Cationic Complexes of Technetium-99m This invention relates to cationic complexes of technetium-99m (Tc-99m) useful as body imaging agents. Radiopharmaceuticals may be used as diagnostic or therapeutic agents by virtue of the physical properties 5 of their constituent radionuclides. Thus, their utility is not based on any pharmacologic action. Most clinically used drugs of this class are diagnostic agents incorporating a gamma-emitting nuclide which because of physical or metabolic properties of its co- ■ _] 0 ordinated ligands, localises in a specific organ after intravenous injection. The resultant images can reflect organ structure or function. These images are obtained by means of a gamma camera that detects the distribution of ionising radiation emitted by the - _j c radioactive molecules. The principal isotope currently used in clinical diagnostic nuclear medicine is metastable technetium-99m (tj 6 hours).

It is well established that neutral bidentate ^' ligands of the general type R Q(CH ) QR_ (where Q may _Q be phosphorus or arsenic, and n is 2) form stable well characterised cationic complexes with 99Tc and

99m _Tc [1] _^ Several patents, including US 4481184, US 4387087, US 4489054, US 4374821, US 4451450 and US 4526776 describe various ligand species in which the co-ordinating atoms are phosphorus or arsenic, with predominantly alkyl and/or aryl substituents.

The present invention concerns neutral bidentate donor ligands based on Phosphorus or Arsenic and which contain ether linkages; and cationic complexes of Tc- 0 99m comprising these ligands. It is found that such complexes show suprising properties which may make them superior body imaging agents, particularly heart imaging agents, to comparable complexes not containing ether linkages. 5 In one ^' aspect the invention provides a ligand having the formula:-

Y ₂ QZQY ₂

~~\

— t_ -

where each Q is phosphorus or arsenic, the groups Y may be the same or different and each is

H or C1-C8 saturated hydrocarbon or saturated fluoro- hydrocarbon which may contain up to 3 ether oxygen atoms.

Z is a -CC- or -CCC- or -COC- chain or an o-phenylene which may be substituted by at least one

C1-C8 saturated hydrocarbon or saturated fluorohydro- carbon group which may contain up to 3 ether oxygen atpms, provided that the ligand contains at least one < - COC- ether linkage. Preferably, the ligand contains 2 or 3 -COC- ether linkages. ^'

Examples of C1-C8 saturated hydrocarbon groups optionally containing up to 3 ether oxygen atoms are

Alkyl

Cyclohexyl • Alkoxy

Alkoxyalkyl

Alkoxyalkoxyalkyl

Alkoxyalkoxyalkoxyalkyl

-CH ₂ 0C(CH ₃ ) ₂ 0CH ₂ -

-CH ₂ CH ₂ 0CH ₂ CH ₂ -

2-Tetrahydrofuryl

Saturated fluorohydrocarbon groups may be, for example., any of the above in which one or more hydrogen atoms are replaced by fluorine atoms. h groups Y may be the same of different and each is preferably H or C1-C4 alkyl which may be substituted by C1-C4 alkoxy. Those groups Y which contain no ether oxygen atom are preferably H, methyl or ethyl. Z is preferably a -CC- or -CCC- or -COC- chain which may be substituted by C1-C4 alkoxy or alkoxyalkyl or spirocyclic ether.

More preferably each Q is phosphorus, Z is a -CC- or -CCC- chain, each Y is H or methyl, and either one or more methoxy substituents is attached to one or more groups Y, or one or more methoxymethyl or -COC- spiro¬ cyclic ether groups is attached to a carbon atom of Z.

In another aspect the invention provides a cationic complex of Tc-99m with the ligand as defined. The cat¬ ionic complex preferably has a formula selected from:-

te ligand for Tc A an is anion n is 1 or 2 and m is correspondingly 1 or 0, and L is the ligand.

In the cationic complexes , X is a monodentate ligand for Tc , generally a halide such as F , Cl , Br or I, or a pseudohalide such as SCN, N-., CN or RS. A is an anion whose nature is not critical, but which- may conveniently be selected from the list given for X.

The ligand is characterised by containing at least one -COC- ether linkage. This may be provided by means of a -COC- chain linking the two arsenic or phosphorus atoms. More usually, the -ether linkage is provided by means of an alkoxy or alkoxyalkyl substituent ^' on one or more of the groups Y and Z. Preferably, Z is a -CC- or - CCC- chain, each Y is a H or methyl , and one or more methoxy or methoxymethyl substituents is attached to one or more groups Y or Z. Various substitution patterns are envisaged, for example: a) Backbone substitution which may be single or multiple for example:

b) Phosphorus functionalisation which may be unsymmetrical or symmetrical for example:

CH2—CH2 CH2- CH2

I

1* P P—Me 2* —P P—Me

I i

Me-0-CH2 MeOCH2 CH2θMe (i) (ii)

c) A mixture of functionalisation at the phosphorus atom and in the backbone. d) The spirocyclic type, for example:

In all these examples, P may.be replaced by As.

As indicated in the Examples below, these cationic complexes of Tc-99m are of interest as heart imaging agents. The size of the groups Y and Z, and the number and size of the alkoxy, alkoxyalkyl or spirocyclic ether substituents, can be chosen to make the complex as lipophilic as may be desired for this purpose (or for any other purpose for which the complexes are to be used). But the ether linkages are not there merely to provide a desired hydrophilic/lipophilic balance. it has surprisingly been found that complexes formed from ligands with an ether linkage have significantly higher heart uptake than similarly hydrophobic complexes formed from corresponding ligands without an ether linkage. It appears that the ether linkages in the ligand modify the biologicial behaviour of the Tc-99m complex, compared to the unsubstituted analogue, by providing increased blood and liver clearance which are essential to acheive good target organ to background ratios. Preferred cationic complexes technetium-99m with bidentate ligands of this ivention have formulae : a) [Tc(N0)XL ₂ ] ⁺ A ^" where X is a monodentate ligand for Tc, A is an anion, and L is a ligand having the formula

Me ₂ P PMe.

b) [TcL ₃ ] ⁺ A ^" where L is a ligand having the formula

Although this invention is concerned with results rather than with mechanisms, applicants offer the following as a possible explanation of mechanisms. Broadly, for compounds of similar structure, there is a ^' relationship between lipophilicity and protein binding. Compounds of high lipophilicity are more strongly bound to proteins than compounds of low lipophilicity. For

99m Tc cations, the effect of high protein binding is that they remain a long time in circulation, so that the image of the heart muscle at convenient imaging times post-injection is obscured by the blood pool activity. A further generally observed tendency for the more highly lipophilic cations is that they possess slow clearance through the hepatobiliary system, so that heart imaging can be impaired by liver activity.

Substantially increasing the hydrophilicity of a

99mTc complex has the desired effect reducing protein binding but also reduces heart uptake. It appears that there is, however, a region of intermediate lipophilicity where the heart uptake is retained and there is also absent, or sufficiently weak, protein binding to permit rapid clearance from blood.

It appears that there may be a ranking in contribution to polarity of TcO greater than MeO greater than EtO. By means of this background understanding, it may be possible to achieve the required lipophilic/hydrophilic balance through the additive hydrophilic effects of the oxygen substituents balancing out the lipophilic effects of the hydrocarbon moietes in the molecule.

These phosphine and arsine ligands are not easy to make. They are toxic, the compounds or their precursors often spontaneously flammable in air, and their preparation frequently entails hazards of explosion or difficult to control reactions. Conditions may need to be carefully chosen to avoid the risk of side-reactions. The following reaction schemes are available: A. MeP(CH ₂ 0Me)-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

THF _ _ i) MePH-(CH ₂ ) ₂ -PHMe + 2MeLi . MeP-(CH ₂ ) ₂ -PMe RT -_ THF ii) MeP-(CH ₂ ) ₂ -PMe + 2BrCH ₂ 0Me . A

-78°C This route is available for other symmetrical ligands which are mono-substituted at*-each P (or As) atom. B. Me ₂ P-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

NH ₃ (I)

<____>

Me ₂ P-(CH ₂ ) ₂ -PHMe + MeLi Me ₂ P-(CH _{2 2} -PMe -78°C or ether

Me ₂ P-(CH ₂ ) ₂ -PHMe + MeLi Me ₂ P-(CH ₂ ) ₂ -P e

NH ₃ (1) Me ₂ P-(CH ₂ ) ₂ -?Me + ClCH ₂ 0Me . B

-78°C

This route is available for other unsymmetrical ligands which are mono-substituted at one P (or As) atom.

