CA2188565A1 - Thioether compounds for use in preparing bifunctional chelating agents for therapeutic radiopharmaceuticals - Google Patents

Abstract

A compound consisting essentially of a multidentade ligand including at least two thioether groups for being complexed to Rhodium-105 in specific activities that are sufficiently high for use in formulating therapeutic 105Rh-radiopharmaceuticals.

Description

~ w09sl29925 21 88~65 r~
-..LU~L-~ uuO,~ FOR ~8~ IN pr~r~P~--r CB1aTING AGENTS FO~ T~ERaPElJTIC
RADIOP~P~r "e~TICaL8 This invention was made with Government support under Contract No. DE-FG02-89ER60875 awarded by the Department of Energy. The Government has certain rights in the invention.

TEC~NICAL FIELD

The present invention relates to '- which chelate radioactive atoms and have rhP~icAl properties which can be used in designing 15 radiotherapeutic agents. More specifically, the present invention relates to a chelate which can complex preferably with rhodium-105, a radioactive for~t of rhodium for use as a radiotherapeutic.

~t~ K(~ UNIJ OF T}IE INVENTION

Radiotherapy using "non-sealed sources"
by way of r~A~tinlAhpled rhA~-~e~tticals has been employed for several decades for cancer tL~ai --L
25 [1,2]. Unfortunately, only a very limited number of therapeutic radiorh~rr--P~lticals are currently in routine use, as being approved by the FDA.

Sl~BSTIIUTE SHEET (RULE 26~

wo sS/2992~ 2 l 8 ~ ; 6 5 ~ r ~

There is a great deal of interest in developing new agents due to the emergence of more sophisticated biomolecular carriers that have high af f inity and high specificity for ~ vivo targeting of tumors.
5 Several types of agents are being developed and investigated at a rapid pace, including monoclonal antibodies (MAbs), antibody f. ~c or Single Chain Antibodies (SCAs) and peptide-based and non-peptide receptor-avid molecules [ 3-7 ] .
lO Radiolabeling of these types of molecules with gamma- or positron-emitting ra~ n~lclides have ~-udu~ d effective agents for scintigraphic and PET
imaging for diagnostic utility in cancer patients [8--10] .
~evelopment of radiotherapeutic agents is also occurring, however, at a much slower rate and is more problematic. For example, the choice of the particle emitting r~Ainrlllrlides for labeling of biomolecules for specific applications is not 20 trivial tll-13]. Many factors must be considered when selecting the appropriate therapeutic radionuclide, including particle energies, matching of half-life with rh ~ nkin~tics, availability, specific activity, suitability of an appropriate 25 chelation system for coupling the radionuclide to the vector, ~a v vo stability, etc. [13,14].

SUBSTITUTE SHEET ~RULE 26~

~ wogsngg2s 2~ &85~5 ~ u~ ~ ~5 Most specific molecular probes target tumor cell populations that have a limited number of binding sites or receptors which in turn limits the quantity (i.e., usually greater than 100 nmoles 5 and often less than 20 nmoles) of the rh~ eutical that can be administered [15,16].
As a result, the specific activity of these radiolabeled drugs must usually be high (i.e., >
100 m (i~nmole) [15,16]. Thus, the most attractive and perhaps the only functional ra~l;onuclides that can be used with these types o~ pharmaceuticals are those readily available in high specif ic activities. Relatively few beta-particle emitting r~linnlln~ q can be produced in sufficient quantities for L-~ai L of very large numbers of patients [11-13]. One of these rA~linn~ s is rhodium-105 (105Rh) [13,1~].
The moderate energy beta particles [E~-(max) -560 keV (70%) and 250 keV (30%) ] emitted by 105Rh make it attractive for therapy while the 306 and 319 keV gamma rays emitted in relatively low ;~h~ n~e (5% and 19% respectively) could be used for imaging in ~ ju..cLiOn with therapy applications, if desired. The half-life of 10~Rh is 25 1. 44 d which could be a good match for the pharr-cnlrin~ti~c of receptor binding agents or MAb SUBStITUTE SHEET (RULE 26) W095/29~25 2`1 88~6~

~L_, ~ts It can be readily produced in larye quantities "indirectly" in a no-carrier-added (NCA) form by bombardment of 704Ru (>99% enriched) to produce 10sRu which decays (th = 4 . 4 hr) to l05Rh.
5 The 105Rh can be separated ~rom the Ru to obtain the high specific activity 10sRh [18]. It is also possible to obtain samples containing high activities (i.e., 103 curies) of 105Rh as a fission product, i f required t 17 ] .
Ther~peutLc agents have been primarily labeled with beta-particle emitting r~Airn~tC~ P5 r~ost of the promising ra~lionllc]icl~pc are y~ ced in nuclear reactors, however, some are accelerator produced [11-13]. Several different chelatin~
15 ~LU- L ~:S have been employed to ~-1nt~in the association of these beta emitters with the drug [19-23~. Many of these :~LU~i~UL~ are not sufficiently stable and most, if not all, do not provide appropriate routes or rates of clearance of 20 radioactivity from non-target tissues [23,24].
Accordingly, there is a delivery of high radiation doses to normal tissues and a rPd~lr~ i on of the therapeutic ratio. This lowers the amount of radiation doses that can be safely delivered to a 25 target tissue. Development of new radionuclide chelates that link the radioactive metal to a SU~S~ SHEET ~LJLE 261 ~ wo95/29925 2 ~ 8~565 1 .,. - ~

