'also be prepared by condensation of a compound of formula: HOOC CM 5CH COO CM COOM /N *n\ 3 /I N R N H 1 "00C '"M

I

R

L

V,

-9in vtiicb I is a radical of formula; 4CH2 M-CH CM R"6 7 m, and R7 having the meanings given previously and 1R6 being selcted from the~ gru xOnistlng of C 1

-C

1 4 alkyl, C l-C 4 lyIraqca1kyl andC polyhydroxyalkyl, with a compound of formula: X RI1 2- X X having the meaning given previously and RI12 representing a possibly protected R 1 2 group.

Thus are obtained compounds of the fbrrmila: HOOC -CH

R

CM -COOH

R

I S MOO C C CM COON 4~I~ I I I I 4* 4 I ii £1 I

I

4 14 I I 2_ N 12 HOOC -C4 /1 1\ CH COOM xvI In add itioi'.. the present invention relates to cxxnlexes formed by ligands of formula I with metal ion~s chosen from .among the lanthanide ions with atomic numbers 57 to 71, transition metal ions 2. 3. 3+ with atomic numbers 21 to 29, In particular Mn Fe and Cr andl retal ions with atomic numbers 55, 50. 82 and 83, as well as the salts of these complexes with pharmaceutically acceptable mineral or organic-bases or basic amino acids.

j O Cl 00 o00SC 00 0 ob00 I In such complexes, the metal ions are preferably gadolinium, europiua, dysprosium, iron (Fe 3 and manganese (Mn As examples of salts mention may be made of those forsed with sodium hydroxide, N-methylglucamine, diethanolamine, lysine and arginine.

The complexes can be prepared by reaction of Uganda with a metal salt or metal oxide in an aquLeous solvent and possibly neutralisation In order to form a salt.

It should be evident that the present invention includes not only the ligands of formula I and the complexes previously defined in the form of racemic mixtures but also the stereoisomers of these ligands and complexes.

The complexes according to the invention can, in addition, be attached to a macromolecule which can become bound preferentially to 15 an organ. Thus, the complexes according to the invention can be linked to proteins and in particular to antibodies.

In addition, they may also be encapsulated, particularXy in liposomes.

The complexes according to the invention formed by the ligands of formula I with paramagnetic ions and their salts with pharmaceutically accepted bases can be used as magnetic resonance imaging agents and as chemical shift reagents in vivo.

The complexes according to the invention formed by ligands of formula I with lanthanide ions of aS.omic numbers 57 to 71 or ions of metals with atomic numbers 55, 56, 82 and 83 and their salts with pharmaceutically accepted bases can be used as X-ray contrast a g e n t s For this purpose, the complexes formed with the following metal ions are particularly preferred: Gd, Er, Dy, Th, Ce, La, Ba and Cs.

-Consequently, the present invention relates also to diagnostic composition which ,:an be administered to man, comprising at least one complex formed by, a ligand of formula I with metal ions selected from the lanthanide ions with atomic numbers 57 to 71, the transition metal ions with atomic numbers 21 to 29 and the metal iokS with atemlc numbers 55, 56, 82 and 83, as well as the salts of these complexes with pharmaceuitically acceptable mineral or organic bases, or with basic amino acids;

A

'I

0 a 0900 040.

S 0 4 I C o Ifl I te~ These compositions can be constituted in particular by slutions of a complex according to the irnention in a physiologically acceptable aqueous solvent.

The diagnostic compositions acoordi j to the invention may be administered by the parenteral route including the intravenour route, the intraarterial route, the intra-lyrrphatic route, the sub-cutaneous route by the oral route, by the sub-arachnoid route, by the intrabronchial route in the form of an aerosol, t O by the intraarticular route, locally for the visualisation of cavities (for axemple, the uterus) I Ain imaging by magnetic resonance, the doses are very variable depending on the routes of admninistration.

a a. !5 For the intravenous or intra-arterial route, the dose is about 0,01 to 2 m/kg.

For the oral route, this dose may be as much as 10 MA/kg.

For the other routes of administration, the doses used are usually lower than 1 rrtkg and for the sub-arachnoid route it is usually even 20 o1.wer than 0.05 r/kg.

a The doses are the same for their utilisation as chemical shift reagents in vivo and as contrasting agents in radiology using X rays except by the intravenous or intra-arterial routes where the doses may be higher than or equal to 5 TM/kg.

In addition, the complexes according to the invention formed by the ligands.of 'formula I with radioactive ions as well as their salts Swith pharmaceutically acceptable bases can be used as diagnostic agents or therapeutic agents in nuclear medicine. Examples of radioactive ions are radioisotopes of elements such as copper, cobalt, gallium, gemanian, indium and, above all, technetiin(Tc 99 m).

The following exanmples illustrate the preparation of the conrounds according to present application.

In these examples the NR spectra were carried out on a Varian EM 360 machine at 60 Mz with IMs as internal reference. Unless otherwise indicated the lvent is CDC13 t3' *1ire II ~I I e r

RI

I t II t~ I 7R .,IItt toed 'Sal C C at Cl., tLCC -12-- 1 The IR spectra were carried out on a Perkins-Elmer 1320 apparatus.

The spectra of the solids were recorded in the form of KBr disks. In the case of liquids (oils) they were recorded in the absence of solvent.

The term "buff er" used in thin layer 'chromatography designates a mixture of 1.5 M NH4 OR and 1. 5 M (N9 4 2 C0 3 The melting poinLa were measured on a Kofler block.

The terms used relating to analyses during complexation: "absence of free Gd 3.and of, free ligands" are to be understood within the limits of detection of the methods used, i.e. 01 ppm and 4 ppmf~r d 3+and ligand, respectively.

Example I Preparation of 2 6-dime thl 4, 7, 0-etraazacyclododcaneN, N' -etacei acid a) Preparation of N-tosyl-bis (2-tosyloxy propyl) amino.

is A solution of 53.2 g (0.4 mole) of diiao-propanolenine in 50 Ca of pyridine is added dropwise with cooling to a solution of 248 (1.3 mole) of tosyl chloride in 200 cm 3 of pyridine at ObC so ihat* the temperature is maintained between 0 and 5SoC. The mixture Is lef t at this temperature for 72h. It is then poured into 2 1 of water plus ice and 250 cm 3 of concentrated hydrochloric acid.

The tosylated derivative is extracted with 2 1 of methylene chloride. The organic phase is dried over sodium sulphate, filtered them decolourised with 3 SA charcoal and ref iltered through a bed of silica. After evaporation of the salt 193.5 g of a yellow oil remain 25 (yield 81Z1 Rf 0.7 silica/CH Cl /acetone/98/2) which is used In the next step without purification.

I 10MR spectrum: 6H CH 3 (doublet 1.2 and 1.3 ppm); 9H CHR 3 tosyl (singlet 2.5 ppm) 4H1 CH 2 (multiplet centred on 3.3 ppm); 2H1 Cl (quadruplet between 4.7 and 5.1 ppm); 12H1 aromatics (multiplet 7.3 and 8 ppm) b) Preparation of N-tosyl-bis(2-azido propyl) amine.