C. Me ₂ P-CH ₂ -CH(CH ₂ 0Me)-CH ₂ -PMe ₂

NH ₃ (1) i) Me ₂ PH + n-BuLi LiPMe.

-60°C

10 NH ₃ (1) ii) LiPMe ₂ + ClCH ₂ -CH(CH ₂ 0Me)-CH ₂ C1_

-

-60°C

This route is available for other ligands where the ^■ '-' backbone is substitued by alkoxy or alkoxyalkyl or spirocyclic ether.

These and other routes are described in more detail in

Examples 1, 2 and 8 to 12 below.

The cationic complexes of Tc-99m may be prepared by 0 methods well known in the art. For example:

- Complexes of the type [Tc pL-] A~ may be prepared by methods as described in US Patents 438708 and 4489054.

- Complexes of the type [Tc(NO) X L ₂ ] ⁺ A~ may be 5 prepared by methods as described in European Patent

Application 88304239.2.

- Complexes of the type [TcL-,] A~ may be prepared by methods as described in US Patent 4481184.

The following Examples illustrate the invention. 0 Synthesis of Methylether Substituted Diphosphines

All reactions and manipulations were performed under vacuo or oxygen free nitrogen atmospheres. Solvents were dried and degassed by nitrogen purge or freeze/thaw cycles prior to use. The reagents BrCH-OMe and CICH-OMe 5 were purchased or obtained from Amersham (BrCH_0Me and

CICH _p OMe were distilled prior to use) and were deoxygenated by freeze thaw cycles. MeLi was prepared from MeCl in diethylether and was estimated and used in diethylether solution. Ammonia was dried by distillation from sodium. The phosphines MePH-(CH ₂ ) ₂ -HPMe [Ref. 1], and Me ₂ P-PMe ₂ [Ref. 2] were prepared according to established procedures. Me ₂ P CH ₂ ) ₂ P(H)Me [Ref. 3] was prepared from Me ₂ P CH ₂ ) ₂ PH ₂ [Ref. 4]. Abreviations used are: THF = tetrahydrofuran; R.T..= ambient temperature; ether = diethylether = Et O; Me = methyl.

Example 1

MeP(CH ₂ 0Me)-(CH ₂ ) ₂ -(Me)P(CH ₂ 0Me)

(See .reaction scheme A above) Procedure:

To a 250ml 3 neck round bottom flask equipped with a condenser, a pressure equalising dropping funnel and a magnetic stirrer, was transferred the phosphine MePH-(CH ₂ ) ₂ -PHMe, ((6.76g; 5.53 x 10~ ² moles) in 35ml THF. MeLi (11.67 x 10 ^" moles in ether solution) was taken into the dropping funnel and added dropwise to the phosphine with stirring at room temperature, which changed colour to light yellow. The dropping funnel was rinsed with lOcm THF and charged with BrCH _? 0Me (13-83g, 11.07 x 10~ ² moles) in 20ml of THF. The reaction flask was cooled to -78°C and the solution of BrCH ₂ 0Me was added dropwise with stirring, stirred at this temperature for 1h, and allowed to warm to room temperature. The resulting suspension was hydrolysed, the organic layer separated and dried over magnesium sulphate overnight. The dried organic layer was distilled. As the temperature was raised gradually,

ether together with the THF distilled at 38-70°C.

Finally, the product distilled at 74-82°C under dynamic vacuum (about 0.1mm Hg) as a colourless liquid.

Yield = 2.96g (25.5%).

Example 2

Me ₂ P-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

(See reaction scheme B above) Procedure 1 :

In a 250ml 3 neck round bottom flask, equipped with a dry ice condenser (with a T-adapter at the top as the N ₂ inlet-outlet system), a pressure equalising dropping funnel and a magnetic stirrer, was condensed about 150ml anhydrous liquid ammonia.

The liquid ammonia was kept at -78°C and the phosphine Me ₂ P-(CH ₂ ) ₂ -PH e (2.06g; 1.51 x 10~ ² moles) was transferred into the reaction flask. Required amount of MeLi (1.51 x 10 moles) was added dropwise to the phosphine from the dropping funnel, which produced a deep orange colour. The dropping funnel was rinsed with 10ml ether and charged with a solution of ClCH ₂ 0Me (1.22g, 1.51 x 10 ^"2 moles) in 20ml ether. This solution was added dropwise to the reaction mixture until the deep orange colour disappeared. The reaction mixture was allowed to warm to room temperature and after the ammonia had evaporated, about 50ml ether was added and the resulting slurry hydrolysed. The ether layer was removed, dried over magnesium sulphate overnight. The dried ether layer was distilled and after removing ether, the product distills at approx. 80 C under dynamic vacuum (O.lmmHg) s a colourless liquid.

Yield = 0.61g (22.5%)

Procedure 2 . Note: As the starting phosphine Me ₂ P-(CH _p ) _p -PHMe was contaminated with a small amount of M ₂ P- CH ₂ ) ₂ -PMe ₂ , it was purified by precipitation of the salt Me~P- (CH ₂ ) ₂ -PMeLi by reacting with MeLi in ether, which would keep Me ₂ P-(CH ) ₂ -PMe ₂ in solution and enable easy removal.

In a 500ml 3 neck flask equipped with dry ice condenser (with a T adapter at the top as N _p inlet- outlet system) , a pressure equalising dropping funnel and a magnetic stirrer, was transferred the phosphine Me ₂ P-(CH ₂ ) ₂ -PHMe (4.9g; 3.6 x 10~ ² mole) in 30ml ether. MeLi (3 ₌ 6 x 10~ moles in ether) was added dropwise from the dropping funnel to the phosphine solution. As there was no immediate precipitation, most of the ether was evaporated when precipitation occured. The remaining solution was filtered off, and the precipitate was washed twice with 10iql portions of ether. The reaction flask was then cooled to -78 C and about 200ml anhydrous liquid ammonia was condensed to the reaction flask. The pr#cipitated colourless solid forms an orange slurry in contact with liquid ammonia. The dropping funnel was rinsed with 10ml portions of ether and charged with a solution of ClCH ₂ 0Me (2.9g; 3.60 x 10~ ² moles) in 20ml ether. This solution was added dropwise to the stirred orange slurry until the colour just disappears. The reaction mixture was allowed warm to room temperature and after all the ammonia has evaporated, about 50ml ether was added and the resulting slurry was hydrolysed. The ether layer was separated, dried over magnesium sulphate overnight. The dried ether layer was distilled and after removing ether, the product distills at about 80 C/dynamic vacuum (0.1mm Hg) as a colourless liquid. As the product contained traces of water, it was dried over KOH for 4h and .redistilled. Amount = 1. g (26%)

References

1. M. Baake , 0. Stelzer and V. ray, Chem. Ber., 113, 1356 (1980); or by addition of Mel (2 mole equivalents) to H _p P(CHp)pPH _p in methanol or ethanol followed by isolation of the disphosphonium salt [Me ₂ P(H _p )-(CH _p ) _p - P(H _p )Me] .21 ^" and liberation of the free disecondary phosphine by neutralisation.

2. D. . Meek et al . , Inor ^' g. Synth., j_4, 15 (1973).

3.. Prepared by deprotonation of Me ₂ P(CH _p ) -?E - with either MeLi or Bun Li in ether or hydrocarbon solvents followed by formation of the required tertiarty- secondary phosphine by addition of Mel. 4. R.B. King and J.C. Cloyd, J. Amer. Chem. Soc, 9J7, 46 (1975). R.C. Taylor and D.B. Walters, Inorg. Synth., 1973, 14, 10.

