CA2009585A1 - Technetium (111/11) imaging agents - Google Patents

Abstract

Technetium (III/II) Imaging Agent Abstract A ligated technetium (III/II) complex is useful as a brain perfusion imaging agent. The complex has a technetium (III/II) center surrounded by six ligating moieties. The complex preferably has a reduction potential Tc(III) to Tc(II) which is less than about +0.6 volts vs Ag/AgCl (3M NaCl) and at least low enough to be oxidized in vivo after crossing the blood brain diffusion barrier. The technetium complex has six ligating moieties, four of which use phosphorous, arsenic, or nitrogen as ligating atoms.
The remaining two ligands use sulfur or selenium to complex to the Tc center. These ligands are modified to establish an effective oxidation potential of the Tc(II) thus providing the necessary redox potential for in vivo oxidation.

Description

o~

Technetium (III~II) Imaa1n~ A~ents Funding for the research leading to this invention was provided in part by the National Institute of Health. ~herefore, the United States Government is hereby granted a paid up non-exclusive royalty free license to practice the present invention.
Technetiu~i-99m complexes are used for a wide range of imaging purpose~. ~oth cationic and anionic complexes are used to image various organs of the human bodv. The ability o~ 99m-Tc complexes to produce dete~table gamma radiation as well as the ready availabili~y of this isotope makes 99m-Tc an important diagnos~ic ~ool.
The potential of single photon emission computed tomography imaging techniques and diagnosis in management of cerebral vascular disease has been studied e~tensLvely in the context of brain perfusion.
Xe-133 has been used ~or some ~ime to determine regional cerebral blood ~low. But tomographic ima~ing with this isotope requires specially dedicated ~ .

~ ~ . : :. : .

instrumentation which is not optimal for other ~Q~o~ 5 graphic applications.
Much more useful results ha~e been obtained in the past few years with I-123 labelled amines.
These amines exhibit high brain uptake, lon~ cerebral retention time and clinical studies with these have clearly demonstrated the ultimate utility of single photon emitting radiopharmaceuticals that monitor regional cerebral ~lood flow. However, since I-123 must be produced in a cyclotron and possesses a physical half life of 13.3 hours, it is expensive and not universally available for daily use. ~ -Clearly, the ideal brain perfusion imaging agent should be based on the readily available, i~expensive Tc-99m isotope. The search for a radio-pharmaceutical which would penetrate the blood-brain barrier (BBB) and have prolonged retention time in the brain has been pu~sued vigorously since 1978.
While Tc-99m brain perfusion imaging agents have been systematically sought since 1978, it has only been in the last three years that significant progress toward such complexes have been achieved.
Neutral Tc(V) complexes containing derivatives o~
tetradentate N252 dithiol ligand ~
HSC~2CE2NHCH2CH2NHC~2CH2SH have been shown to cross the ~BB. But the underivatized complexes are not suf~iciently retained in the brain. Attachment of . , l~ -2- ~

1: ` ' ,:

_3_ 2~
.
pendent amine functionalities to the core Tc(V) complexes leads to significant brain retention in primates similar to the brain retention observed for the I-123 labelled amines. But these do not function well in humans.
A totally different class of neutral Tc(V) complexes containing derivatives of tetradentate-N4 bis(oxime) ligand has been also shown to cross the BBB
and severaL of these compLexes are retained in the brain long enough to allow clinically useful images to be obtained in humans. These compounds are inherently unstable and must b~ used within 30 minutes after formulation. This presents inherent problems in clinics. Further, these present cer~ain interpreta-tional problem~.
Further~ a series of neutral Tc(II) com-plexes and cationic Tc(III) complexas which are reduced to theix neutral Tc(II) forms in vivo, have been evaluated as brain imaging agents. However, none of these complexes are suficiently retained in the brain to provide an effecti~e image~ See "Neutral Tc(II) 99m Complexes as Potential Brain Perfusion Imaging agents", ~ur.~ di-ire ~i~l , Vol. 14, 5 pages 503-510 (1987).
Others have investigated the potential utility of redox reactio~ occurring after crossing the BBB. Bodor, PCT application no. PC~/US85/01334 :` :

' :

~, .

,: . . ` . . , .: ~ : :- .

