Functionalized polyamine chelants and rhodium complexes thereof and process for their preparation

The rhodium complexes prepared according to the invention have the formula (II):

/ RhChP₁P₂Wherein Rh represents a rhodium atom, Ch represents a compound of formula (I).

<img file="PT87802B_D0001.tif" />

wherein R represents, for example, alkylene of 2 to 10 carbon atoms; R¹ represents H or alkylene of 1 to 10 carbon atoms; X and X¹ represent, for example, Η; n is 0 or 1; y is 1 to 3; L is a group of formula

R²

<img file="PT87802B_D0002.tif" />

ch₂—

I

<img file="PT87802B_D0003.tif" />

-3who isOoul; to 20; R^Z is an electrophilic or nucleophilic moiety that permits binding to an antibody or antibody fragment;

<img file="PT87802B_D0004.tif" />

represents, for example, an aliphatic cyclic moiety; and P₂ represent, for example, monodentate ligands; and A represents one or more anions.

The preparation process consists, for example, of reacting

RhA.nH ^ O with ChP- ^ P₂

ί.

<img file="PT87802B_D0005.tif" />

The present invention relates to a process for the preparation of functionalized polyamine chelators and their rhodium complexes.

Functionalized chelators, or bifunctional coordinators, are known to be covalently linked to an antibody with specificity for cancer or tumor cell epitopes or antigens. Radionuclide complexes of such antibody / chelator conjugates are usable in diagnostic and / or therapeutic applications as a means for displacing the radionuclide to a cancer or tumor cell. See, for example. Meares et al. , Anal Biochem., 142, pp 68-78, (1984); and Krejcarek et al., Biochem. and Biophys. Res.

Comm., 77, pp. 581-585 (1977).

Standard methodology among those skilled in the preparation of such complexes comprises treating an antibody / chelator conjugate with excess radionuclide to form a complex, followed by purification of the complex. A major drawback of this methodology is that the radionuclide (lanthanide or transition metal) must be kinetically unstable to be rapidly sequestered by the antibody / chelate conjugate. This feature is inconvenient because kinetic instability (or substitution instability) gives rise to problems associated with the stability of complex serode. That is, the radionuclide easily dissociates from the complex in the presence of serum.

The poor serum stability of these complexes leads to lower therapeutic and / or diagnostic efficacy (representation) and creates greater potential for general radiation damage to normal tissue.^_Cole et al., J. Nucl. Med., 28, pp. 83-90 (1987) -7- More specifically, it has been observed that serum stability is a problem with Cu-containing complexes, 90 57 111

Y, Co and In (Brechbeil et al., Inorg. Chem. 25, pp. 2772-2781 (1986)).

Another drawback associated with the use of unstable radionuclides for antibody labeling

<img file="PT87802B_D0006.tif" />

-5 is that substitutably unstable (non-radioactive) marker metals are often incorporated into chelate.

Competition from these non-active marker metals decreases the biological efficacy of the antibody / chelate complex as less radionuclide is supplied to the target site.

Most of the bifunctional or functionalized chelating coordinators known to those skilled in the art to sequester radionuclides are carboxymethylated amine derivatives, for example functionalized forms of ethylenediaminetetraacetic acid (EDTA) (EU Patent 4,622,420) or diethylenetriaminapentaacetic acid (DTPA) (EU patents 4,479). 930 and 4,454,106). US Patent 4,622,420 generally states that EDTA derivatives may also sequester rhodium ion species. However, rhodium, especially rhodium (III), is known to be a replacement inert transition metal and extreme temperature and duration conditions are required to form its EDTA complex (Dwyer et al. , J. Amer. Chem. Soc., Pp. 4823-4826 (1960)). In addition, ethylenediaminadisuccinic acid has been indicated not to form rhodium (III) complexes at any pH below temperatures of 100 ° C (JA Neal and NJ Rose, Inorg. Chem., 12, 1226-1232 (1972)).

Chelating tetraaza [Troutner et al., J. Nucl. Med., 21, pp. 443-448 (1980) 7 and alkylene amine oximes (EU Patent 4,615,876) to sequester

99rn_m . x,. ,. . . ,

Tc, an isotope with nuclear properties suitable for diagnostic work only.

Rhodium-105 is a gamma emitter (suitable for diagnostic work) and a short half-life beta emitter (suitable for therapeutic work). Since rhodium-105 can be used for both diagnosis and therapy, and since rhodium is inert in substitution, it would be very desirable to have functionalized chelators capable of forming complexes with radioactive rhodium

<img file="PT87802B_D0007.tif" />

-6 bound to an antibody. Naturally existing rhodium (III) tetraaza complexes are described in the literature in relation to linear amines (eg, Bosnich et al., J. Chem. Soc. Sec. A, pp. 1331-1339 (1966) and macrocyclic / EJ Bounsall and SR Koprich, Canadian Journal of Chemistry, 48 (10), pp. 1481-1491 (1970), 7 however, the functionalized polyazole chelates suitable for complexing radioactive rhodium and subsequent binding to an antibody have been unknown so far.

The present invention relates to bifunctional chelators that form rhodium complexes. Bifunctional chelators are preferably used for complexing radioactive rhodium, for example rhodium-105 (^ Rh)._andrhodium-101^m (101^mRh). These bifunctional chelators would also be usable for complexing technetium and rhenium. The complexes thus formed may be linked (covalently joined) to an antibody or fragment thereof and used for therapeutic or diagnostic purposes. More specifically, the present invention relates to a compound of the formula:

<img file="PT87802B_D0008.tif" />

I)

X

<img file="PT87802B_D0009.tif" />

in which:

each R independently represents a straight or branched chain alkylene group having from 2 to 10 carbon atoms inclusive (preferably 2 or 3 carbon atoms), provided that for any two contiguous nitrogen linked by an R group, the R group has providing at least three simple bonds between the nitrogen that they are bound by;

each R 3 independently represents hydrogen or a straight or branched chain alkylene group having from 1 to 10 inclusive carbon atoms (preferably hydrogen or methyl);

X and X¹ represent hydrogen, or X and X 2 taken together complete a straight or branched chain alkylene group bridge of 2 to 10 inclusive carbon atoms or an aralkylene group bridge in which alkylene is a straight or branched chain alkylene group of 2 to 10 carbon atoms. to 10 inclusive carbon atoms (preferably X and X4 represent hydrogen, or, when taken together, a benzyl or alkylene group having 2 or 3 carbon atoms), provided that when X and X 2 are taken together, the group they represent must provide at least three single bonds between the contiguous nitrogen attached thereto;

n is an integer 0 or 1, provided that when group L is linked to it where:

<img file="PT87802B_D0010.tif" />

Nitrogen atom n must be 0, in other cases it must be 1;

y is an integer from 1 to 3 inclusive (preferably integer 1); and

L is a linker / spacer group that is covalently bonded to a hydrogen atom and replaces either of the nitrogen or carbon atoms, said linker / spacer group being represented by the formula

R

<img file="PT87802B_D0011.tif" />

ts represents an integer 0 or 1;

t represents an integer from 0 to 20 inclusive (preferably 0 to 6 inclusive);

R represents an electrophilic or nucleophilic moiety that provides covalent attachment to an antibody or fragment thereof, or a synthetic linker that may be attached to an antibody or fragment thereof; and © represents a cyclic aliphatic moiety, aromatic moiety (preferably phenyl)

I

<img file="PT87802B_D0012.tif" />

ί

Aliphatic heterocyclic moiety, or aromatic heterocyclic moiety, each of said moieties optionally substituted by one or more groups that do not interfere with binding to an antibody or antibody fragment;

provided that the compound of formula I is not 3- (4-aminobutyl) -1,5,8,12-tetraazacyclotetradecane.

