CA1266864A - Radiopharmaceuticals and chelating agents useful in their preparation - Google Patents

Abstract

Abstract of the Disclosure A dihydropyridine?pyridinium salt type of redox, or chemical, delivery system for the site-specific and/or site-enhanced delivery of a radio-nuclide to the brain is provided. A chelating agent capable of chelating with a radionuclide and having a primary, secondary or tertiary amino function can be converted to the corresponding analogue in which said function is replaced with a dihydropyri-dine?pyridinium salt redox system and then complexed with a radionuclide to provide a new radiopharmaceutical that, in its lipoidal dihydropyridine form, penetrates the blood-brain barrier ("BBB") and allows increased levels of radionuclide concentration in the brain, particularly since oxidation of the dihydropyridine moiety in vivo to the ionic pyridinium salt retards elimination from the brain while elimination from the general circulation is accelerated.
This radionuclide delivery system is well suited for use in scintigraphy and similar radiographic techniques.

Description

6~

NOVEL RADIOPHARMACEUTICALS AND CHELATING
AGEN~S USEFUL IN THEIR PREPARATION

Field of the Invention The present invention relates to a dihydro-pyridine~ pyridinium salt type of redox, or chemical, delivery system for the site-specific and/or site-enhanced delivery of a radionuclide to the brain and other organs. More particularly, this invention re-lates to the discovcry that a chelating agent capable of chelating with a radionuclide and having a primary, secondary or tertiary amino function can be converted to the corresponding analogue in which said function is replaced with a dihydropyridine~ `pyridinium salt redox system and then complexed with a radio-nuclide to proYide a new radiopharmaceutical that,in its lipoidal dihydropyridine form, penetrates the blood-brain barrier ("BBB") and allows increased levels of radionuclide concentration in the brain, particularly since oxidation of the dihydropyridine moiety in vivo to the ionic pyridinium salt retards elimination from the brain while elimination from the general circulation is accelerated.

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The present radionuclide delivery system is well suited for use in scintigraphy and s;milar radio-graphic techniques.

Backqround of the Invention Radiographic techniques such as scintigraphy, and the like, find application in biological and medical procedures for diagnosis as well as research. Scinti-graphy involves the use of radiopharmaceuticals; i.e., compounds containing (or labeled with) a radioisotope (i.e. radionuclide) which upon introduction into a mammal become localized in specific organs, tissue>
or skeletal material that are sought to be imaged.
When the radiopharmaceutical is so localized, traces, plates, or scintiphotos of the existing distribution of the radionuclide may be made by various radiation detectors known in the art. The observed distribution of the localized radionuclide can then be used to detect the presence of pathological conditions, ab-normalities, and the like. Radiopharmaceuticals are thus often referred to as radiodiagnostics.
In many cases, radiopharmaceuticals are prepared using target-specific chelating agents which provide a bridge connecting a radionuclide, such as a radio-active metal like technetium-99m, or the like, and a material which will temporarily localize in the organ, tissue, or skeletal material which is to be imaged. Typical chelating agents for such purposes are: polydentate ligands that form a 1:1 or 2:1 ligand:
radioactive metal complex; macrocyclic ligands of lZ6~

appropriate ring size and preferably where all coordina-ting atoms are in a planar configuration; and bicyclic or polycyclic ligands that can encapsulate the radio-active metal.
It is a well established fact that the delivery of drugs, including radiopharmaceuticals, to the brain is often seriously limited by transport and metabolism factors and, more specifically, by the functional barrier of the endothelial brain capillary wall deemed the blood-brain barrier or BBB. Site-specific delivery and/or sustained delivery of drugs to the brain are even more difficult.
A dihydropyridine---' pyridinium redox system has now been successfully applied to delivery to the brain of a number o~ drugs. Generally speaking, ac-cording to this system, a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the blood-brain barrier following its systemic administration.
Subsequent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
Two maSn approaches have been used thus far for delivering drugs to the brain using this redox system.
The first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component. This approach was first applied to delivering to the brain N-methylpyridinium-2-carb-aldoxime chloride (2-PAM), the active nucleus of which constitutes a quaternary pyridinium salt, by way of the dihydropyridine latentiated prodrug form 1266~