-4~ S8~
discusses a redox reactlon of ligated Tc(Vt wherein the ligand con~ain.s a dihydropyridine moiety which is oxidized to a pyridinium salt. See also PCT applica-tion no. PCT/US85/01333. These referenc~s discuss the oxida~ion of the ligand as oppose~ to the metal center and of course do not suggest that the oxidation or reduction of the metal center will have any effect on the biological fa~e of the complex. Unfortunatel~, these have not been found to provide an effective imaging agent for humans.
There has been a great deal of wor.~ con-ducted with respect to cationic technetium blood imaging agents and from a chemical point of view, this art has been well developed. Various potential ligands and systems are discLosed in Deutsch, U.S~
P~t. No. 4,489,054, Jones, U.S. Pat. No. 4,452,774, Dean, U.S. Pat. No. 4,582,900, Glavan, U.S. Pat. No.
4,374,B21, Linder, U.S. Pat. No. 4,51~, ~67. Further, Deutsch application Serial No. 172,969, filed March 11, 198~, discloses a Tc(III) complex which is non re~ucible in vivo to provide an improved heart imaging agent.
Of coursè, none of these haar~ Lmaging agents are effective brain imaging agen~s. In light of this, there still remains a need for an effective brain imaging agent which provides an accessible source of technetium99m, which crosses the blood brain .~
l -4-'~:
~ ' : . ~: - : : , : . , -5~ 2 ~ ~ ~ S8 5 diffusion barrier and remains in the brain for a period o time effective ~o obtain a good image of the brain. Of course, it must be one which also does not have any adverse side effects.
Summarv of the In~ention The present invention is premised on the realization that such an effective single photon emission computed tomographic imaging agent can be obtained using the neutral Tc~ 99m complex. The invention is further premised on the realization that these complexes are particularly effective as brain imaging agents where the reduc~ion poten~ial Tc~III) to Tc(II) of the Tc(II) complex is such that upon crossing the blood brain diffusion barrier, th~ Tc(II) complex is oxidized to a Tc(III) complex. This oxidation state of the Tc(III) complex imp~des the crossing of the blood brain diffusion barrier, thus keepin~ the imaging agent in the brain for an efec-~ tive period of time.
;~ The present invention is further premised on the discovery of a new class of 99m-technetium com-plexes. These said complexes ha~e the following general formula:
~ ~ Tc(III/II)LlL2L3L~L5L6] Xz ; and more particularIy , ;
_5_ , ': :
: - ' ~.:

:. ~ ::
; . i~ :

-6~ i8~; .
L5 ~/0 Ll +/0 6 ~ ¦ ~L3 Tc(III/II) Xz Tc(III/II~ Xz 3 ¦ L2 L5 ~ L~
L6 L~
' (L5 and L6 trans) (L5 and L6--Cis~

In this formula X is a pharmaceutically acceptable anion and Z is O or 1 depending on the oxidation state of the Tc center.
Wherein Ll-L4 are ligands which coordinata . through phosphorous, arsenic, or nitrogen and L5 and L~ are ligands which coordinate through sulfur or .
: : selenium atoms. The choice o Ll-L4 and L5, L6 ~- ligands controls the effactive redox potential of the c~II)/Tc(III) redox complex and pro~ide a reduction ~potential Tc(III~ to Tc(II) which is low enough to permit oxidation in vivo.
X represents a parentally acceptable anion such as a halogen. ~ the Tc complex is in the plu5 two state, z is O and the complex is neu~ral. If the Tc center is in the plug three state,~ Z is one and th~
complex is 8 cation. As will be demonstra~ed further, the complex can be easily converted from a Tc~II) : complex to a Tc(III) complex and back again.
' `
;, , : ~ :
'~, :
:, _7_ 2~g~
The invention will be further appreciated in light of the following detailed description.
Detailed Descriptlon The complex of the present invention is a 99mTc~III/II) complex. The octahedrally coordinated Tc center has six coordination bonding sites, two designated trans and four desiqnated Ci9. These bonding sites are occupied by six ligating moieties.
A ligating moiety is a complex which has an atom ~rhich has electron density available for donation to the technetium center. The ligating rnoieties may be bonded together providing two atoms with electron density available to the technetium center. These could be multidentate ligands such as bidentate ` ligands.
~- The complex of the present invention can be ~ expres~ed by one of the foIlowing general ~ormulae:

`` L5 ~/0 L1 ~/0 L4 ` ~ ~1 6 ` ¦ ~ 3 Tc99m(III/II) Xz Tc99m(III/II) Xz L3 ¦ L2 L5 ¦ L4 Formula I Formula II
(Trans) ~Cis) represents a parentally acceptable anion 1 : :
,: ~ ~ - ;

- : : .~ , : ::

-8~ g S8 S
such as a halo~en. If the Tc center is in the plus two state, Z is ~ero and there is no anion present.
I the Tc center is in the plus thxee state, Z is one.
According to the present invention, the ligands L1-L4 shall be referred to as the primary ligands. This appellation is used only as a means to distinguish these ligands from L5 and L6 which will be referred to as the secondary ligancts. This reference, primary and secondary, however, has no scientific significance.
The primary ligands have a formula (Ri)3-A:
wherein A represents P,As, or N. In this formulation~
i represents an integer from 1-4 corresponding to the sub-numeral for the respective ligands L. Rl-R4 inde-pendently represen~s the same or different radical : includin~ hydrogen, C1 C20 alkyl which is intended to inalude substi~uted and unsubstituted alkyl 9 OXy alkyl ~Cl-C10) (i-e- j -0-CH3), cycloalkyl ~C3-C10), as well as aryl which is also intended to include substituted and unsubstltute~ aryl~guch as arylene alkyl, aryl halide, and heterocyclic aromatics.
Further, two, three or four Rs may be bonded together farming a bi, tri or tetradentate ligand.
Examples of ligands useful in the~present invention are disclosed in a series of di~ferent applications including Linder, U.S. Pat. No. 4,512,967, Glavan, :: :

: , .