The present invention also relates to rhodium complexes and rhodium chelate / antibody conjugates formed with the chelators of formula I; however, for these rhodium complexes and rhodium chelate / antibody conjugates there is no limitation on 3- (4-aminobutyl) -1,5,8,12-tetraazacyclotetradecane. Furthermore, the present invention includes chelate / rhodium antibody compositions consisting of the chelate / rhodium antibody conjugates according to the invention and a pharmaceutically acceptable carrier, and typically in these compositions the pharmaceutically acceptable carrier is in liquid form. The invention also includes a method for the diagnosis or treatment of a disease state in a mammal by administering the rhodium chelate / antibody composition (s) and is particularly suitable for the diagnosis and treatment of cancer.

at present for a question

<img file="PT87802B_D0013.tif" />

will often be referred to as the 3e reference facility simply by Cic. Of the Cic portions, phenyl and substituted phenyl are preferred, with phenyl being the most preferred Cic portion.

In the present description, the terms given below have the following meanings.

With respect to the definition of R, electrophilic moieties include, but are not limited to, isothio-

<img file="PT87802B_D0014.tif" />

Cyanoate, bromoacetamide, maleimide, imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyl disulfide, and phenyl azide; suitable nucleophilic moieties include, but are not limited to, carboxyl, amino, acyl hydrazide, semicarbazide and thiosemicarbazide; Synthetic linkers include any synthetic organic or inorganic linkers capable of being covalently linked to an antibody or antibody fragment, and preferred synthetic linkers are biodegradable synthetic linkers that are stable in a patient's serum but have an enzymatic fission potential within an organ. for the radiosotope, for example peptides or groups containing biodegradable peptides. Of the electrophilic moieties, isothiocyanate is preferred, and of the nucleophilic moieties, carboxyl, semicarbazide and thiosemicarbazide are preferred. It is desirable that the nature and / or position of R allows it not to interfere appreciably with the chelation reaction.

The term alkylene represents a group

- (CH „) - where n is an integer from 1 to 10 inclusive. The group may also be branched chain but not exceeding a total of 10 carbon atoms.

The term aralkylene represents a group

- (CH) - where n is an integer from 2 to 10 inclusive, n

I

R

R is an aryl moiety, for example benzyl, phenyl, or phenyl substituted by one or more hydroxy, C1 -C4 alkyl, C1 -C4 alkoxy, or halo (preferably chloro or bromo) groups.

The term mammal means animals that feed their offspring with milk secreted by mammary glands, preferably warm-blooded mammals, more preferably humans.

Antibody refers to any anti-

<img file="PT87802B_D0015.tif" />

Polyclonal, monoclonal, chimeric or heteroantibody, preferably a monoclonal antibody, antibody fragment includes Fab fragments and F (ab ') ^ fragments, and any portion of an antibody that has specificity for a desired epitope or epitopes. When the term rhodium chelate / antibody conjugate is used, antibody encompasses all antibodies and / or antibody fragments, including their semi-synthetic or genetically engineered variants.

Rhodium complex indicates a complex of the compound of formula I in which at least one rhodium atom is chelated or sequestered; chelate / rhodium antibody conjugate indicates a rhodium complex that is covalently linked to an antibody or antibody fragment; nature, when used in conjunction with rhodium, indicates the element in a form that is obtained when the element is purified from natural sources using generally accepted processes, that is, in a form containing several isotopes, which for the most part are not radioactive; radioactive, when used in conjunction with rhodium, indicates one or more isotopes of the element emitting alpha, beta and / or gamma particles,

105 for example Rh; Rhodium means radioactive rodium or rhodium in nature or mixtures thereof.

The terms bifunctional coordinator and functionalized chelator are interchangeably used and indicate compounds that have a chelium rhodium-capable chelating moiety and a linker / spacer moiety covalently linked to the chelating moiety that can serve as a means for covalently binding to an antibody or antibody fragment.

Used herein, BA-cyclam means 3- / 4- (aminophenyl) methyl-1,5,8,12-tetraazacyclotetradecane; BA-2,32-tet means 6- [4- (aminophenyl) methyl] -1,4,8,11-tetraazaundecane; PA-2,3,2-tet means 6- (3-aminopropyl) -1,4,8,11-tetraazaundeca-

<img file="PT87802B_D0016.tif" />

-12&lt;

at the; and BA-N-cyclen denotes the compound 1,4,7,10-tetraaza-1- (4-aminophenyl) methyl] cyclododecane. BA-cyclem, BA-2,3,2-tet, BA-N-cyclen and PA-2,3,2-tet are represented by the following formulas:

<img file="PT87802B_D0017.tif" />

<img file="PT87802B_D0018.tif" />

<img file="PT87802B_D0019.tif" />

H₂no

<img file="PT87802B_D0020.tif" />

-13^,- ^ ea »sse *^cr3

Preferred compounds of formula I are BA-cyclam, BA-2,3,2-tet, PA-2,3,2-tet and BA-N-cyclen and their respective Rh (III) complexes.

Preferred rhodium complexes according to the present invention are salts which are represented by the formula / RhChP₁P₂_7A in which:

Ch represents a compound of formula I;

Ρ_χ^{and P}2 have monodentate ligands, and if taken together, a bidentate ligand (PpP2), ^P1 P^{the &lt;}^^{and be}same or different from P2, provided, however, that:

(a) P₂ is absent if y in formula I is 2; and (b) P₁ and P₂are absent if y in formula I is 3; and

A represents one or more anions with sufficient charge to make the whole complex neutral. Examples of P ^ and P₂ are F, Cl Br^-, I^-, CN^-, NCO^-, SCN^-, N3 ~ OH^-, and H2 O;

examples of P ^ P₂ are ^Ç2°4^^{- and et}ii^{not &lt;}I^:*-^ami^at · Elements of A include F^-, Cl^-, Br^-, I^-, CN^-, NCO^-, SCN^-, N3^_, C104^_,

BF₄^-, BPh4^-, N03, and PF₆^-.

In general, these rhodium complexes of formula II are prepared by refluxing in aqueous solution a single rhodium starting material RhQ.nH₂O (Q is halide (in sufficient quantity to compensate for Rh), and nH ^ O is hydrate (when necessary), eg rhodium halide hydrates) with the bifunctional coordinator. The pH may be titrated to about pH = 7 or controlled to approximately pH = 7 using a buffer. Generally, the ligands P ^ and P₂ will be the halide used in the rhodium or H starting material₂0 The counterion (s) A will be the halide used in the rhodium starting material. Other ligands,^Pi ^{and P}2 ' ^or counterions A may be substituted either by adding them to the initial reaction

<img file="PT87802B_D0021.tif" />

Subsequent reflux. The complexes are purified by column chromatography.

The functionalized polyamine described herein (i.e. compounds of formula I) may be used to chelate or sequester rhodium to form rhodium chelates (also referred to herein as rhodium complexes). Rhodium complexes, due to the presence of the functionalizing moiety (represented by L in formula I), may be attached to functionalized supports, for example functionalized polymeric supports, or preferably covalently linked to antibodies or antibody fragments. Antibodies or antibody fragments that may be used in the chelate / rhodium antibody conjugates described herein may be prepared by techniques known to those skilled in the art. Very specific monoclonal antibodies can be produced using hybridization techniques known to those skilled in the art, see, for example, Kohler and Milstein (Nature, 256, pp. 495-497 (1975); and Eur. J. Immunol., 6, pp. 511-519 (1976) 7 · Such antibodies usually have very specific reactivity In chelate / rhodium antibody conjugates with labeled antibodies, antibodies directed against any desired antigen or hapten may be employed. Preferably, the antibodies that are used in the chelate / rhodium antibody conjugates are monoclonal antibodies, or fragments thereof that have great specificity for one or more desired epitopes. Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs and any molecules. biologically active. For a more complete list of antigens cf. US Patent 4,193,983. Rhodium chelate / antibody conjugates are particularly preferred for the diagnosis and / or treatment of various cancers. Rhodium complexes and chelate / antibody conjugates

<img file="PT87802B_D0022.tif" />

Rhodium compounds described herein have excellent serum stability and / or excellent in vivo biolocalization. The rhodium gelate / antibody conjugates described herein may be administered according to procedures known to those skilled in the art.

