AU683213B2 - Bicyclopolyazamacrocyclocarboxylic acid complexes, their conjugates, processes for their preparation, and use as contrast agents - Google Patents

Description

01it/

PCT

WORLD IIVrELI AL PROPERTY ORGANIZATION International Bureau 1NTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) international Publication Number: WO 94/26313 A61K 4/00 Al(43) International Publication Date: 24 November 1994 (24.11.94) (21) International Application Number: PCrUS93I04322 (81) Designated States: AU, BR, CA, Fl, HU, JP, KR RU, UA, European patent (AT, BE, CIL DE, DK, ES, FR GB3, GR, (22) International Filing Date: 6 May 1993 (06.05.93) lE, IT, LU, MC, NL, PT, SE).

(71) Applicant: THE DOW CHEMICAL COMPANY [USIUS]; Published 2030 Dow Center, Abbott Road, Midland, MI 48640 With international search report.

(72) Inventors: KIFER, Garry, 114 Juniper, Lake Jackson, TX 77566 SIMON, Jaime; Route 1, Box 120-G, Agnieton, TX 77515 (US).

(74) Agent. KIMBLE, Karen, The Dow Chemical Company, 3 2 1 Patent Dept., P.O. Box 1967, Midland, MI 48641-1967

(US).

n'RECEIVED

SRA

12 DEC 1994 PROPERTY OM{GtU'ATTON (54) Title: BICYCLOPOLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, THEI CONJUGATES, PROCESSES FOR THEI PREPARATION, AND USE AS CONTRAST AGENTS (57) Abstract Complexes of bicyclopolyazamacrocyclocarboxylic acid with Gd, Ma or Fe ions are disclosed. The complexes can be covalently attached to a biologically active molecule, e.g. an antibody or antibody fragment, to form conjugates. The complexes and conjugates are useful as contrast agents for diagnostic purposes. Processes for preparing both the complex and conjugate are disclosed.

WO 94/26313 PCT/US93/04322 BICYCLOPOLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, THEIR CONJUGATES, PROCESSES FOR THEIR PREPARATION, AND USE AS CONTRAST AGENTS This invention concerns complexes that contain as the ligand bicyclopolyazamacrocyclocarboxylic acids, and conjugates thereof, for use as contrast agents in magnetic resonance imaging (MRI). Processes for preparing these complexes and conjugates are also provided. To better understand this invention, a brief background on MRI is provided in the following section.

Background MRI is a non-invasive diagnostic technique which produces well resolved crosssectional images of soft tissue within an animal body, preferably a human body. This technique is based upon the property o, certain atomic nuclei water protons) which possess a magnetic moment [as defined by mathematical equations; see G. M. Barrow, Physical Chemistry, 3rd Ed., McGraw-Hill, NY (1973)] to align in an applied magnetic field. Once aligned, this equilibrium state can be perturbed by applying an external radio frequency (RF) pulse which causes the protons to be tilted out of alignment with the magnetic field. When the RF pulse is terminated, the nuclei return to their equilibrium state and the time required for this to occur is known as the relaxation time. The relaxation time consists of two parameters known as spin-lattice (T1) and spin-spin (T2) relaxation and it is these relaxation measurements which give information on the degree of molecular organization and interaction of protons with the surrounding environment.

Since water content of living tissue is substantial and variations in content and environment exist among tissue types, diagnostic images of biological organisms are obtained which reflect proton density and relaxation times. The greater the differences in relaxation times (T1 and T2) of protons present in tissue being examined, the greater will be the contrast in the obtained image Magnetic Resonance 33, 83-106 (1979)].

It is known that paramagnetic chelates possessing a symmetric electronic ground state can dramatically affect the T1 and T2 relaxation rates of juxtaposed water protons and that the effectiveness of the chelate in this regard is related, in part, to the number of unpaired electrons producing the magnetic moment [Magnetic Resonance Annual 231-266 (Raven Press, NY (1985)1. It has also been shown that when a paramagnetic chelate of this type is administered to a living animal, its effect on the T1 and T2 of various tissues can be directly observed in the magnetic resonance (MR) images with increased contrast being observed in the areas of chelate localization. It has therefore been proposed that stable, non-toxic paramagnetic chelates be administered to animals in order to increase the diagnostic information obtained by MRI [Frontiers of Biol. Energetics I, 752-759 (1978); J. Nucl. Med. 506-513 (1984); Proc. of NMR Imaginq Symp. (Oct. 26-27, 1980); F. A. Cotton et al., Adv. Inorg.

WO 94!26313 PCT/US93104322 Chem. 634-639 (1966)]. Paramagnetic metal chelates used in this manner are referred to as contrast enhancement agents or contrast agents.

There are a number of paramagnetic metal ions which can be considered when undertaking the design of an MRI contrast agent. In practice, however, the most useful paramagnetic metal ions are gadolinium (Gd iron (Fe 3 manganese (Mn and (Mn 3), and chromium (Cr* 3 because these ions exert the greatest effect on water protons by virtue of their large magnetic moments. In a non-complexed form GdCI these metal ions are toxic to an animal, thereby precluding their use in the simple salt form. Therefore, a fundamental role of the organic chelating agent (also referred to as a ligand) is to render the paramagnetic metal non-toxic to the animal while preserving its desirable influence on T1 and T2 relaxation rates of the surrounding water protons.

Art in the MRI field is quite extensive, such that the following summary, not intended to be exhaustive, is provided only as a review of this area and other compounds that are possibly similar in structure. U.S. Patent 4,899,755 discloses a method of alternating the proton NMR relaxation times in the liver or bile duct of an animal using Fe 3 -ethylene-bis(2hydroxyphenylglycine) complexes and its derivatives, and suggests among various other compounds the possible use of a pyridine macrocyclomethylenecarboxylic acid. U.S. Patent 4,880,008 (a CIP of U.S. Patent 4,899,755) discloses additional imaging data for liver tissue of rats, but without any additional complexes being shown. U.S. Patent 4,980,148 disclose gadolinium complexes for MRI which are non-cyclic compounds. C. J. Broan et al., J. Chem. Soc., Chem. Commun., 1739-1741 (1990) describe some bifunctional macrocyclic phosphinic acid compounds. C. J. Broan et al.,J. Chem. Soc., Chem. Commun., 1738-1739 (1990) describe compounds that are triazabicyclo compounds. I. K. Adzamli et al., J. Med. Chem. 2, 139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging.

At the present time, the only commercial contrast agent available in the U.S. is the complex of gadolinium with diethylenetriaminepentaacetic acid (DTPA-Gd 3

MAGNEVIST'"

by Shering). MAGNEVIST" is considered as a non-specific/perfusion agent since it freely distributes in extracellular fluid followed by efficient elimination through the renal system.

MAGNEVIST'" has proven to be extremely valuable in the diagnosis of brain lesions since the accompanying breakdown of the blood/brain barrier allows perfusion of the contrast agent into the affected regions. In addition to MAGNEVIST'", Guerbet is commercially marketing a macrocyclic perfusion agent (DOTAREM") which presently is only available in Europe. A number of other potential contrast agents are in various stages of development.