: :

U.S. Pat. No. 4,374,821, Dean, U.S. Pat. No.
4,582,700, Deu~sch, U.S. Pat. No. 4,387,0E7.
The ligands which are conventionally used in technetium heart imaging are generally usaful as primary ligands. These ligands include:
D~P~((CH3)2p-cH2cH2-p~cH3)2) diars (O-C6H4(AS(cH3)2)2 diphS ((C~H5)2-CH2CH2 P(C6~5)2) tris (l-pyrazolyl)borato porphyrin cyclam, 1,4,8,11-tetraazacylotetra decane and derivatives . . tetraphos P(C~2CH2P(C6H5~2)3 DAE~(C6~5)2As-CH2C~2~HCH~C~2NHZ) EN(~2N-cH2cH2NH~H2c~NH2) TRIEN H2NCH2C~2N~CH2cH2NHc~2 2 2 1, 2-bis Iditoluylphosphino) ethane 1,2-bis(di(~rifluoromathyl)phosphino)ethane 1,2-bis(dimethylphosphino)~ difluoroethane ;~ 1, 2-bis (dimethylphosphino~ fluoroethane : 1,2-bis(dimethylphosphino)propane : .
1,2-bis(di(trifluoromethyL)phosphino)-1,1,2,2-: tetrafluoroethane 1,2-bisIdi(trifluoromethyl)phosphino)propane ; 2,3-bis(di(trifluoromethyl)phosphino)butane 1,2-bis(di~trifluoromethyl)phosphino)butane 1,3-bis(dimethylphosphino)butane _g_ F
.~ .

-10~ 9S85 1,3-bis(dimethylphosphino)propane 1,3-bis(di~trifluoromethyl)phosphino)propane 1,2-bis(dimethylphosphino~-1,1-dichloro-2,2-difluoroethane 1,~-bis(diethylphosphino)ethane 1,2-bis(diisopropylphosphino~ethane 1,2-bis(dipropylphosphino)ethane 1-dimethylphosphino-2-diisopropylphosphino-ethane 1,2-bis~diisobutylphosphinolethane l-dimethylphosphino-2-dimethylarsinoethane.
The secondary ligands ha~e the formula Rn~Y : wherein Y represents sulux or selenium and Rn represents H or an organic radical. The numeral n represents 5 or 6 to indicate correspondence to liqand L5 or L6. These monodentate ligands have a charge of minus one. More specifically, R5 and R6 can represent the same or different radical includin~ C1-C O alkyl (substituted and unsubstituted~, aryl (substituted and unsubstituted), alkylene aryl, substitu~ed carbonyl such as amides, carbamates as well as alkyl substi tuted carbonyls. Radicals which accep~ electron den~ity such as h~lides are particularly suitable substitutes for both alXyl and aryl compounds.
Further the R can represent a sulfanyl containing radical and thus represent a xanthal or sulfonamide. -~
Specific groups represented bv R include-:

. .

--ll--~CF3 2009S85 (CH2)1-19 CH3 -C-R' (R' represents Cl-C10 alkyl) o -C-R' (R' represents Cl-C10 alkyl) S R' -C-N (R' represents Cl-C5 alkyl) O R~

: R' -C-N (R' represents Cl-C5 alkyl) S R' Cj-OR' ~R' represen~s Cl-C10 alkyl~
O
-C-OR' (R' represents Cl~C10 alkyl) S
: Making the radical bonded to the su}fur or selenium either more or less electro negative makes the complex itself more easily or more difficultly oxidized.
: A complex is designated cis where the two , : : secondary ligands are adjacent each other (E~ormula .
~:~ II). A trans complex indicates the secondary ligands : are opposlte each other (Formula I).
;~ The single photon emiss1on~ computed ~~
tomographic lmaging agent or the present invention is a Tc(III/II) complex ~hich preferably has a redu~tion potential Tc(III~-~c(II~ of less than about +0.6 volts : ?

.
, r -12- 2~9~85 vs. Ag/AgCl (3M NaCll and also low enough to provide for oxidation o~ the Tc(II) complex upon crossing the blood brain diffusion barrier. Further, this complax, since it is injected into the blood, must be stable enough in the TCtII) st~te to avoid oxidation in the blood stream during the few seconds (at least about 2 seconds) required for transport to the brain. For this reason, the reduction potential Tc(III) to Tc(II) should generally be from about +0.6 to about -Q.4 volt~, and optionally from about ~0.4 to about +0.2 volts as measured versus a Ag/AgCl (3M NaCl) reference electrode by electrochemical techniques that are well known in the art.
The groups R5 and R6 attached to the sulfur or selenium have a substantial influence on the reduction potential of the ~9mTc center. Basically, , making these radicals more electron donating increases ~; the reduction potential of Tc(III) to Tc(II). An R
group which is elec~ron withdrawing (for example, a halog~nated methyl group -CF3) has the opposite effect.
The radicals Rl-R4 have a similar althouqh ;less pronounced effect~ `
These Tc(III/II) complexes can be made in a two step process. These complexes are prepared from pertechnetate-99m which is obtained from a 99-molybdenum generator. This can be further purified ~. ~
: ~i ~ -12-.~ . .
: : :
~;, according to the method disclosed in Deutsch ~ ~ ~ ~ 5 U.S. Serial No. 802,779 filed November 27, 1985 which uses a lipophillic counter ion bound to the pertechnetate-99m which is then separated from im-purities by preferential sorption such as elution from a Sephadex column.
The obtained 99m-pertechnetate can be complexed with the primary ligands to form a Tc(V) complex having the following general formula:
[ Tc(V)O(OH)Ll L4]
The Tc(V) complex is then reduced to the Tc~III/II) complex by reducing the ~echnetium ~V~
complex in the presence of the secondary ligand.
Alternately ~he pertechnetate is reacted with the secondary ligand first to form a Tc~III) ;
complex. This is then reac~ed with the primary ligand to ~orm a mixed ligand Tc(III) complex. Where the primary ligand is a tetradentate ligand it takes up four of the six available bonding sites leaving two which must be filled with the monodentate secondary ligand. Because the primary ligands bond more strongly to the ~c cen~er, this method is most useful ~, when the primary ligands are tetradentate.