The compounds of formula I may be prepared by applying processes known to those skilled in the art. For example, compounds of formula I may be prepared by synthetic methodology such as the following Synthesis Schemes AD:

<img file="PT87802B_D0023.tif" />

-16Synthesis Scheme A:

<img file="PT87802B_D0024.tif" />

BA-2,3,2-tet

<img file="PT87802B_D0025.tif" />

Synthesis Scheme B:

<img file="PT87802B_D0026.tif" />

Synthesis Scheme C:

<img file="PT87802B_D0027.tif" />

<img file="PT87802B_D0028.tif" />

8

<img file="PT87802B_D0029.tif" />

PA-2,3 »2, -tet

<img file="PT87802B_D0030.tif" />

Synthesis Scheme D:

<img file="PT87802B_D0031.tif" />

<img file="PT87802B_D0032.tif" />

chci₃

->

25'C

<img file="PT87802B_D0033.tif" />

Pd / C Cal

->

Η, ΛίεΟΗ

<img file="PT87802B_D0034.tif" />

BA-N- cyclen

The four bifunctional ligand systems that were synthesized during this work (ie,

BA-2,3,2-tet, PA-2,3,2-tet, BA-cyclam and BA-N-cyclen) are specific examples of the generic bifunctional structure represented by formula I. There are two main types of polyza compounds (number of chelating nitrogen atoms = 4-6) that are representative of the generic structure: 1) Linear polyza compounds with a spacer / linker group covalently attached to this moiety (eg, BA-2,3, 2-tet or PA-2,3,2-tet); and 2) macrocyclic polyamide compounds with a covalently joined spacer / linker (eg, BA-cyclam or BA-N-cyclen).

Both major types may be further subdivided as to how the spacer / linker group may be covalently joined to the chelating polyza moiety. Conceptually, the union can be done by

<img file="PT87802B_D0035.tif" />

Linking to a carbon atom (eg, BA-cyclem, BA-2,3,2-tet, PA-2,3,2-tet) or to a nitrogen atom (eg, BA-N-cyclen).

Synthesis Scheme D represents a methodology that is applicable to the synthesis of any nitrogen ring linker / spacer group. Most of this pathway has been recently documented in the literature /^-E. Kimura et al.,

J. Chem. Soc. Chem. Comm. , pp. 1158-1159 (1986) 7 θ provides a method for monoalkylating any polyazamacrocycle with an appropriate electrophile (ie, linker / spacer) that may contain a latent functionality that allows antibody conjugation. There are several polyacity macrocycles on the market or have been prepared using the tosylate displacement / macrocyclization techniques mentioned in the literature (TJ Atkins et al., Org. Synth., Vol. 58;

Ed. WA Sheppard, John Wiley and Sons, New York, 1978 pp. 86-97). Of course, the N-alkylation pathway provides the greatest versatility through a convergent synthetic pathway.

Linear or macrocyclic ligands can be reached which are attached to the linker / spacer by a carbon atom bond, following mainly three established methodologies. Macrocyclic amines containing four to five nitrogen atoms were produced from condensation of the appropriately substituted malonate ester with linear tetraamines or pentaamines (Tabushi et al., Tetrahedron Letters, 12, pp. 1049-1052 (1977) and Machida et al. al., Inorg, Chem., 25, pp. 3461-3466 (1986). The article by Tabushi et al. mentioned above describes 3- (4-aminobutyl) -1,5,8,12-tetraazacyclotetradecane compound. A second route to carbon ring macrocycles, for example BA-cyclam, involves displacement of malonate ester with a large excess of diamine (Schemes A and C) and ring closure with acrylate or malonate (Scheme Β). The versatility of this process has been mentioned in the literature (E. Kimura et al., Inorg. Chem., 23,

<img file="PT87802B_D0036.tif" />

-20ρρ. 4181-4188 (1984)) and may also be used to produce linear polyza compounds with varying ligand / metal adhesion dimensions.

Both of these pathways involve nucleophilic attack of the amine or aza compound on an ester or acyl functionality. Thus, a reduction phase is required to transform the amide into polyamine.

A third potential method for the synthesis of macrocycles containing a carbon ring linker / spacer group would entail macrocyclization via S_no2 or simple aliphatic displacement chemistry. So far, this strategy has only been applied for the synthesis of mono-N-substituted tetraazamacrocycles (N. Alcook et al., J.

Chem. Soc. Dalton Trans ,, pp. 2371-2376 (1984)). However, this technique could also be applied in carbon ring system synthesis:

<img file="PT87802B_D0037.tif" />

Ts-n

Θ

Na © ch₂ich₂:

I /, -CH ¹ ι

N-Ts

Θ

Na © n = 0-3

Ts = phenylsulfonyl

TsO-CHp

I pCH₂)

<img file="PT87802B_D0038.tif" />

CH₂) , ^The n '

CH₂-OTs n '= 0-4 m = 2-4

Ts

DMF dimethyl formamide

Ts-N N-Ts | DMF / Δ Z

CH₂- (CH₂)_no-CH

<img file="PT87802B_D0039.tif" />

Ts

Tosylate groups can be easily removed by various methods known to those skilled in the art. It should be noted that most of any specific bifunctional coordinator generally encompassed by formula I could be prepared using one of the general routes described briefly in the present description. Surprisingly, no examples of an antibody conjugated rhodium tetramine complex are documented in the literature.

The following examples are presented

<img file="PT87802B_D0040.tif" />

These are intended to describe the invention, but should not be considered limitations on the invention.

General Conditions of Experimentation

Mass spectra were obtained on a Finnigan TSQ mass spectrometer (MA Q1 mode) or a high resolution VG ZAB-MS mass spectrometer (Xenon Fast Atomic Bombardment). He C NMR was obtained using a Varian VXR-300 spectrometer. Infrared (IR) spectra were recorded on a Nicolet S5X FT / / IR instrument.

All solvents used were Fisher quality CLAP materials which were used without further purification. All preparative chromatography of organic compounds (AD schemes) was performed using the instantaneous chromatography technique mentioned in the literature © (Merck Quality 60, 230-400 mesh silica gel, 6oS - Aldrich Chemical Company) employing the following solvent systems:

(1) 1 -CHClg / MeOH / NH dissolving system₄OH -2 / 2/1;

(2) 2 -CHC1 dissolving system₃/ MeOH / NH₄OH-12/4/1;

(3) 3 -CHCl3 / MeOH / NH4 OH-16/4/1 v / v solvent system.

Rf values ​​are indicated with the use of these commercially available Analtech solvent systems and silica plates (250 microns, Analtech Inc.).

(D

WC Still, M. Kahn and A. Mitra, J. Org. Chem., 43, p. 2923-2925 (1978)

Example 1

2-Carbomethoxy-3- (4-nitrophenyl) propenoic acid methyl ester (p-nitrobenzyl malonate dimethyl ester).