It would be advantageous if contrast agents were developed that could have site specificity for the tissue desired to be imaged, rather than non-specific/perfusion agents. The present invention is directed to just such novel complexes comprising a ligand that is a bicyclopolyazamacrocyclocarboxylic acid of the formula WO 94/26313 WO 946313CTIUS93/04322 Q

A

lz R-N N

N-R

wherein: R is hydrogen, x

-C-CO

2

H

Y

x -C Q R 4 or CO02H where: X and Y are independently H, OH, CI-C 3 a lkyl or COOH; R' is H or OH; and

R

4 is H, NO 2 ,N 2' isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least two R terms must be x

-C-CO

2

H

y A CH, N, C-Br, C-Cl, C-OR 1 C-OR 2 N-R 3 or c-c= C

R

4 H, C I-C 5 alkyl, benzyl, or benzyl substituted with at least one R 4 R' is C 1 -C 1 6 alkylamino;

R

3 is C 1

-C

16 alkyl, benzyl, or benzyl substituted with at least one R4;

R

4 is defined as before; X- is C1-, Br-, I or H 3 CC02-; Q and Z independently are CH, N, N+-R 3

C-CH

2

-OR

1 or C-C(O)-R 5 s R 1 and R 3 are defined as above;

R

5 is -O-(C 1

-C

3 alkyl), OH or NHR 6

R

6 is C 1

-C

5 alkyl or a biologically active material; X- is defined as above; and with the provisos that: a) when Q, A or Z is N or N+-R 3 then the other two groups must be CH; b) when A is C-Br, C-CI, C-OR 1 or C-OR 2 then both Q and Z must be CH; c) the sum of the R 2

R

4 and R 6 terms may not exceed one and one such group must be present; and d) only one of Q or Z can be C-C(O)-R 5 and when one of Q or Z is C-C(O)-R 5 then A must be CH; and complexed with a metal ion selected from Gd+ 3 Mn+ 2 or Fe+ 3 or pharmaceutically-acceptable salts thereof.

Bifunctional complexes of Formula are desirable to prepare the conjugates of this invention. Such ligands must have: 20 one R term is

R

7 V

I

R

CO

2 H CO R where R 4 and R 7 are defined as above; or A is C-OR 1

C-OR

2 where R 1 and R 2 are defined as above or C-C=C R4 where R 4 is defined as above; or A is CH, and one of Q or Z is CH and the other is C-C(O)-R 5 or C-CH 2

-OR

1 where R 1 and R 5 are defined as above; especially those ligands where R 5 is NHR 6 where I is a biologically active material.

The ligands of Formula are complexed with various metal ions, such as gadolinium (Gd+ 3 iron (Fe+ 3 and manganese (Mn+ 2 and Gd+ 3 being preferred.

The complexes so formed can be used by themselves or can be attached, by being covalently RANI 4A TO 'NrALIBZ)006B4iSAK WO 94/26313 PCT/US93/04322 bonded, to a larger molecule, such as a dextran, a polypeptide or a biologically active molecule, including an antibody or fragment thereof, and used for diagnostic purposes. Such conjugates and complexes are useful as contrast agents.

The complexes and conjugates of this invention can be modified to provide a specific overall charge. For example, when the metal ion is 3 the following can be obtained: an overall neutral charge-when Ris

X

-C-C0 2

H

I

Y

and X and Y are all equal to H; an overall 1 charge-when one of A, Q or Z is N -R 3 X, where R 3 and X are defined as above; and the three R terms are

X

I

-C-CO2H

I

Y

and X and Y are all equal to H; or when A, Q and Z are CH; X and Y are H; and one R term is H.

Both the complexes and conjugates may be formulated to be in a pharmaceutically acceptable form for administration to an animal.

Use of the ligands of this invention with other metal ions for diagnosis of disease states such as cancer is possible. The use of those'complexes and conjugates is discussed in another copending application.

The complex has the ligand of Formula numbered for nomenclature purposes as follows: WO 94/26313 PCTIJS93/04322 13

A

14 Q A~ Z 12 N2 51 (I) R-N 3 6 9 N-R 14 N 81

N

R

The present invention concerns development of contrast agents having a neutral or 1 charge which enables site specific delivery of the contrast agent to a desired tissue. The advantage being increased contrast in theareas of interest based upon tissue affinity as opposed to contrast arising from non-specific perfusion which may or may not be apparent with an extracellular agent. The specificity of the ligand of Formula may be controlled by adjusting the total charge and lipophilic character of the complex. The overall range of the charge of the complex is from 1 to 0. For example, for a complex having a 1 overall charge has heart and/or bone uptake expected; whereas when the overall charge of the complex is 0 (thus neutral), the complex may have the ability to cross the blood brain barrier and normal brain uptake may be possible.

Tissue specificity may also be realized by ionic or covalent attachmentof the chelate to a naturally occurring or synthetic macromolecule having specificity for a desired target tissue. One possible application of this approach is through the use of chelate conjugated monoclonal antibodies which would transport the paramagnetic chelate to diseased tissue enabling visualizaton by MRI. In addition, attachment of a paramagnetic chelate to a macromolecule can further increase the contrast agent efficiency resulting in improved contrast relative to the unbound chelate. Recent work by Lauffer Patents 4,880,008 and 4,899,755) has demonstrated that variations in lipophilicity can result in tissuespecific agents and that increased lipophilic character favors non-covalent interactions with blood proteins resulting in enhancement of relaxivity.

Additionally, the present contrast agents of Formula which are neutral in charge are particularly preferred for forming the conjugates of this invention since undesirable ionic interactions between the chelate and protein are minimized which preserves the antibody immunoreactivity. Also the present neutral complexes reduce the osmolarity relative to DTPA- Gd

J

which may alleviate the discomfort of injection.

While not wishing to be bound by theory, it is believed that when a charged complex of the invention is made 1 for heart), the variations in that chelate ionic charge can influence biolocalization. Thus, if the antibody or other directing moiety is also specific for the same site, then the conjugate displays two portions to aid in site specific delivery.

WO 94/26313 PCT/US93/04322 The terms used in Formula and for this invention are further defined as follows.

alkyl", "C,-C 5 alkyl", alkyl", include both straight and branched chain alkyl groups. An "animal" includes a warmblooded mammal, preferably a human being.

"Biologically active material" refers to, for example, a dextran, peptide, or molecules that have specific affinity for a receptor, or preferably antibodies or antibody fragments.

"Antibody" refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody, preferably a monoclonal antibody; "antibody fragment" includes Fab fragments and F(ab')2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes. When using the term "radioactive metal chelate/antibody conjugats' or "conjugate", the "antibody" is meant to include whole antibodies and/or antibody fragments, including semisynthetic or genetically engineered variants thereof.

Possible antibodies are 1116-NS- 19-9 (anti-colorectal carcinoma), 1116-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 (anti-human lung cancer), CC49 (anti-TAG-72), CC83 (anti- TAG-72) and B72.3. The hybridoma cell lines 11 16-NS-19-9, 1116-NS-3d, 703D4, 704A1, CC49, CC83 and B72.3 are deposited with the American Type Culture Collection, having the accession numbers ATCC HB 8059, ATCC CRL 8019, ATCC ~B 8301, ATCC HB 8302, ATCC HB 9459, ATCC HB 9453 and ATCC HB 8108, respectively.

As used herein, "complex" refers to a complex of the compound of tne invention, e.g. Formula complexed with a metal ion, where at least one metal atom is chelated or sequestered; "conjugate" refvs to a metal ion chelate that is covalently attached to an antibody or antibody fragment. The terms "bifunctional coordinator", "bifunctional chelating agent" and "functionalized chelant" are used interchangeably and refer to compounds that have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the chelant moiety that is capable of serving as a means to covalently attach to an antibody or antibody fragment.

The bifunctional chelating agents described herein (represented by Formula I) can be used to chelate or sequester the metal ions so as to form metal ion chelates (also referred to herein as "complexes"). The complexes, because of the presence of the functionalizing moiety (represented by R 2 R4 or R 6 in Formula can be covalently attached to biologically active materials, such as dextran, molecules that have specific affinity for a receptor, or preferably covalently attached to antibodies or antibody fragments. Thus the complexes described herein may be covalently attached to an antibody or antibody fragment or have specific affinity for a receptor and are referred to herein as "conjugates".

As used herein, "pharmaceutically-acceptable salt" means any salt or mixtures of salts of a complex or conjugate of formula which is sufficiently non-toxic to be useful in therapy or diagnosis of animals, preferably mammals. Thus, the salts are useful in accordance with this invention. Representative of those salts formed by standard reactions from both 8 organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, gluconic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids. Also included are salts formed by standard reactions from both organic and inorganic sources such as ammonium or 1-deoxy-l-(methylamino)-D-glucitol, alkali metal ions, alkaline earth metal ions, and other similar ions. Particularly preferred are the salts of the complexes or conjugates of formula where the salt is potassium, sodium or ammonium. Also included are mixtures of the above salts.