To 193.8 g (0.32 mole) of the compound obtained in a ini 1.2 1 of 3 acetonitrile and 300 cm of water are added 65.1 g of sodium azide (1 mole). The mixture is stirred and heated at 75 0 C for 48h. After coig h ctntiei vprtdi aum colnteaI-nieiseaoae i aum -13- The realidue is taken up in 1 1 of methylene chloride. The organic phase Is washed with water. dried and filtered through-a bed of silica (200 g) After evaporation 82 g of a clear yellow oil remain (yield 75%j RE 0.85 silica CH 2

C

2 /acetone/92/2) sufficiently pure to be useA directly.

IR spectrum N 3 a2100 cm 1 I intense.

c) Preparation of N-tosyl-bis(2-amino propyl)amins.

82.2 g (0.244 mole) of the compound obtained inb are dissolved in 500 cm 3 of ethanol containing 8 g of 5Z palladium on charcoal at 50% humidity.

The mixture is stirred vigorously while a gentle stream of hydrogen is passed through (evacuation of nitrogen which Is released). After 8h at ambient temperature, TLC shows the absence of the azido function.

The mixture is then filtered and evaporated. 68,4 g of a clear yellow Oaf oil. are obtained (yield 98.5%; Rf a 0.6 silica/MeOH/NH 4 0HR.95/5) which is used without purification.

4~a oat NMR spectrum: 6H CH 3 (doublet 0.9 and 1 ppm); -3H CH 3 tosyl (singlet at 2.4 ppm); 6H CH~ and CH %'c-omplex multiplet bitwein 2.7 and 3.2 ppm); 4H aromatics (multiplet betweez 7.1 and 7.7 ppm).

d) Preparation of N-tosyl-bisiZtosylamino)propyi7aaine.

93 g (0.5 mole) of tosyl chloride are added in portions to 68.4 g 9.,3 a (0.24 mole) of the amine obtained in c in 500 cm 3 of methyleme chloride and 70 cm 3 (0.5 mole) of triethylamiae'at 0 C. After the addition is 4complete, the mixture is stirred for 6h at ambient temperature. The 3.

reaction mixture is then washed with 600 cm of water, the organic togs phase is dried, evaporated to dry-ness and the residue is chromatographed on a column of silica with pure methylene chloride, then with a methylene chloride/methanol/98/2 mixture. The fractions of interest are evaporated and the solid residue is recrystallised from ethanol.

filtration and drying the mass obtained is 99.1 g (yield NMR sipectrum: 6H CH 3 (doublet 0.9, 1 ppm) 9H CH 3 p-tosyl (singlet 2.4 ppm); 4H CH 2 (triplet centred on 2.9 ppm); 2H CH (doublet 3.3 and ppm); 12H aromatics (multiplet centred on 7.4 ppm).

e) Preparation of I-tosyl-bis(2-tosyloxyethyl) amin.

A solution of 32.5 g (0.31 mole) of diethanolamine in 60 cm 3 of L,,pyridine are added 3slowly to a solution of 185 g (0.97 mole) of tosyl W~1 hloride In 220 cm of pyridine at 0 0 C so that the temperatura does not excee

<U

O~ C. After the addition is complete, the mixturfk is maintained at this 00~000

COO

0.00 0*09 499044 o C 9400 0040 0 CO 0'0 0 9 C 000* 0eCt~9C Z~ jot t aOL t I temperature for lh, then it is poured into 220 cm 3of ice-cold water with viborous stirring. After filtration, washing mid drying 148.4 g of precipitate are obtained (yield 85%; Pf 0.6 silica/C1 2 C1/acetone/ 98/2).

NMR spectrum: 9H1 CH 3 tosyl (singlet 2.4 ppm); 4H CH 21 (triplet at 3.4 ppm); 4H CH2 (triplet at 4.1 ppm); 12H1 aromatics (multiplet between 7.1 and 7.7 ppm).

f) Preparation of N,N',N'',N'''-tetra.tosyl-2,6-dimethyl-l, 4, 7, 655 g (0.11 mole) of the compound obtained in d dissolved in 500 cm 3 of dry DM? are added dropwise to 8.8 g (0.22 ihole) of a suspension of NaH in oil in 50 cm 3 of DWG. The addition ise carried out at ambient temperature and in such a manner:that there Is a steady release of hydrogen. After the addition is complete, the mixture is 15 heated to 100 0 C and a solution of 68.1 g (0.12 mole) *o the compound obtained in e dissolved in 500 cm 3 of dry DMF are added drkpwioe. The reaction mixture is then maintained at this temperature for 24h with.

vigorous stirring.

The solvent is then evaporated in a vacuum and the residup, is tken p ina CH2 1l 2

M

2 0 mixture. The oraanic. phase Is washed with water, dried and evaporated to dryness. The residue (100 g) is recrystallised from isopropanol, then from toluene to give, after filtration, washing with isopropyl ether and drying, 36 g of a white solid (yield 40%; Ri a 0.5-0.6 silica/CH 2 Cl 2 /acetone/98/2).

25 NMR spectrum: 611 CH, (doublet at 1 and.1.2 ppm); 12H1 CH3 tosyl ,(singlet 2.4 ppm); 14H1 CH 2 and CH (multiplet between 3 and 4.5 ppm); 1611 aromatics (nultiplet between 7.1 and 7.7 ppm).

g) Preparation of N, N'''-tetratosyl-2,6-disethyl-1, 4,7,10-tetraazacyclododecane (variant).

A freshly prepared solution of sodium ethylate (60 mie1es) in 200 cm 3 of dry DM? is added rapidly to a solution of 17 g (28.7 mmoles) of the compound obtained in d in 100 cm 3 of ethanol at reflux. The mixture obtained becomes clear and Is ref luxed for 1-2h. The solvents are then evaporated to dryness, the residue is taken off in 200 cm 3 of D1!E and heated to 1O 0 C. A solution of 17 g (30 mt~oles) of the compound obtained in e in 100 cm 3 of DM? is added to this solution during 1/2h. The reaction mixture is maintained at 106 0 C overnight.

S

-Gi 4

''U

tM Ii *4Z m Te rDG io then evaporated and the residue is taken up in a H 2 0/CH 2 C1 2 mixture. The product derived from the organic phase is cbromatographed on a column of silica with the mixture-CH 2 Cl1a/ethyl acetate/98/2 as eluant. The product is recrystallised from isopropyl ether and weighs 13.5 g after drying (yield 58%; R1i a 0.5-0.6 silica/CH 2 C1 2 /acetone/98/2).

Spectrum identical with that obtained in f.

h) Preparation of 2 ,6-dimethyl-1 14,7 ~3 of the compound obtained in f or are suspended in 80 cVA Of 98% sulphuric acid and heated at 100 0 C in an argon atmosphere for 72b.

After being cooled, the reaction mixture is added dropuiss to 1 1 of ethyl ether at O 0 C. The sulphate of 2, 6 -diiethyl-l,4,7,lO-tetraazacyclododecan* obtained Is f iltred off, taken up in water, neutralised with sodium hydroxide, then extracted with CH 2 C1 2 The orlanic phases are combined and evaporated to dryness, and the resulting 6 g of solid are used without IS further purification (yield 75%; Ri 0.65 al.uminia/butanol/vater/acetic acid/50/25/11).