thereof. Thus, a hydrophilic compound (2-PAM) was made 1ipoidal (i.e. lipophilic) by making its dihydro-pyridine form (Pro-2-PAM) to enable its penetration through lipoidal barriers. This simple prodrug approach allowed the compound to get into the brain as well as other organs, but this manipulation did not and could not result in any brain specificity. On the contrary, such approach was delimited to relatively small molecule quaternary pyridinium ring-containing drug species and did not provide the overall ideal result of brain-specific, sustained release of the desired drug, with concomitant rapid elimination from the general circulation, enhanced drug efficacy and decreased toxicity. No "trapping" in the brain of the 2-PAM formed in situ resulted, and obviously no _ brain-specific, sustained delivery occurred as any consequence thereof: the 2-PAM was eliminated as fast from the brain as it was from the general cir-culation and other organs. Compare U.5. Patents Nos.
Z 3,929,813 and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5, pp. 685-687 (1978); Bodor et al, Science, Vol. 190 (1975), pp. 155-156; Shek, Higuchi and Bodor, J. Med. Chem., Vol. 19 (1976), pp. 113-117. A more recent extension of this approach i5 described by Brewster, Dissertation Abstracts International, Vol.
43, No. 09, March 1983, p. 2910B. See a1so Bodor et al, Science, Vol. 214, December 18, 1981, pp. 1370-1372.
The second approach for delivering drugs to the brain us~ng the redox system lnvolves the use of a pyridinium carrier chemically linked to a biologically 126~

active compound. Bodor et al, Science, Vol. 214, December 18, 1981, pp. 1370-1372, outlines a scheme for this specific and sustained delivery of drug species to the brain, as depicted in the following Scheme A:

12f~

LlNG REDUCTION ID-DHCl DELlVERY ~ / K7 ~ TO BODY \ / ELIMINATlON
lD-DHCl lD-DHCl Ih THE BRAIN IN tlRCuLAToRr SYSTE~
AND ORGANS
Kl IN VlVO K~ IN YIVO
OX I DAT I ON OX I DAT I ON

ID-aCI~ - ENZYMATIC ENZYMATIC - ID-OCl~
IN ~HE SRAIN CLEAVAGE CLEAVAGE IN CIRCULATORY SYSTE~

IDl ~ IOC]1 ID1 ~ lOC11 K3 5 ¦ 6 ~' ` , ELI~INATION

StHEME_A. BBB. BLOOD-BRAIN BARRIER

12~

According to the scheme in Science,a drug tD] is coupled to a quaternary carrier [QC]+ and the LD-QC]+ which results is then reduced chemically to the lipoidal dihydro form rD-DHC]. After administration of [D-DHC]
in vivo, it is rapidly distributed throughout the body, including the brain. The dihydro form [D-DHC]
is then in situ oxidized (rate constant, kl) (by the NAD~ NADH system) to the ideally inactive original tD-QC] quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the blood-brain barrier should prevent its elimination from in (k3+>~ k2; k3 >> k7)- Enzymatic cleavage of the [D-QC] that is "locked" in the brain effects a sustained delivery of the drug species [D], followed by its normal elimination (k5), metabolism. A properly selected carrier [QC]+ will also be rapidly eliminated from the brain (k6 ~ k2). Because of the facile elimination of [D-QC~+ from the general circulation, only minor amounts of drug are released in the body (k3 >> k4); tD] will be released primarily in the brain (k4 > k2). The overall result ideally will be a brain-specific sustained release of the target drug species. Specifically, Bodor et al worked with phenylethylamine as the drug model. That compound was coupled to nicotinic acid, then quaternized to gi~e compounds of the formula ~ CONHCH2CH2~ ~R - CH3 cr CH2 ~

126~

which were subsequently reduced by sodium dithionite to the corresponding compounds of the formula ¢;~CO~ zCN2~ (F ~ Cl~s or ~U2~3 Testing of the N-methyl derivative in vivo supported the criteria set forth in Scheme A. Bodor et al speculated that various types of drugs might possibly be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring-sub-stituted derivatives was being studied for deliveryof amino- or hydroxy1-containing drugs, including small peptides, to the brain. No other possible specific carriers were disclosed. Other reports of this ~ork with the redox carrier system have appeared in The Friday Evenin~ Post. August 14, 1981, Health Center Communciations, University of Florida, Gainesville, Florida; Chemical & Engineering News, December 21, 1981, pp. 24-25; and Science News, January 2, 1982, Vol. 121, No. 1, page 7. More recently, the present inventor has substantially extended the redox carrier system in terms of possible carriers and drugs to be delivered; see, for example International Patent Application No. PCT/US83/00725, filed by UNIVER5~TY
OF FLORIDA on May 12, 1983 and published under In-ternational Publication No. W083t03968 on November 24, 1983.