H NMR spectra were recorded on a Bruker WH360 operating at 360 MHz and were referenced on the p otio impurity of the CgDg used as solvent (7.27 ppm).

Phosphines/Assignments

P-Me P-CH ₂ 0 P-CH ₂ -CH ₂ -P 0-Me

MeOCH ₂ P(Me)-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

Me ₂ P-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

ι . Complicated multiplet

2. Quintet

Mass Spectrometry Data

MeOCH ₂ P(Me)-(CH ₂ ) ₂ -P(Me) (CH ₂ 0 e)

MW 210

M. ^' 210

M-15 [Me0CH ₂ P(Me)-(CH ₂ ) ₂ -P(CH ₂ 0Me)] ⁺ 195

M-45 [MeOCH ₂ P(Me)-(CH ₂ ) ₂ -P(Me)] ^' 165

M-105 [MeOCH ₂ P(Me)-CH ₂ ] ⁺ 105

M-165 [MeOCH ₂ ] ⁺ 45 base peak

IR Data

MeOCH ₂ P(Me)-(CH ₂ ) ₂ -P(Me) (CH ₂ 0Me)

(Neat)

2900(m) 2δ10(m) 1465(W) 1450(W) 1425(m)

1310(w) 1280(w) 1l80(m) 1090(s) 950(m)

700(m)

B. Synthesis and Properties of Cationic Technetium-99m Complexes of Ligands of Examples 1 and 2

Example 3

[TcX _p L _p ] Technetium III disphosphine dihalide complex.

Materials

8mg _. EGTA

120mg NaCl

eluate (at 1.49

GBq/ml)

These constituents were placed in a sealed glass vial under ~ and heated at 120°C for 70 minutes, to which 3ml of 50% Et0H=saline, and 0.15ml 1M KHC0-. were added, final pH = 6. The resulting solution was submitted to various analytical techniques, summarised below.

Chromatography Data

The resulting solution (above) contains no free ^y TcOw ^" and no colloid, and indicates that the technetium complex is present in solution in approximately 95% yield.

Saline rf = 0.88

Methylethylketone rf = +0.76 Acetonitrile/water 50:50 rf = 0.80 (broad)

388

- 15 -

Gel Electrophoresis Data

The complex moved as a single band towards the cathode rf = -0.53 (- indicating movement towards the cathode).

HPLC Data

The compound elutes as a single band with a retention time of approximately 7.0 minutes.

EXAMPLE 4

Synthesis of [Tc(N0)X(L) ₂ ] ⁺ X ^" Nitrosyl

Compl ex X»C 1 L

θMe

2.5mg

0.8ml at 6.6x10~ ⁵ M solution (aq.)

5μl generator eluate at 3 6mCi/ml

0.4ml -

The components were mixed in a sealed, N purged vial and heated at 120°C for 1 hour. The crude preparation was then subjected to HPLC purification, and the major component (retention time 6.3 minutes) collected, any remaining THF was removed and the resulting solution analysed in the usual way. A sample of this material was submitted for animal biodistribution studies, final pH = 7.4.

Chromatography

The resulting preparation contains no colloid or free TcO,-, and indicates that the desired species is present in approximately 85% yield.

Saline rf = 0.02

Methylethylketone rf = 0.69

Acetonitrile:Water, 50:50 rf = 1.0 Gel Electrophoresis The complex moves as a.single band towards the cathode rf = -0.75 (- indicates movement towards cathode) .

HPLC Data

The complex elutes as a single band with a retention time of approximately 6.3 minutes.

Animal Biodistribution Data

See Table I.

Table I

[Tc (N0)X(L) ₂ ] + ⁺ C_-,l-_ -

Animal Biodistribution Data in Rats

EXAMPLE 5

Synthesis of [Tc(L)- X ^" tris phosphine-complex

X=C1 L= Me Me

\,

MeOCH L2 I H2θMe

Materials Ethanol 2ml NaOH 0.05ml, 10M aq.solution Saline 0.6ml

L 20μl 99m TcCvNa Generator Eluate 0.4ml at 3.73 GBq/ml

Method

The components were mixed in a sealed, _p purged vial and heated at 120°C for 30 minutes. This solution was then diluted with 3ml 66% saline/ethanol, and the pH adjusted to 6 with 0.1M HCl. The resulting solution was then submitted to chromatographic analysis and animal biodistribution study.

Chromatographic Data

The resulting preparation contains no free TcO^ ^" or ^" reduced technetium colloid, and indicates that the desired species is present in solution approximately 90% yield.

Saline rf = 0.00

Methylethylketone rf = 0.75 (broad)

Acetonitrile :Water 50:50 rf = 0.98

HPLC Data

The complex elutes as a sharp band at approximately 8.1 minutes (^-10% minor component at ~6 minutes = 10%).

Animal Biodistribution Data

See Tables II and III

Table II

CTc ⁱ (L) ₃ ] ⁺ X ^" Animal Biodistribution in Rats

Table III

<s>

[Tc(L)-- ⁺ X Animal Biodistribution in Guinea Pigs

Exampl e 6

Synthesis of [Tc(NO) X (L) _? ] ⁺ X ^" Nitrosyl Complex

where X = Cl L =

Method

The components were mixed in a sealed N _? purged vial and heated at 120°C for 1 hour. The crude preparation was then subjected to HPLC purification and the major component (retention time 6.5 minutes) collected, any remaining THF was removed and the resulting solution analysed in the usual way. A sample of this material was submitted for animal biodistribution studies, final pH = 7.4.

Chromatography

The resulting preparation contains no reduced technetium colloid or free Tc0 ₄ ^~ , and indicates that the desired species is present in approximately 95% yield.

Saline rf = 0.04

Methylethylketone rf = 0.86 Acetonitrile:water 50:5.0 rf = 0.90

Gel Electrophoresis Data

The complex moves as a single band towards the cathode rf = -0.78 (- indicates movement towards the cathode).

HPLC Data

This complex elutes as a single band, with a retention time of approximately 6.5 minutes.

Animal Biodistribution Data

See Table IV

Table IV

Animal Biodistribution Data

CTc(N0)Cl(L) ₂ ] ⁺ (in rats)

Me Me

\

L =

Me "CH ₂ OMe

Examp l e 7

Synthesis of [Tc(L)2_] ⁺ X~ Tris-diphosphine Complex

Method

The components were mixed in a sealed N« purged vial and heated at 120°C for 30 minutes; pH of the solution was adjusted to 7-8 with 0.1M HCl. The resulting solution was them submitted to chromatographic analysis and animal biodistribution study. .

Chromatography

The resulting preparation contains no free TcO^ ^' or reduced technetium colloid , and indicates that the desired species is present in approximately 95% yield.

Saline rf = 0.00

Methylethylketone rf = 0.67

Acetonitrile.-water- 0:50 • rf = 0.75

HPLC Data

This complex elutes as a sharp peak at approximately 8.4 minutes ( 5% minor components between 4.5 to 6 minutes) .

Animal Biodistribution Data

See Tables V and VI

Table V

Animal Biodistribution Data in Rats

©

[Tc ^J (L). 3 ⁺ X

Me Me

L = ,p p X = Cl

Me 'CH ₂ O e

Table VI

Animal Biodistribution Data in Guinea Pigs

[Tc(L) ₃ ]^( ^a

Me Me

\

L = X = Cl

Me CH ₂ OMe

Comparative Data

The bidentate phosphine ligand di- (dimethylphosphino) ethane (dmpe) is well known, and various Tc-99πι complexes have been proposed for use as heart imaging agents. The following Tables VII, and VIII compare the biodistribution data of two such prior art complexes with the data given above in respect of the complexes of Example 4, 5, 6 and 7 (derived from the ligands of Example 1 and 2). The Tables permit the following conclusion.