NMR spectrum (D 2 6H CH 3 (doublet 0.9 to 1 ppm); 141 CH 2 and CH (multiplet centred on 2.5 ppm).

i) Preparation of 2 ,6-dimethyl-l ,4,7,10-tetraazacyclododecane-N, N', Nit, N'''-tetraacetic.acid.

A mixture of 5.7 g (60 nmnoled) of monochloroacetic acid and 3.4 g mmoles)in 25 cm 3 of water'is added to a solution of 3 S (15 mmoles) of the compound obtained in hi in 25 cm 3of water. The mixture obtained is heatext to 60 0 C and a solution of potassium hydroxide (3.4 g, 60 mole) in 25 cm 3 of water is added so that the pH is maintained between 9 and The addiction -requires 8h. Af ter the addition of potassium hydroxide is complete, heating is maintained for 24h. Af ter cooling, the pH is brought to 2.5 with concentrated HCl. The precipitate formed is fi 9 ed off, washed with ice-cold water and weight 3 after being dried (yield 3!2; Rf 0.33 silica/ethyl acetate/ isopropanol/ammonia/12/35/30). This compound corresponds to the complex of 2,6-dimethyl-1,4,7,0-tetraazacyclododecane- N, It'''-tetr~acetic acid with 2 KCl.

g of this compitx are eluted with 200 cm 3 of 10% acetic acid from an ion exchange resin IRA 958 (OH) which has been regenerated beforehand 3S with IN NaOH and washed with water until it became neutral. The fractions obtained are evaporated to dryness and taken up 3 times in 50 cm 3 of water in order to eliminlate traces of acetic acid. The re~~due obtained is.

K 3 Utriturated with ethyl ether (100 cm to give, after f iltration and drying, 63g-of a white solid. Yield: 89%.

U

fl -16- I MM pectrum: (D 0) 6H CH 3 (doublet 1.4 and 1.5 ppm); l4H CH 2 anad CH (compLia mltiplet centred on 3.6 ppm); 8H 2 2 COOH (doublet at 3.8 ppm).

Example 2: Preparation of the ladolinium complex of 2,6-<timethyl-1,4,7,10tetraazacyclododecane-N, N'''-tetraacetic acid (metbylglucamine salt) Suspension of -5.425 g C 2.54 mmole) of 2,6-dimethyl-l,4,7,l0tetraazacyclododecane-N, N'''-tetraacetiC acid obtained in example 1 1 and 2.27 g of Gd 2 0 3 (6.27 mmo1e) in 125 ca 3 of water in heated at 65 0 C for 24h. The pH is then adjusted to 7.4 by the addition of methylglucarnine. After determination of free Gd3+ by the xylenol orange/EDTA method, 650 mg of 2, 6-dimethyl-l ,4,7 N, N1, N'''-tetraacetic acid (1.5 mmole) are added to complex the remaining gadolinium. The completion of complexation ise confirmed by the is absence of free Gd 3*(determination with xylemol orange) and of frees 4ligands (complexometric determination with Cu 2. The determination of total gadolinium in the solution is carried out by atomic emission spectroscopy in DCP on a Spectrospan 4 Becmknn apparatus. Quantitative yield Rf 0,.49 'silica/ethyl acetate/ isopropanol/amonia/12/35/30).

Example 3 Preparation of 2-hexyl-1,4_,7,10-tetraazacyclododecane-N, N'''-tetraacetic acid a) Preparation of N- (2 -hydroxye thyl) 2-hexyl-2-hyd roxye thyl) amine 50 g (0.39 mmle) of 1,2-epoxy-octane are added dropwise to 250 cm 3 (4 moles) of ethanolamine at 100 C. Heating is maintained for lb after the addition is complete, then the excess ethamblamine is distilled in a vacuum. The residue is recrystallised from 600 cm 3 of hexane, after filtration and drying. The solid residue obtained weighs 69 g (melting point 45 C, yield -93% Rf z0.62 silica/butanol/H 2 0/acetic acid/50/25/ll).

~NMR spectrum% 3H CH 3 (triplet 0.9 ppm); 10H CH 2 chain (large singlet at 2.2 ppm); 7H CU 2 and CH (2 badly resolved masses at 2.8 and 3.8 ppm).

b) Preparation of N-tosyl-N-(2-tosyloxyethyl) -N-(2-hexyl-l-tosyloxyethyl) amine.

47.3 g (0.25 mole) of the compound obtained in a are added in small portions to a solution of 156 g (0.82 mole) of tosyl chloride in 300 cm 3 of pyridine~ at 0 0 C during lh. The mixture is maintained at 0 0 C for' 2 days.

p~lq then it is poured into an ice/HCl mixture The product is extracted with CH C1 2 then chromatographed on a column of silica with CHl 2 C1 2 as eluant.

2 2 2 0 17 I The mass obtained is 118 g (yield 722; Rf 0.6 silica/CB 2 Cl 2 /acetone/ 98/2).

NM spectrum: 3H CH 3 chain (triplet at 0.9 ppm); 10 CH 2 chain (large singlet at 1.3 ppm) 9H CH 3 tosyl (singlet at 2.4 ppm) 4H CH 2

N

(poorly resolved triplet at 3.4 ppm). 3H C20and CH (indtiplet at 4.2 ppm); 12H1 aromatics (multiplet between 7 and 7.7 pp.).

c) Preparation of N-tosyl-N-(2-azido-ethyl) -N-(2-bexyl-2-azidoethyl) amine.

87 g (0.133 mole) of the compound obtained in b and 29.25 g mole) of sodium azide are mixed with 350 cm 3 of acetonitrile and 80 c4 3 of water. The mixture is heated at 65 0 C for 3 days. The acetonitrile Is then evaporated in a vacuum, the residue is taken up in C8 2 C1 2 the organic phase is washed with water, dried and evaporated; 502 of a yellow oil are recovered and used without purification (yie.ld: 952; Rf 0.75 silica/C11 2 C1 2 /acetone/98/2).

NRR spectrum: 3H CH 3 chain (triplet at 0.9 ppm); 10 CR2 chain (mitiplet at 1.4 ppm) 3H CH 3 tosyl (singlet at 2.4 pp2); 5H C and CE (complex multiplet at 3.4 ppm); 4H aromatics (multiplet between 7.1 and 7.7 ppa).

spectrum N 3 2100 cm- intense.

d) Preparation of N- tosyl-N(2-aminoe thyl) 2-hoql-2-aninoe thyl) amine 71 g (0.18 mole) of Lhe diazide obtained in c are dissolved in 500 ca 3 of ethanol to which 5 g of palladium on charcoal at 502 humidity has been added. The suspension is stirred* very vigorously under a stre~a of hydrogen at ambient temperature for 24h. The catalyst is removed by filtration; after evaporation of the ethanol,,61.5 g of diamine are recovered and used without purification (yieldt quantitative; RI z 0.51 silica/XeOH.- NH1 4 0H/ 95/5)t e) Preparation of N-tosyl-N (2-tosylaminoe thyl) -N-(2-hexyl-2-tosyl- S aminoethyl)anine.