The complexes or conjugates of the present invention contain a ligand of Formula The ligands are prepared by various processes. Typical general synthetic approaches to such processes are provided by the reaction schemes given below. In the following schemes, Schemes 1, 2, 3, 5, 6, 7, 8 and 9 describe the preparation of starting materials for compounds of the invention. Schemes 4, 10, 11 and 12 describe the preparation of 1i compounds of the invention or precursors thereof from which compounds of the invention may be obtained by ester hydrolysis.

In Scheme 1, the compounds of Formula are prepared wherein Q, A and Z CH, and either one R H and the other two R the formula below or all three R

X

-C-CO

2

H

Y

*C)

A r IN:\LIBZ00684:SAK Scheme 1

N

SOC1 2 80 0 C, 2 hours

N

1 (2) F-j F-I NaN N NNa I I I Ts Ts Ts DMF, 100 0 C 1 Ts-

X-CH

2

-CQ

2

H

(X=Br or Cl) Ni N-Ts

NNA

Ts (3) ux N NH

N

Br/AcOH ref

HO

2 c-H 2

C-N

N-CH

2

CO

2 H NO/ 2

N

CH

2

CO

2 H a compound of Formula (I) Scheme 1 Cont'd

HOCH

2

SO

3 Na NaOH/H 2 0 NaQ 3

S-H

2 cr N NH N-CH.-SO,Na HIN~ NaCN NaOI/ H 2 0 12M HCl/A

HO

2

C-H

2 C-N N-CH 2

CO

2

H

N

H

a compound of Formula (I)

NC-H

2 C-N N-CH 2

-CN

H

WO 94126313 WO 9426313FCTIUS93/04322 Scheme 2 prepares the compounds of Formula wherein A C-Br, and Q and Z CH.

-11- Scheme 2

H

N 1) PBr4 2) EtOH HO OH E tOH Br .7 N SOC1 2 HO OH 80 0 C, 2 hrs. EtO OEt (7) Br

N

F-I NaN N NNa I I I Ts Ts Ts DMF, 100 0

C

Br

N

Ts-j N-Ts HBr/AcOH ref lux Br

N

H

(11) Scheme 2 Cont'd Br ~-1

N

H0 2

C

C0 2

H

C0 2

H

(12) BrCH 2

CO

2

H

AaOH/H 2 0 .N N 1 __N (11) a coumpound of Formula (I) WO 94/26313 rCT/US93/04322 Scheme 3 prepares the compounds of Formula wherein A R= H, NO 2 N H 2 or SCN; and Qand Z CH.

-14- Scheme 3 Pd(PPh 3 2 Cl2, CuI, Na/Hg amalgam

N

N NH

N

Ts-N N-Ts

~NX

Ts (13) (14) Scheme 3 Cont'd O~i Ii' BrCH 2

CO

2

H

(14) 1 NaOH/H 2 0 H0 2 C C0 2

H

a compound bf Formula (I) WO 94126313 WO 9/26313 CTIUS93/04322 Scheme 4 prepares the compounds of Formula wherein A C-OR 2 where R' CI-C 5 alkylamino; and Q and Z CH, -17- Scheme 4 OHOH

CN

NaH/Br(CH2),CN EtOH/H+

AN-I

0 N 0 0 0 000 HO OH EtO OEt EtO OEt (18 j (16) 0 (17)

N

IaBH 4 /EtOH El 4 L~INaN N NNa -r N Ts Ts Ts s- N-Ts DMF, 1O 0

C

Ts (21 I 80 0 C, 2 hours Ts-N N-Ts N

I

Ts (22) (19) Scheme 4 Cont'd 0/\7\A'1

HBOC

HBr/AcOH HO-N02C 2 OH C0 2

H

(22) N0 HN-M1 0r N KCO 2 H0 reflux HN NH IN NHC2 L (23) H (24)H Scheme 4 Cont'd

F

3

CCO

2 I

N

H011 2 C I I C0 2

H

(26) a compound of Formula (I) WO 94126313 PCT[(JS93/04322 Scheme 5 prepares the compounds of Formula wherein A CH; and one of Q or Z CH and the other Q or Z C-C(O)-R 6 or C-CH,-R 6 where R' is def-ined as before.

Scheme 0C 2

H

5 H3 N CH 3 (27)

OH

Ts-N N-Ts NCS/CC1 4 (HCIi2

CH

reflux cli Cl (28) rn I- NaN N NNa I I I Ts Ts Ts 0C 2

H

DMF, 1o 0 C N Ts-N N-Ts Ts (29) 1) NaBH 4 /EtOH I OH flxHN

NH

H

(31) LiAlH 4

/THF

Ts Scheme 5 Cont'd

OH

N

H0 2 C C2

CO

2

H

BrCH 2

CO

2

H

NaOH/H 2 0 (31) (32) a compound of Formula (I) WO 94/26313 WO 9/26313 CTfUS93IO4322 Scheme 6 prepares the compounds of Formula wherein Z C-CH 2 -013z or C-C(O)-R'where R' -O-(CI-C 3 alIkyl), OH or NHR where is defined as before; and QandA CH, -24- Scheme 6 BzCl, CHC1 3

/K

2 C0 3 -00 Tsa 0 111 Ph 1 j-

T

s (33) HBr/AcQH reflux 0 Ph HI~ I H N (34) 0 Ph H0 2 C I\ ^C0 2

H

N X-C11 2

-CO

2

H

(X=Br or Cl) NaOH/H 2 0 C0 2

H

a compound of Formula (I) Scheme 6 Cont'd 0 Ph

X-CH

2

-CO

2 H, <N >dH

H'

(3(X=Br or Cl), 3,0 C 'N N dH C0 2 H 0 Nr-UiT/H 2 0 O N I OH 0 2 C 2 (37 C0 2

H

C0 2

H

C0 2

H

(36) a compound of Formula (I) a compound of Formula (I) WO 94/26313 WO 9426313PCT1US93/04322 Scheme 7 prepares the compounds of Formula wherein A N or N R' C-C C 16 alkyl and is Xhalide; and Q and Z CH.

-27- 0 Scheme 7

;NI

N

NCS/CC1 4

(H

3

CCO

2 2 ref lux

(N

-I NaN N NNa I I I Ts Ts Ts DMF, 100 0

C

H

3

C

CH

3

N

<N

Ts-N N-Ts Ts (38) (39) r_ N HN NH 1 1 (41)

H

HBr/AcOH ref lux Scheme 7 Cont'd

N

X-CH2-CO2H N (41) (X=Br or Cl). N -"1CO 2 H X-(CH 2 )n-CH3 pH0 2 C I (42) n=1-16, C02H1 x=c1, Br, I

N

<N

H10 2 C C0 2

H

(43) C0 2

H

a compound of Formula (I) WO 94/26313 WO 9426313PCTTUS93/04322 Scheme 8 prepares the compounds of Formula wherein Q N'-RsX',where R' C 1 -C 16 al kyl and X* halide; and A and Z CH.

0 Scheme 8 IN M NCS/CC 14,

(H

3

CCO

2 2

CH

3 ref lux >1 I c:i~ Cl F-I FI NaN N NNa I I I <N

H

3

C

TsT T o Ts-N N-Ts DMF, 100 0

C

Ts (46) (44)

NX-

HN NH H (48) HBr/AcOH ref lux X Ts-N N-Ts Ts (47) X- (CI1 2

-CH

3 n=1-16, X=C1, Br, I 0 Scheme 8 Cont'd X-CH2-CO2H (48) (X=Br or Cl)- NaOR/H2U' H0 2

C

C0 2

H

(49) C0 2

H

a compound of Formula (I) WO 94/26313 WO 9426313PCTIUS93/ 04322 Scheme 9 prepares the compounds of Formula wherein Q =N or N+-115X-,where R' CI-C 16 alkyl and halide; and A and Z CH.