From Table VII, the complex formed from the ligand of Example 1 shows: '-* i) significantl -higher heart uptake and retention at 2' and 60' ii) reduced liver retention at 60' iii) significantly increased heart/liver ratio at 60' . ^~ - ~* From Table VIII, the complex formed from the ligand of Example 1 shows: i) increased heart uptake and retention at 2' and 60' ii) much lower liver retention at 60' " iii) better heart/blood, heart/muscle and heart/liver ratios at 60'. Complexes formed from the ligand of Example 2 show the same general trend.

30

35

Table VII

[Tc(N0)Cl ₂ L ₂ ] in Rats

Organ

Ligand or Ratio 2min p.i . 60 min p. i

% injected dose dmpe Heart 0.46 0.32 Liver 24.92 3.05

Counts/Gram ratio

Heart/Blood 1.71 24.18

Heart/Muscle 1.84 2.08

Heart/Liver 0.24 1.52

% injected dose

Ex. 1 Heart 1.71 1.42 Liver 12.6 1.53

Counts/Gram ratio

Heart/Blood 4.19 25.7

Heart/Muscle 6.61 5.46

Heart/Liver 1.81 10.8

% injected dose

Ex, Heart 0.96 0.94 Liver 20.45 1.62

Counts/Gram ratio

Heart/Blood

Heart/Muscle

Heart/Liver

Table VIII [TcL-,] ⁺ in Guinea Pigs

Organ

Ligand or Ratio 2min p.i. 60 min p.i

% injected dose dmpe Heart 0.53 0.38 Liver 38.0 36.8

Counts/Gram ratio

Heart/Blood 0.42 3.27 Heart/Muscle 6.11 4.12 Heart/Liver 0.30 0.13

% injected dose

Ex. 1 Heart 0.98 0.83 Liver 24.0 13.3

Counts/Gram ratio

Heart/Blood 1.63 10.5 Heart/Muscle 8.18 4.29 Heart/Liver 0.82 0.72

% injected dose

Ex. 2 Heart 0.96 0.43 Liver 24.85 26.13

Counts/Gram ratio

Heart/Blood 1.59 7.82 Heart/Muscle 9.19 4.60 Heart/Liver 0.51 0.30

C. Synthesis of Backbone-Substituted Diphosphine Ligands

EXAMPLE 8 Preparation of Bis(dimethylphosphinomethyl)ether. i. Bis(bromomethyl) ether

This was prepared by the method of Stephen et al, J. Chem.Soc, 1920, 117, 515.

ii. Bis(dimethylphosphinomethyl) ether

To a solution of dimethylphosphine (5g) in diethyl ether (110 cm ) at -70°C was added portions of a solution on n-butyl lithium (2.5M in hexanes) until formation of the phosphide anion was complete (as monitored by P nmr spectroscopy) . The mixture was stirred for 10 minutes at -70°C and bis(bromomethyl) ether (5g) then added. The mixture was allowed to warm to room temperature and then stirred at this temperature for several hours. The resulting mixture was extracted with portions of dilute hydrochloric acid (2M) and the combined aqueous extracts washed with diethyl ether. The aqueous layer was slightly basified with aqueous sodium hydroxide (30%) and then extracted with chloroform. Removal of the solvent from the chloroform layer under reduced pressure (40 C at 150 mmHg) gave the diphosphine (2.8g) in a substantially pure state. £ ³¹ P (CDCl -51.1 & ¹³ C (CDC1 ₃ ) 10.0(d, J=12), 74.2(dd, J=11, 5)

EXAMPLE 9

Preparation of 1 ,3-Bis(dimethylphosphino)-2, - bis(methoxymethyl)propane i. 1 , 3-Dibromo-2,2-bis(methoxymethyl)propane

To 2,2-bis(bromomethyl)propane-1 ,3-diol (100g) was added potassium bicarbonate (8θg) and dimethyl sulphate (170g). The mixture was stirred and heated to 100 C.

After about 30 minutes at this temperature the reaction became very vigorous (Care!). After a further 2 hours heating and stirring the mixture had become very viscous. The mixture was cooled, made slightly basic 5 by the addition of sodium bicarbonate, and then extracted with chloroform (4 x 200 cm-). The chloroform extracts were combined and the solvent removed under reduced pressure to give the crude product. Repeated distillation under reduced pressure gave the pure ° product (25g) as a colourless- liquid (b.p. 65-70°C at 0.1 mmHg) .

£ ^{1 3} C ( CDClg ) 3 . 9 ( x2 ) , 43. 7 , 58 .9 (x2) , 71 .0 ( x2 )

ii . 1 , 3-Bis ( dimethylphosphinyl ) -2 , 2-bis( methoxymethyl ) 5 propane

To a stirred solution of dimethylphosphine (24cm ) in dry diethyl ether (150 cm ) at -4θ°C was added a solution of n-butyl lithium (44 cm , 2.5M in hexanes). After about 30 minutes the temperature had risen to - 0 20°C and 1 ,3-dibromo-2,2-bis(methoxymethyl) ropane

(15g) was added^ causing an exothermic reaction. The reaction mixture was allowed to warm to room temperature and then left stirring overnight. The ether solution was extracted withseveral portions of dilute hydro- 5 chloric acid (2M) , the combined aqueous extracts basified with aqueous sodium hydroxide, and the liberated phosphines extracted with chloroform. Nmr spectroscopy showed the formation of both the required diphosphine and a substantial quantity of 2,2-bis(meth- 0 oxymethyDpropyldimethylphosphine. Addition of excess aqueous hydrogen peroxide (6%) converted both phosphines to their corresponding oxides. The aqueous layer was then separated and evaporated under reduced pressure. The resulting viscous oil was purified by chromatography 5 on Florisil using methanol/ethyl acetate mixtures as

■eluants. The pure 1 ,3-bis(dimethylphosphinyl)-2 ,2- bis(methoxymethyl)propane (2.8g) was isolated as a semicrystalline material. £ ³¹ P (CDC1-) 41.8

£ ¹³ C (CDC1 ₃ ) 19.4(d, J=69), 19.45(d, J=69), 33.9(dd, J=68, 5), 43.1(t, J=5) 58.7(s), 58.7(s), 75.5(t, J=8)

iii. 1 ,3-Bis(dimethylphosphino)-2,2-bis(methoxymethyl) propane

The previously prepared bis(phosphine oxide) (2.7g) was added to lithium aluminium hydride (3-0g) in dioxan (200 cm ) and the mixture heated under reflux for 2.5 hours. After cooling, the mixture was hydrolysed by the careful addition of aqueous dioxan (25 cm , 1:1), and then aqueous sodium hydroxide (4 cm , 50%) was added. The resulting mixture was filtered and the bulk of the solvent then removed under reduced pressure (60°C at 100 mmHg) to give the diphosphine. ³ P nmr showed no presence of other phosphorus-containg components.

£ ³¹ P (CDCl ₃ /dioxane) -63 S ¹³ C (CDC1-) 15.7(m), 38.3(dd, J=16, 10), 42.2(t, J=10), 58.7(s), 76.7(t, J=9)

The diphosphine was converted to its crystalline disulphide derivative for analysis. Found: C, 41.9; H, 8.3- ^c n ^H 26°2 ^P 2 ³ 2 ^{re uires} C, 41.75; H, 8.3%.

EXAMPLE 10

Preparation of 1 ,3-Dimethoxy-1 ,3-bis(dimethylphosphino) propane i. 1 ,3-Dichloro-1 ,3-dimethoxypropane

To a stirred quantity of 1 , 1 ,3 ,3-tetramethoxy- propane (10g) was cautiously added thionyl chloride

(10g) over a period of 10 minutes. The reaction was then stirred at room temperature until the reaction was complete (as indicated by nmr specttroscopy) . The excess thionyl chloride was removed under reduced 5 pressure (20 mmHg) and the residue distilled to give the dichloride (7.7 g) (b.p. 40°C at 5 mmHg). Nmr spectroscopy showed the presence of approximately equal quantities-of two diastereoiso ers.