68.6 g (0.36 mole) of tosyl chloride are added in portions to a solution of 61.5 g (0.18 mole) of the compound obtained in d In 500 cm 3 of CH 2 C1 2 and 52.5 cm 3(0.38 mole) of triethylanine at 09C. After being stirred for 2h at ambient temperature, the reaction mixture is treated with 500 cm 3 of water. The organit phase is washed with water, L1q dried, evaporated; the oily residue is chromatographed on a column of L silica with CH Cl as eluant. The oil obtained after evaporation of the 2 2 i solvent and being taken up In isopropyl ether gives 60 g of wihite solid (melting point '120OC; yield 51%; RI 0,6 silica/CH Cl /MeOH/98/2).

NM3R spectrum: 13H1 Hexyl chain (poorly resolved niul tiplet centred at I ppm); 9rH C113 tosyl (singlet at 2.4 ppm); 7H CH 2 and CH (multiplet centred at .3.1 ppm).

Preparation of N. N' N1' N'''-tetra tosyl-2-hexyl-1,4,7, tetraazacyclododecane A mixture of 46.5 g (71.5 murole) of the compound obtained in d aibove, 41.5 g (73 numole) of the compound obtained in example 1 a and 24 g-(70 mmle) of tetrefutyla nnium hydrogen sulphate are suspended in 400 ca~ of toluene and 200 cam 3 of 20% sodium hydroxide. The mixture is stirred very vigorously at 70 0C for 24h. After cooling, the organic phase Is washed :with water, dried and evaporated. The residue Is crystallised in ethanol, then chromatographed on a column of silica with CH 2 C1 2 as eluent. 35 g of solid are obtained (melting point 154/161 0 Yield 56%; Rf a 0.55 silica/CH 2 Cl 2 facetone/98/2.

N)R spectrum: 3H1 CH 3 chain (triplet at 0.9 ppm); 10H C! 2 chain (iultiplet at 1.3 ppm); 12H1 CH 3 tosyl (singlet at 2.4 15H C0 2 and CH ring (multiplet at 3.3 ppm); 16H1 aromatics (multiplet between 7.1 and 7.7 ppm).

g) Preparation of 2-hexyl-l.4.7,l0-tetraazacyclododecane 12 g (13 mmoles) of the compound obtained in f are heated at 100 0

C

u ~In 40 cma Of 982 sulphuric acid under argon for 24h. After being cooled, the mixture Is added dropwise to 500 iwl of ethyl ether at 0 0 C. The obttined Is filtered, then neutralised by a 102 solution of i odiua hydroxide and extracted with CH 2 Cl 2 The organic phase is dried over sodium sulphate, then evaporated to dryness to give 2 g of a cream solid (yield: 57%; Rf a 0.75 alum in/ butanol /water/ace tic acid/50/25/11).

The compound Is stored in the form of the oxalate by reacting an ethanolic solution of oxalic acid with 2 -hexyl-l.

4 ,7,10-tetraazacyclododecane overnight at ambient temperature. The oxalate precipitates In the form of a white solid.

h) Preparation of 2-hexyl-1,4,7,1O-tetraazacyclododecane-N, N1, N"''-tetraacetic acid.

19- A solution of 1.09 g (2.8 mmols) of the oxalate obtained in in 13 cum of water and 20 ml of etbanol is neutralised with 470 mg. (8-.4 nmoles)of potassium hydroxide. To this solution is added potassium monochioroacetate prepared from 1.063 g (11.25 amoles)of monochioroacetic acid and 630 mg (11.25 mnoleB) of potassium hydroxide in 20 cm1 3 of water.

reaction mixture is heated to 60 0 C and the pH is maintained between 8 and 10 by the addition of potassium hydroxide. The addition requires 3h during which 10 cm 3 of water containing 630 mg of potassium hydroxide are added. After a reaction-time of 3h, 141 mg (1.5 mmoles) of chioroacetic acid and 84 mg (1.5 rrmoles)of potassium hydroxide are added.

The mixture is then maintained at 66 0 C for 2 days. After being cooled and acidified to pH 2.5 O6N HUl) the solution is passed through a column of a strongly basic resin IRA 958. Elution with 100 cam 3 of 10% formic acid yields 700 mg of product. The flow-through fractions (product not ret~ined) IS are concentrated and retreated on an identical col-. After the same treatment, 2.5 g of product are recovered (yield: 38.41; Rf a 0.65 silica/ ethanol/buffer/2/l).

1{HR spectrum: 3H CH 3 chain (triplet 0.9 ppm); 10 CH2 chain (multiplet at 1.4 ppm); 15H CH 2 and CH ring (multiplet at 2.3 ppm); all CE 2

CO

(singlet at 3.9 ppm). Spectrum carried out in D 0.

Example 4: Preparation of the gadolinium complex of 2-hex!yl-I,4,7,l0tetraazacyclododecane-N. N'''-tetraacetic acid 488.6 mg (1 nmole) of the compound obtained in example 3 and 181.3 mg (1 meq of metal) of gadolinium oxide are suspended in 40 cm 3 of water and tieated at 650C for 2 days. After 2 hours the solution is clear. The progress of the complexation is monitored during the reaction by determination of free gadolinium. When it is complete, the solution is filtered through a Millipore filter paper, then evaporated to dryness and crystallised from ethyl ether. 550 mg of white solid are thus recovered (yield: 85.52; Rf a 0.65 EtOH/buffer/2/1).

Example 5: Preparation of the gadolinium complex of 2-hexyl-1,4,7,10- _7 tetraazacyclododecane-N, N19"-tetraacetic acid (methyIglucamine 480.6 mg (I =wsle) of the compound obtained in example 3 and 181.3 mg (1 meq of metal) of gadolinium oxide are suspended in 40 c= 3 of water and heated to 65 C for 12h. Methylgiucamine is added to the clear solution to bring the pH to 7.4. Additions of ligands are made depending on the results of the analyses. The completion of complexation is confirmed by the absence of Gd 3+(determination by xylenol orange) and of free ligands (complexometric determination with copper). The determination of total gadolinium in the solution is carried out by means of atomic emission spectroscopy on a Spectrospan 4 Beckmann apparatus. Rf a 0.65 Ini EtOH/buffer/2/l.

Example 6: Preparation of 2 -methyl-1l,7,10-tetraazacyclododecane-N, N', N'''-tetraacetic acid a) Preparation of N, N'-ditoayl-1,2-diaminopropane.

In a 1 1 3-necked flask fitted with a magnetic stirrer, a thermmter and a guard tube, 14.8 g of 1,2-diaminopropane are dissolved In 500 ml of C2C2and 58 cm 3 of Et 3 g of tosyl chloride are introduced in portions during 1 hour.

Cooling in an ice bath is necessary in order to maintain a~ temperature of The reaction mixture Is then stirred overnight at room temperature.

The reaction mixture is transferred to a 1 1 separating funnel and 3 then washed with 2 x 260 cm of water.

The organic fraction is dried over Na SO 4 evaporated to dryness, then crystallised from isopropyl ether.