radiopharmaceutical is nPcPcc~ry. Further, new 2pproaches must be taken in order to identify radiolabeling terhniqnoc that produce chelates that are highly stable ' v vo but have improved clearance characteristics ~rom normal tissues.
Bifunctional chelating agents have been used to form stable metal complexes that were designed to minimize ~ y vo release of the metallic r~-l; nrl~clide ~rom the radiorh~rr~~e-~ltical.
For example, diethylenetr;~minnrPntaacetic acid (D~PA) forms rather stable chelates with a variety of metals. However, COI~rl in~ of this ligand to monoclonal antibodies by one of its five carboxyl groups resulted in unacceptable ~ ~y~ stability with a variety of radion~rl i,lPc [14] . Linking of this ~ _ ' by a side group attached to one of the carbon atoms on an ethylene bridging group provides i r.,v~:d stability n ~Q and ~,~ vivo.
U..fo.Lui.~tely, the stability characteristics of 20 this chelate and its analogues with all radioactive metals are not ideal resulting in poor clearance of activity from certain non-target organs.

~ he fact that Rh (III) forms a variety of 25 complexes that are rhPm;r~lly inert under physiologic conditions [25] makes 1a5Rh (III) suBsTlTurE SHEET ~RULE 2~

W0 95/29925 2 1 8 ~ 5 ~ 5 J ~,1/0.. , _.'.- - ~r particularly attractive for formulating new 1C5Rh-labeled bifunctional chelating agents to form new therapeutic rA~9;o~h~ euticals. Unfortunately, the formation of desirable Rh (III) chelates in 5 aqueous media usually requires rather harsh conditions (e.g. refluxing for greater than two hours) [26-29]. In addition, polymerized forms of Rh are often produced, even with a large excess of the ligand [30,31].
Complexation of Rh with a variety of thioether _ -c has been reported recently [28-32]. Rh (III), considered a moderately soft acid, will usually form stable complexes with ligands containing "soft" donor atoms (e.g., thioethers) [ 3 2 , 3 3 ] . Blake et al ., ( 198 9 ) [ 3 2 ] showed that 1, 5, 9 ,13-tetrathiacyclAh~Y~la~~An~ ( 16-ane-S~ ) forms trans Rh(III) C12 complexes with Rh(III) chloride.
The ability of thioether ~ '- to complex Rh(III) in high yields at low ligand ~u.._cn~L~.tions 20 has not been investiga~ed. Unfortunately, al~ost all metal chelates with high ther~odynamic stabilities are not sufficiently stable vivo and will not form in high yields using low quantities of ligand. ~he applicant investigated the 25 possibility of 1~5Rh to form well-defined 1~5Rh chelates with tetrathiomacrocycles or their SUBSTITUTE SHEET ~RULE 2~

~ WO 95129925 2 1 ~ ~ 5 6 5 F~ t' _ 7 _ analogues using quantities of the ligand in the nanomole range under relatively mild conditions.
Unlike other prior art chelates, these chelating agents show the ability to form highly stable 1~5Rh 5 chelates in high yields and high specif ic activities using very low quantities o~ the ligand (i.e., < 20 nmoles).

SU~MARY OF l'~E lNV~h~lON

In accordance with the present invention, there is provided a ,_ ' consisting essentially of a multidentate ligand including at least two thioether groups for being complexed to rhodium-15 105. The present invention further provides stablecomplexes of Rhodium-105 with 16-ane-S~-diol, 14-ane-NS3 and 14-ane-N2Sz ligand.

BRIEF DESCRIPTION OF T~E FIGURES

Other advantages o~ the present invention will be readily appreciated as the same becomes better understood by re~erence to the following detailed description when considered in connection 25 with the A~-_ nying ~rawings wherein:
SUBSTITUTE SHEET (PULE 26~

wo 95ng92s 2 1 8 8 S 6 ~

Figure 1 is a r2diochromatogram of electrophoresis analysis of (a) ~05Rh-chloride as received from MURR
: 5 in acidic solution, and (b) lsRh (III) after complexation to 16-ane-54-dlol;
Figure 2 shows the complexation yield of 1sRh-l6-ane-S4-diol at different temperatures as a function of heating time, studies being performed using 10 ~Lg 16-ane-S4-diol in 0. 5 ml solutions at pH
4 containing 15% ethanol;
Figure 3 shows the stability of l5Rh-l6-ane-S4-diol at pH 7 . 4, 8 . 5 and in human serum at pH
7.4-7.8 for greater than 4 days, samples being maintained at (a) pH 7 . 4 in . 09% saline (N. saline) at room temperature tR~) using 0 . O5M sodium phosphate; tb) pH 8.5 in N. saline at RT using O.lM
sodium bicarbonate and tc) pH 7 . 4-7 . 8 in human serum at 37-C;
Figure 4 is a radiochromatogram of electrophoreses of:
Sl~8ST~ E Sl iEE~ E 2 . _ .. . . .