2-Carbomethoxy-3- (4-nitrophenyl) propenoic acid dimethyl ester was prepared from the condensation

Knovenagle of dimethylmalonate and p-nitrobenzaldehyde according to the method of Baker and Eccles; Observed melting point (mpg)⁼= 133-134 ° C. Melting point mentioned in the literature (mp) = 136-137 ° C.² 2-Carbomethoxy-3- (4-nitrophenyl) propenoic acid methyl ester (23.0 grams (g), 86.7 millimoles (mmol)) dissolved in 175 milliliters (ml) of methanol (MeOH) under 3 nitrogen atmosphere and sodium cyanoborohydride (6.0 g, 95.5 mmol) was cautiously added to the stirred solution with cooling. The pH was adjusted to 4.0 with concentrated hydrochloric acid and the solution was stirred at 25 ° C overnight. During the first eight hours, the pH was adjusted from 6 to 4 on several occasions. The yellow methanol solution was poured into 700 ml of water and extracted with 3x200 ml portions of methylene chloride. The combined organic fractions were washed with 400 ml saturated sodium bicarbonate and 400 ml water, dried over magnesium sulfate and evaporated to a pale yellow oil on a rotary evaporator. The oil crystallized (mp_obs = = 82-83 ° C; mp. = 82.5-83.5 ° C) upon standing and gave 2-carboxymethoxy-3- (4-nitrophenyl) propanoic acid methyl ester (p-nitrobenzyl malonate dimethyl ester) in 93 percent yield (21 3 g, 81 mmol).

JW Baker and A. Eccles, J. Chem. Soc. (1927), pp. 2125-2133.

RO Hutchins, D. Rotstein, N. Natale, J. Fanelli and D. Dimmel, sL. Org. Chem. . 41, p. 3328 (1976).

<img file="PT87802B_D0041.tif" />

Example 2

3- (4-Aminophenyl) -2-carbomethoxypropanoic acid methyl ester (p-aminobenzyl malonate dimethyl ester), /

The 2-carbomethoxy-3- (4-nitrophenyl) propanoic acid methyl ether (p-nitrobenzyl malonate dimethyl ester) (2.00 g, 7.55 mmol) was dissolved in ml of ethyl acetate containing 6% by weight. palladium on carbon (1.0 g - Aldrich Chemical Company) catalyst 2 and was hydrogenated in a Parr shaker using 3.51 kg / cm3 (50 psig) hydrogen at 22 ° C. Hydrogen evolution was rapid (15 minutes) and the mixture was kept under hydrogen pressure for a further three hours. 0 Pressure vessel was vented and flushed with nitrogen (^). The suspension was filtered through a pad of celite and the solvent was separated in vacuo using a rotary evaporator to afford 3- (4-aminophenyl) -2-carbomethoxypropanoic acid methyl ester (p-aminobenzylmalonate dimethyl ester) (1 76 g, 7.41 mmol) as a pale yellow oil in 98 percent yield. The structure was confirmed by nuclear magnetic resonance (RMP) and nuclear magnetic resonance (CMR) as well as mass spectroscopy (SM) spectral analysis.

Example 3

6- (4- (Aminophenyl) methyl) -1,4,8,11-tetraaza-5,7-dioxoundecane, /

3- (4-Aminophenyl) -2-carbomethoxypropanoic acid methyl ester (p-aminobenzylmalonate dimethyl ester) (30.0 g, 0.126 mmol) was added dropwise to an ethylene diamine solution (75 g, 1 mL). 25 mmol) in 150 ml methanol under a vigorously stirring nitrogen atmosphere (25 ° C). The solution was allowed to stand for 4 days until the reaction was considered complete by

<img file="PT87802B_D0042.tif" />

Thin layer chromatography (CCD). At this point the solvent and excess amine were separated in vacuo and the tan residue dried overnight (70 ° C / 0.1 mm), which afforded 36.3 g of the desired compound 6- (4- (Aminophenyl) methyl) -1,8,8,11-tetraaza-5,7-dioxoundecane as a tan solid in 98 percent yield. An analytical sample was prepared by recrystallization from chloroform / hexane, mp = 157-159 ° C, as a white crystalline powder. The structure was confirmed by RMP, RMC and SM.

Analyze

Calculated for ^çi4^H23°2^N5^:Found:

C Η N

57,3 7,90 23,87

57,16 7,48 23,65

Example 4

6- (4-Aminophenyl) methyl) -1,4,8,11-tetraazaundecane, (BA-2,3,2-Tet), 4

Compound 6- (4- (aminophenyl) methyl) -1,4,8,11-tetraaza-5,7-dioxoundecane (7.0 g, 23.9 mmol) was placed in a 3 neck round bottom flask with 250 ml capacity, equipped with a stirrer and reflux condenser under a nitrogen atmosphere. Borane / tetrahydrofuran (TIF) complex (150 ml, 150 mmol) (Aldrich Chem., Co.) was slowly added to the solid while stirring under a positive pressure nitrogen cannula. Short-term exotherm was observed, and after it disappeared, the stirred solution was refluxed for 48 hours. From the clear solution the solvent was separated in vacuo leaving a semi-solid glassy material. Methanol (100 ml) was added cautiously and hydrogen evolution was observed. The resulting solution was dried in vacuo. At this point, 100 ml of methanol was added and the solution was saturated with

<img file="PT87802B_D0043.tif" />

Anhydrous hydrogen chloride. The solution was refluxed under nitrogen for 1 hour and the solvent was separated using a rotary evaporator. This cycle was repeated and the resulting crude hydrochloride salt of the compound was dissolved in 15 ml of water. This fraction was extracted with 2x20 ml portions of chloroform (CHCl3) and the aqueous phase was then made basic (pH> 12) by the addition of 50 percent aqueous sodium hydroxide with cooling under argon. The basic solution was extracted with 6 x 75 ml portions of chloroform. These fractions were combined (without drying) and the chloroform was separated in vacuo to afford 5.8 g of impure amine as a yellow oil (91 percent). The crude material was purified by flash chromatography using a 16: 4: 1 solvent of chloroform: methanol: concentrated ammonium hydroxide and silica gel (Aldrich Chemical Co. / Merck quality 60 mesh 230-400), (Rf = 0, 33 solvent 1). The structure of the purified product was confirmed by RMP, RMC and SM.

Analyze

Calculated for ^çi4^H27^N5th * ^ HCl Found:

37,56

37.5

7,20 6 .42

15,64

15.83

Example 5

3- (4- (Aminophenyl) methyl) -1,5,8,12-tetraaza-2,4,9-trioxocyclotetradecane, (BA-cyclamtriamide), 5a

6- (4- (Aminophenyl) methyl) -1,4,8,14-tetraaza-5,7-dioxotetradecane (15.0 g, 51.1 mmol) and methylacrylate (4.29 g, 51.1 mol) in 800 ml methanol (MeOH) under a nitrogen atmosphere with stirring. After 40 hours at room temperature (25 ° C), the solution was refluxed for 13 days. After cooling, a white precipitate formed. The solvent was separated,

<img file="PT87802B_D0044.tif" />

employing a rotary evaporator, and the resulting waxy solid was chromatographed using a 350: 35: 5 chloroform: methanol: concentrated ammonium hydroxide solution and the instant chromatography technique. The desired compound 3- [4- (aminophenyl) methyl] -1,5,8,12-tetraaza-2,4,9-trioxocyclotetradecane was obtained (7.5 g, 21.6 mmol) in 42 percent yield. as a white solid. (Rf = 0.62 / solvent;) mp = 250-252 ° C.