Weight obtained a 66 g' Yield =86% Melting point 98/100 0

C

I ppm doublet O3H) 3.1 ppm multiplet (lIH) 2.4 ppm, singlet (61) 5.5 ppm exchangeable singlet 2HJ 2.9 ppm doublet (2H1) 7.1 to 7.8 ppm aromatics (8H)

TLC

A1 SiO 2 eluant CH1 2 C1 2 90 VeH 10 Rf 2 0.2 4b 1\ eH l R -21- I b).Preparation, Of N, N'-ditosyl-bis (2-tosyloxycehyl) ethylene d ismine.

in a 500 cm~ 3-neck'ed flask fitted with a thermometer, a guard tube and a magnetic stirrer ai solution of 162 g of tosyl chloride in 300 a~l of pyfidine is cooled to 0 0 C by means of an ice-salt bath.

29.6 g of this (2-hydroxyethyl) ethylene diamina. are added In portions during 2h at this temperature. At no time must the temperature exceed 5 0 C. ]rhe reaction mixture is stirred for 4h at this temperature, left for 48h at 6/8 0 C in the refrigerator and then for 4h at room temperature.

*The reaction mixture is poured into 1 1 of ice plus. water end 300 al of concentrated HC1. The product is extracted with 500 ml of CH C1.

This organic phase is dried over Na 2 S0 4 then evaporated to dryness.. The residue Is takdn up in 250 cm 3 of ethanol by warming. The product crystallises. It is filtered off on a glass frit and dried at 60PC for 48h.

Weight obtained a 107.5 g Yield a Melting point a 138/140 0

C

Nn 2.4 ppm singlet (12H) 3.3 ppm singlet 4 triplet (8H1) 4.2 ppm triplet (4H1) 7.2 to 7.8 ppm multiplet (16H1)

TLC

9i1 plt0eun toluene 80 RE 0.6 acetone c) Preparation of N. N' N' '-tetratosyl-2-methyl-1,4,7,10tetraazacylododecane In a 1 1 3-necked flask, a solution of 17.5 g of N, N' ditosyldiamino 1,2-propane in 500 ml of dry DMF is stirred for 1/4 hour at ambient temperature. 33 g of Cs 2 C0 3 are powdered and added In suspension to this solution. This suspension is heated to 55 0 C by means of an oil bath In *an Inert atmosphere.

22 A solution of 35 g of N, N'-ditosyl-bis (2-tosyloxethyl) ethylene diaminefn 200 cam of DII? in added dropvise at this temperature during 2h. After the addition is complete, heating is continued for 48h. The DMF is then removed by distillation in a vacuum. The residue is taken up in a water/CH 2 Cl 2 mixture.

The organic phtase is dried over Na 2

SO

4 The solvent is removed by distillation in a rotary evaporator.

The residue ia heated and stirred in 200 al of ethyL acetate. The expected product crystaillises. It is filtered off, then dried at 60 0

C

in a vacuum for 24h.

Weight obtained 22.5£ Yield 61% Melting point 274/275 0

C

1 ppm doublet (2H) 2.2 ppa singlet (12H) 3 to 3.8 ppm multiplet 7.2 to 7.9 ppm multiplet (16R) aromatics

TLC

SiO 2 eluant toluene u acetone a2C Rf 0.56 d) Preparation of 2-methyl-l,4,7,1O-tetraazacyclododecane in a.-1 1 3-necked flask fitted with a thermometer, a balloonx of argon and a mechanical stirrer, a solution of 72.5 g of the compound obtained *in c) In 300 ml of 98% H S0 4 is heated at 100 C for 48h by means of an oil bath in an inert atmosphere.

The reaction mixture is cooled' to ambient temperature and 800 ml of Et 2 O, qooled to 0 0 C by means of a bath of ethylene glycol and Dry Ice, is added during lh.

The very hygroscopic sulphate precipitates. It is filtered off carefully on a glass frit under nitrogen, then quickly dissolved in 200 al of water. This solution is made alkaline (pellets of NaOH), then extracted with 5 x 100 m1 of CH C1 2 -23- IThe combined organic phase* are dried over Na 2

SO

4 then evaporated on standing.

CDCl 3 1.1 ppm doublet (2H1) Spectrum in D 2 0 2.7 ppm 2 singlets as a m~atiplet (19H. 4 of which are exchangeable) Al2 03 plate eluant BuOli 50 Rf a 0.8 H 2 0 AcOH 11 'Ve) Preparation of the complex of 2-mtbl44,4,7,10-tetr~azacyclododecan.it N, MI, N'''-tetraacetic acid with 2 KCl.

in a 250 ml 3-necked flask a solution of 34 g of chloroacetic acid In 150 cma 3 of water Is cooled to 10 0 C in an ice bath. 20 g of potassium hydroxide are added at this temperature to neutralise the acid.

The compound obtained in d is then dissolved in this solutioia. The reaction mixture is then heated to 65 0 C by means of an oil bath.

A solution of 20 g of KOH in 50 cam 3 of water Is added cautiously during 6h at this temperature while the pH is maintained between 8 and t 4 1 Heating is then continued for 72 hours, then the reaction mixture is acidified to PH -2.5 with concentrated HUl.

The complex precipitates. It is filtered off onto a glass (nit, 30 rinsed with 50 cia of water, then dried at 600C for 18h in an oven.

Weight obtained -30 g Yield 66% Mlmting point IP 3 00 C f) Purificatioa of 2-methyl-1,4,7,l0-tetraazacyclododecane-1,4,7,10tetraacetii acid.

g of the complex obtained in a are suspended in the presence of 150 cm 3 of IRA 958 resin, which has been regenerated beforehand.

After dissolution of the complex, this.suspension ii applied to the head of a column containing 150 cm 3 of IRA 958 resin.

,450-Ax The elution is carried out by a 52 acetic acid solution in water.

24 The fractions containing the expected pure 'product are evaporated todrynessiu'order to remove the acetic acid completely.

W6ight obtained a 18.4 9 Yield a 892 100.3% (4 equivalents) titrated with 0.1 II NaOK.

Sbo eluant AcOEt 12 Rf 0.36 2 Isopropanol

NH

3 2 FAB mass spectrum: mass peak at M 1 *419.

ExAmple 7: Preparation of a solution of the gadolinium colx of 2--ethytl-l.4.7.10-ttraazacyclododecCaS. N1, N11' 9 I' -tetraacetic 0 4 0 acid (methylalucastinesat 21 g (50 miles)of the compound obtained in ex mle 6 and 9.05 S 0 (25 nimoles) of gadolinium oxide are dissolved in mli of twice:Tdistilled, de-aerated water at 70 0 C. After one hour,, dissolution. is complete NAn the pl 8 is close to 3. After cooling, the pH io adjusted to 7.3 with methyglucamina.

The solution Is adjusted to 100 al and filtered through a Millipore menbrane 0.2.2 pm. A solution having a Gd content of 0.5 mole/i is thug obtained. This solution has &,viscosity at 20 0 C of less than 4 .Pa.s9.

(Free Gd not detected).

Exam~le 8: 2-hydroxyiethyl-l.4,*7. 10-tetraazacyclododecane.4 7,10triacetic acid.

a) Preparation of N, N'-ditosyl-2,3-diaminopropionic acid.