The 99m-pertechnetate solution is obtained from a 99-Mo generator. This method o obtaining 99m-Tc is well known to those skilled in the art and : . , ~ . . . . ~

2 0~ 35 is disclosed for example in Deutsch et al U.S. Patent No. 4,489,054 incorporated herein by reference and Glavan et al U.S. Patent No. 4,374,821 also incor-porated herein by reference. ~he 99m-pertechnetate is eluted from the 9~-Mo generator and is diluted to the desired concentration of 99m-Tc activity of 10-lOOmCi/mL with normal saline.
Pertechnetate-99m i5 extracted from saline (generator eluent) as (C4Hg)4N99ml'c04 using a reversed phase IC18) cartridge tWaters). The method involves the addition of an excess (C4Hg)4NBr ~O.OlM~ to the saline solution containing Na99mTcO4. The mixture is passed through the reverse~ phase car~ridge and the retained (C4H9j4N99mTco~ is washed thoroughly with water to remove the interfering ions and then eluted with 0.5 to 1 mL of ethanol.
0.5 mL of an ethanolic solution containing the desired amount of activity ~mCi's) is transferred to a 5 mL borosilicate vial. The solution is acidified with 10 microliters of lM tri~luoromethyl sulfonic acid~rLflic acid, CF3S03H). The solution degaased with:argon for abou~ 3 minutes and while .~
: degassing, 50 microliters of neat thiol (RSH) are added /RSH represents separately for example benzyl mercaptan, p-methoxybenæyl mercaptan, l-mercaptopropane, ethyl mercaptan, methyl , :

~ -14-thioglycolate, ethyl thioglycolate or 2~mercap~oethanol).
The mixture is degassed with argon for about 30 seconds then is treated with 50 microli~ers of 1%
primary ligand solution ~in this general example Diars) in absolute ethanol. (The primary ligand solution was prepared in a dry box under argon by diluting 30 microliters of neat ligand to a volu~,e of

3.0 mL with well degassed absolute ethanol.~ The borosilicate vial is quickly capped and treated in an oil bath at 80C for about 5 minutes.
Preparation of 99mTc compLex for use in the present inven~ion is also disclosed in the following particular examples.
Example 1 99mTc(Diars) (thiog~ucose-H)~ Complex The complex Tc(Diars~2(thioglucose-H)2 where thioglucose-H is a deprotonated thioglucose is prepared in the same manner described immediately above except tha~ the synthesis requires 5 mg of solid basic sodium ~-D-thioglucose (Si~na) and 40 microliter of lM CF3SO3H. The borosilicate vial containing the mixture of pH=2 is heated at 85C for 5 minute~.
Exam~e 2 99mTctDiars) (thiocholesterol-H?
The complex is also prepared in the same manner but in the presence of about 20 mg o~
:, ~ . -15-~; .

' . , ~' , ', . '` ` ', '` , :' , ' -. . ~

-16~ 5~S
thiocholesterol-H. The mixture is heated at 90-100C
for 20 minutes. (Thiocholesterol-~ represents deprotonated thiocholesterol.) Example 3 99mTc(Diars) (SCH3)~
An ethanolic solution (O.S mL) containing the desired activity of (C4Hg)4N99~Tc04 is acidified with 10 microliters of concentratec~ CF3SO3H. The soLution is degassed (argon) in a S mL borosilicate vial for about 5 minute~. While degassing, 80 microliters of 1~ Diars solution in absolute ethanol are added followed by a quick addition of 3 mg of sodium ~hiomethoxide ~in a hood). The vial is quickly capped. The generated CR3SH which is essential Eor the synthesis of the complex is a gas at room temperature (B.p. = 6C). The vial i9 heated a~ ~5C
for 10 minutes.
For use as brain imaging aqents, the complexes made according to these examples are reduced prior to injection by making the p~ of the solution alkaline and allowing the excess thiol ligand to reduce the complex to Tc(II). ~lternately, reductants such as sodium borohydride can be added.
Se~ e~ e Cationic mTc(Diars)2¦SRj2 can also be formed from the reaction of tr-99mTc(Diars)202 and ' :`

,: . . ~ . .

- . .. . . . ~ .
: ~ ,: . . . .
~ , . . , ~ . . .
:,- : . .
. . .. . .
, . .

thiols (RSH) where RSH - benzyl mercaptan~ C6H5CH ~ H
or p-methoxybenzyl mercaptan, CH30~C6~4-CH2SH.
Ethanolic solutions (pH = 2~3) of tr-Tc(Diars)2O2 are treated with 20-50 microliters of neat thivls then degassed for 3 minutes~ The boro-silicate vial containinq the above mixture is heated at 80C for 10 minutes; then allowed to cool to room temperature. This method can be applied for the preparation of other cationic Tc (Diars)2(SR~2 as an alternative method.
Example 4 Synthesis of ~tr- Tc~Diars) O
2 2] ln ethanol and in The 0.5 mL of (C4Hg)4N99mTco4 in ethanol is transferred to a 5 mL borosilicate vial. The solution is acidified with 10 microliters of lM CF3SO3H then degassed for 3 minutes. While degassing, S0 micro-liters of 1~ Diars solution in absolute ethanol is added. The capped vial is heated at 85C for S
minutes then allowed to cool to room temperature.
This complex is next reduced to Tc(III) complex bv adding the thiol ligand in the presence of a reducing agent as is discussed below.
The preparation of 99Tc(V) and 99-Tc(IIItII) complexes i~ shown by the followi~g examples. These experiments~are designed ~o provide information relevant to the 99mTc counterparts.