Analyze

Calculated for ^ 2γ^Η25^Ν5θ3^:Found:

C Η N

58,77 7,25 20,16

58,03 7,26 19,81

Example 6

3- (4- (Aminophenyl) methyl) -1,5,8,12-tetraazacyclotetradecane, (BA-Ciclam), 6

3- / 4- (Aminophenyl) methyl7-1,5,8,12-tetraaza-2,4,9-trioxocyclotetradecane (2.5 g, 7.20 mmol) was refluxed in 200 ml of a complex 1 M borane / TIF in a nitrogen atmosphere for 50 hours. Analogous treatment to Example 4 provided the crude hydrochloride salt. The salt was dissolved in 20 ml of water and extracted with 2 x 100 ml portions of chloroform. The aqueous layer was cooled to 0-5 ° C in an argon atmosphere, then treated with 50 percent sodium hydroxide (pH = 11.5), after which a white precipitate formed. The material was extracted with combined 3 x 100 ml portions of chloroform, filtered through a glass wool plug and evaporated to dryness (high vacuum) to give 2.1 g (7.0 mmol) of. desired product 3- [4- (aminophenyl) methyl] -1,5,8,12-tetraazacyclotetradecane as a white solid in 97 percent yield (mp = 156-158 ° C). The structure was confirmed by RMP and RMC.

<img file="PT87802B_D0045.tif" />

Analyze

Calculated for ^çq7^H3i^N5 · ^H2°

CH

63,12 10,28

63,65 9,92

N

21,65

21,60

Example 7

3- / 4- (Aminophenyl) methyl7,1,5,8,12-tetraaza-2,4,9,11-tetraoxycyclotetradecane, (BA-Cyclamtetraamide), 5b

The compound 6- [4- (aminophenyl) methyl7-1,4,8,11-tetraaza-5,7-dioxoundecane (7.03 g, 24 mmol) and dimethylmalonate (3.17 g, 24 mmol) in 50 mL methanol were warmed to mild reflux with stirring in an atmosphere of N₂ for 4 days. The mixture was cooled and the colorless precipitate obtained was filtered off. This material was then chromatographed on silica gel by flash chromatography eluting with an 85: 10: 2 v / v / v mixture of chloroform: methanol: concentrated ammonium hydroxide. The crude material was recrystallized from methanol and gave 3- [4- (aminophenyl) methyl] -1,5,8,12-tetraaza-2,4,9,11-tetraoxycyclotetradecane as colorless crystals (2.01 g, 24 percent) mp 288-290 ° C (dec) which was characterized by IR, RMP and CMR techniques.

Example 8

BA-Cycle 6

The 3- / 4- (aminophenyl) methyl7-1,5,8,12-tetraaza-2,4,9,11-tetraoxycyclotetradecane compound prepared in Example 7 was reduced with diborane (reflux, 18 hours) in tetrahydrofuran (TIF) to give 4-aminobenzyl cycle in 55.3 percent yield. The material exhibited properties essentially as described in Example 6.

<img file="PT87802B_D0046.tif" />

Example 9 1, 4,8,11-Tetraaza-6- (2-cyanoethyl) -5,7-dioxoundecane, 8

Diethyl 2- (2-cyanoethyl) malonate (5.0 g, 23.5 mmol - Aldrich Chemical Company) was added dropwise over one hour to a stirred portion of freshly distilled ethylene diamine ( 15 g, 0.25 mol) which was kept in a nitrogen atmosphere at 0 ° C. The stirred solution was allowed to warm to room temperature (25 ° C) and stirring was continued over a period of four days. At this point, excess ethylene diamine was separated in vacuo carefully to avoid heating above 40 ° C. The resulting crude clear oil was subjected to flash chromatography using Solvent System 3 as eluent to give 2.3 g (8.81 mmol) of 1,4,8,11-tetraaza-6- (2-cyanoethyl) -5.7 -dioxoundecane as a clear viscous oil in 37 percent yield (R_f = 0.39 / Dissolving System 3); NMR (CDCl 3) δ 7.59 (t, 2H, J = 3.1 Hg, amide H), 3.29 (m, 5H, methine H and cg starch CH₂ ), 2.82 (dt, 4H, J_x = 4.0 Hg, J₂ - 0.9 Hg), (S starch CH₂) 2.48 (t, 2H, Jg = 7.1, nitrile CH₂ ), 2.21 (q, 2H, Jg = = 7.1 Hg, p nitrile CH₂ ), 1.39 (S, 4H, amino NH); ¹³C NMR (CDCl3) 169.6 (carbonyl amide), 118.9 (nitrile), 52.9, 42.3, 41.2, 27.1, 15.4.

<img file="PT87802B_D0047.tif" />

Example 10

6- (3-Aminopropyl) -1,4,8,11-tetraazaundecane,

PA-2,3,2-Tet,

The 1,4,8,11-tetraaza-6- (2-cyanoethyl) -5,7-dioxoundecane compound (1.6 g, 6.14 mmol) of Example 9 was refluxed under a nitrogen atmosphere in a solution 1 Borane / TIF (200 ml) for 40 hours. Refluxing methanol / hydrogen chloride as indicated in Example 4 and appropriate treatment afforded 1.5 g of the 6- (3-aminopropyl) -1,4,8,11-tetraazaundecane crude hydrogen chloride salt. This material was dissolved in 1.5 ml of water and 50 percent sodium hydroxide was added. It was observed (pH = 13) with gas evolution. The free base was extracted with 3 x 7 ml portions of acetonitrile using a Mixer extractor (Liddex Corporation, Ltd., Haifa, Israel). The combined organic phase was reduced using a rotary evaporator and the clear oil was applied to a short block of instant silica gel as chloroform solution. 6- (3-Aminopropyl) -1,4,8,11-tetraazaundecane was isolated as a clear oil using Solvent System 1 as eluent after solvent separation (R_f = 0.04 / Dissolving System 1). Free base was dissolved in 5 ml of methanol which was subsequently saturated with anhydrous hydrogen chloride. Evaporation to dryness afforded 350 mg of 6- (3-aminopropyl) -1,4,8,11-tetraazaundecane as the hydrochloride salt (yield 15 percent): TMN, pH = 1.5) 6 3.47 (m (8H), 3.34 (m, 4H), 3.06 distal amine), 2.41 (δ, 1H, 1.67 (m, 2H);¹³C NMR (D₂O, 28.1, 25.7.

(t, 2H, J = 3.8 methylene at

J = 3.8, methine), 1.78 (m, 2H), pH = 1.5) is 51.5, 47.7, 37.9, 36.1,

<img file="PT87802B_D0048.tif" />

Example 11 1, 4,7,10-Tetraaza-1-^-(4-nitrophenyl) methyl7-cyclododecane, 11

1,4,7,10-Tetraazacyclododecane, 10 (270 mg, 1.57 mmol) prepared by the Richman method (4) and Adkins (5 ml) was dissolved in 5 ml of chloroform. P- Bromide was added. nitrobenzyl (113 mg, 0.52 mmol - Aldrich Chemical Company) was added to this solution and stirring was started and maintained for 14 hours. Thin layer chromatography revealed a strongly positive nyhydrin speck (Rf = 0.58 / Dissolvent System 3) different from the starting materials. The solution was applied to a 17 χ 1 centimeter (cm) column of flash silica gel and eluted with Dissolvent System 3. Fractions without starting materials were combined and evaporated to afford 109 mg of analytically pale yellow crystals. pure 1,4,7,10-tetraaza-1- (4-nitrophenyl) methyl7-cyclododecane in 68 percent yield, mp = 128-129 ° C. The structure was confirmed by NMR.

Analyze

Ç

Calculated for ^çj5^H25^N5°2^: 58,61

Found: 58.4

H

8,20

8,30

N

22,78 22,80

JE Richman and TJ Adkins, J. Amer. Chem. Soc. 96, 2268-2269 (1974).