-33- Scheme 9

<N

H0 2 C C0 2

H

(51) C0 2

H

a compound of Formula (I) X- (CH 2 -CH3 n=1-16, X=Cl, Br, I

NX-

<N

H0 2 C 1'\N N C0 2 H1 (52) C0 2

H

a compound of Formula (I) X-CH2-CO2H (X=Br or Cl) NaOH /H20 H0 2 C N C0 2

H

HBr/AcOH (46) ref lux WO 94/26313 PCTIUS93/04322 Scheme 10 preparethe compounds of Formula wherein R term at the 6 position is x Q-

R

4 C0 2

E

where R 4 NO 2 or NH 2 and A, Q and Z CH.

ArC Scheme N NH

N

r L" 1 02H N N

N-

H

0O 2 (53) (4)

CH

3

CN/K

2 C0 3

CO

2

CH

3 N0 2 (54)

NO

2 Schem 1 e 10 Cont'd

X-CH

2

-CO

2

H

(X Br or Cl) NaOH/11 2 0 (54) P1O 2

/H

2 H0 2

C

H0 2

C

N

N C0 2

H

H0 2 C N NO 2 NY0 2

H

2 C 4 H (56) a compound of Formula (I) WO 94/26313 WO 9426313PCTIUS93/04322 Scheme 11 prepares the compounds of Formula wherein the R term at the 9 position is x -Cr where R 4 NO2 or NH 2 and A, Qand Z CH.

-38- Scheme 11 (53)

X-CH

2 -C0 2 11 (X Br or Cl) NaOH/11 2 0

N

I' C0 2

H

N NJ H02C1 NN (57) a compound of Formula (I) Pt0 2 /H1 2 H0 2

C

/P-N N"

C

2

H

N

2

C

NH

2 (58) a compound of Formula (I) i Scheme 12 prepares the methyl ester of a compound of Formula (II) wherein the R term at the 6 position is where R 4

NO

2

R

7

=OCR

3 and Qand Z =CH.

S

*0 OS IN;\L18Z100684:SAK Scheme 12

N

H

2 0II

HOCH

2

SO

3 Na P. IpH=9 NaO 3 S Nz1 0N Na CN (59)

FN

N N

CN

H

-4 -J o *9 0.

0* 0 C *e C 0.00 C *00 00 0 *0 Scheme 12 Cont'd

H

3 C BrII

CO

2

CH

3 N02

THF

N

1 NaCN

CN

HC 1 heat/ ref lux (61) H0 2

C

C0 2

H

N02

CQ

2

CH

3 (62) WO 94/26313 PCT/US93/04322 In the above Schemes, the general process description illustrates specific steps that may be used to accomplish a desired reaction step. The general description of these process steps follows.

The synthetic Scheme 1 begins with a halogenation of commercially available bispyridyl alcohol using thionyl chloride. Similar procedures for converting an alcohol to an electrophilic substrate, such astreatment with toluenesulfonyl chloride, HBr or HCI, should also result in a similarly reactive product which would work well in subsequent ring closure reactions. Macrocyclization procedures are numerous in the literature and the desired tetraazamacrocycle was prepared according to the method of Stetter et al., Tetrahedron 37, 767-772 (1981). More general procedures have since been published which give good yields of similar macrocycles using milder conditions D. Sherry et al., J. Org. Chem. 54 2990-2992 (1989)]. Detosylation of the intermediate macrocycle to yield was accomplished under acidic conditions in good yield. Reductive detosylation procedures are also well known in the literature and can be adapted to the present reaction sequence.

Schemes 10, 11 and 12 delineate a synthetic approach which introduces an aromatic nitrobenzyl substituent at one of the macrocyclic nitrogen positions. Typically, the macrocyclic amine is mono-N-functionalized in an organic solvent such as acetonitrile or DMF at room temperature using a non-nucleophilic base such as potassium carbonate. Additional functionalization of the remaining nitrogen positions is then preformed by methods and conditions described in previous Schemes. After the introduction of the desired chelating moieties, the nitro group is reduced using platinum oxide and hydrogen in water. In this form, the chelating agent is compatible with conjugation techniques which will enable attachment to larger synthetic or natural molecules.

The metal ions used to form the complexes of this invention are Gd Mn", Fe'3 and available commercially, e.g. from Aldrich Chemical Company. The anion present is halide, preferably chloride, or salt free (metal oxide).

A "paramagnetic nuclide" of this invention means a metal ion which displays spin angular momentum and/or orbital angular momentum. The two types of momentum combine to give the observed paramagnetic moment in a manner that depends largely on the atoms bearing the unpaired electron and, to a lesser extent, upon the environment of such atoms.

The paramagnetic nuclides found to be useful in the practice of the invention are gadolinium iron (Fe* 3 and manganese (Mn* 2 with Gd* 3 being preferred.

The complexes are prepared by methods well known in the art. Thus, for example, see Chelating Agents and Metal Chelates, Dwyer Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Synthetic Production and Utilization of Amino Acids, (edited by Kameko, et al.) John Wiley Sons (1974). An example of the preparation of a complex involves reacting a bicyclopolyazamacrocyclophosphonic acid with -43- WO 94/26313 PCT/US93/04322 the metal ion under aqueous conditions at a pH from 5 to 7. The complex formed is by a chemical bond and results in a stable paramagnetic nuclide composition, e.g. stableto the disassociation of the paramagnetic nuclide from the ligand.

The complexes of the present invention are administered at a ligand to metal molar ratio of at least about 1:1, preferably from 1:1 to 3:1, more preferably from 1:1 to 1.5:1.

A large excess of ligand is undesirable since uncomplexed ligand may be toxic to the animal or may result in cardiac arrest or hypocalcemic convulsions.

The antibodies or antibody fragments which may be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see for example, Kohler and Milstein [Nature, 256, 495-497 (1975); and Eur. J. Immunol., 6, 511-519 (1976)]. Such antibodies normally have a highly specific reactivity. In the antibody targeted conjugates, antibodies directed against any desired antigen or hapten may be used. Preferably the antibodies which are used in the conjugates are monoclonal antibodies, or fragments thereof having high specificity for a desired epitope(s). Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. Some examples of antibodies or antibody fragments are 1116-NS-19-9, 1116-NS-3d, 703D4, 704A1, CC49, CC83 and B72.3. All of these antibodies have been deposited in ATCC. A more complete list of antigens can be found in U.S. Patent 4,193,983. The conjugates of the present invention are particularly preferred forthe diagnosis of various cancers.

This invention is used with a physiologically acceptable carrier, excipient or vehicle therefor. The methods for preparing such formulations are well known. The formulations may be in the form of a suspension, injectable solution or other suitable formulations. Physiologically acceptable suspending media, with or without adjuvants, may be used.

An "effective amount" of the formulation is used for diagnosis. The dose will vary depending on the disease and physical parameters of the animal, such as weight. In vivo diagnostics are also contemplated using formulations of this invention.

Other uses of some of the chelants of the present invention may include the removal of undesirable metals iron) from the body, attachment to polymeric supports for various purposes, e.g. as diagnostic agents, and removal of metal ion by selective extraction.

The ligands of Formula having in at least two R terms T equal to P(O)R'OH may be used for metal ion control as scale inhibitors. It is likely that these ligands could be used in less than stoichiometric amounts. Similar uses are known for compounds described in U.S. Patents 2,609,390; 3,331,773; 3,336,221; and 3,434,969.

WO 94/26313 PCT/US93/04322 The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the present invention.

Some terms used in the following examples are defined as follows: .C liquid chromatrography, purifications were carrier out at low pressure using Dionex 2010i system fitted with a hand-packed Q-Sepharose'" anion exchange column (23 x 2 cm).

DMF dimethylformamide.

AcOH aceticacid.