21 8~565 Wo 95l29925 r~l~v. . . t~
_g_ A. I05Rh-chloride and B. 1sRh-14-ane-NS3;
Figure 5 shows complexation yield of 5 10sRh(III) with 14-ane-NS3 as a function of quantity o~ 14-ane-NS~ used, lowest quantity of ligand - 1 ~g (about 4 nmoles), prepared by heating 10sRh-chloride and 14-ane-NS3 at pH 4 in 20% ethanol for 1 hour and analyzed by TLC and ele- L~pl.ul~.3is; and Figure 6 shows complexation yield oP
sRh-14-ane-NS3 as a function of temperature, 12 ~Lq 14-ane-NS3 being incubated in aqueous solutions at pH 4 c~ntAini~ 20% ethanol for one hour at 55, 60, 70 or 80 C.
r)T~TT.Tn DESCRIPTION OF THE INVENTION
Generally, the present invention provides a - consisting eslientially of a multidentate ligand including at least two thioether groups for being complexed to rhodium-105. That is, the _ can contain an 105Rh core and a multidentate ligand containing two or more 25 thioether (TE) groups for bonding to the metal.
The resulting 10sRh-TE complexes have high n vitro SU~ST~IUT~ SHFET (nULE 2~

~,1 8~5 WO95/29925 1 ~

and ~ vivo stability and are formed using low quantities of the ligand (i.e., < 50 nmoles).
The TE containing ligand is complexed to lsRh to form a chelate where the metal to ligand 5 ratio is 1:1. The method of complexation permits the formation of the 10sRh-chelate in a one step, high yield reaction (~ , lifi~ in the Experimental Section), especially with 1CsRh-synthons that are currently available or can be 10 made commercially available. The ~sRh-chelates are produced in high yields (i.e. greater than 85%) under relatively mild conditions ( i . e ., heating at 60-80 C for one hour at moderate pHs) following mixing of the 10sRh (III) synthon and the thioether 15 ligand in aqueous 601utions containing very small quantities of the ligand ( i . e., less than 50 nmoles). This is critical to solve the problems o~
prior art. 10SRh chelates req~ired more severe conditions (e.g., refluxing for greater than two 20 hours) and the use of larger quantities of the , l~Yinq ligands. Synthesis of 1C5Rh chelates under these conditions will normally destroy the speci~icity or binding affinity of sensitive biomolecules and produce 10sRh ~ with 25 specific activities that are usually too low for use as radioFhA~-rot~ticals.

SU~STIME SHEET (RULE 2 ~ W0 9S/2992S ~ 3 5 6 5 P~

~ he lsRh chelates made in accordance with the present invention have been found to be stable in aqueous solutions and in human serum at 37 ' C.
These chelates are also stable over a wide pH range 5 (i.e., pH 1-10). This high stability is critical with regard to permitting localization of the ulld in areas of the body having different pH's as well as being stable through different administration routes.
More specifically, the thioether (TE) containing multidentate ligands used for complexing 5Rh can be characterized by the following ~ormulas:

A. ~acrocyclic ligands containin two or more thioether crrol~n~::
--R~
4 `2 X3 X?

Essentially X ' s are or contain "donor"
atoms that will complex Rh-105 (i.e., S, 0, or N).
A wide variety of thioether macrocycles have the above aLLU-;~UL_ and can be made using well described methods [34,35]. R1, R2, R3, and R~ are SU~STITUTE SHEE~ (RU~E 2~1 W0 9~29925 :2 1 ~ 8 5 6 J . ~ . 15 all the same or different and are selected ~rom the group consisting of -(CHz)z-, -(CH2)3-, -CH2CH ( C3CH2-, - ( CH2 ) 4 -, -CH2CH ( R5 ) -, -CH2CH ( Rs ) CH2 ) -, -CH ( R5 ) CH2CH2- and -CH2CH ( CH2Rs ) CH2- .
Rs is -H, or any side chain containing groups commonly used for linking (e.g. ,-OH, NH2, COOH
-NCS, activated ester). Rs also can be selected from the group consisting of -OCH3, -OC2Hs and groups for attaching a linking group used to modify ~;ro~hilicity for conjugation of the uncomplexed ligand or the ligand chelated to lSRh to a biomolecular targeting agent and Rl 4 can also contain another coordinating atom, e.g., R6-X4-R6, wherein R6 is -(CH2)2-, -(CH2)3-, -CH2CH(CH)3CHz-, -(CH2CH(Rs) CH2) -, -CH(Rs) CH2CHz-, -CH2CH(CHzRs) CHz-, wherein R~ is -OH, NE12, COOH. X1 4 is an atom or group containing S, O or N donor atoms that can also coordinate 1CsRh(III), wherein X~, X2, X3 and X4 are all the same or different in which one o~ the X's is an -S- and the others are -S, -O-, NH, NR7, wherein R7 i5 H, -CH3 or a group attached to the N-atom to alter 1 i rFh; 1 i~ity or to link the uncomplexed ligand or the "preformed" 1CsRh-chelate to the biomolecular targeting agent.
As stated a~ove, the ligand may ~e uncomplexed; that is, not complexed to the Rhodium-SU~iSTITUrE S~IEE~ ~RULE 2'-~