£ ¹³ C (CDC1 ₃ ) 48.3(x2), 48.7(x2), 57.5(x4), ° 96.2(x2), 96.4(x2) ^'

This material was found to deteriorate on standing and it was therefore prepared immediately prior to use.

ii. 1 ,3-Diιπβthoxy-1 ,3-bis(dimethylphosphino)propane 5 To a stirred solution of dimethylphosphine (18 cm ) in hexane (150 cm ) at -50 C was added a solution of n- butyl lithium- (37 cm , 2.5M in hexanes) . As the phosphide anion precipitated the mixture became very viscous and difficult to stir. After about 15 minutes 0 1 ,3-dichloro-1 ,3-dimethoxypropane (7.7g) was added and the mixture allowed to warm to room temperature. The viscous mixture was agitated by bubbling dry nitrogen and then allowed to stand overnight. The resulting mixture was extracted with portions of dilute hydro- 5 chloric acid (2M), the combined aqueous extracts basified with aqueous sodium hydroxide solution, and the liberated phosphines extracted into chloroform. Removal of the chloroform under reduced pressure gave a mixture of the two diastereoisomers of 1 ,3-dimethoxy- 0 1 ,3-bis(dimethylphosphino)propane in a satisfactory yield.

S ³¹ P (CDC1 ₃ ) -43.6 and -44.6 £ ¹³ C (CDC1 ₃ ) 8.2(d, J=14), 9.2(d, J=14), 9.4(x2)(d, J=14), 32.6(t, J=13), 5 32.8(t, J=13), 58.7-59.0Cm),

79.0(dd, J=10, 8), 80.0(cc, J=12, 9)

EXAMPLE 11

Preparation of 1 ,3-bis(dimethylphosphino)-2-

(2 '-methoxyethoxymethyl)-2-(methoxymethyl)propane i. 5 ,5-Bis(bromomethyl)-2-phenyl-1 ,3-dioxane

2 ,2-Bis(bromomethyl)propane-1 ,3-diol (250g) and benzaldehyde (110g) were added to benzene (500 cm ) containing concentrated sulphuric acid (2 cm ) and the mixture boiled. Water was removed using a Dean Stark apparatus. After about 6 hours the reaction was complete. The mixture was cooled and the sulphuric acid neutralised by the addition ^' of excess sodium bicarbonate. The resulting solution was filtered and the benzene removed under reduced pressure to give an oil which solidified on standing. Recrystallisation from petroleum ether gave the 1,3-dioxane (296g) as a white solid (m.p.66°C).

£ ¹³ C (CDC1 ₃ ) 34.4, 36.0, 37.2, 7l ^" -7(x2), 102.1, 125.9(x2), 128.2(x2), 129.2, 137.2.

ii. 5-Bromo-5-(2 '-methoxyethoxymethy1)-2-phenyl-1 ,3- dioxane

Sodium (3-3g) was added to 2-methoxypropanol (50 cm ) and the mixture was then stirred and warmed" until the metal had dissolved. 5 ,5-Bis(bromomethyl)-2- phenyl-1 ,3-dioxane (50 g) in 2-methoxypropanol (150 cm ) was then added. The mixture was then boiled under reflux for 24 hours in an atmosphere of dry nitrogen. The excess 2-methoxypropanol was removed under reduced pressure and diethyl ether (550 cm ) added. The sodium bromide was filtered off and the ether removed under reduced pressure to give the product as an oil (49.3g, 95%). This material was sufficiently pure to be used in the subsequent reaction without further purification. <S ¹³ C (CDC1 ₃ ) 36.1, 38.3, 58.8, 70.7(x2), 70.9, 71.1, 71.6, 101.8, 125.8(x2), 128.0(x2),

128-.8, 137.7.

iii. 5-(2'-Methoxyethoxymethyl)-5-(methoxymethyl)-2- phenyl-1 ,3-dioxan

Sodium (1.4g) was dissolved in dry methanol

(20 cm ) and placed in a Teflon-lined autoclave (Berghof, 150ml) together with 5-bromo-5-(2'-methoxy- ethoxymethyl)-2-phenyl-1 ,3-dioxane (20g) in dry

^~ > methanol (30 cm ) . The reaction mixture was then heated at 150°C for 6 days. The solvent was removed from the reaction mixture under reduced pressure and the organic residue taken up ^' into ether. Sodium bromide was filtered off and the ether removed under reduced pressure to give the crude product (17g). This material was sufficiently pure to be used without purification. ¹³ C (CDC1 ₃ ) 38.7, 58.8, 59.1, 69.8(x2), 70.9,

71.0, 71-2, 71.6, 101.5, 125.9(x2), 128.1 (x2) ,- 128.7, 138.2. -

i . 2-(2'-Methoxyethoxymethy1)-2-(methoxymethyl)propane

-1 ,3-diol

5-(2'-Methoxyethoxymethyl)-5-(methoxymethyl)-2- phenyl-1 ,3-dioxan (17g) was heated under reflux for 4 hours with a mixture of ethanol (130 cm- ³ ), water

(50 cm ~) and concentrated sulphuric acid (1.5 cm3) . T e mixture was then cooled, neutralised by the addition of sodium bicarbonate, filtered and the volume reduced to about 50 cm under reduced pressure. The aqueous residue was extracted with methylene chloride and the organic layer then dried over anhydrous sodium sulphate. Removal of the volatile components under reduced pressure gave an oil (9.5 g) which was shown to be largely the required diol. This was used without further purification.

£ ¹³ C (CDC1 ₃ ) 44.9, 58.8, 59.4, 64.4(x2), 70.6, 71.5, 72.1 , 74.3.

v. 1 ,3-Dichloro-2-(2'-methoxyethoxymethyl)-2-

(methoxymethyl)propane

2-(2'-Methoxyethoxymethy1)-2-(methoxymethyl) propane-1 ,3-diol (5.5g), triphenylphosphine (24g) and dry carbon tetrachloride were heated under reflux in an atmosphere of dry nitrogen until the reaction was complete (about 2.5 hours). The mixture was cooled, filtered and the solid residue washed with carbon tetrachloride. The carbon tetrachloride solutions were combined and the solvent removed under reduced pressure. Petroleum ether (250 cm ³ , b.p. 4θ-6θ°C) was added to the residue and the resulting suspension filtered. Removal of the petroleum ether under reduced pressure gave the crude product (6g) as an oil. The dichloride was isolated by chromatography on Kieselgel 60 (Merck) using ethyl acetate/petroleum ether mixtures as eluants. Final purification was achieved by distillation under reduced pressure. The pure dichloride (1.4g) was isolated as an oil (b.p. 97 C at 0.06 mmHg). £ ¹³ C (CDC1 ₃ ) 44.7(x2), 45.7, 58.8, 59.2, 68.9, 70.4, 70.8, 71.6.

vi. 1 , 3-Bis(dimethylphosphino)-2-(2'-methoxyethoxy- methyl)-2-(methoxymethyl)propane

- To a__ stirred solution of dimethylphosphine (5 cm ^J ) in dry liquid ammonia (80 cm ) was added a solution of n-butyl lithium (10 cm ³ , 2.5M in hexanes). This mixture was stirred at -6θ°C for 10 minutes and

1 ,3-dichloro-2-(2'-methoxyethoxymethyl)-2-(methoxy- methyDpropane (1.4g) in a little dry diethyl ether was then added. The resulting mixture was stirred for 15 minutes at -60 C and then allowed to warm up slowly to room temperature. When the ammonia had evaporated diethyl ether (150 cm ) was added and the resulting mixture extracted with portions of dilute hydrochloric

acid (2M) . The combined aqueous extracts were basified with aqueous sodium hydroxide and the liberated phosphine extracted into chloroform. The solvent was removed from the combined chloroform extracts under 5 reduced pressure to give the required phosphine (0.4g). £ ³¹ P (CDC1 ₃ ) -63.1 ^{' 3} C (CDC1 ₃ ) 15.5(t, J=5), 15.9(t, J=5),

38.1(dd, J=16, 11), 42.2(t, J=10), 58.6(s),

58.8(s), 70.3(s), 71.6(s), 74.0(t, J=9),

10 76.5(t, J=9).