~: , . . . . . . . .. . .

-18~ 39S~s Exam~
trans-Oxohydroxobis~1,2-bis(dimethylphosphino)ethane~-te~hnetium(V) Hexafluorophosphate, trans-[Tc~OH)O(DMPE)2](PF6)2.
To a solution con~aining 100 mg of NH4Tc04 (5.5 x 10 4 mol) in 4 mL of degassed 0.05 M NaOH was added 420 mg of neat DMPE (2.8 x 10 3 mol), followed by 4 mL of degassed 95~ ethanol. After stirring at room temperature for 15 min, 0.4 mI, of concen~ra~ed CF3SO3H was added to the yellow-orange solution. The reaction solution was stirred at room temperatura for another 15 min and became deep orange-brown in color.
Addition of ~ g of NH4PF6 in a small amount of water, followed by cooling in a refrigerator for 1 day, produced an orange precipitate of trans-~rc(O~)O(DMPE)2~PF6)2. ~ield: 350 mg.; 88%.
Example 6 trans-Oxohydroxobis(l,~-bis(die~hylphosphino)etha.ne)-technetium(V) Hexafluorophosphate, trani-[Tc~OH)O(DEPE)2](PF6)2.
To a solu~ion containing 100 mg of NH4Tc04 in 22 mL of degassed 0.1 M NaO~ was added 800 mg of neat DEPE 13.88 x 10 3 mol) followed by 4 mL of degassed ethanol. After stirring at room temperature for one hour under an argon atmosphere, G.4 mL of concentrated CE'3S03H was added to the yeLlow solution.
The reaction solution turned deep orange in color upon : ~ ~ : . . .............................. .
- . . . . .

: . . ~ . .

as~5 stirring 30 min longer at room tempera~ure. When 2 g of NH4PF6 in a small amount of water was added to the deep orange solution, a white precipitate appeared.
After removal of the white precipitate by filtration, the filtrate wa~ kept in a refrigerator for 1 day.
Orange crystals of trans-[TctOH)O~DEPE)2](PF6)2 were collected hy filtration. Yield: 300 mg: 65~. Anal.
Calcd for trans-[Tc(OH)O(DEPE)2]~PF6): C, 28.79; H, 5.92; F, 27.32; S, 22.27. Found: C, 2~.58; H, 5.57;
F, 27.98; S, 21.55.
To form a Tc(III/II) complex of the present invention the Tc(V) complex is reduced in the presence - of a monodentate secondary ligand. This reduct:ion is conducted in anaerobic conditions in the presence of a mild reducing agent. Generally, the ligand itself acts as a mild reducing agent. Other mild reducing agents such as sodium dithionite, NaSC~3, or stannous chloride can be added.
The Tc(V) complex previously described is added to degassed ethanol with a fivefold molar excess of the secondary ligand. This mixture is hea~ed to about 60~C until a dramatic deep purple color change occurs.
e ' trans-bis(methanethiolato~bi5(1,2-bis(dimethylphosphi-no)ethane)technetium~III) Hexafluorophosphate, trans-~Tc(SC~3)2(DMPE)2]PF6-,,' ~ --19--` ' ~, ,, :: :. , . ~ . , , ~
:; - ~ : :~ : :: : . : :

s~s To a suspension containing 100 mg of trans-[Tc~OH(O)DMPE)2](P~G)2 (1.4 x lO 4 mol) in 20 mL
of degassed ethanol was added 100 mg of NaSCH3 ~1~4 x lO 3 mol) in 51 mL of degassed ethanol. The mixture was stirred at 60C for 30 min under an argon atmosphere whereupon the solution became deep purple.
To this was added 0.5 mL of saturated NH4PF6 in water and the solution turned blue almost immediately. When the blue solution was cooled to room temperature a blue precipitate appeared. The blue precipitate was dissolved in a small amount of CH3CN, and kept in a refrigerator for one day. The resulting crystals of tran5-lTc(SC~3)2(DMPE)2~pF6 were collected by filtration. Yield: 30 mg; 34%.

:~ ~
trans-bis(methanethiolato)bis(1,2-bis(dimethylphosphi-no)ethane~technetium(III) Trifluoromethylsulfonate, trans-[Tc(SCH3)2(DMPE~2]CF3S03 The TFMS salt was obtained by the addition of NaCF3503 to an almost saturated solution of trans-[Tc(SCH3)2~DMPE)2]PF6 in acetone, followed by cooling in a refrigerator for one day.
Re~rys~allization from acetone in a rerigerator produced crystals suitable for x-ray analysis.
Example 9 .' .