WO ss/2ss2s 2 1 ~3 ~3 5 ~ r 105. Alternatively, the ligand may be complexed to the Rhodium and referred to as l~L~_ , lexed" .
Hence, either an uncomplexed or ~L~_ , lexed ligand can be linked to a receptor avid molecule.
several rh--mir~Al methods for conjugating ligand or metal chelates to biomolecules have been well described in the literature [36,37] and one or more of these methods will be used to link the uncomplexed T~ ligands or l05Rh-TE complexes to the receptor avid biomolecular targeting molecules.
These include the use of acid anhydrides, aldehydes, arylisothiocyantes, activated esters or N-hydlu~cy~ ;nimi~-~C [36, 37].
The ligands can also be linear, open chain-ligands, containing at least one thioether group . The _ - are I i f ied by the following formula:
R4~ ~Rl ~ ~ R2~ ~ R3~ ~ RS

wherein R1, R2 and R3 are all the same or different and are selected from the groups consisting of 2~ -(CH2)2-, --(CH2)3-, -CH2CH(CH)IcH2-~ (CH2)~, -CH2CH t R5 ) -, -CH2CH ( Rs ) CH2) -, -CH (Rs ) CH2CH2- and ~IJBS~ITUTE SHEE~ (RULE 2~) Wo 95/29925 ' ~ ~ 8 8 5 6 5 -CH2CX(CH2Rs)CH2 . R~ and Rs can be the same or different and can be H or an alkyl group or a linking group containing f~nrtionAl groups such as -OH, NH2, COOH. -OCH3, -OC2H2 and other functional 5 groups for attaching a linking group used to modify l iroFhi l irity for conjugation of the uncomplexed ligand or the ligand (chelated or not to 10sRh) to the b; -l~c~ r targeting agent. X1 ~ is an atom or group that can also coordinate 1CsRh(III). X7, X2, 10 X3, and X~ are all the same or dif~erent wherl at least one X is a S-atom and the r i n i n7 are selected from thQ group consists of -S-, -0-, -SH, NH and NR7. R7 is selected from the group consisting of H, -CH3 and a group attached to the N-15 atom to alter l; Porh i 1 i r-ity or to link the uncomplexed ligand or the ligand ~~ ,1f~Y~ to 105RH
to the biomolecular targeting agent.
The above formulas characterize the present invention as providing capabilities ~or 20 ligand modifications in order to tailor the ligands, that when chelated to 105Rh, can be designed to optimize ~ yiYQ binding and pharmacokinetic properties for specif ic localization on target tis6ues (i.e., cancerous 25 cells or tumors).
S~STITUTE SHEET (RULE 2~1 ~ W095/29~25 2 1 8 ~ ~ 6 . I~lru~
For example, the uncomplexed ligand or COIL~71~...A;n~ 10sRh-chelate can be conjugated to peptides and other receptor avid molecules ~targeting molecules) such as antibodies and 5 antibody fragments by using side chains previously used for cu..juyAtion to bioactive molecules [36,37]. Conjugation reactions can involve reactive groups such as benzyl isothiocyanate, b. --~etamide, N hy-lL~y-succin;m;~lo~, activated 10 esters and aldehydes (15).
Other side chains can be added to functional groups to make the chelate more polar or more hydrophilic. Charged groups, such as carboxyl or hydroxyl groups can be added to either the C-15 h~ khnn~ or other sites (i.e., N-atoms) to increase the hydrophilic character of the resulting chelate.
Alternatively, non-polar groups (e.g., alkyl, alkoxy, etc. ) can be added to the ligand b~khnn~ in a similar manner to increase the 20 l~y.l.v~ Jbic character of the resultant lCsRh-chelate. Also, if one of the terminal groups on the linear ligand is a thiol group, a neutral-l;roFh;lic l05Rh chelate should be formed. These modifications can be systematically tuned to 25 provide a 10sRh pharmaceutical that has properties that maximize selective and maximal binding SUBSTITL1TE SHEE7 (RULE 2~