ICP inductively coupled plasma.

g gram(s).

mg= milligrams.

kg kilogram(s).

mL milliliter(s).

pL microliter(s).

pH Stability General Procedure A stock 5 GdCIg or 1S'SmCI 3 solution was prepared by adding 2 pL of 3x10'M 5 GdCI3 in 0.1 N HCI to 2 mL of a 3x10M GdCI 3 carrier solution. Appropriate ligand solutions were then prepared in deionized water. The 1:1 ligand/metal complexes were then prepared by combining the ligands (dissolved in 100-500 iL of deionized water) with 2 mL of the stock 9 GdCI solution, followed bythrough mixing to give an acidic solution (pH The pH of the solution wasthen raised to 7.0 using 0.1N NaOH. The percent metal as a complex was then determined by passing a sample of the complex solution through a Sephadex'" G-50 column, eluting with 4:1 saline (85% NaCI/NH,OH) and collecting 2 x 3 mL fractions. The amount of radioactivity in the combined elutions was then compared with that left on the resin (noncomplexed metal is retained on the resin). The pH stability profile was generated by adjusting the pH of an aliquet of the complex solution using 1M NaOH or 1M HCI and determining the percent of the metal existing as a complex using the ion excha ige method described above.

The Sm results are known by expermintal comparison to be identical for complexation and biodistribution of the ligands of this invention.

STARTING MATERIALS Example A Preparation of 2,6-bis(chloromethyl)pyridine.

To 100 mL of thionyl chloride that was cooled (ice bath) was added 24 g (0.17 mol) of 2,6-bis(hydroxymethyl)pyridine. After 30 min, the reaction mixture was warmed to room temperature, then refluxed for 1.5 hrs. After cooling the reaction mixture to room temperature, the solid which formed was filtered, washed with benzene and dried in vacuo.

WO 94/26313 PCT/US93/64322 The solid was then neutralized with saturated NaHCO filtered and dried to yield 23.1 g of the titled product as an off-white crystalline solid, mp 74.5-75.5"C, and further 'haracterized by: 'H NMR (CDCI) 6 4.88 4H), 7.25-7.95 3H).

Example B Preparation of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1 11,13triene.

A DMF solution (92 mL) of 6.9 g (11.4 mmol) of 1,4,7-tris(ptolylsulfonyl)diethylenetriamine disodium salt was stirred and heated to 100 0 C under nitrogen.

To the solution was added dropwise over 45 min 2 g (11.4 mmol) of 2,6bis(chloromethyl)pyridine (prepared bythe procedure of Example A) in 37 mL of DMF. When the addition was completed the reaction mixture was stirred at 40°C for 12 hrs. To the reaction mixture was then added 50-75 mL of water, resulting in immediate dissolution NaCI, followed by precipitation of the title product. The resulting slurry was then filtered and the solid washed with water and dried in vacuo. The title product was obtained as a light-tan powder, 6.5 g mp 168-170°C dec. and further characterized by: 'H NMR (CDCI3) 6 2.40 3H), 2.44 6H), 2.75 4H), 3.30 4H), 4.28 4H), 7.27 2H), 7.34 4H), 7.43 2H), 7.65 4H), 7.75 1 and 3 C NMR 621.48,47.29,50.37,54.86, 124.19,127.00, 127.11,129.73, 135.04, 135.74,138.95,143.42, 143.73, 155.15.

Example C Preparation of 3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1 11,13-triene.

A solution of HBr and AcOH was prepared by rm,xing 48% HBr and glacial AcOH in a 64:35 ratio. To 112 mL of the HBr/AcOH mixture was added 5.5 g (8.2 mmoi) of 3,6,9-tris(ptolylsulfonyl)-3,6,9, 1 5-tetraazabicyclo[9.3.1 ]pentadeca-1(15), 11,13-triene (prepared by the procedure of Example B) and the reaction mixture was heated at mild reflux with constant stirring for 72 hrs. The reaction mixture was then cooled to room temperature and concentrated to approximately 1/10 of the original volume. The remaining solution was stirred vigorously and 15-20 mL of diethyl ether was added. A off-white solid formed which was filtered, washed with diethyl ether, and dried in vacuo. The dry tetrahydrobromide salt was then dissolved in 10 mL of water, adjusted to pH 9.5 with NaOH (50% w/w) and continuously extracted with chloroform for 4 hrs. After drying over anhydrous sodium sulfate, the chloroform was evaporated to give a light-tan oil which gradually crystallized upon standing at room temperature to yield 1.2 g of the title product, mp 86-88°C and further characterized by: WO 94/26313 PCT/US93/04322 'H NMR (CDCI 3 8 2.21 4H), 2.59 4H), 3.06 3H), 3.85 4H), 6.89 2H), 7.44 1H); and 3

CNMR

848.73,49.01, 53.63,119.67,136.29,159.54.

Example D Preparation of 3,6,9,15-tetraazabicyclo[9.3. 1 ]pentadeca-1(15), 11,13-triene-3,6,9-triacetic acid

(PCTA).

An aqueous solution (15 mL) of 2.1 g (15 mmol) of bromoacetic acid was added to 0.8 g (3.8 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1(15), 11,13-triene (prepared by the procedure of Example C) with stirring at room temperature. After complete dissolution, the reaction mixture was cooled with an ice bath and the pH adjusted to 9 by the slow addition of NaOH The pH was held constant at 9 throughout the reaction by adding small aliquots of NaOH. After 1.5 hrs the reaction mixture was warmed to 60 0 C with continued monitoring of pH. When no further drop in pH could be detected, the reaction was cooled to room temperature and the aqueous solution freeze-dried to give a white solid. The solid was then dissolved in a minimum of hot water and allowed to stand at room temperature for 12 hrs. The resulting crystals were filtered and dried in vacuo to give 1.2 g of the title product as the trisodium salt, mp 378-380 0 C dec. and further characterized by: 'H NMR(D 2

O)

8 2.76 4H), 3.36 4H), 3.47 2H), 4.10 4H), 7.31 2H), 7.84 1H); and 3

CNMR

8 53.83, 57.31, 57.40, 59.48, 62.36, 125.47, 143.72, 152.67, 172.15, 177.41.

Example E Preparation of 3,9-bis(sodium methylenesulfonate)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1(15),11,13-triene (PC2S).

A solution of 10 mL of an aqueous solution of 1.03 g (5.0 mmol) of 3,6,9,15tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (prepared by the procedure of Example C) and 0.5 mL of concentrated HCI was stirred for 10 min at room temperature. The solution had a pH of 8.6. To the solution was then added 1.37 g (10.2 mmol) of HOCH 2

SO

3 Na and 5 mL of deionized water. The solution was then heated at 60 0 C for 10 min and the pH was 5.6. After cooling, the pH was adjusted to 9.0 with 1M NaOH, followed by freeze-drying to give the desired product as a white solid (quantitative yield), and further characterized by: 'H NMR (D,O) 6 2.87 4H), 3.18 4H), 3.85 4H), 4.11 4H), 7.03 2H), 7.55 1 and 3 CNMR(D0O) 648.52,54.04,58.92,75.09, 123.90,141.37, 161.89.

WO 94/26313 PCT/US93/04322 Example F Preparation of 3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3. 1]pentadeca-1(15),11,13triene.

To an aqueous solution of 10 mL of 3,9-bis(sodium methylenesulfonate)-3,6,9,15tetraazabicyclo[9.3. 1 ]pentadeca-1 (1 5),11,13-triene (prepared by the procedure of Example E) was added 10 mL of 0.06 g (12.24 mmol) of NaCN. The reaction mixture was stirred for 3 hrs at room terr.Perature. The solution had a pH of about 10. Upon adjustment of the pH to greater than 13 by with concentrated NaOH, the product precipitated, was extracted with chloroform (3 x 20 mL), dried over magnesium sulfate, and filtered. Upon removal of the solvent and concentration in vacuo, the desired product was isolated as a waxy white powder, 1.00 g and further characterized by: 'H NMR(CDCI 3 8 2.03 (s br, 4H), 2.64 4H), 3.82 4H), 3.90 4H), 7.14 2H), 7.62 1H); and 3 C NMR(CDC3) 8 46.64, 52.89, 60.78, 115.31, 122.02, 137.57, 157.33.