: , .
';' ~ ~ ' `: :

; : . : : .

trans~bislmethanethiolato)bis(1,2-bis~diethylphosphin-o)ethane)technetium~ Hexafluorophosphate, trans-[Tc(SCH3)2(DEPE)2](PF6).
The trans-[Tc(scH3)2(DEpE)2]pF6 P
prepared by a method similar to that described abo~e for trans-~Tc(SCH3)2tDMPE)2]pF6~ using txanc-~Tc(OH)O~DE2E)2]~PF6)2 instead of trans-[Tc~OH)O)(DMPE)2](PF6)2. After keeping the hlue solution in a refrigerator for one day, the resultant crystals of trans-[Tc(SCH3)2(D~PE)2P~6 were collected by filtration. Yield: 50 mg; 48%. The crystals used for x-ray analysis were obtained by slow evaporation fxom an ethanol solution at room temperatura.
ExPmplary composikions which can be made by either of the above methods include:
99mTc-compound Reduction Potential tran~-~Tc(SC~3)2(dmpe)2] / ~0.550 trans-~Tc(SCH3)2(depe?2] /
trans-~Tc(SCF3)2 (diars)2] / : `

trans-ETc(SCH3)2(diars)~]+/0 trans-[Tc(SEt)2(dmpe)2] / -0.566 trans- [Tc (SPr) 2 (dmpe)2] / -0.622 trans-~Tc(SBz)2(dmpe)2] -0.513 trans-~Tc(SCH2C6R4OCH3)2(dmpe)2] ~0-559 trans-~Tc(sc6H4cl)2(dmpe)2] /
cis-[Tc(sc6H4cl)2~dmpe)2]
cis-[TclSC6H5)2(dmpe)2] /
ciS-[Tc(sc6~I4cH3)2(dmpe)2]

:
: ,r ~ .
~ , ~

cis-[Tc~SC6H4OCH3)2~dmpe)2] /
cis-~Tc(SC~H4tBu)2(dmpe)2]
cis-[Tc(sc6H4cl)2(dmpe)2]
trans- [ Tc (SC6H5)2(diars)2]
trans-[Tc(S8z)2(diars)2] / -0.362 cis-[TctSPh)2(diars)2]+/0 trans-[Tc(SPh)2(diars)~] / -0.322 [Tcl3,4-toluenedithiol-2H)(dmpe)2:l /
Cis-[Tctsc6H4No2)2(dmpe)2]
trans-[TclSC6H4C1)2(diars)2]
cis-lTc(SPh)Cltdmpe12] /
[Tc(thioglucose-H)2(diars)2] /
[Tc(thiocholesterol-H)2(diars)21 /
[Tc(SCH3)2)diars)2] / -0.465 [TC(scH2c6H4ocH3~2(diar5)2]
[Tc(SPr)2(diars)2] /
[Tc(SEt)2(diars)2] /
[TctSCH2CO2Me)2(diars)2] /
[Tc(SCH2CO2Et)2(diars)2] /
Tc(SCH2CH20H)2(diars)2] /
lTc(diars)2(SPh)2~/0 [Tc(S~z)2(dppe)2] /
[Tc(SPh)2(dppe)2]+/0 [Tc(SC6H4Cl)2(dppe)2] /
[Tc(SCH~CO2Me)2~dppe)2¦ /
[Tc(SCH2CO2Et)2(dppe321 [Tc~SCH2C02Et.)(SCH2C02 )~dppe)2] /

.- :

J -23- Z~9~8~
[Tc(SCH2C02Mc)(SCH~COq )(clppe)2~ /
[Tc(SBZ)2(dtpe)2] /
[Tc(SPh)2(dtpe)2]+/0 In the above Bz represent benzyl, et ethyl, me methyl, t~u tertbut~l and Ph phenyl, diars O-phenylene, bis(dimethylarsine), D~.PE l,2-bis (dimethylphosphino) ethane, DPPE
l,2-bis(diphenylphosphino) ethane and dtpe l,2-bis(ditoluylphosphino) ethane.
The complexes are designated Tc(III/II~ but are generally obtained in the form of Tc(III) complexes which then can be reduced by the addition of a few drops of a mild reducing agent under anaerobic conditions. For example, the Tc~III) complex can be dissolved in acetoni~rile and a few drops of tetrabutylammoniumborohydride or sodium methylsulfide added.
To test this,:anaerobic solutions of the 3~2(D)2]PF6 where D equals DMPE or DEPE
were dissolved in acetonitrile and a few drops of (C4Hg)4NBH4 and a small amount of ethanol were acided under an argon atmosphere. The pre~iously blue solu~ion almost immediately turned a purple color.
The purpIe color could also be obtained by adding NaSCH3 in a small amount of ethanol to the blue solutionO Contact with the air caused the exposed surfaces of the purple so1ution to turn blue and . , , 1 :
!, .", ~ ` : - ~ ~
. .
" : . i , ~ ~ . . . - .. .

-24- 2~ 8S
bubbling air through the purple solution immediately causes the color change to a pale yellow-brown. When several drops of NH4PF6 in a small amount of water or an acid such as CF3S03H, HPF6 or HC104 were added to the purple solution undex an argon atmosphere, the color reverted back to the original blue. No color change occurred when water alone was added to the purple solution. The con~ersion between blue and purple solution is reverqible for at least four times.
Ln these solutions the purple complex is attributed to tile Tc(II) complex Tc(SC~3)2D~. The deep blue solu-tion is attributed to the Tc(III) complex.
To determine the reduction potential of the complexes according to the present invention, electro-chem~cal studies were performed in 0.5 mol TEAP/DMF at a P~ disc electrode. A summary of these potential measurement: is found in Table 1.
The methanethiolato technetium complexes are characterized by two reversible redox couples corre-sponding to the reactions ~TcIII(SC}~3~2D2] + + e ~?
tTcII(S OE 3)2D2] and [Tc~I(scH3?2D2] + e ~
TcI~SC~3`2D2] . Electrochemical reversibility of this~Tc(II_)/(II) couple ls es~ablished from the ob~ervations that the peak current is proportional to the square r~ot of the scan rate, the ratio of anodic to cathodic peak occurrence is nearly unity and the separation be~ween related cathodic and anodic peaks , :