Wo 95129925 2 1 8 ~3 ~ 6 3 P_l/u~.. J4' ~

af~inity to tumor cells while minimizing non-speci~ic binding and localization in normal, rA-i i osPncitive tissues .
All of the afoL Lioned modifications 5 demonstrate the flexibility o~ ~_ '- made in acc~L~anc~a with the present invention and further the ability to modi~y these __ to alter binding, elimination and absorption of the _ullds in order to tailor the resulting 10 radiopharmaceutical for speci~ic ~n vivo targeting, dosing and metabolism.
The thioether ligands used in accordance with the present inventions were purchased commercially or could be made by methods similar to 15 those outlined in the literature t34,35].
Attachment of side chains to function~li7~lhl~ atoms on the ligand bA~rl~hor~ (e.g., N-atoms) or attached to the ligand barkhnn~ (e.g., -OH, amines or carboxyl groups) are pe~ol ' by standard methods.
20 For example, the avai~able 14-ane-NS3 macrocyclic ligand shown below is derivatized by reaction of an alkyl halide with the lone ring N-atom to produce a variety of thioether derivatives.

SUBStlTUTE SH~ET (RULE ~fi) 2 ~ ~ ~ 5 6 5 wo ssnss2s ~ Ir S;m; l 7rly~ the commercially available 16-ane-S4 diol (1,5,9,13 tetr;~th; ~-yclo-hexane-3,11-diol) can be ~;fiPr' as shown below for ~ttz~h;n~
a single side chai~ (R) to the ligand. The R group 10 can then be used ~or cu..j uyation to bioactive 1 erlll pc [36, 37] .

u ~ J,7 ~}_ By rr~'; Fh-~rr -~P~lt; cal, it is meant that the chelate linced to a targeting r le~~lll ~ can be used to lo~ l; 7e sufficient level6 of lsRh at a 20 site to provide rA~'; nthPr;7r~llti~ properties.
Accordingly, the chelate ;n~ 7;ng the lsRh is bound to a targeting 1P~I11P~ such as a peptide, antibody or other receptor avid 1 e~ 1 e directed towards a specific antigen or other receptor on a 25 target cell. Such - ~o~ln~C formed in high specific activities (i.e., greater than 100 curies/nmole) with 5l7ff;r;Pnt stability in SUBSTITUTE SHEET (RULE 2B) W095l29925 2 i ~; 8 ~ 6 5 accordance with the present invention can be injected, circulate through the patient's system, and bind at target tissue to then provide radiotherapy at that site. The preferred compounds 5 of the present invention, as designated above, contain two or more thioether groups that form high specific activity complexes with the rhodium-105 at high yields, as d LL~ted below. The tetrathiamacrocycle (16-ane-54 diol) which is an 10 example of the present invention as indicated above, chelates rhodium-105 to form a single species at low ligand concentrations permitting production of high specific activity chelates.
As d ~ c.ted below, the formed rhodium-105-16-ane-5~,-diol chelate is st~ble for up to and greater than four days at physiological pH.
This model 54 ligand used in the experimental studies below includes two hydroxyl groups which can be used for linking either the macrocycle alone 20 or the rhodium-105 chelate to biomolecules. Hence, there is signif icant potential for 54 ligands and analogs thereof for use in formulating new rhodium-105 therapeutic radiopharmaceuticals.
The advantages of the present invention 25 are numerous. The _.I..d m~de in accordance with the present invention forms a stable, well defined, SI~BSTITUTE SHEET (RULE 26) wo 95n992~ 5 6 5 r~

single species . The rhodium-105 ( III ) -16-ane-5~,-diol chelate is formed in high yield under relatively mild conditions ~i.e., 65-C for 60-90 minutes).
Since these mild conditions will not result in 5 significant lsRh(III) complexation with other groups on proteins, such as amines, carboxyls, or hydroxyls etc. or most other molecules, the lQ5Rh(III) is able to selectively chelate to 5~, moieties already linked to biomolecular targeting 10 molecules. The resulting l05Rh-pharmaceutical can be used to selectively localize the l05Rh on target cells. In addition to the 5~ ligand system, other examples follow ' ~L~-ting that substitution of N-atoms for the thioether groups also results in 15 high '~i~. , lexation yields.
The following are examples of the use of three different specific thioether macrocyclic ligands to form l05Rh chelates in high yields using exceptionally low quantities (less than 50 nmoles 20 and often less th~n 20 nmoles of the thioether ligands) in accordance with the present invention.
Several chemical methods for conjugating ligand or metal chelates to biomolecules have been well described in the literature [36,37] and one or 25 more of these methods is used to link the uncomplexed TE ligands or l05Rh-TE complexes to the SUBSl ITUTE SHEET (RULE 2Ll WO 95l29925 ' 2 1 8 ~ 5 6 ~

biomolecular targeting molecules. These include the use of acid anhydrides, aldehydes, arylisothyiocyantes, activated esters or N-IIYdL~I~YS~UCC; nimi~s [ 14] .
rAMpLE 1 Formation of lCsRh-l6-ane-S4-diol The 16-ane-S4-diol ( 1, 5, 9, 13-tetrathiacyrlnh~YA~r In~-3 ,11-diol) ligand, obtained ~