Example G Preparation of 3,6,9,15-tetraazabicyclo[9.3. 1 ]pentadeca- 1(15), 11,13-triene-3,9dimethylenenitrile-6-(2-methoxy-5-nitrophenyl)methy acetate.

To 7 mL of a THF solution of 200 mg (0.73 mmol) of 3,6,9,15tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,9-dimethylenenitrile (prepared by the procedure of Example F) was added 223 mg (0.73 mmol) of nitrophenyl)methyl acetate. The resulting solution was stirred at room temperature for 12 hrs.

To the reaction mixture was added 100 mg of K 2

CO

3 and the mixture stirred for an additional 2 hrs. The reaction mixture was then filtered and the filtrate concentrated in vacuo. The resulting crude product was then purified by column chromatography (silica gel,

CH

3 0H/CHC13).

Example H Preparation of 3,6,9,15-tetraazabicyclol9.3. lpentadeca-1(15),11,13-triene-3,9-acetic acid-6-(2acid.

3,6,9,15-Tetraazabicyclo[9.3.1 ]pentadeca-1 (15),11,13-triene-3,9dimethylenen;trie-6-(2-methoxy-5-nitrophenyl)methyl acetate (prepared by the procedure of Example G) was stirred for 12 hrs at reflux in 6N HC. The solution was then cooled and concentrated in vacuo. The residue was then dissolved in water and lyophilized to give the desired product.

Example

I

Preparation of 3,Sl-diacetic acid-3,6,9,1 5-tetraazabicyclo[9.3. I )pentadeca-1(15),11,13-triene (PC2A).

49 A concentrated aqueous solution of 30mL of HCI and mg (2.5 mmol) of 3,9bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3. 1]pentadeca-1(15),11,13-triene (prepared by the procedure of Example F) was heated at reflux for 2 hrs. After cooling, the aqueous solution was evaporated to dryness, followed by coevaporation with deionized water (2 x 10 mL) to eliminate excess HC1. The pH of the reaction mixture was adjusted to 7 with concentrated NaOH. The resulting neutral solution chromatographed on cation exchange (SP-Sepharose") column (1.5x50cm), elution with first deionized water, then with IM HCI. The acidic fraction containing product was evaporated to dryness, followed by coevnporatiun with deionized water (3x10mL) to eliminate excess HCI. The final product was isolated as a white solid upon freeze drying c' 'he concentrated aqueous solution, and characterised by: 1H NMR (D 2 0) 2.84 (s br, 4H), 3.18 4H), 3.77 4H), 4.35 4H), 7.63 2H), 8.23 1H); and 1 3 C NMR (D 2 0) S47.45, 54.33, 59.73, 60.36, 127.20, 149,31, 155.60, 177.74.

Example J Example of a procedure for preparation of a complex Preparation of the complex of 15'? -3,6,9,15-tetraazabicyclo[9.3.1]pentadeca- 1(15),11,13-triene-3,6,9-trimethylenecarbox1 acid 153 Sm-PCTA).

A solution of the ligand of Example D was prepared by dissolving 3.8mg of ligand/0.517mL of deionized water A 1:1 ligand/metal complex was then prepared by combining 401IL of the ligand solution with 2mL of aqueous SmCl 3

.H

2 0 (3x10- 4 M in 0.01N HC1) containing tracer 1 53 SmC1 3 After thorough mixing, the percent metal as a complex was determined by passing a sample of the complex solution through a Sephadex"' column, eluting with 4:1 saline (0.85% NaCl/NH 4 OH), and collecting 2x3mL fractions. The amount of radioactivity in the combined elutions was then compared with that left on the resin. Under these conditions, complex was removed with the eluent and non-complexed metal is retained on the resin. By this complexation was determined to be 92%. A sample of the solution that was passed through the resin was used for pH studies. The pH stability was then determined using the General Procedure above.

Complexation for the title product after passing through the resin was determined to be greater than 98% at the 1:1 ligand to metal ratio.

BIODISTRIBUTION

General Procedure Sprague Dawley rats were allowed to acclimate for five days then injected with 100piL of the complex solution via a tail vein. The rats weighed between 150 and 200g at the time of injection. After 30min. the rats were killed by cervical dislocation and dissected. The amount of radioactivity in each tissue was determined by counting in a Nal scintillation counter coupled to a multichannel analyzer. The counts were compared [N:\l.iBZ100684:JVR to the counts in 100iL standards in order to determine the percentage of the dose in each tissue or organ.

The percent dose in blood was estimated assuming blood to be 7% of the body weight. The percent dose in bone was estimated by multipuling the percent dose in the femur by 25. The percent dose in muscle was estimated assuming muscle to be 43% of the body weight.

In addition to organ biodistribution, chelates of the compounds of Formula were evaluated for efficiency of bone localisation since phosphonates are known for their ability to bind to hydroxyapatite.

Example I i'he percent of the injected dose of complex of Example J 153 Sm-PCTA) in several tissues are given in Table I. The numbers represent the average of 5 rates per data point.

TABLE I INJECTED DOSE IN SEVERAL TISSUES FOR Sm-PCTA o i o

I

eo sc s 1 e o Tissue Average Bone 2.77 Liver 0.80 Kidney 1.50 Spleen 0.12 Muscle 0.87 Blood 0.39 15 The bone to blood ratio dose) was 7. The bone to liver ratio was 3.5. The bone to muscle ratio was 4.8.

IMAGING EXPERIMENTS o General Procedure Injectable solutions were first prepared (0.5M) by dissolving the appropriate amount of each complex in 2mL of deionized water. The pH of the solutions were then adjusted to 7.4 using IM HC1 or NaOH as needed. The total Gd content of each solution was then determined by ICP analysis.

An anaesthetized Sprague Dawvley rat was injected intramuscularly with one of the metal solutions described above at a dose of 0.05-0.1mmol Gd/kg body weight. Images were then taken at various time intervals and compared with a non-injected control at time 0.

EXAMPLE II The Gd-PCTA complex (prepared in Example J) was rapidly taken up by the renal system with brilliant enhancement of the kidney cortex as well as peripheral kidney tissue.

C"

VT O IN:\LIBZ)006B4:JVR 51 FINAL PRODUCT EXAMPLE N N ON H-N N-H ON -N N-/ 2) NaCN NC N H H A. Preparation of 3, 9-bis (methylene nitrile)-3, 6,9, 15-t etraazabi cyclof9. 3. llpentadeca- 13-tr-iene.

An aqueous solution (2OmL) of 3,6,9, 15-tetraazabicyclo 1]pentadeca-l 11,13triene (2 grams, 9.7mmol) was stirred and the pH4 adjusted to 5 using 6N 1101. The bisulfite-formaldehyde adduct (,2.65 grams, 19, 77mmol) was added in one portion with constant stirring. The pH was maintained at 7.5 using IN KOH. When no further drop in pH4 was observed, the pH was raised to 8.5 followed by the addition of solid sodium io cyanide (1.2 grams, 24mmol) in one portion. After stirring for 18 hours at 250C the :solution was extracted with chloroform (3x5OmL), dried over anhydrous sodium sulfate, filtered. The resulting filtrate was concentrated to give the bis-nitrile as a white solid (1.82 grams, 114 NMR (CDC1 3 8 2.03 014), 5,7-cyclic, 4 H4), 2.64 (in, 0142, 4,8-cyclic 4,H4), 3,82 N-014 2 -CN, 4 14), 3.90 0142, 2,10-cyclic, 4 14), 7.14 pyH, J=7.6 H-z, 2 7.62 4-pyll. J=7.6 H4z, 1 H4); 13 CIIH} NMR (CD1 3 546.05, 46.64, 52.89, 60.78, 115.31, 122.02, 137.57, 157.33.