<img file="PT87802B_D0049.tif" />

32Example 12

1,4,7,10-Tetraaza-1- (T-4-aminophenyl) methyl7-cyclododecane, (BA-N-Cyclen), 12

1,4,7,10-Tetraaza-1- (4-nitrophenyl) methyl-7-cyclododecane (170 mg, 0.55 mmol) was dissolved in 5 mL of methanol and 100 mg palladium on carbon was added to 10% (Lancaster Synthesis Ltd.) to this solution while stirring. Steady stream hydrogen gas bubbles were passed through the stirred mixture. Thirty minutes later, thin layer chromatography analysis indicated complete transformation of 1,4,7,10-tetraaza-1- / (4-nitrophenyl) methyl-7-cyclododecane, (Rf = 0.28 / Dissolving System 3). The solution was purged with nitrogen and filtered through a short plug of celite. Evaporation of the solvent and flash chromatography (Solvent System 3) afforded 103 mg of 1,4,7,10-tetraaza-1-^-(4-Aminophenyl) methyl-7-cyclododecane in 67 percent yield.

13

The structure was confirmed by He C analysis. The free base was transformed into the tetrahydrochloride salt / mp = 255-260 ° C (dec) 7 - pale yellow powder.

Example 13

Rh was obtained using a system in which five balloons were interconnected via ground glass junctions in the following order: a first balloon (a catcher used as a gas trap), a second balloon (the reaction vessel) , a third balloon (trap No. 1), a fourth balloon (trap No. 2), and a fifth balloon (trap No. 3).

In the reaction vessel was placed 10 ml of 2% NaOH. Trap No. 1 was added 150 ml of

CC1. to trap No. 2 was added 150 ml of 2 M NaOH and

<img file="PT87802B_D0050.tif" />

No. 3, 150 ml of 2 M NaOH was added. An amount of ruthenium metal (5.18 mg) which had been irradiated for 30 minutes in the P-tube row wool in the University of Missouri Research Reactor (MURR). Research from the University of Missouri) the day before was added to the reaction vessel. Glass stoppers were placed on top of the first four balloons. Cl 2 bubbles were passed through the apparatus for approximately 10 minutes, the solution in the reaction vessel turning bright yellow. An air stream was then passed through the apparatus for 20 minutes and the reaction vessel was refluxed for approximately 5 minutes using an incandescent jacket. During this process, the solution in the reaction vessel became clear and the CCl4 in trap No. 1 turned bright yellow. The solution was taken from the reaction vessel and filtered through a 0.2 mm filter. An amount of solution was taken from the reaction vessel (1.0 ml) and diluted to 10 ml in a scintillation vial for counting. An amount of 10 ml of each of the solutions contained in traps No. 1, No. 2 and No. 3 were also taken for counting. The solution in the reaction vessel contained Rh.

Example 14

The following example describes the preparation of rhodium chelated complexes using methods analogous to those indicated by SA Jonhson and F. Basolo, Inorg. Chem. (1962), 14, pp. 925-932.

<img file="PT87802B_D0051.tif" />

-34A. Materials and Techniques

Rhodium hydrate and lithium hydroxide trichloride (LiOH) (99.3 percent, anhydrous, 4 + 14 mesh) were obtained from Jonhnson Mattney and Alpha respectively. The chelators BA-2,3,2-tet. 5HCl and BA-cycle. 5HCl were prepared as described in Examples 4 and 6.

Sephadex-SP

<img file="PT87802B_D0052.tif" />

PharmaC-25 cation exchange resin was purchased from Aldrich. The chromatography glass columns were approximately 2.5 χ 70 cm and were fitted with a 29/42 frosted glass joint at the top and a thick glass tap-and-tap tap at the bottom. The cation exchange resin was prepared by adding 40 g of dry gel to 300 ml of 0.3 N aqueous HCl with gentle stirring to form a paste. The paste was then transferred to a large graduated cylinder and allowed to swell over a period of 1.5 hours. At various intervals during this period, a portion of 0.3 HCl was decanted off (in an effort to separate fines), an additional 0.3 N HCl was added, and the slurry slowly mixed. The column was poured by attaching a 1 liter Kugelrohr flask to the top of the column and transferring the paste in one go.

The gel was transformed into form H⁺ and stacked by running 2-3 liters of 0.3 N HCl through the column.

Samples (0.1 to 1.0 g) were chromatographed by dissolving in 5-10 ml of distilled water and applying the solution directly to the top of the column. The solution was washed on the gel with several small portions of 0.3 N HCl and eluted from the top down the column with the same solvent. Solvent flow through the column was maintained with a Gilson Miniplus 2 Γ 3-4 ml peristaltic pump per minute (min¹) _7 and sample peaks eluted at 254 nanometers (nm) were detected with an Isco model UA-5 absorbance monitor with a model 1132 multiplex dilator and optical unit type 6. Neutrally charged and charged species.

<img file="PT87802B_D0053.tif" />

Mono-positively separated by elution from the column rapidly (0.5-1.5 hours), positively charged species by elution after 5-8 hours, and more intensely positively charged species. stood at the top of the column.

B. / Rh (BA-2,3,2-tet) C1₂7C1. HCl

With minor modifications, the method of Martins and Sheridan (Martins, E.; Sheridan, PS, Inorg. Chem. (1978), 17, pp. 2822-2826) for the preparation of dichloro chloride (J.S. p -triamine triethylamine) rhodium (III)

RhClg was added. 3H₂O (0.308 g, 263.309 g mol \ 1.17 mmol) to a solution of BA-2,3,2-tet. 5HCl (0.524 g, 447.71 g mol¹0.17 mmol) in 30 ml of 0.1 N LiOH. The red solution was refluxed for 5 minutes and then slowly titrated to pH = 6 over 45 minutes using 0.1 N LiOH. (A total of 64.1 ml or 5.5 equivalent) was used. The pH was observed with the use of colorpHasl indicator strips (attached to Macalaster Bicknell Co.). 1 hour of reflux,

After a total of approximately the brownish-yellow mixture was cooled, filtered and the solvent separated on a rotary evaporator. The solid was dissolved in 10 ml of distilled water, (Èr) filtered through a Celite® block over a fine porous glass frit filter and a Gelman Acrodisc® disposable syringe tip filter. Fischer Scientific), and applied to the top of a Sephadex-SP '' - ^ column (cf. above). The positively charged species were separated by elution from the column in a single lane with 0.3 N HCl and a fraction collected. The solvent was separated on a rotary evaporator and the yellow solid was dried at 30 ° C in a vacuum oven yielding 0.205 mg of product (34.3 percent). The material was characterized by He C NMR and fast atom bombardment mass spectroscopy. NMR spectroscopy indicated that the product existed in three forms.

<img file="PT87802B_D0054.tif" />

-36isomeric.

Analyze

Calculated for 1 NgClgRh. HC1.2 ^ 0:

Found:

C Η N

30.73 5.90 12.8C

30,5 5,4 12,5

C. / Rh (BA-cyclam) C1₂7C1. HC1

The method was the same as described above except that 0.50 g RhClg was employed. 30% (1.90 mmol), 0.93 g BA-cyclam. 5HCl (1.91 mmol), and 102.5 mL of 0.1 N LiOH (10.3 mmol, 5.39 equivalents), yielding 0.385 g of product (36.8 percent). The product was characterized as described above. NMR spectroscopy indicated the presence of multiple isomers.