from Aldrich Chemical Co., was used to react with the "105Rh-chloride reagent" that ~as obtained from 20 the Missouri University Research Reactor (MURR).
The lsRh-chloride reagent contains a mixture of lsRh (III) species, ~Le_ -' to include lCSRhCl3(Hz0)3~
1SRhcl4 (HzO) 2-, lsRhCls (H2O) -2 and l5RhC16 3 [ 38, 40 ] .
Electrophoresis of this reagent typically 25 rl I LclteS a mixture primarily _ . - ' of t~hree different anionic 105~ e cies, presumably tetra, SUBSTITLIIE SHEET ~RULE 26) .

..... ....... ... . . . _ .. ...

WO 95/29925 2 ~ 5 6 5 r~ r penta- any hexachloro-l05Rh(III) anions. These samples contained only no-carrier-added (NCA) or trace guantities of 1asRh(III) in HCl acidified solutions (- o . l-l ~ HCl) . After adjusting the pH
of the 105Rh-chloride reagent, containing 1-10 mCi (37-370 M8g) of 10sRh, to a desired pH between 1. 5-8.5, 0.5 ml aliguots were added to 0.1-0.2 ml solutions (containing some ethanol) containing between 0.2 to 10 ~g (1-50 nmoles) of 16-ane-S~-diol. After heating the samples at temperatures ranging from 55- to 80- for up to 3 hours, the ~
l~hPl ing efficiency was detP~ino~l. It was shown that the ~sRh-complex with 16-ane-S~-diol is cationic and is a single species as ~PtP~inp~ by electrophoresis performed at 300 V for 1 hour on paper strips saturated with 0.075E~ sodium phosphate buffer at pH 4 . 5 (Figure 1) . Silica-TLC also was used to routinely measure complex yield. The silica-TLC plates were developed with 0. g% agueous NaCl (i.e., N saline) on which the uncomplexed lsRh-chloride reagent has a R~ o f 0 . 9-1. 0 while the Rf of the 1CsRh-16-ane-S4-diol is 0. 05-0.10.
Development of the C-18-reverse-phase TLC plates with 0 . 02 ~ hexane sulfonic acid in 10% CH3CN in water showed l5Rh-16-ane-S4-diol ~ ; nPtl at the origin while the ~sRh-chloride reagent exhibits an SUBSTITUTE SHEET (RULE 26~

W0 95/29925 ~ 6 ~ ~ r in 5096 CH3CN, the Rf, o~ l05Rh-16-ane-S4-diol was 0 .70 .
The results of a series of Gl rPr; ~c in which systematic, ;nfl-~ flG~I variation of 5 t _ - L t:, quantity of ligand, percent of ethanol ~nd pH are ~l 7~Gfl in Tables 1-3 and Figure 2.
These results show that GYrPl 1 Gnt yields of 105Rh-16-ane-S4-diol are achieved using as low as 0 . 5 /~g ~2.5 nmoles) of ligand and heating for 1 to 1.5 10 hours at tl - _tllreS greater than 60C. Maximal yields, under these rnnfl;t;nnA are achieved at pH
ranging between 3-6, which can be used to ;nrl~hAte most bioactive I ler~l GC ~ Also, only small s~uantities of ethanol (i.e. lO'oL) are reSIuired to 15 produce optimal 105Rh-complex yields.
sased on ~ LI to~rArh; C comparisons with a non-radioactive cheiate o~ Rh(III) ~lGypfl with 16-ane-S4-diol made under similar rnnfl;t;rnc~ the 1o5Rh-l6-ane-s4-diol is assigned to be [l05Rh(III) (16-ane-S4-diol)Cl2]+ as shown below:
~+l 25 s~{$~

SiJBSTIT1~E SI~En (RULE 2 W0 9s/2992s r~

[Rh ( I II ) 16 -ane-S4 -diol ) Clz ] + was ~Le~aL ~:d by a method similar to that tlf~R~r;hed by Blake, et al. [32] for a similar S4-macrocycles. The PF6-salt of this chelate was crystAl 1; 7C~ and fully 5 characterized by NMR, W-b~euLLo8uu~yJ Pl ~ Al analysis and X-ray crystA~70~rarhy. Since the m;srAt;nn digtance of this yellow [Rh(III~16-ane-S4-diol)Cl2]+ on electrophoresis and the silica and reverse-phase TLC Rf was ;rl~nt;rAl to 15Rh-16-ane-10 S4-diol, the assignment of the l05Rh chelate as [105Rh- (16-ane-S4-diol) Cl2] ~ could be made with a high degree of cnnf; rl~nre . This chelate was shown to be stable for, 4 days in aqueous 8n.1~lt;nnq near phy~inl o~ 1 p~ and at room t , ~ and in 15 human serum at 37C (Figure 3).