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g Nevertheless, serious need also has existed in this art for new, centrally acting drugs which can be site-specifically and sustainedly delivered to the brain, while at the same time avoiding the aforesaid noted and notable disadvantages and drawbacks associ-ated with penetration of the blood-brain barrier, with dihydropyridine latentiated prodrug forms of drug species themselves comprising a pyridinium salt active nucleus, with the necessity for introduciny critically coordinated and designed, release rate-controlling sub-stituents onto any particular drug carrier moiety, and/or with the limitation of delivery of only known drug entities. This need has led to a new approach for delivering drugs to the brain using the redox system.
This novel approach provides new derivatives of cen-trally acting amines in which a primary, secondary or tertiary amino function has been replaced with a dihy-dropyridine/pyridinum salt redox system. These new dihydropyridine analogues are characterized by the structural formula D-N ~

wherein D is the residue of a centrally acting primary, secondary or tertiary amine, and -N ~ is a radical of the formula lZ6~

~ (R)q ~ or (R)n (R)p (R)m (I) (Il) (111) (Iv) wherein the dotted line in formula (i) indicates the S presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system;
m is zero or one; n is zero, one or two; p is zero, one or two, provided that when p is one or two, each R in formula (ii) can be located on either of the two fused rings; q is zero, one, or two, provided that when q is one or two, each R in formula (iii) can be located on either of the two fused rings; and each R is independently selected from the group con-sisting of halo, Cl-C7 alkyl, Cl-C7 alkoxy, C~-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, Cl-C7 haloalkyl, Cl-C7 alkylthio, Cl-C7 alkylsulfinyl, Cl-C7 alkylsulfonyl, CH=NOR''' wherein R''' is H or Cl-C7 alkyl, and -CONR'R'' wherein R' and K'', which can be the same or different.
are each H or C~-C7 alkyl.
~ he new dihydropyridine analogues described in the preceding paragraph act as a delivery system for the corresponding quaternary compounds in vivo; the quaternary derivatives, which also are chemical inter--. :

: , ~ - . , .. '' ~ ' ,. ' ~ .
~ ~ ' lZ66~

mediates to the dihydro compounds, are pharmacologically active and are characterized by site-specific and sustained delivery to the brain when administered via the corresponding dihydropyridine form. Nevertheles,, a serious need still exists for an effective general method for the site-specific and/or sustained delivery of a desired radionuclide to the brain. It would therefore be desirable to adapt the analogue concept to the radiopharmaceutical area.

Summary of the Invention It has now been found that a chemical delivery system based upon a dihydropyridine ~ `pyridinium salt type redox system is uniquely well suited for an effective site-specific and/or sustained andlor lS enhanced delivery of a radionuclide to the brain or like organ, via novel redox system-containing radio-pharmaceuticals, and novel redox system-containing chelating agents and novel redox system-containing precursors thereto, useful in the preparation of said radiopharmaceuticals. In one aspect, the present invention thus provides novel redox system-containing chelating agent precursors having the formula ]sX~ttl) lZ66~

wherein f ,-is the residue of a chelating agent capable of chelating with a metallic radionuclide, said chelat-ing agent having at least one primary, secondary or tertiary amino functional group, said functional group being not essential for the complexing properties of said chelating agent, said residue being character-ized by the absence of at least one of said primary, secondary or tertiary amino functional groups from the chelating agent; y is 1 or 2;-N ~ is a radical of the formula ~ , ~ or (R)n ~R)p ~R)"
ta) ~b) (c) wherein n is zero, one or two, p is zero, one or two, provided that when p is one or two, each R in formula (b) can be located on either of the two fused rings;
1~ q is zero, one, or two, provided that when q is one or two, each R in formula (c) can be located on either of the two fused rings; and each R is independently se7ected from the group consisting of halo, Cl-C7 alkyl, C~-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, Cl-C7 haloalkyl, C1-C7 alkylthio, C1-C7 alkylsulfinyl, Cl-C7 alkylsufonyl, -CH=NOR''' wherein R"' is H or C1-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are each 1266B~

H or Cl-C7 alkyl; X is the anion of a pharmaceutically acceptable organic or inorganic acid; t is the valence of the acid anion; and s is a number which when multiplied by t is equal to y.
In another aspect, the present invention provides novel redox system-~ontaining chelating agents having the formula and the non-toxic pharmaceutica71y acceptable salts thereof, wherein' ,~ and y are defined as above, and -N ~ is a radical of the formula tR)q~
~R)n (R)D ~R)m wherein the dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system;
.

~.