.i ~

-25- ~09~5 is close to the Mernstian value of 59mV _or a one equi~alent redox process. The observed peak sepa-rations are 60mV and 59mV for D equals DMPE and DEPE
respectively ~or Tc(III/II) reaction which occurs at about -0.55V ~s Ag/AgCl ~3M NaCl). The difference in reduction potentials resulting from the differing phosphine subs~ituen~s of these complexes is slight for this reaction. E is only 4mV more negative for the DEPE complex than the DMPE complex. The effect is more profound for the Tc(II/I) reaction were the difference is 90mV.
The potential at which the central Tc atom undergoes reduction is an inherent component of the energy of the ligand to Tc chaxged transfer transition (ELTMCT). For a series of closely related complexes a linear correlation between reduction poten~ial and ELTMCT can be anticipated. The easier a complex is to reduce the lower will be ELTMC~. Such linear corela-~ tionships have been observed.
:
, . ~

,;~ ' .

: 25-., ;', . : ~ ~ '~'' , ~

,: ~r ~ ~ ` F
: . . ~ . , : ~ . . , ; . ~ , . .

: , -26- 20~9~85 Table I
Analytlcal Characteriz~lons of trans-[Tc(SC~3)2D2)] Complexe~
with D - DMPE and DEPE.
Elemental Analvses C% HZ F% P% S%
[Tc(SCH3)2(DMPE)2]PF~ calcd.26.34 6.00 17.86 24.26 10.04 found26.365.8616.75 23.40 10.74 [Tc(SCH3)2(DEPE)2]PF6 calcd.35.20 7.25 15.18 20.63 8.54 found35.297.2014.89 20.68 8.36 Mass Spectral Data Comple~ Fragment Ion, M-~) Ion, M (CH3) (2C~3) (SC~3) (CH3,SC~3) (D) (D,2C~3) ~Tc(SCH3)2(DMPE)2] 4931 478 463 446 430 [Tc(SC~3)2(DEPE)2~ 605 558 399 369 ~ ~ D a 2 (max)/lo3cm-l (E/1o3M lcm [Tc(SC~3)2(DMPE)2] 16.81(12.96),28.49(1.97),29.68(4.85),46.0~(11.64) [Tc(SCH3)2~DEPE)2] 16.61(12.43)~28.33(1.81),38.61~8.09),46.08sh(19.71) 3 Tc(III/II),E Tc(II/I),E oxidation,Epc [Tc(SC~3)(2(DMPE)2] -.559 -1.72 +0.925 [Tc(SCH3)2(DEPE)2] -.554 -1.81 +0.9545 ~, Intense peak.
2In acetonitrile sh denotes a shoulder.
At 25Ç in O.S M TEAP/DMF at PDE and scan rate =
100 mV/s. E = (E + E )/2 in V vs. Ag/AgCl (3 M
NaCl) from cyclic ~ltam~eatry.
4Irreversible at 25C.
~ S
' Becomes reversible at -70C; see text.

.
.

Method of Use = ~ .
The composition of the present invention wa~
tested for imagin~ the brains of rats and guinea pigs.
The various sodium borohydride reducing 99mTc(II) agents were screened for their ability to cross the BBB in anesthetized (Metofane), female Sprague-Dawley rats of ca 200g weight. Approximately 1 mCi of 99mTc activity per 200 g body weight was injected into the jugular vein. The complexes injected were Tc(II)¦diars)2(SCH2C6~5)2, TctII)(diars)2(SCH2COOCH3)2 and 99~ clII)(diars)2(thiocholesterol)2. After intrajugular injection of the agent, the rats were sacrificed by cervical dislocation, blood samples were collected, and the brains were excised. Tis~ue samples were weighed (average brain weight = 1.5g) and as~ayed ~or 99mTc by s~andard techniques. Analysis indicated passage of the complex through the BBB into the brain and retention at periods of from 30 seconds to 30 minutes or more with peak concentration between 30 seconds and one minute. To image the ra~ brain, 1 mCi/per about 200g body weight of the above 99mTc(II) complexes are injec~ed in the jugular vein and the brain is imaged using a tomo~raphic scintillation camera.

The Tc(~I) comp~exes of ~he present inven-tion cross the b1Ood-brain di~fusion barrier. These -.
.

~; . ~ . .
. . ~ . - . , , -28- ~0~095~5 compounds should remain in the brain for a period of time sufficien~ to permit tomographic analysis of blood flow to the brain. This provide~ a useful method of imaging the brain which can be practiced in virtually any radiopharmacy~
Further, these complexes, regardless of their reduction po~ential, may be used as blood pool imaging agents as well as kidney and liver imaging agents by well known methods~
This has been a general description o this invention as well as the best mode of practicing this invention. However, the invention is deflned by tha following claims wherein:
We claim:

~, -2B- ~

~ I .
' '.

, --~', : ' .