To a olutin of46 g f sodum hdroxie in 00 c3ofwtr Tor atoui'o 6go oimhyrxd n50c fwtr of the monohydrochioride of 2,3-diaminopropronic acid are added with vigotous stirring. 200 cm 3 of ethyl ether are added followed by 110.5 g of tosyl chloride in portion during lh. Stirring Is maintained for 12h, the precipitate formed is filtered off, then washed with water and ethyl ether. The solid obtained is suspended Inm I 1 of water, then acidifiled with 6N Nd1. After f iltration and washing with water and ethyl ether, the solid Is dried for 24h at 60 0 C in a vacuum.

.<~PSL1/kWeight obtained *76 g <Yield a652 cc)) Melting point 3200-201 0

C

T!LC: Sto 2 CH 2 Cl 2 80/MeOf Rf: 2.4 ppm singlet 6H (CH of the tosyl group) h3 2.8 ppm mutiplet 35 (CH 2 CH of the diamino chain) to 5 ppm extended multiplet 3H exchangeable with D 0 -7.2 to 7.8 jpm multiplet 8H aromatics.

b) Preparation of N, N'ditosyl-2,3-diminopropanol.

In a 2 1 3-necked f lask, a Suspension of 40 S of the compound 3 obtained in b) in 600 cm of THP is stirred at 20 C in an inert and anhydrous atmosphere. (argon).

A solution of 500 al of 1M BE 1 TUF is added in an lzert atmosphere during 1/2h. The temperature of the reaction mixture rises to 30 0

C.

Stirring is continued for 48h. Hydrolysis Je carried out cautiously with 20 ml of water. The TOE Is removed by distillation in a vacuum.

The residue Is extracted with a water/ether mixture. The organic ph&s" is washed with water, dried over Na 2 90 4 v then evaporated to dryness.

The residue Is triturated with ie'opropyl ether untl It crystallises.

Ater filtration and drying 35 g of product are obtained.

Yield: Melting point: 126-12'7 0

C

TLC: SiO 2 CH Cl 2 90/ MeOi Rf: 0.6 -2.7 ppm singlet "CH of tosyl (6H) ~3 to 3.7 ppm multiplet (7H, 2 of which are exchangelble) T.6.9 ppm triplet, exchangeable alcoholic OH to 7.9 ppm vultiplet aromatics (8H) d) Preparation of N, N' N'"I -tetratosyl-2-hydroxympthyl- 1,4, in a 2 1 3-necked flask under irgon,35 g of the compound obtained in b) are dissolved in 1 1 of anhydrous D)W, tilien 58.6 g of anhydrous CS 2 C0 3 are added.

This suspension Is stirred for lh at ambient temperature, then heated ,"to 650C by means of an oil bath. A solution containing 69 g of N'-

N'L

-26

J

B

C~E

4000 o 0*I o .1)4' 4e~f 0P90 o* 0 P044 0 *4 4 4 04b I dicylJ-bS (2-tosyohyoxyehyl) ethylene diamine in 600 ca 3 of anhydrous DMF arecc~&ddropvise at this temperature during 6h. Heating at 65 0 C is maintained overnight; the DMF is removed by.distillation in a vacuum.

The residue is taken up in a mixture of- 400 ml of water and 400 cm 3 of 5 dichloromethane. The organic phase is decanted, washed with 200 cm3 of water, dried over Na 2

SO

4 t.en evaporated to dryness. The residual oil is dissolved at 80 0 C in 200 cm 3 of toluene, then kept in the refrigerator for 4 8h.for'crystallisation to occur. 24 g of proddct:are obtained.

10 Yield: 322 Melting point: 143-145 0

C

TLC: S102 CH 2 C1 2 90/AcOEt Rf:

NMR

2.4 ppm singlet 12H CH 3 tosyl 3.2 to 4.1 ppm multiplet 17H CH 2 ring CH 2

-OH

7.2 to 8.1 ppm multiplet 16H aromatics d) Preparation of 2-hydroxymethyl-1,4,7,10-tetraazacyclododecane.

g of the compound obtained in c) are dissolved in 100 ea 3 of 20 98% 82SO 4 This solution is heated to 100 0 C for 48h in an inert atmosphere. The reaction mixture is cooled, then added dropise to 1 1 of ethyl ether cooled by means of a Dry Ice/acetone bath. The precipitate of the sulphate of the amine is filtered off onto a glass frit, then washed with ethyl ether. The solid is Immediately dissolved in 200 ca 3 of water, the solution is adjusted to pH 12 with NaOH, and 'evaporated to dryness. After drying the residual solid in a vacuum in the presence of P 2 0 5 the product is extracted with 2 x 100 ca 3 of refluxing (THF). Evaporation of the fractions obtained after extraction lead to a colourless oil.

Weight obtained: 4.5 g of base Yield: 902 TLF: A1 2 0 3 BuOH 50 Water 25 AcOH 11 Rf: 0.8 lMR (CDC13 spectrum) 2.8 ppm singlet (17H) triplet I. i*i -27- L 3.8 PPs ehangeable siaglet (SH) a Preparation of 2-hydroxymethyl- ,4 ,7 4,7,10-trisatic acid.

In a 250 cm 3 3-necked flask equipped with a heater-magnetic stirrer, s a temperature probe and a *pH electrode connected to an analog pH meter and a system for the addition.of--reagent correlated with the pH of the medium, a solution of 8.5 g of the compound obtained in 15.8 g of 2-chloroacetic acid and 100 ml of water Is neutralised to pH a9.5 with the aid of a solution of 15.8 g of potassium hydroxide In 50 cm 3 off water. The reaction mixture is then heated at 50PC by means of an oil bath for 72h. The pH is, moreover, continuously adjusted to 9.5 by the addition-odf a solution of potassium hydroxide. The mixture is cooled, *acidified to pH 5, diluted toi 500 cm 3 and applied to a column of 500 cm 3 of the strongly basic anion exchange resin IRA 958, which has is been regenerated beforehand. The alkylation product* bind to the resin.

This latter is rinsed with water, then eluted with fractions of 52 acetic acid. 'The fractions are evaporated to dryness. The residue Is a crude powder which Is purified on a preparative HPLC column of diameter 40 loaded with RP.18 grafted silica.

Weight obtained: 3.5 g of pure product Yield: 22Z MIelting points 142-144 0

C

TLC: S10 2 AcOEt 12 Isopropztxol 35 /NH 4 0H R1: 0.35 Acidity: 198.7% (2 waves) titration -Ath 0.1 M NaOH corrected for H 0 FAB mass spectrum: peak at M 4- 1 a 377 Exmle_ Preparation of a solution of the gadolinium complex of 2-hydroxiymethyl-,47,0-tetraazacyclododecanC-4.7,10-triacetic acid.

A suspension of 11.05 g of 2-hydroxymethyl-1,4,7,10-tetraazacyclododtcane- 4,7,10-triacetic acid and 5.07 g of gadolinium oxide in twice distilled water Is heated at 80 0 C for lh.

After cooling, the pH is adjusted to 7.3 by the addition of sodium hydroxide and the volume-is adjusted to 100 21. The determination of -28toa j adoliium~ is carried! out by atomic emi.ssion spectroscopy (0.2 )af 1 Example* 10: Pre2aration of 2-hydroxymethyl-1,4, 7,1O-tetraazacyclododecane- 1,4 ,7,10-tatraaeetic acid.