~ I\qpT.l;~ 2 lsRh-C ~lP~t;nn with 14-ane-NS3 [~

, . I~S3 Between 1-60 ~g of 14-ane-NS3 (4,8,11-trithia-8-amino-cyclotetradecane), obtained as a gift, was reacted with the 105Rh-~-hlor;~lP reagent obtained from Mt7RR by the method previously described for SUBSTITUTE SHEET (RULE 26~

W09S/29925 ~ )5 reacted with the t05Rh-chloride reagent obtained from MURR by the method previously described for complexation of 1CsRh with 16-ane-54-diol (See Example 1).
The results of 105Rh(III) complexation with 14-ane-NS3 are summarized in Figure 4-6. These data show that the 14-ane-SN3 ligand forms cationic complexes with 1~5Rh in yields > 90~ at pH 4-5 using only 1 I g of the NS3 ligand (Figure 5) . High yields are obtained at 60 C incubation te~perature . The temperature ~r~ e (Figure 6) i5 observed with 1sRh-l6-ane-S~-diol (Figure 2). Results with this ligand ' ~ ~te that the presence of one N-atom in the chelate in combination with three thioether groups will form a well defined 105Rh-chelate in yields similar to the tetrathia ligand. The ~sRh-14-ane-NS3 chelate has the same electrophoretic migration distance as 1sRh-16-ane-S4-diol, indicating the two chelates have the same overall charge (i.e., +1). 1sRh-14-ane-NS3 was also shown to be stable in aqueous solutions for greater than 4 days.
SUBSTITUTE SHEET (RlJLE Z6 WO 95129925 ;~ 5 6 5 r ~ r F,Y~AMP~ 3 ~sRh-complexation with 14-ane-N252-f~

Between 10-50 ~q of 14-ane-N2S2(l,4,8,ll dithia-8, ll-diamino-cyclotetradeCane) was mixed with the lsRh-chloride reagent from ~URR by the method previously described for complexation with 1sRh-16-ane-54-diol (see ~Yample 1). After heating the solution at pH 4 at 80-c for 1 hour, a cationic lsRh-complex (with a similar migration distance in elecLL~,~horesis as 105Rh-16-ane-S~,-diol) was produced in ~80% yield. The results of these studies ,1 L~te that high 10sRh-complexation yields can be achieved using a ligand with two thioether group and two amine functionalities.
The above experimental re~ults ~1- t. lte that the ligands made in accordance with the present invention that contain at least two thioether groups that ~orm complexes with rhodium-105 can be made in high yields. The spe~ifjc ligands PY~lm;n~ form a single species on complexation to Rhodium-105, in low ~ o..c ~-L~Itions~
SU~STITUTE SHEE~ ~ULE 26) W095/29925 ~ - 2~ 6r thereby permitting production of high specific activity ~55Rh-complexes. The ligands are stable for greater than four days at physiological pH.
The ligand used having at two hydroxyl groups S attached to the ligand bAckhon~ can be easily used for linking the macrocycle or rhodium chelate macrocycle to biomolecules as known in the art.
Accordingly, there is great potential for the present invention and analogs thereof in 10 ~ormulating therapeutic rh~rr~ euticals.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of 15 description rather than of limitation.
Obviously, many modifications and variations of the present invention are possible in light of the above tPA~-hinqC. It i5, therefore, to be understood that within the scope of the App~n~l~d 20 claims wherein referençe numerals are merely for convenience and are not to be in any way limiting, the invention may be practiced otherwise than as specifically described.

SUBSTITUTE SHEEt (RULE 261 ~ W095129925 2 1 ~65 Table 1. E~fect of pl-l on 'sRh chloride . , '~ 1 yields with 16S,-diol pH % C , ' ' ~ (+SD) 1.5 93.0 $ 3.6 3.0 99.3 $ ~

4.0 99.7 + 0.6 97.0 $ 1.5 6 97.9 $ 2.9 7 90.0 $ 5.0 8.5 79.3 + 10.0 The , ~ , eA~ ts were performed usin3 10 ,u~ ~6S~-diol ~n 0.5 ml solutions . . ' ,i.,~ 15% ethanol and heated at ~OoC ~or 1 hr. analysis was perForrned usin~ silica-TLC; N ~ between 3-8.