Analyze

<td>Calculated for C C^Hg ^NgClgRh.HCl1H₂O:</td><td>Ç 34.77</td><td>H 6.18</td><td>N 11.93</td>

<td>Found:</td><td> 34,6</td><td> 5,6</td><td> 11,6</td>

Example 15 Preparation of Rh (BA-2,3,2-tet) C1₂ 7⁺

Rhodium chloride (approximately 5 mCi / ml in 0.1 N HCl) was obtained from the University of Missouri research reactor. Three milliliters of this stock solution was neutralized by the addition of 0.4 ml of 1.0 M NaHCOg. BA-2,3,2-tet (0.2 ml of a 10 mg / ml solution was added). ) keeping in agitation. This solution was heated to 90 ° C in a water bath for 1 hour. Rh complexes were purified from any non-chelating and chelating metal by passing the solution through a

Chrompak Hamilton PRP-1 unit. Rh complexes were eluted

<img file="PT87802B_D0055.tif" />

37 to 30 percent. The birth of equal parts of

-105, e / Rh (BA-2,3,2-tet) was converted to a complete 0.5 N in HCl and warming for an additional 30 minutes. The complexed and concentrated with an acetonitrile wash / lysis of this material indicated the complexes / ~ · * · θ ^ ΒΗ (ΒΑ-2,3,2-tet) 01 ^ 7 (01) (^ 0) 2+ . the x-dichloro aguochlorine complex by passing the solution to 90 ° C in a water bath for xo /^_105Rh (BA-2,3,2-tet) C1₂7+ went to Chrompak Hamilton PRP-1. The complex was characterized by comparison with known standard material using cation exchange chromatography and thin layer chromatography. Yields greater than 85 percent were obtained for Rh.

Example 16 Conjugation of / ¹⁰⁵Rh (BA-2,3,2-tet) C1₂ 7⁺ with Antibody

The complex Rh (BA-2,3,2-tet) Cl₂7 + with an antibody via the carbohydrate side chains according to the basic procedure described by Murayama et al. (A. Murayama, K. Shimada and

T. Yamamoto, Immunochemistry, 15, pp. 523-528 (1978)). The antibody used was CC-49, a murine IgG monoclonal, which binds to an epitope of TAG-72, a tumor associated antigen. 1 mg of purified CC-49 IgG (10 mg / ml in 0.05 M sodium acetate pH 5.2) was treated with 1 mmol (0.010 ml of 0.100 M solution) of NalO 4 for 1 hour at room temperature. in the dark. This activated antibody was separated and recovered from excess NalO 4 by centrifugal gel filtration. 0.100 ml of the complex Rh / BA (2,3,2-tet) C1 „7+ (approximately 5 mCi / ml, -4 µM) was added to the activated antibody. ^z x 10 M) and 0.010 ml NaCNBH3 (0.10 M). The joining was allowed to proceed for 2 hours at room temperature. Rh labeled antibody was isolated by repeating the centrifugal gel filtration process. Antibody integrity was verified by established biochemical and immunological processes.

<img file="PT87802B_D0056.tif" />

eidos.

Example 17 Preparation of / (BITC-2,3,2-tet) C1₂ 7⁺ í

Transformed / (BA-2,3,2-tet 7⁺ in the derivative /^-105Rh (BITC-2,3,2-tet) C12_7⁺ reagent (BITC designates p-isothiocyanatobenzyl) by mixing 2 ml of βO4 Rh (BA-2,3,2-tet) C12 7 (approximately 5 mCi / ml, 1 x 10

M) with 0.002 ml thiophosgene. The reaction was allowed to proceed.

for 15 minutes at room temperature. The product was isolated by passing solution through a Chrompak Hamilton PRP-1. O / (BITC-2,3,2-tet) C1₂ 7⁺ eluted with 2 ml acetonitrile. The product was characterized by comparison to known standards using cation exchange chromatography and reverse phase chromatography. Using this process yields between 50 and 85 percent.

Example 17a Conjugation of Rh (BITC-2,3,2-tet) C1₂ 7⁺to antibodies

O /^_105Rh (BITC-2,3,2-tet) C12_7⁺ was ligated to tumor-specific antibody lysine residues (igG) by the following procedure. The antibodies used were CC-49 and B72.3 (hybridoma cell line B72.3 is deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland and has ATCC registration number HB 8108), both monoclonal antibodies. binding to TAG-72 epitopes, a tumor-associated antigen, 1.5 χ 10 ^ mmole (0.5 mCi) from / (BITC-2,3,2-tet) C12 7⁺ It was evaporated to dryness under a nitrogen atmosphere in a 1.5 ml Eppendorff bullet tube. To this dry vessel, 0.10 ml of the appropriate antibody (10 mg / ml in Na₂CC>₂ 0.1 M pH 9.5).

<img file="PT87802B_D0057.tif" />

The coupling was allowed to proceed for 1 hour at room temperature. Rh-labeled antibodies were isolated by centrifugal gel filtration. Antibody integrity was verified by established biochemical and immunological processes.

105

Example 17b In Vivo Localization of Rh-Tagged Antibodies

The usefulness of labeled antibodies was demonstrated by measuring uptake of the materials by a human tumor xenograft in an athymic mouse. Mice (Nu / Nu) subcutaneously (SC) (0.1 ml / source) were inoculated with the human colon carcinoma cell line, LS-174T (approximately 4 x 10 4 cells / animal). Approximately two weeks after inoculation, each animal was injected through the tail vein with 3 uCi (15 ug) of labeled antibody.

Rh (CC-49 or B72.3). The mice were sacrificed at various times, excised and the tumor and tissues chosen were weighed and radioactivity was measured on a gamma counter. Counts per minute per gram of Rh in each tissue (cpm / g) were determined and expressed as a function of the amount injected. Results are shown in the tables below.

<img file="PT87802B_D0058.tif" />

-40Distribution of ¹⁰^ Rh (BITC-2,3,2-tetJC] ^ CC-49 IgG in nude LS-174T tumor bearing mice

<td rowspan="2">Organ</td><td colspan="7">¹⁰⁵Rh</td>

<td colspan="2">17 hrs</td><td colspan="3">40 hrs</td><td colspan="2">66 hrs</td>

<td>Blood</td><td> 10,79 +</td><td> 0,99</td><td> 8,62</td><td> +</td><td> 2,46</td><td> 10,46 +</td><td> 1,65</td>

<td>Heart</td><td> 2,51 +</td><td> 0,30</td><td> 2,16</td><td> +</td><td> 0,45</td><td> 1,96 +</td><td> 0,53</td>

<td>Lung</td><td> 4,51 +</td><td> 0,99</td><td> 4,30</td><td> +</td><td> 1,14</td><td> 3,91 +</td><td> 0,95</td>

<td>Liver</td><td> 10,52 +</td><td> 3,28</td><td> 10,15</td><td> +</td><td> 1,50</td><td> 8,22 +</td><td> 1,30</td>

<td>Spleen</td><td> 5,40 +</td><td> 1,14</td><td> 6,93</td><td> +</td><td> 1,05</td><td> 5,14 +</td><td> 0,73</td>

<td>Kidney</td><td> 3,43 +</td><td> 0,52</td><td> 2 ,97</td><td> +</td><td> 0,36</td><td> 2,70 +</td><td> 0,73</td>

<td>Muscle</td><td> 1,92 +</td><td> 0,23</td><td> 1,14</td><td> +</td><td> 0,30</td><td> 1,15 +</td><td> 0,29</td>

<td>Tumor</td><td> 35,94 +</td><td> 5 ,38</td><td> 62,03</td><td colspan="2"> + 18,6</td><td colspan="2"> 85,89 +23,15</td>

-41τ ns

Rh Biodistribution (BITC-2,3,2-tet) C1₂~ B72.3 IgG in LS-174T tumor-bearing nude mice

<td rowspan="2">Organ</td><td colspan="8">¹⁰⁵Rh</td>

<td> 5,5</td><td>hrs</td><td> 24</td><td>hrs</td><td> 48</td><td>hrs</td><td> 72</td><td>hrs</td>

<td>Blood</td><td> 23,44</td><td> + 1,63</td><td> 18,12</td><td> + 1,14</td><td> 13,46</td><td> + 0,57</td><td> 13,07</td><td> + 1,55</td>

<td>Heart</td><td> 3,98</td><td> + 0,37</td><td> 3,30</td><td> + 0,19</td><td> 2,70</td><td> + 0,46</td><td> 2,90</td><td> + 1,34</td>