In a reactor of 50 cm 3 equipped with a magnetic stirrer, a solution of 0.7 S of 2-hydroxyme thyl-1, 4, 7 .l0-tetra&zacyclododecane-4, 7, triacetic acid and 0.2 g of chioroacetic acid in 15 cm 3 of water is heated at 70 C.

The pH is brought to 10.5 by means of a solution of potassium hydroxide and is maintained at this value for 48h at 70 0

C.

When the reaction is complete, the pH is brought to 5, then the solution is applied to a IRA 958 resin.

The ligand is chromatographed on the resin by elution vith 5% acetic acid.

After evaporaton to dryness, the product is purified by preparative HPLC (RPl8 grafted silica).

0.15 g of ligand are thus recovered in a yield of 182.

TLC (silica) eluent ethyl acetate 22 :Isopropanol 35 Rf a 0.25 %2 H H 2 0 FAB mass spectrum: peak at M 1 a 435.

4 ,Example 11: Preparation of 2- (2-hydroxyme thyl) l0-tetraazacycladodecane- No N1, N'IIttraet acid.il This ligand is prepared according to the procedure described in -'examples 8 a~nd 10 for the synthesis of 2-hydroxymethyl-1,4,7-,10- A' tetraazacyclododecane-N, N'll-tetraacetic acid.starting from 3,4-diaminobutanoic acid (S.Kasina et al., J. Med Chem., 29, 1933, 1986).

Example 12: Preparation of 2-methyl-i ,4 .7 ,l,13-pentaazacyclopentadecane- 4, 7, 0,13-tetraace tic acid (product 12a) and 2-methyl-1.4,7,lO,13pen taa zacyclopentadecane-1 .4.10,13-pentaacatic acid (product 12b).

a) Preparation of 1,4,7,10,13-peltatosyl-2-methyl-1,4,7,10,13- V pentaaZAcyclopentaecale.

7'E 292 1 £0 (0.093 mole) of N, N'-ditoayl-l,2-diaminopropane, 75.7 of cesf unicakonate (0.23 mole) and 800 ml of WEF are loaded into a 2 1 3-necked flask equipped with a coolant and a mechanical stirrer.

The mixture in placed under argon and heated at 75 0

C.

S A solution of 83 g (0.012 mole) of l,ll-mesyloxy-3,6,9-tritosyl- 3,6,9 triazaundecane synthesised according to Richman and Atkins, Organic Synthesis 58, p.86 in 700 ml of DMF is added during 4 hours at 750 C.

The reaction mixture is maintaiad-at;75 0 C for 48 bours, then is filtered and the filtrate is evaporated to dryness.

The residue is taken up in 700 ml of ethanol, the solid is filtered off then taken up in 800 ml of toluene by heating.

The solid is filtered off at room temperature, then dried at 609C.

Weight obtained: 45.4 g Yield: 492 Analysis: TLC: SiO 2 60 F 254 Merck Eluant CH C1 2 Acetone 98.2 FRi 0.45 Mass spectruam 4 DCI method (NE 3 Mass peak at 999.

b) Preparation of 2 -me thyl -1 4,7,10,13 -pen taazacyclo pentadecane.

44 g (0.044 mole) of the compound attained in a) are maintained for 72 hours at 100 0 C in 130 ml of concentrated sulphuric acid.

After being cooled, the- reaction mixture is poured into a mixture of 250 ml of ethyl ether and 250 ml of ethanol at 0 0

C.

The solid is filtered off, then dissolved in 250 al of water and ereated with carbon black. The solution Is made alkaline with cesium hydroxide, then extracted with CH 2 Cl 2 The organic solution is dried 6VwV Be 2 so 4 and evaporated to dryness. The amine thus obtained can be used as suq;h or in the form of its hydrochloride.

Weight obtained: 14 a 8.4 g as the free base m4a 13.2 g as the'5 HUl Yield: 72.8% Analysis of the hydrochloride; TLC: Al 0 F 254 Merck 1 Iluat: Ethanol/Isopropylamine 80/20 Deitiloper: Iodine Rf: 0.85 NMR% 1.7 ppm 3H CH 3 3.7 ppm 19H C62 and CH.

c) Preparation of 2-methyJ-l,4,7,10,13-pentaazacyclopentadecana- 4,7,10,13-tetraacetic acid (product 12a) and 2-metbyl-1,4,7,l0,13pentaazacyclopentadecane-1 ,4 ,7 ,1,13-pentaacetic acid (product 12b).

in a 500 ml 3-necked flask a solution of 25.7 g (0.27 mle) of chioroacetic acid in 50 al of water is neutralised to pR a 5 at T 100 C by means of 5 M potassium hydroxiide.

10 g of the compound obtained in b) (10.043 nmole) dissolved in ml of water are added to this solution. The mixture is heated at C and 50 ml of 5 H potassium hydroxide are added during 48 hours to maintain a pH of 8.5-9. 5. After the addition is complete, heating Is..

maintained overnight. The reaction mixture is cooled and acidified t6 pa 3. The solution is then applied to 200 al of DOW9I 50 V.resin.

Elution of the rosin by means of 1 1 of a 1 H solution of anionia. leads to the recovery of 20 g of crude product. The crude product is *0 dissolved In 150 ml of water and applied to 250 al of 11A 958 resin.

The resin is washed with water, then eluted with 2 1 of 0.1 X acetic acid, followed by 2'l 6f 0.8 M acetic acid.

9 g of crude product 12& are obtained on evaporation of the 0.1 M acetic acid. 2.5 g of crude product 12b are obtained on evaporation of the 0.8 H acetic acid.

products 12a and 12b are then purified by preparative HPLC on 'RP18-silica.

Product obtained: product 17e: H 5 g product. 12b: H 1.1 g Yield: Analysis: TLC: SiO 2 60 F 254 Merck Eluant: ethyl acetatelisopropanol/NH 3 (302)/12/35/30 Developert iodine Product*12. RI: 0.4 is3\ Z~ Product 12b Rf: 0.27 2 f 31 Water analysis apduct 17ai 1.8% Proddct 12b: 2.8% Acidity aal.yhlu by means of 0.1 M NaOH Product 12a: 2 acidity analyses 98.6% and 100.62 titre: 99.62 Proddict 12b: 3 acidity determinations 199.22 and 92.42 titre: 97.3Z P'AB mass spectra: 12a peak at M+1 462 12b peak at M+ 1 520 r i if ~K