N N

OCH

3 NC -NN- N NN N NC NJ N 0 2 N' H 3 00 2 C N 2 H CH 3

CNIK

2 00 3

H

3 00 r B3. Preparation of 3, 9-bis (met hylene ii-e) -6-12-nethoxy-5-niitrophenyl)car-boxymethvU13, 6,9, 1S-tetr-aazabi cyclof9. 3. llpentadeca-1 13-triene.

To an acetonitrile solution (2OmL) of 3,9-bis(methylene nitrile)-3,6,9,15tetraazabicyclo 1]pentadeca-1 11,1 3-triene (900mg, 3. l6mmol) and K 2 C0 3 (900mg, 6.52mmol, 325 mesh) was added methy1[bromot(2-methoxy-5nitrophenyl)] acetate (963mg, 3. l6mmnol). The reaction was stirred for 2 hours at filtered and the filtrate concentrated in7 vacuo. The resultingr solid was dissolved in a minimum. amount of 0CHC13 and purified by column chromatograph on silica gel eluting with 5% 0143014 in 014013 (928mg, 1H NMR (ODC1 3 8 2.62-2.94 (in, C14 2 7&48-yclc,8 14), 3.69 00143. 3 11), 3.87 N-C14 2 -CN, 4 3.93 0142, IN:\UBZ100684:JVR 52 2,10-cyclic, 4 4.46 CHI, 1 6.89 3-phenyl, f=7.5 Hz, 1 7.14 pyH, fJ=7.8 Hz, 2H), 7.62 4-pyH, fJ=7.8 Hz, 1 8.16 (dd, 4-phenyl, fJ=7.5, 2.75 Hz, 1 8.34 6-phenyl, J-2.75 Hz, 1 13 NMR (CDCI 3 8 44.56, 47.97, 51.46, 51.68, 56.23, 60.02, 62.46, 110.2, 115.77, 122.46, 124.54, 125.32, 127.30, 137.47, 141.40, 156.52, 161.94, 161.94, 170.72.

N CN N C0 2

H

N-'HCIIA H0 2

CN

NC

H

3 C0 2 C N0 2 H0 2 C N0 2

H

3 CO

H

3 C0 C. Preparation of 3, 9-diacetic acid-6-[(2-rnetlzoxy-5-nzitrophenyl)acetic acid)]3, 6,9,15tetraazabicyclo[9. 3. llpentadeca-] 11, 13-triene.

A 12 N UCI solution (3OmL) of 3,9-bis(methylene nitrile)-6-[(2-methioxy-5lo 1 nitrophenyl)carboxymethiyl]3 ,6 15-tetraazabicyclo[9.3. 1]pentadeca- 1(15)411, 13-trienie (928mg, 1.S3mmol) was heated to reflux for 2 hours. Water was then added (5O.mL and the solution concentrated to near dryness. Tile residue was redissolved in water *and freeze dried to give the desired product as a cream colored solid (1.04g, 100%): 1 3 NMR (D 2 0) 5 49.10, 56.98, 58.51, 59.45, 62,42, 63.08, 115.20, 124.88, 15 125.99, 129.74, 129.96, 143.02, 143.34, 152.13, 165.63, 171.24, 176.94.

N C0 2 H N C0 2

H

-N PtO 2 p-N N-/ H0 2 C ,Nj H0 2 C ,Nj 0 NO 2 NH 2 H 3 00 2 C H2

H

3 CO

HCO

D. Preparation of 3, 9-diacetic acid-6-[('2-mnetlzoxy-S-amiinopIzeny4)acetic acid)J3, 6,9,15teti-aazabicyclo[9. 3. llpentadeca-1 11, 13-triene.

Water (50mL) was purged with N 2 for 15 minutes followed by the addition of 3,9-diacetic acid-6-[(2-methoxy-5-nitrophenyl)acetic acid)] 3,6 ,9,15 -tetraazab icyclo [9 1]pentadeca- 1(15),11,13-triene (200mg, 0.3mmol) with stirring. After complete dissolution the pH was adjusted to 9.0 using 1N KOH. PtO 2 was then added and the solution stirred under 1 atmosphere of H 2 for 6 hours. The solution was filtered through a 0.2 t filter and freeze dried to give a flocculant, off-white solid (165mg, 'H NMR (D 2 0 29-3.34 INAL IBZiOO684:JVR 53

CH

2 16 3.47 OCH 3 3 4.62 CH, 1 6.57-6.70 (2d, s, 3,4,6phenyl, 3 7.13 3,5-pyH, J=7.5 Hz, 2 7.68 4-pyH, J=7.5 Hz, 1 H).

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indic ated by the following claims.

e o o IN:\LIBZ]O0G84:JVR

Claims (20)