EXAMPLE 12

Preparation of ,4-Bis(dimethylphosphinoπ_ethyl) tetrahydropyran

15 1. _ ,4-Bis(ethoxycarbonyl)tetrahydropyran

Sodium (10g) was dissolved in dry ethanol (300 cm ) and then diethyl malonate (30g) was added slowly. After stirring for 15 minutes, bis(2-chloroethyl)ether (35g) was added dropwise. The mixture was heated under 0 reflux for 72 hours and then cooled to room temperature. After filtering the solvent was removed from the reaction mixture under reduced pressure to give the crude product. An initial purification was achieved by chromatography on alumina (activity 4) using ethyl

^ acetate/petroleum ether mixtures as eluants. The pure product (11.3g) was isolated by distillation under reduced pressure as an oil (b.p. 106-108 C at 0.03 mmHg). £ ¹³ C (CDC1 ₃ ) 13-8(x2), 30.8(x2), 52.1,

61.3(X2), 64.5(x2), 170.6(x2)

30 ii. 4 ,4-Bis(hydroxymethyl)tetrahydropyran

4 ,4-Bis(ethoxycarbonyl)tetrahydropyran (11g) in dry diethyl ether (50 cm ) was added slowly to a stirred suspension of lithium aluminium hydride (2g) in 35 dry diethyl ether (15 cm ) at a rate so as to maintain gentle boiling. Following the addition, the mixture

was heated under reflux for a further 1 hour and then allowed to cool. Ethyl acetate (4 cm ) was slowly added, followed by water (1.8 cm ) , then aqueous sodium hydroxide (1.8 cm ) , 15%, and finally a second portion of water (5 cm ) . The mixture was filtered and the solid washed with diethyl ether. The combined ether extracts were dried over anhydrous sodium sulphate and the ether then removed to give the diol (5.5g).

$ ¹³ C (CDC1,) 29.1(x2), 36.0, 63.3(x2), 67.4(x2)

^' 3 ^'

iii. 4,4-Bis(chloromethyl)tetrahydropyran

4,4-Bis(hydroxymethyl)tetrahydropyran (5.5g) , triphenylphosphine (19.8g), dry carbon tetrachloride

(100 cm 3) and chloroform (15 cm3) were boiled together under reflux in an atmosphere of dry nitrogen ^' until the reaction was complete (about 8 hours, as shown by nmr spectroscopy). The volatile components were removed under reduced pressure and the resulting solid mass extracted with petroleum ether (b.p. 40-60°C). Evaporation of the petrol from these extracts gave the crude dichloride. A sample of the pure dihalide (1.2g) was obtained by vacuum distillation (b.p.60 C at 0.05 mmHg) followed by chromatography on Kieselgel 60 (Merck) using ethyl acetate/petroleum ether mixtures as eluants.

£ ¹³ C (CDC1 ₃ ) 31. x2), 37.2, 48.4(x2), 63.0(x2)

iv. 4, -Bis(dimethylphosphinomethyl)tetrahydropyran

- To a stirred solution of dimethylphosphine (5-cm ) in dry liquid ammonia (60 cm ) was added a solution of n-butyl lithium (10.5 cm , 2.4M in hexanes). After 10 minutes at 60 C 4,4-bis(chloromethyl)tetrahydropyran (1g), in a little diethyl ether, was added. After a further period of 15 minutes at low temperatures the mixture was allowed to warm up slowly to room

temperature. When the ammonia had evaporated diethyl ether (150 cm ) was added and the resulting mixture extracted with dilute hydrochloric acid (2M). The combined aqueous extracts were basified with aqueous sodium hydroxide and the liberated phosphine extracted into chloroform. The solvent was removed from the combined chloroform extracts under reduced pressure to give the phosphine (0.3g). S ³¹ P (CDCI -62.5

1 ^{J 3} C (CDC1 ₃ ) 15.7(x2)(t, ^• J=4), I6.1(x2)(t, J=4)

34.7(t _r J=12), 37.8(x2)(t, J=8),

43.4(x2)(dd, J =16, 12), 63-5(x2)(s).

EXAMPLE 13

By routes corresponding to those described in Examples 8 to 12, the following ligands were prepared.

EtO OEt

MeO P P OMe

MeO O P P' O _v OMe P δ ^X H P63

90

2

EXAMPLE 1 4 [Tc ( I ) L,] Technetium I . Di phosphine complex

Me ₂ P PMe ₂

L = Ligand of Example 8 Materials ° 10mg Na ₂ S ₂ 0 ₄ 1ml Saline 2ml EtOH 0.05ml 10M NaOH

20μl L 5 1ml 9 9<9m Tcθ4 ^" Na generator eluate (at 2.9GBq/ml)

Method

The components were mixed in a sealed, N _? purged vial and heated at 120°C for 30 minutes. After cooling, the pH was adjusted to 6.5 with ^' 0.1M HCl and 0 diluted with 4ml of saline. The resulting solution was then submitted to chromatography analysis and animal biodistribution studies. Chromatographic Data

The resulting preparation contains no free TcO ~ 5 or reduced technetium colloid, and indicates that the desired species is present in solution approximately 85% yield.

Saline rf = 0.09

Methylethylketone rf = 0.60 (broad) 0 Acetonitrile=Water 50:50 rf = 0.90 HPLC Data

The complex elutes as a sharp band at approximately 18.3 minutes (~ 15% minor component at ~ 17.0 minutes) . 5 Biodistribution Results See Tables IX and X.

TABLE IX

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev, Mean Std. ev.

Counts/Gram ratio

Heart/Blood 3.92 0.67 13.6 1.1 Heart/Muscle 3.32 1.00 1.51 0.38 Heart/Liver 0.53 0.08 0.50 0.04 Heart/Lung 1.4 0.3 1-2 0.0

TABLE X

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i, 2 min 60 min in vivo % injected dose/organ

Mean Std.dev. Mean Std.dev,

Heart Blood Muscle Lung Liver Liver+GI Kidney+Urine Brain

Counts/Gram ratio

Heart/Blood 3.58 1.47 Heart/Muscle 1.90 0.14 Heart/Liver 0.23 0.09 Heart/Lung 0.9 0.2

EXAMPLE 1 5

Synthesis of [Tc(N0)X(L) ₅ ] X ^" Nitrosyl complex

NH ₂ 0H.HC1 = 25mg ° SnF ₂ = 1.0ml at 6.6 ^' x 10 ^"5 M solution (aq). L = 10μl

" ^m Tc0 _2i ~Na, 0.7ml, generator eluate at 3-58 GBq/ml Saline 0.3ml Method 5 The components were mixed in a sealed, _? purged vial and heated at 120°C for 1 hour. After cooling, the prep was filtered through a filter (an acrodisc 0.2μm). The resulting solution was then submitted to chromatographic analysis and animal biodistribution 0 study.

Chromatography

The resulting prepartion contain no colloid or free Tc0^~, and indicates that the de-sired species is present in approximately 90% yield. 5 Saline rf = 0.04

Methylethylketone rf = 0.62

Acetonitrile:Water 50:50 rf = 0.98 HPLC Data

The complex elutes as a sharp peak at 0 approximately 17.4 minutes. Gel Electrophoresis

The complex moves as a single band towards the cathode rf = -0.89 (- indicating movement towards cathode) . 5 Biodistribution Results See Table XI

TABLE IX

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev. Mean Std.dev,

EXAMPLE 16 Synthesis of [Tc(L)- ⁺ technetium (I) trisdiphosphine complex.