SU~STITLITE SHEEr (RllLE 26 W095J29925 ~188~ T~

Table 2. EfFect of 16S.-diol C~ .C.ltldtiUII cn ''Rh-4S16-diol comp~ex yie!d at pH 4.
~g 4S16-diol % C~ Yield (+SD)t 10 ~3 97.7 :: 2.6 1,ug 96.3 + 4.3 0.5 pg g4.7 i 5.9 0.2 llg 64.5 i 14.8 This is the #,~19 of ligand present in between 1.2 to 0.5 ml of solution.
tSamples eontaining 15% ethanol were heabd at 80C for 1 hr (N-~ ~ 3-1S).
Table 3. Effect of ethanol col-ccl-tl n on ''Rh-16S,-diol yields at pH 4.
% Ethanol ~VN) % Cu.,., ' " . Yields O ô1.0 + 19.5 95.9 + 2.2 97.6 + 3.8 97.0 + 3.1 g7.5 + 3.7 98.0 + 2.2 Resctions werc- carried out using 10 ~g 16S~-diol in 0.5 ml solutions and heated at 80C for 1 hr (N=4).
SUBSTITUTE SHEET (RULE 26

Claims (40)

What is claimed is:

1. A compound consisting essentially of a multidentate ligand including at least two thioether groups and two or more other donor atoms (including S, N or O) for being complexed to Rhodium-105 in high specific activities to use in formulating therapeutic radiopharmaceuticals.

2. A compound according to claim 1 including three thioether groups.

3. A compound according to claim 1 including four thioether groups.

4. A compound according to claim 1 including three thioether groups and one group containing a nitrogen donor atom.

5. A compound according to claim 1 wherein said ligand is a macrocycle containing at least two thioether groups of the formula:

wherein R1, R, R3, and R4 are all the same or different and are selected from the group consisting of -Ch2-, -(CH2)2-, -(CH2)3-, -CH2CH(C3CH2-, -(CH2)4-, -CH2CH(R5)-, -CH2CH(R5)CH2)-, -CH(R5)CH2CH2-, -CH2CH(CH2R5)CH2- and R5 is -H or a group containing -OH, NH2, COOH, -OCH3-, -OC2H5 and groups for attaching a linking group used to modify lipophilicity for conjugation of the uncomplexed ligand or the ligand chelated to Rh-105 to a biomolecular targeting agent and R1-4 can also contain another coordinating atom (R6-X4-R6) wherein R6 is -Ch2- -(CH2)2-, -(CH2)3-, -CH2CH(CH)3CH2-, -(CH2CH(R5)CH2)-, -CH(R5)CH2CH2-, -CH2CH(CH2R5)CH2- and R5 is a group containing -OH, NH2, COOH, X being an atom or group containing S, O or N donor atoms that also coordinates 105Rh(III), wherein X1, X, X3 and X4 are, all the same or different in which at least one or more X-atoms or groups is -S-, and the others are -S, -O-, NH, NR7, R7 being H, -CH3 or a group attached to the N-atom to alter lipophilicity or to link the uncomplexed ligand or the "preformed"
105Rh-chelate to the biomolecular targeting agent.

6. A compound according to claim 1 wherein said ligand is an open-chain multidentate ligand containing at least two thioether groups of the formula:

wherein R1, R2, and R3 are all the same or different and are selected from the groups consisting of -(CH2)2-, -(CH2)3-, -CH2CH(CH)3CH2-, -(CH2)4-, -CH2CH(R5)-, -CH2CH(R5)CH2)-, -CH(R5)CH2CH2-, -CH2CH(CH2R5)CH2- -CH2CH(CH2R5. R4 and R5 can be the same or different and can be H or an alkyl group or a linking group containing functional groups such as OH, NH2, COOH -OCH3, -OC2H2 and a functional group for attaching a linking group used to modify lipophilicity for conjugation of the uncomplexed ligand or the ligand chelated to said Rhodium-105 to the biomolecular targeting agent; X being an atom or group containing S, N.
Or O donor atoms that can also coordinate 105Rh(III), X1, X2, X3 and X4 being all the same or different and at least two are -S- and the others being selected from the group consisting of -S-, -O-, -SH, NH, NR7, R7 being selected from the group consisting of H, -CH3 and a group attached to the N-atom to alter lipophilicity or to link said uncomplexed ligand or said ligand complexed to said Rhodium-105 to the biomolecular targeting agent.
R4 and R5 can also contain a group containing one or more atoms that will complex 105Rh selected from the groups containing O, S or N donor items.

7. A compound according to claim 6 or 7 wherein R5 is selected from the group consisting of benzyl isothiocyanate, bromacetamide, an activated ester, N-hydroxy succinimides, a cleavable ester and an aldehyde.

8. A compound as set forth in claims 6 or 7 wherein any one or all of R1-7 are carboxylated, hydroxylated, alkylated or alkoxylated to render said ligand more or less polar.

9. A compound according to claim 1 including a 1 to 1 metal to ligand ratio.

10. A compound according to above claims wherein said ligand is linked to a targeting molecule for selective in vivo targeting of preselected cells.

11. A compound according to claim 10 wherein said targeting molecules are selected from the group consisting of peptide and non-peptide receptor-avid molecules, single chain antibodies where antibodies and antibody fragments.

12. A compound according to claim 11 wherein said targeting molecules have high binding affinity and specificity for cancer cells.

13. The use of a compound as set forth in claim 1 complexed to Rhodium-105 in high specific activities at low quantities of ligand or ligand conjugates as a therapeutic radiopharmaceutical.

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