1. 3. A complex of claim 1 wherein A, Q and Z are CH; and X and Y are H.
2. 4. A complex of claim 1 wherein X and Y are H.
3. 5. A complex of claim 1 wherein A, Q and Y are H. 5. A complex of claim 1 wherein A, Q and Z are CH.
4. 6. A complex of claim 1 wherein Q, A and Z are CH; and one R term is R7 X Sor C 0 2 H CO 2 H R4 where: X, Y, R 2 and R 4 are defined as in claim 1.
5. 7. A complex of claim 1 wherein A is C-OR 1 C-OR 2 where R 1 and R 2 are defined as in claim 1 or -c C) R 4 where R 4 is defined as in claim 1. T [N:\LIBZ100684:SAK 'err 5-6
6. 8. A complex of claim 1 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-RS or C-CH11 2 -OR, where RI and R 5 are defined as in claim 1.
7. 9. A complex of claim 8 wherein R 5 is NHR 6 where R 6 is a biologically active material. 6 10. A conjugate comprising a bicyclopolyazamacrocyclocarboxylic acid compound of the formula ,A" QQZ R-N N-R N R wherein: is hydrogen, R7 H X X -R-4 -C-CO2H R CO, O 0 CO 2 H R4 to or C where: and Y are independently H, OH, C 1 -C 3 alkyl or COOH; R 7 is H -Ior OH; and R4 is H, NO 2 NH 2 isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; with the proviso that at least two R terms must have a CO 2 H group present A CH, N, C-Br, C-C1, C-ORI, C-OR 2 N+-R 3 or C-C=C Q) R 4 RI H, C 1 -C 5 alkyl, benzyl, or benzyl substituted with at least one R4 R 2 is C 1 -C 16 alkylamino; R 3 is C 1 -C 16 alkyl, benzyl, or benzyl substituted with at least one R4; R 4 is defined as before; X- is Cl-, Br-, I- or H 3 CC0 2 Q and Z independently are CH, N, N+-R 3 C-CH 2 -OR 1 or C-C(O)-R 5 R 1 and R 3 are defined as.above; R 5 is -O-(C 1 -C 3 alkyl), OH or NHR 6 R 6 is C 1 -C 5 alkyl or a biologically active material; IN:\LIBZ]00684:SAK I X is defined as above; and with the proviso that: a) when Q, A or Z is N or N+-R 3 then the other two groups must be CH; b) when A is C-Br, C-CI, C-OR 1 or C-OR 2 then both Q and Z must be CH; c) the sum of the R 2 R 4 and R 6 terms may not exceed one and one such group must be present; and d) only one of Q or Z can be C-C(O)-R 5 and when one of Q or Z is C-C(0)-R 5 then A must be CH; complexed with a metal ion selected from Gd+ 3 Mn+ 2 or Fe+ 3 and covalently attached to a biologically active material.
8. 11. A conjugate of claim 10 wherein the biologically active material is a dextran, a peptide, a molecule that has specific affinity for a receptor, or an antibody or antibody fragment.
9. 12. A conjugate of claim 11 wherein the antibody or antibody fragment is a 15 monoclonal antibody or fragment thereof.
10. 13. A conjugate of claim 12 wherein the antibody or antibody fragment is B72.3.
11. 14. A conjugate as claimed in any one of claims 11-13 wherein the metal ion is Gd+3 S. 15. A conjugate of claim 10 wheiein X and Y are H. 20 16. A conjugate of claim 10 wherein A, Q and Z are CH.
12. 17. A conjugate of claim 10 wherein Q, A and Z are CH; and one R term is x 0S* R CO 2 H where: X and R 4 are defined as in claim
13. 18. A conjugate of claim 10 wherein Q, A and Z are CH; and one R term is R 7 H -W CO 2 H R4 where: R 4 and R 7 are defined as in claim
14. 19. A conjugate of claim 10 wherein A is C-OR 1 C-OR 2 where R 1 and R 2 are defined as in claim 10, or C-CmC R 4 where R 4 is defined as in claim N:\LIBZ00684:SAK .s 5' 8 A conjugate of claim 10 wherein A is CH, and one of Q or Z is CH and the other is C-C(O)-R 6 where R 6 is defined as in claim
15. 21. A conjugate of claim 20 wherein R 6 is NHR 7 where R 7 is a biologically active material.
16. 22. A conjugate which comprises a bicyclopolyazamacrocyclocarboxylic acid compound complexed with a metal ion and covalently attached to a biologically active material.
17. 23. A pharmaceutical formulation comprising a complex as claimed in any one of claims 1 to 9 with a pharmaceutically-acceptable carrier.
18. 24. A pharmaceutical formulation comprising a conjugate as claimed in any one of claims 10-22 with a pharmaceutically-acceptable carrier. A method for the diagnosis of cancer in an animal which comprises administering to said animal an effective amount of the formulation of claim 23.
19. 26. A method for the diagnosis of cancer in an animal which comprises 15 administering to said animal an effective amount of the formulation of claim 24.
20. 27. A kit for use as a diagnostic agent having as an ingredient a complex as claimed in any one of claims 1-9 and a container containing at least one pharmaceutically acceptable carrier, vehicle or excipient at a physiologically acceptable pH. A process for preparing a complex as claimed in claim 1 which comprises 20 reacting a bicyclopolyazamacrocyclocarboxylic acid compound as defined in claim 1 with a metal ion selected from Gd+ 3 Mn+ 2 or Fe+ 3 under aqueous conditions at a pH from *V to 7. Dated 3 July, 1997 SThe Dow Chemical Company Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON ^[N:\LIBZT 000684: [N:\LIBZI00684:SAK INTURATIOAL SARCH EPOR intrnational 6plicatio1 No. PCT/US 93/04322 A. CLASSIFICATION OF SURJECrMA'ITER IPC 5 6K90 According to International Patent Classification (IPC) or to both national classification and [PC B3. FIELDS SEARCH-ED Minimum documnentation searched (classification system followed by classification symbols) IPC 5 A61K C07F C7 Documentation searched other than rninium documentation to the extent that such documnents are included in the fields searched Electronic data base consuilted during the international search (name of data base and, where practical, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT_________ Category Citation of document, i4th indication, where appropriate, of the relevant pas:;ages Relevant to claim No. XY EP,A,0 391 766 (GUERBET 10 October 1-30 1990 see page 2, line 3; claims Y WO,A,91 10669 (COCKBAIN J. 25 July 1-30 1991 see page 10; claims 1,2; figures IK, IN, IS, IQ XY WO,A,91 10645 (SALUTAR INC.) 25 July 1991 1-30 see page 4 see page 8; claims; figure ID XY EP,A,0 438 206 (SCHERING AG.) 24 July 1991 1-30 see page 1 page 2; claims XY EP,A,0 430 863 (SCHERING AG.) 5 June 1991 1-30 see claims; figure IC rM Further documents are listed in the continuation of box C. Mv Patent family members are listed in arnex. Specal ateonc of ite doumets t' later document published after the international filing date document derunng the general state of the art which is not or iri~toritydat ad nth pincinplec wit the ailicain t consideredl to be of particular relevance inedntiondrtnthpicplorheyudryge E' earlier document but published on or after the international W* document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority clairrs) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another 'Y document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, tuse, exhibition or document is combined itih one or more other such docu- other means ments, such combination being obvious to a person slilled document published prior to the international filing date but in the art. later than the priority date claimed "W documrent member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report January 1994 1i. 0z 91h Name slid mailing address of the ISA Authorized officer European Patent Office, P.13. 5818 PAtentlin 2 NL 2280 HV Rijswsik Tel.( +31-70) 340-204, Tx. 31 651 epcnl, B re Fax 31.70) 340-3016 B re Form PCT/ISA'2ill (tid iheet) (Juiy 1992) page 1 of 2 1NTEtNAEIONAL~ SCIt~ll II REOWI Internationa~l applicatiort No. PCT/US 93/04322 C4(Continuatuon) DOCUMENT'S CONSIDERED TO BE RELEVANT Catgoz r itton of docwmcnt, with indication, where appropriate, of the relevant pam-es Reent to claim No. EP,A,O 352 218 (SCHERING AG.) 24 January 1990 see claims JOURNAL OF HETEROCYCLIC CHEMISTRY vol. 27 1990 PROVO US pages 167 169 H. TAKALO ET AL. 'PREPARATION OF NEW MACROCYCLIC POLYAMINES CONTAINING 4-(PHENYLETHYNYL)PYRIDINE SUBUNIT.' see figure 6 TETRAHEDRON vol. 37 1981 OXFORD GB pages 767 772 H. STETTER ET AL. 'DARSTELLUNG UND KOMPLEXBILDUNG VON POLYAZACYCLOALKAN-N-ESSIGSAUREN.l see figure 19 1-30 1-30 1-8, 22-26, 29,30 Form PCT/15A1210 (contlaustion or ,eond theet) (July 1992) page 2 of 2 _I I INTERNATIONAL SEARCH REPORT International application No, PCT/ US 93/ 04322 Box I Observations where certain claims were found unsearchable (Continuation of item I of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. X Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: REMARK: Although claims 24, 25 are directed to a method of treatment of (diagnostic method practised on) the human/animal body the search has been carried out and based on the alleged effects of the compound/composition. 2. Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: In view of the large number of compounds which are designed by the general formulas of claim 1 the search has to be restricted for economic reasons. The search was limited to the compounds for which pharmacological data was given and/or mentioned in the examples. (guidelines, Part B, Chapter III, 3. ii Z3-WAH ?.4s P,7A-A1V n- 6f9 /6 V l-S>011- because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment rf any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos: 4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest The additional search fees were accompanied by the applicant's protest 0 No protest accompanied the payment of additional csarch fee. Form PCTIISA/210 (continuation of first sheet (July 1992) II(nronn1uon On V~~lt(MI r~~r nternation~1 l plication No, IPCT/US 93/04322 Patent document I Publication Patent family I Publication cited in search report date Imember(s) :E LEP-A-U391766 10-10-90 FR-A- 2644785 28-09-90 WO-A- 9021282 04-10-90 FR-A- 2660977 18-10-91 JP-T- 4506955 03-12-92 WO-A-9110669 25-07-91 AU-A- 7146391 05-08-91 EP-A- 0511275 04-11-92 JP-T- 5503519 10-06-93 WO-A-9110645 25-07-91 AU-A- 7060291 05-08-91 EP-A- 0527131 17-02-93 EP-A-0438206 24-07-91 0E-A- 4001655 25-07-91 AU-A- 6981291 22-08-91 JP-A- 5112567 07-05-93 EP-A-0430863 05-06-91. DE-A- 3938992 23-05-91 AU-A- 6685990 30-05-91 CA-A- 2030472 22-05-91 JP-A- 3246234 01-11-91 EP-A-0352218 24'-01-90 OE-A- 3825040 25-01-90 -AU-B- 637052 20-05-93 AU-A- 3827589 01-02-90 JP-A- 2104588 17-04-90 Io.. PCT/ISA/210 (patnt family azrncx) (July 1992)