Claims

(1) A brain perfusion imaging agent comprising a ligated Tc-99m complex wherein (1) said Tc is Tc(II);
(2) and said complex has an reduction potential Tc(III) to Tc(II) said reduction potential being (a) at least low enough to be oxidized to Tc(III) in vivo after crossing the blood brain diffusion barrier;
(b) high enough to allow it to remain unoxidized to Tc(III) while in human blood for a period of at least about 2 seconds.

(2) The agent claimed in claim 1 wherein said reduction potential is less than about +0.6 volts vs Ag/AgCl(3M NaCl).

(3) The agent claimed in claim 1 wherein said complex has the following formula [TC(II)L1L2L3L4L5L6]0 wherein L1-L4 represent neutral ligands having the following formula (Ri)3-A, wherein A represents the same or different atoms selected from the group consisting of P,As, and N; and i represents an integer from 1-4; and L5 and L6 represent anionic ligands having the following genexal formula Rn-Y
wherein Y represents S or Se and wherein n represents an integer 5 or 6 and R1, R2, R3, R4 represent the same or different radical selected from the group consisting of hydroqen, C1-C20 alkyl, oxy alkyl (C1-C10), C3-C10 cyclo alkyl and aryl, and R4 and R5 the same or different radical selected from the group consistinq of hydrogen, C1-C20 alkyl, C3-C10 cyclo alkyl, carbonyl, sulfonyl, aryl, and alkylene aryl.

(4) The agent claimed in claim 3 wherein Y
represents S.

(5) The complex claimed in claim 4 wherein Rn-Y
represents a ligand selected from the group consisting of methyl thiolate, benzyl thiolate, and 4 halo benzyl thiolate.

(6) The agent claimed in claim 3 wherein Y

represents Se.

(7) The agent claimed in claim 3 wherein two of said R1 R2, R3, R4 combined represent neutral ligands selected from the group consisting of DMPE, DEPE, DIARS, DIEN, DPPE and DTPE.

(8) A method of imaging the brain comprising injecting an effective amount of a brain perfusion imaging agent comprising a ligated Tc-99m complex wherein said Tc is Tc(II) and said complex having a reduction potential of Tc(III) to Tc(II) said reduction potential being at least low enough to be oxidized in vivo after crossing the blood brain diffusion barrier and high enough to remain unoxidized in human blood during transfer from a source of injection to said blood brain diffusion barrier.

(9) The method claimed in claim 9 wherein said complex has a reduction potential Tc(III) to Tc(II) of at least about +0.6 volts vs Ag/AgCl (3M NaCl).

(10) The method claimed in claim 9 wherein said complex has the following general formula [Tc(II)L1L2L3L4L5L6]0 wherein L1-L4 represent neutral ligands having the following formula (Ri)3-A wherein A
represents the same or different atoms selected from the group consisting of P,As, and N; and i represents an integer from 1-4; and L5 and L6 represent neutral ligands having the following general formula Rn-Y
wherein Y represents S or Se and wherein n represents an integer 5 or 6 and R1, R2, R3 R4 represent the same or different radicals selected from the group con-sisting of hydrogen, C1-C20 alkyl, oxy alXyl (C1-C10), C3-C10 cyclo alkyl and aryi, and R4 and R5 the same or different radical selected from the group consisting of hydrogen, C1-C20 alkyl, C3-C10 cyclo alkyl, carbonyl, sulfonyl aryl alkylene, aryl and alkylene aryl.

(11) The method claimed in claim 10 wherein Y
represents S.

(12) The method claimed in claim 11 wherein (Ri)3-A represents ligands selected from the group consisting of DMPE, DEPE, DIARS, DIEN, TRIEN, DPPE and DTPE.

(13) The method claimed in claim 8 wherein said complex is selected from the group consisting of trans-[Tc(SCF3)2(diars)2]+/0 trans-[Tc(SBz)2(diars)2]+/0 trans-[TctSPh)2(diars)2]+/0 wherein said Tc is 99mTc.

(14) A composition of matter having the following general formula [99mTc L1L2L3L4L5L6]Xz-wherein L1-L4 represent neutral ligands bonded to said Tc center by an atom selected from the group consisting of N, P and Ar and mixtures thereof and L5 and L6 represent ligands said ligands selected from the group consisting of sulfur group containing ligands and selenium group containing ligands wherein L5 and L6 are bonded to said Tc by either said sulfur group or said selenium group and X is a parentally acceptable anion and Z is 0 or 1.

(15) The composition claimed in claim 14 wherein L1-L4 represent neutral ligands having the following formula (Ri)3-A, wherein A represents the same or different atoms selected from the group consisting of P,A5, and N; and i represents an integer from 1-4: and L5 and L6 represent anionic ligands having the fol-lowing general formula Rn Y wherein Y represents S or Se and wherein n represents an integer 5 or 6 and R1, R2, R3, R4 represent the same or different radical selected from the group consisting of hydrogen, C1-C20 alkyl, oxy alkyl (C1-C10), C3-C10 cyclo alkyl and aryl, and R4 and R5 the same or different radical selected from the group consisting of hydrogen, C1-C20 alkyl, C3-C10 cyclo alkyl, carbonyl, sulfonyl, aryl, and alkylene aryl.

(16) The composition claimed in claim 15 wherein Y represents S.

(17) The composition claimed in claim 15 wherein Y repre ents Se.

(18) The composition claimed in claim 15 wherein two of said L1, L2, L3, L4 combined represent neutral ligands selected from the group consisting of DMPE, DEPE, DIARS, DIEN, DPPE and DTPE.