L = Ligand of Example 10. Materials

10 ^"4 M solution (aq)

TcO _j ,- Na generator eulate at

The components were mixed in a sealed, N~ purged vial and heated at 120°C for 1 -hour. After cooling, the preparation was submitted for animal biodistribution studies. Chromatography

The resulting preparation contains no colloid or free TcO.-, and indicates that the desired species is present in approximately 70% yield.

Saline rf = 0.02

Methylethylketone rf = 0.-69 (70%)

0.03 (30%) Acetonitrile:Water 50:50 rf = 0.98 HPLC Data

The complete elutes as a single band with a retention of approximately 19-8 minutes (two minor components at 14 minutes and 18 minutes). Gel Electrophoresis

The complex moves as a single band towards the cathode rf = -0.53 (with two minor components with rf = +0.04 and -1.07, where - indicates movement towards cathode) . Biodistribution Results See Tables X11 and XI11

TABLE XI 1

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev, Mean Std.dev,

TABLE XI 1 1

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i. 2 min 60 min in vivo % injected dose/organ - Mean Std.dev. Mean Std.dev,

;

EXAMPLE 17

[Tc N0(X)(L) ] +T _-l_ *

X = Cl

MeO OMe

⁵ Me ₂ P PMe ₂ L = Ligand of Example 10

Materials NH ₂ 0H.HC1 25mg ^T0 SnF ₂ 1ml. of 6.6 x 10 ^"5 M solution. Saline 2.0ml L 10μl

" ^m TcO _ii ~Na ⁺ 2.0ml Generator eluate (6.1 GBq/ml)

5 Methods

The components were mixed in a sealed nitrogen ^" purged vial, and heated at 120°C for 1 hour. The cooled prepartion was filtered through 0.2μm acrosdisc filter (Gelman) and diluted with 8 ml of saline. The 0 resulting solution was submitted for chromatography analysis and animal biodistribution studies.

Chromatography

The resulting preparation contains no colloid and free Tc0^~, and indicates that the desired species is present is approximately 80% yield.

Saline rf ^' = 0.06

Methylethylketone rf = 0.74 (80%)

0.01 (20%) 0 Acetonitrile:water 50:50 rf = 0.99

Gel Electrophoresis

The complex moves as a single band towards the cathode rf = -0.78, (- indicates movement towards cathode) 5 Biodistribution Results See Tables XIV and XV

TABLE XIV

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ ^'

Mean Std.dev. Mean Std.dev.

TABLE XV

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Stdidev. Mean Std.dev.

EXAMPLE 1 8

Synthesis of CTc(I)(L),] Technetium trisdiphosphine

The components were mixed in a sealed, N _? purged vial and heated at 120°C for 1 hour. After cooling the preparation was submitted for chromatography analysis and biodistribution studies. Chromatography

The resulting preparation contains no colloid or free Tc0^ ^~ , and indicates that the desired species is present in approximately 90% yield.

Saline rf = 0.06

Methylethylketone rf = 0.72

Acetonitrile:Water 50:50 rf = 0.99 ■ HPLC Data The complex elutes as a broad band with a retention time of approximately 7.1 minutes. Gel Electrophoresis

The complex moves as a single band towards the cathode rf= -0.27 (- indicates movement towards cathode) Biodistribution results See Tables XVI and XVII

TABLE XVI

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev. Mean Std.dev,

TABLE XVII

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev, Mean Std.dev,

Counts/Gram ratio

Heart/Blood 5.7 0.22 56.1 7.2 Heart/Muscle 6.5 2.49 6.14 1.07 Heart/Liver 1.12 0.07 5.63 0.36 Heart/Lung 0.6 0.1 1.9 0.3

EXAMPLE 19 Synthesis of [Tc ¹ (NC)X(L) ₂ 1 ⁺ C1 Technetium Nitrosyl diphosphine complex

L

The components were mixed in a sealed, N _? purged vial and heated at 120°C for 1 hour. After cooling the preparation was filtered through 0.2μm acrodisc (Gelman). The resulting preparation was then submitted for chromatography analysis and biodistribution studies.

Chromatography

The resulting preparation contains no colloid or free TcO^-, and indicates that the desired species is present in approximately 90% yield .

Saline rf = 0.01

Methylethylketone rf = 0.78

Acetonitrile:Water 50:50 rf = 0.99

HPLC Data

The complex elutes as a single band with a retention time of approximately 6.7 minutes (a minor component is at approximatly 5.8 mintues)

Gel Electrophoresis

The complex moves as a single band towards the cathode rf = -0.64 (- indicates movement towards cathode)

Biodistribution results

See Tables XVIII and XIX

TABLE XVIII

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i, 2 min 60 min in vivo % injected dose/organ

Mean Std.dev, Mean Std.dev,

TABLE XIX

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev, Mean Std.dev,

EXAMPLE 20 Synthesis of [Tc(I) (L)-] ⁺ X~ Technetium (I) trisdiphosphine complex MeO

L = Ligand of Example 11. Materials

EtOH 0.5ml

Saline 3ml

L 15μl

^99m Tc0 _1| Na 0.35ml Generator eluate at 4.26 GBq/ml, Method

The components were mixed in a sealed, N _? purged vial and heated at 60 C for 1 hour. After cooling the resulting preparation was submitted for chromatography analysis and biodistribution studies. Chromatography

The resulting preparation contains no colloid or free TCOH ^" , and indicates that the desired species is present in approximately 90% yield.

Saline rf = 0.00 Methylethylketone rf 0.71

Acetonitrile:Water 50:50 rf = 0.01

HPLC Data

The complex elutes as a single peak with a retention time of approximately 6.3 minutes. Gel Electrophoresis

The complex moves eluates as a single band towards the cathode rf = -0.60 (- indicates movement towards cathode) .

Biodistribution results See Tables XX and XXI

TABLE XX

ANIMAL BIODISTRIBUTION DATA - GUINEA PIG

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev. Mean Std.dev.

Counts/Gram ratio Heart/Blood 11.9 1.3 55.8 10.3

Heart/Muscle 9.61 5.2 6.12 2.15

Heart/Liver 1.12 0.32 12.7 3-3

Heart/Lung 1.5 0.4 1.1 0.1

TABLE XX 1

ANIMAL BIODISTRIBUTION DATA - RAT

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std.dev. Mean Std.dev.

5

0

5

EXAMPLE 21

Preparation of P46:

(MeOC ₂ H ₁ OCH ₂ )MePC ₂ H ₄ PMe(CH ₂ 0C ₂ H ₄ 0Me)

Reaction Scheme: i) Me(H)PC,H _u P(H)Me 2n-BuLi . Li,[MePC.H„PMe) ^{d μ} petrol ^~

2CH ₃ 0C ₂ H ₄ 0CH ₂ C1 ii) Li ₂ [MePC ₂ H ₄ PMe] P46

Exper ental:

All reactions and manipulations were performed under vacuo or oxygen-free nitrogen atmosphere. Solvents were dried, and degassed by nitrogen purge prior to use. CH OCH-CH OCH Cl and n-

BuLi were purchased from Aldrich. Me(H)PC,H ₁ .P(H)Me was prepared

(1) according to a published method

1. M Baake, 0 Stelzer, and V Wray. Chem.Ber. 113, 1356 (1980) Procedure:

In a 250 cm , 3-necked round bottomed flask, equipped with a dry ice condenser, pressure equallising dropping funnel and a teflon stirring bar, was placed in a solution of Me(H)PCpH _i .P(H)Me (3.1δg, 26.06 mmol) in petrol (4θ-6θ°C, 50cm ³ ). To this solution _W as added n-BuLi (35ml, 57.0mmol, 1.6M in hexane) at -78°C.