<td>Lung</td><td> 7,11</td><td> + 0,91</td><td> 5,96</td><td> + 0,73</td><td> 4,95</td><td> + 0,26</td><td> 4,65</td><td> + 0,76</td>

<td>Liver</td><td> 6,08</td><td> + 0,85</td><td> 4,81</td><td> + 0,51</td><td> 3,86</td><td> + 0,26</td><td> 3,77</td><td> + 0,25</td>

<td>Spleen</td><td> 4,60</td><td> + 0,64</td><td> 3,95</td><td> + 0,33</td><td> 3,27</td><td> + 0,32</td><td> 3,38</td><td> + 0,54</td>

<td>Kidney</td><td> 3,00</td><td> + 0,24</td><td> 3,18</td><td> + 0,29</td><td> 2,35</td><td> + 0,36</td><td> 2,20</td><td> + 0,52</td>

<td>Muscle</td><td> 1,21</td><td> + 0,24</td><td> 1,53</td><td> + 0,06</td><td> 1,77</td><td> + 0,41</td><td colspan="2"> 1,52+ 0,50</td>

<td>Tumor</td><td> 13,74</td><td> + 2,02</td><td> 28,07</td><td> + 1,90</td><td> 28,46</td><td> + 4,28</td><td> 34,70</td><td> +10,78</td>

<img file="PT87802B_D0059.tif" />

<td>in</td><td colspan="2">CC antibody</td>

<td> 1,5</td><td>x 10 ⁵</td><td>mmole</td>

<td>you</td><td>drying</td><td>in at</td>

<td> 1,5</td><td>ml. THE</td><td>This one</td>

Example 18 Conjugation of / (BITC-2,3,2-tet) C1₂ 7⁺ with antibody fragments /^_105Rh (BITC-2,3,2-tet) C1₂_7⁺ was bound to the lysine residues of the F (ab ') g fragment CC-49 antibody by the following procedure. Evaporated (0.5 mCi from /^_105Rh (BITC-2,3,2-tet) C1₂_7⁴ mosphere N₂ In a Eppendorf bullet tube to the dry container, 0.10 ml of CC-49 fragments were added.

F (ab¹ )₂ (10 mg / ml in Na₂CC>₂ 0.1 M, pH 9.5) prepared by the enzymatic digestion method described by Lamoyl and Nisonoff (E. Lamoyl and A. Nisonoff, J. Immunol. Methods, 56, pp. 235-243, (1983). if the reaction proceeds for 1 hour at room temperature.

Rh were isolated by centrifugal gel filtration. Antibody integrity was verified by established biochemical and immunological processes.

Example 19

CC-49 In Vivo Location (Fab 'Marked ^^ r ^

105.

The utility of the Rh-labeled antibody fragments has been demonstrated by measuring uptake of the material by a human tumor xenograft in an athymic mouse. Athymic mice (Nu / Nu) subcutaneously (SC) (0.1 ml / origin) were inoculated with the human θ colon carcinoma cell line, LS-174T (approximately 4 x 10 6 cells / animal). Approximately two weeks after inoculation, each animal was injected through the tail vein with 3 µCi (15 µg) CC-49 105 (Fab¹)₂ labeled Rh in saturated sodium chloride solution with phosphate buffer. Mice were sacrificed at various times, the chosen tumors and tissues were excised and weighed, and radioactivity was measured in a gamma counter.

105

Counts per minute per gram of Rh in each tissue (cpm / g) were determined and expressed as a function of the amount injected. The results are shown in the table below.

<img file="PT87802B_D0060.tif" />

-43 follows.

(BITC-2,3,2-tet) Biodistribution 0 ^ -CC-49 F (ab ')_? in nude LS-174T tumor bearing mice

<td rowspan="2">Organ</td><td colspan="9">¹⁰⁵Rh</td>

<td> 24</td><td colspan="2">hrs</td><td> 48</td><td colspan="2">hrs</td><td> 72</td><td colspan="2">hrs</td>

<td>Blood</td><td> 1,32</td><td> +</td><td> 0,21</td><td> 0,23</td><td> +</td><td> 0,09</td><td> 0,07</td><td> +</td><td> 0,01</td>

<td>Heart</td><td> 2,53</td><td> +</td><td> 0,36</td><td> 1,04</td><td> +</td><td> 0,12</td><td> 1,00</td><td> +</td><td> 0,15</td>

<td>Lung</td><td> 1,64</td><td> +</td><td> 0,08</td><td> 0,93</td><td> +</td><td> 0,09</td><td> 0,79</td><td> +</td><td> 0,42</td>

<td>Liver</td><td> 5,43</td><td> +</td><td> 0,65</td><td> 3,53</td><td> +</td><td> 0,76</td><td> 2,00</td><td> +</td><td> 0,43</td>

<td>Spleen</td><td> 2 , 79</td><td> +</td><td> 0,41</td><td> 2,03</td><td> +</td><td> 0,29</td><td> 1,00</td><td> +</td><td> 0,23</td>

<td>Kidney</td><td> 37,23</td><td> +</td><td> 3,27</td><td> 17,19</td><td> +</td><td> 2,09</td><td> 8,12</td><td> +</td><td> 1,85</td>

<td>Muscle</td><td> 0,94</td><td> +</td><td> 0,23</td><td> 0,67</td><td> +</td><td> 0,14</td><td> 0,45</td><td> +</td><td> 0,10</td>

<td>Tumor</td><td rowspan="2"> 26 ,45</td><td> +</td><td rowspan="2"> 4,53</td><td rowspan="2"> 22,82</td><td> +</td><td> 3,00</td><td> 12,76</td><td> +</td><td> 2,04</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

The biodistribution data presented clearly show the usefulness of chelate / rhodium antibody conjugates in localizing tumor tissue. The chelate / rhodium antibody conjugates quickly find the tumor tissue and the remnant exits the body through the kidneys. The proportions of tumor tissue to normal tissue are large, indicating that immunodetection and / or therapy is possible.

<img file="PT87802B_D0061.tif" />

Example 20

Similarly to that described in Example

19, athymic tumor-bearing mice were injected

LS-174T with chelate / rhodium antibody conjugates (labeled 105

Rh), both antibody fragments (ie, (F (ab ')) θ complete IgG monoclonal antibodies were respectively tested. At various times after injection, gamma-ray images of the whole animal were obtained using Kev 319 gamma rays). and 306. The images showed rapid disappearance of blood radioactivity and tumor uptake according to the quantitative results obtained in Example 19.

In the use of the chelate / rhodium antibody conjugates of the present invention for the diagnosis or treatment of a disease state in a mammal, the chelate / rhodium antibody conjugates are preferably administered in the form of a composition comprising the chelate / rhodium antibody conjugate. mixed with a pharmaceutically acceptable carrier (ie a carrier that is inert to the active material and has no significant harmful side effects or toxicity under conditions of use). The rhodium chelate / antibody composition is appropriately administered for each particular application, generally parenterally, for example by intraperitoneal, subcutaneous or intravenous injection. In these applications, an effective quantity (ie an amount sufficient to provide the desired effect) of one or more of the chelate / rhodium antibody conjugates is used in the composition. The choice of particular rhodium chelate / antibody conjugate or conjugates to be employed in a particular composition is dictated by considerations such as ease of administration, stability, compatibility with appropriate carriers, etc. In particular cases, the amount to be administered may be determined by methods known to those skilled in the art.

<img file="PT87802B_D0062.tif" />

-45 of the specialty. Compositions which are administered are generally in liquid form as sterile injectable suspensions or solutions. Pharmaceutically acceptable carriers for use in any particular situation may be readily determined and known to those skilled in the art, and may additionally optionally contain other active materials and / or excipients.