Claims

1. A ligand having the formula R Q HOOC CH CH -COOn I I N n] ~N~R 2 HoOC CH in which R 1 represen ts a radical of the formula -(cR -CH-CH R RA R 6 being selected from the group consisting of C 1 -C 1 4 alkyl, C 1 -C 4 hydroxyalkyl C 1 -C 4 polyhydroxyalkyl and a group of the formula R 1 CH-COON CR-COON I I C- RC II 2 N IC-COON 33 R selected from the group consisting of the groups A and 11 the groups of the formula (CH2)t Y A Y (CH 2 )t A bergw-let framn the group consisting of C 1 -C 8 alkylene, C 1 -C 8 hydroxyalkylene and C 1 -C 8 polyhydroxyalkylene, Y being selected fr om O and O and t 1 to 4, 0 #iR7 eirse2adEd fam the group consisting of hydrogen, C 1 -C 14 alkyl, C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl m 0 or 1 lC R 2 R 3 R 4 identical or different represent a radical of the formula -CH- I I R R9 8 9 Ranmd R, identical or different, being selected frmz hIdroge C 1 C 14 alkyl, hydroxyalkyl C1-C 4 and C 1 -C 4 polyhydroxyalkyl,. p 1 or 2 n 0, 1 or 2 and is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, a C 1 -C 4 hydroxyalkyl and C 1 -C 4 polyhydroxyalkyl, and 0 Z is selected from the group consisting of oxygen and a group of the formula SR 10 being selected from the group consisting of hydrogen, C'-C 14 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 polyhydroxyalkyl, a group of the formula CH COOH, R 5 having the meaning given R previously, and a group of the formula CH-CH CH-COO C-COOH R R 12 N -R 2 N 3 CH-COON R_ A

34- R 1 2 being selected from the group consisting Of Cl-C 8 alkylene, C 1 -C 8 hydroxyalkylene and Cl-C 8 polyhydroxyalkylene, as well as salts thereof, with the proviso that when R 10 represents a macrocycle; R 6 is selected from the group consisting Of CI-. 14 alkyl, Cl 1 4 hydroxyalkyl and Cl 1 4 polyhydroxyalkyl. 2. A coapound as claimed in claim 1 which is 2,6-dimethyl-1, 4,7, 1O-tetraazacyclododecane-N, N' ''-tetraacetic acid., 3. A compound as claimed in claim 1 which is 2-hexyl-1,4,7, 1O-tetraazacyclodedane-N, NO'''-tetraacetic acid. 4. A compound as claimed in claim 1 which is 2-methyl-i, 4, V 7,1O-tetraazacyclododecane-N, NO', N'''-tetraacetic acid. A co~mpound as claimed in claim 1 which is 2-hydroxymethyl- 1,4 10-tetraazacyclododecane- -4,7,10-triacetic acid. 6. A compound as claimed in claim 1 which is 2-hyezoxymethyl- 1,4,7, 1O-tetraazacyclododecane-1,4,7,10-tetraacetic acid. 7. A compound as claimed in claim 1 which is 2-hydroxyethyl- 1,4,7, 1O-tetraazacyclododecane-1,4,7, iO-tetraacetic acid. 8. A process for the preparation of a compound according to claim 1, comprising the reaction of 900M2,dblet036db12249655pC34 NH I_ I' It >1 t L I t II in which R 2 R 3 R 4 and n have the meanings given above, RI 1 represents a radical of formula: -(CH 2 IM -CM -CM RI6 7 R 6 being selected from the group consisting of C I-C 14 alkyl, C 1 -C 4 hydroxyalkyl, C I-C 4 polyhydroxyalkyl and a group of the formula: H /;/fN-RJn R 3 R -CH -R -CH (CH 2) Z' -R 2- N R 2 P R 3 R 4 R 7 R 1 mo n, having the meanings given above, and Z' is selected from the group consisting of an oxygen atom and a group of tbs foruia N RIt -36- R 10 being selected from the group consisting of hydrogen, C 1 -C14 alkyl, C 1 -C 4 hydroxyalkyl, C 1 -C 4 polyhodroxyalkyl, and a group of the formula: H H R R N R N 1 2 2 R 1 R 2 R 3 R 4 R 12 and n having the meaning given above with the proviso that when RI 0 represents a macrocycle; R 6 is selected from the group consisting of C1-1 4 alkyl, C 1 4 hydroxyalkyl and C1- 4 polyhydroxyalkyl. with either a compound of the formula: X CH---COO II 20 I in which R 5 has the meaning given in Claim 1 and X represents a labile group, or an aldehyde of formula: «R CHO IIa in the presence of hydrogen canide or cyanide ions in which R 5 has the meaning given in Claim 1. 9. Process as claimed in claim 8, comprising the preparation of the compound of formula III by reaction of polyamine of formula: R'H N J-I R' ,-NHI' IV -n p" in which n, R 1 and R 2 have the meanings give in claim 8 Edblet03 b22 spec <Y26,dbletO36,db1224965_TM36 r -37- and R' represents a tosyl, mesyl or benzene sulfonyl group, with a compound of formula: X- R 2 N R3- X v R' in which R 3 R 4 have the meanings given in claim 8, R' as defined above, X represents a labile group. A process as claimed in claim 8, comprising the 10 preparation of the compound of formula III by reaction of a diamine of formula: r R' HN R 1 NH R' x in which R' and R' have the meanings given in claim 8, with a compound of formula R' R' X 3 N R -X xi n in which n, R 2 R 3 R 4 and R' have the meanings given in claim a 8 and X represents a labile group. 11. Complexes formed by ligands of the formula I as claimed in claim 1, with metal ions selected from the group consisting of the lanthanide ions (atomic numbers 57 to 71) the ions of j 414 St t Stransition metals (atomic number 21 to 29) and the ions of I metals wi t h atomic number 55, 56, 82 and 83, as well as the salts of these complexes with pharmaceutically accepta- ble mineral or organic bases or with basic amino acids. 12. Complexes asclaired in. claim 11, in which the metal ion is selected from the group consisting of gadolinium, europium, 3+ 2+ dysprosium, iron (Fe and manganese (Mn 13. A complex as claimed in claim 12, which is the methylgluca- mine salt of the gadolinium complex of 2,6-dimethyl-1,4,7,10- tetraazacyclododecane-N,N' ,N '-tetraacetic acid. 14. A complex as claimed in claim 12 which is the methylglu- camine salt of the gadolinium complex of 2-hexyl-1,4,7,10-te- traazacyclododecane-N,N' ,N '-tetraacetic acid. ,LIM/ 15. A complex as claimed in claim 12 which is the methylglu- camine salt of the gadolinium complex of 2-methyl-l,4,7,10- tetraazacyclododecane-N,N',N N '-tetraacetic acid. 900926,dbIeLO36,db1224965.spe,37 I -3 S I ij clr- t V j r 16. A complex as claimed in claim 12 which is the gadolinium complex of 2-hydroxymethyl-l,4,7,10- tetraazacyclododecane-4,7,10-triacetic acid. 17. A diagnostic composition which can be administered to man, comprising at least one complex as claimed in any one of the claims 11 to 16, together with a physiologically acceptable aqueous solvent. 18. A composition for magnetic resonance imaging comprising at least one complex formed by a ligand as oic:med i-n claim 1 with paramagnetic metal ions, and salts thereof with pharmaceutically acceptable bases, together with a physiologically acceptable aqueous solvent. 19. An X-ray contrast composition comprising at least one complex formed by a ligand as claimed in claim 1 with lanthanide ions of atomic numbers 57 to 71, and ions of metals of atomic numbers 55, 56, 82 and 83 and salts thereof with pharmaceutically acceptable bases together with a physiologically acceptable aqueous solvent. Ligands of the formula or metal complexes thereof, methods for their manufacture or compositions containing them, substantially as hereinbefore described with reference to the Examples. DATED this 22th day of October, 1990 Guerbet S.A. By Its Patent Attorneys DAVIES COLLISON j41 1 L.I I 4h,dbbeL36,db1224965.spec,38 h