After warming back to room temperature a white precipitate was filtered and washed with petrol (40-60°C, 2x25cm ). Sodium-dried liquid ammonia (150cm ) was then condensed into the above reaction vessel and an orange solution was formed. To this a solution of CH^OCgH^OCHgCl (6cm ³ , 52.1mmol) in diethyl ether (20 cm ) was added dropwise until the orange colour just disappeared. Then the ammonia was allowed to vaporise at ambient temperature. Diethyl ether (30cm ) was added to give a white suspension. This subsequently was hydrolysed, and the organic layer was separated and dried over anhydrous magensium sulphate. Diethyl ether

(30cm ) was added to give a white suspension. This subsequently was hydrolysed, and the organic layer was separated and dried over anhydrous magnesium sulphate. Diethyl ether was removed by distilation at atmospheric pressure. The rest of the volatile materials were removed by distillation under vacuo at 120°C. The colourless liquid left was pure by NMR. Yield = 4.7g, 50.3% NMR ³¹ P(H) = 39-54 ppm and -39.65ppm, singlets

1.5ml SnF_ aqueous solution 32μg per ml 2ml Ethanol 0.05ml 10M NaOH 10μl P46

2ml Tc ^99m O _ij ^(") Na ⁽⁺⁾ generator eluate at 1.78GBq per ml Method:

The components were mixed in a sealed, ~ purged vial and left standing at room temperature for 2 hours. Then pH was adjusted to 7 with diluted HCl acid (1M). The resulting solution- as then sub¬ mitted to chromatographic analysis and animal biodistribution study. Chromatography Data:

The resulting solution (above) contains no colloid or free

99m Tc O - and indicates that the Tc complex present in solution is approximately 70% pure.

Saline rf = 0.00 Methyl Ethyl ketone rf = 0.00 (30%) rf +0.62 (70%)

Acetonitrile /water 50:50 rf = +0.98

HPLC Data:

The complex elutes as a sharp peak at approximately 6.7 minutes plus peaks at 5.5 and 6 minutes.

Gel Electrophoresis Data:

The complex moved as a single band towards the cathode rf = -0.34

(- indicating movement towards cathode.)

Biodistribution Results: See Tables XX11 and XX111

Experimental and Chromatography, Electrophoresis, HPLC and in vivo biodistribution are exactly the same as previously described.

8 _ ₆ ^_ P T

TABLE TT

Animal Biodistribution Data (P46) [TcL.] ⁺ in rats L = P46

Time p.i. 2 min 60 min in vivo % injected dose/organ

Mean Std. dev. Mean Std. dev.

Counts/Gram Ratio

Heart/Blood Heart/Muscle Heart/Liver Heart/Lung

ANIMAL BICDIST IBLITICN DATA IN GUINEA FIG

CTc(P46) ₃ ] ⁺

TRHL£ XX111

EXAMPLE 23 [TcCl ₂ L ₂ ]+ Technetium 111 diphosphine dichloro complex

Materials :

5mg FeCl ₃ .6H ₂ 0 ) 15mg EGTA

2ml Ethanol ) added into 100mg NaCl

10μl P46 ) 1.7ml saline

99m 1.3ml 100^3+ generator eluate at 5.37 GBq/ml

EGTA = Ethylene glycol - 0,0'-bis(2 aminoethyl)-N,N,N' ,N'- tetraacetic acid.

Method

Firstly, the FeCl,.6Hp0 and P46 in ethanol were mixed in a p6 vial. A purple solution was formed instantaneously. Then this purple solution was transferred into a P11 vial which contained

NaCl, EGTA, saline and ^OO^,-. The reaction mixture was then heated at 120°C for 60 minutes (pH=4). The resulting solution was submitted to various analytical techniques, summarised below:

Chromatography Data:

The resulting solutions (above) contain no colloid or free

99ro

TcO.- and indicates that the technetium complex is present in solution in approximately 80% yield.

Saline rf = 0.01

Methyl Ethyl ketone rf = 0.59, 0.02

Acetonitrile/water rf = 0.99 HPLC Data:

The complex elutes as a sharp peak at approximately 7.3 minutes, plus two small peaks at 5.2' and 6.1'. Gel Electrophoresis Data:

The complex moved as a single band towards the cathode rf = -0.67 (- indicating movement towards cathode). Biodistribution Results:

See Table XX1V

ANIMAL BICDIST IHTΠCM DATA IN RAT tTcCl ₂ (P46) ₂ _] ⁺ 'P46' = CH ₃ P-3

\

/

MeOC2H40CH2 ^' CH2θC2H OMe

ΏBLE ^~~ T~Ύ

EXAMPLE 24

By methods generally as described in Examples 14 to 20, ligands listed in Example 13 were used to prepare cationic Technetium-99m complexes which were subjected to biodistribution testing in rats. The results are summarized in the following Tables XXV and

XXVI.

TABLE XXV

TABLE XXVI

RAT BIODISTRIBUTION RESULTS

EXPERIMENTAL

The experimental techniques used to characterise and evaluate these new radiopharmaceutical complexes are outlined below:

Chromatography

Samples were supplied by needle approximately

2.5cm from the bottom of two Gelman ITLC/SG strips

(2.5cm x 20cm) and one Whatman No.1 strips (2.5cm x

20cm) and then immediately placed in prepared ascending chromatography development tanks containing fresh solvent (1cm): a) saline, b) methyl ethyl ketone, and c) 50:50 acetonitrile.-water respectively. After

15cm elution the strips are removed, solvent fronts marked, dried and the distribution of activity determined using suitable equipment.

Electrophoresis

^• 3 An 0.1g agarose/10cm ^J phosphate buffer pH 7.4 gel was run at an applied potential of 300V for approximately 35 mins, using bromophenol blue indicator

(this indicator moves towards the cathode). The resulting distribution of activity was determined using suitable equipment.

HPLC

For Examples 3 to 7 and 18 to 23, a solvent gradient HPLC system was used, in conjunction with: a) 20mM phosphate buffer pH 7.4 b) Tetrahydrofuran (THF) Samples are applied initially at 100% (a), the gradient changing to 100% (b) in approximately 17 minutes. Flow rate = 2ml/min Hamilton PRP column (15cm x 4.0mm) ambient temperature. Identical HPLC systems were used for " ^m Tc and ⁹⁹ Tc determinations, " Tc detected

99 by emission, ^Tc detected via liquid scintillation method.

For Examples 14 to 17 the system was the same except solvent gradient system

(a) 100 nM sodium acetate pH 5.5

(b) Acetone

Samples are applied at 1Q0% (a), which is held for 5 minutes and then gradient changing to 100% (b) in 20 minutes. Held at 100% (b) for further 5 minutes. Flow rate = 2 ml/min Hamilton PRP column.

ANIMAL BIODISTRIBUTION In vivo studies In vivo biodistribution: 0.1ml Tc 9 m prep was injected i.v. into a laterial tail vein of 6 anaesthetised rats. At 2 minutes and 60 minutes post-injection, 3 rats were sacrificed by decapitation, bled from the neck and dissected. The ^" following organs were removed at dissection: kidney; bladder(+ urine), lung, liver, spleen, stomach, small intestine, large intestine, brain - (weighed), heart (weighed), thyroid and samples of blood (weighed) and muscle (weighed), the residual carcass and the tail (injection site). Subsequently samples were counted in an automatic twin crystal gamma counter. Percentage biodistribution of injected material 5 _W s calculated (after correction for background) for all organs using the formula:

% injected dose = counts/organ x 100 total count in animal - count in tail

° Since only samples of muscle and blood were taken, the percentage in these tissues was calculated assuming blood and muscle to represent 5.8 and 43% of total animal weight respectively using the formual: % injected dose = counts/organ x 100 5 total count in animal - count in tail

Since only samples of muscle and blood were taken, the percentage in these tissues was calculated assuming blood and muscle to represent 5.8 and 43% of total animal weight respectively using the formula:

% injected dose in Counts/gram tissue x CF x tissue = Bodyweight x 100

Total counts in animal - total counts in tail

where CF = 0.058 for blood 0.43 for blood

References

1. Deutsch, E., Libson, K., Jurisson, S., Lindoy, L.F., Technetium Chemistry and Technetium Radiopharmaceuticals Prog.Inorg.Chem. 1982, Vol.30, p.175