' ~ 2 6 m is zero or one; n is zero, one or two; p is zero, one or two,provided that when p is one or two, each R in formula (ii) can be located on either of the two fused rings; q is zero, one, or two,provided that 5 when q is one or two, each R in formula (iii) can be located on either of the two fused rings; and each R is independently selected from the group consisting of halo, C1-C7 alkyl, Cl-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, Cl-C7 alkylthio, C1-C7 alkylsulfinyl, C1-C7 alkylsufonyl, -CH=NOR''' wherein R " ' is H or Cl-C7 alkyl, and -CONR'R " wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl.
In yet another aspect, the present invention provides, as an effective radionuclide delivery system, novel redox system-containing radiopharmaceuticals of the formula and the non-toxic pharmaceutically acceptable salts thereof, wherein M is a metallic radionuclide and the remaining structural variables are defined as before; in other words, (III) is the chelated, or com-plexed, counterpart of (II), formed by complexing the novel redox system-containing chelating agent of formula (Il) with a radioactive metal. When a i266~

radiopharmaceutical of formula (III) is administered, due to its lipoidal nature it readily penetrates the B~B. Oxidation of (III) in vivo affords the ~orrespondin~
pyridinium salt of the formula '` '1 ,`~ ~ sx~t (ly) wherein the structural variables are as defined above.
Bec~use of its hydrophilic, ionic nature, the formula (IV) substance is "locked-in" the brain, thus allowing radiographic imaging of the radionuclide present in the complex ~IV). There is no readily biologically cleavable bond between the redox portion of the formula (IV) complex and the radiolabeled chelate portion thereof.
Consequently, it is not expected that the quaternary "locked in" form will gradually cleave to release the redox moiety and the chelate portion of the molecule.
Rather,sustained levels of the formula (IV) quaternary will be present at the desired site.
It is generally considered most desirable, from the standpoint of patient and technician safety, to image the target area as soon as possible after ad-ministration and to use relatively short-lived radio-isotopes. Under these circumstances, or indeed even when longer lived radioisotopes are utilized, the "locked-in" quaternary ~orm is not expected to be metabolized 12t~6 or to exit the brain until after the radioactivity has decayed to a considerable extent. Thus, the present invention does not in fact provide a system for delivery and imaging of previously known radiopharmaceuticalsi by the time the present delivery system would no longer be in its "locked in" quaternary form, it would generally no longer be sufficiently radioactive for practical imaging. Thus, in contrast to the teachings of the Bodor et al publications, e.g. Bodor et al, Science, Vol. 214, December 1&, 1981, pp. 1370-1372, which emphasize the desirability of an inactive quaternary form locked in the brain, the present invention pro-vides, and indeed requires, an active quaternary form locked in the brain in order to allow effective radionuclide imaging.
Technetium-99m is a preferred radionuclide for diagnostic purposes because of its favorable radiation energy, its relatively short half-life, and the absence of corpuscular radiation, and is preferred for use in the present invention. Other radionucliaes that can be used diagnostically herein in a chelated form are cobalt-57, gallium-67, gallium-68, indium-111, indium-lllm, and the like.

Detailed Description of the Invention The following definitions are applicable:
The term "drug" as used herein means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in man or other animal.

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The expression "non-toxic pharmaceutically ac-ceptable salts" as used herein generally includes the non-toxic salts of products of the invention of structures ~Il) and (111) hereinabove formed with non-toxic, pharmaceutically acceptable inorganic or organic acids of the general formula HX. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts pre-pared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, fumaric, methanesulfonic, toluenesulfonic and the like. The expression "anion of a pharmaceutically acceptable organic or inorganic acid" as used herein, e.g. in connection with structures (I) and (IY) above, is intended to include anions of such HX acids.
The term "halo" encompasses fluoro, chl-oro, bromo and iodo.
The term "Cl-C7 alkyl" includes straight and branched lower alkyl radicals having up to seven carbon atoms. When R, R', R'' and/or R''' are Cl-C7 alkyl, they are preferably methyl or ethyl.
The term "Cl-C7 alkoxy" includes straight and branched chain 70wer alkoxy radicals having up to seven carbon atoms. Uhen R is C~-C7 alkoxy, it is preferably methoxy or ethoxy.
The term "C2-C8 alkoxycarbonyl" designates straight and branched chain radicals of the formula 12~

(Cl-C7 alkyl) - 0-C-wherein the C1-C7 alkyl group is defined as above.
When R is alkoxycarbonyl, it is preferably ethoxy-carbonyl or isopropoxycarbonyl.
S The term "C2-C8 alkanoyloxy" designates straight and branched chain radicals of the formula (Cl-C7 alkyl) -C-0-wherein the Cl-C7 alkyl group is defined as above.
When R is alkanoy10xy, it is preferably acetoxy, piva-lyloxy or isobutyryloxy.
The term "C1-C7 haloalkyl" designates straight and branched chain lower alkyl radicals having up to seven carbon atoms and bearing one or more halo substituents (F, Cl, Br or I), which can be the same or different. Preferably, when R is haloa;kyl, the group contains 1 or 2 carbon atoms and bears 1 to 3 ha10~en substituents, e.g. chloromethyl or tri-fluoromethyl.
The term "C1-C7 alkylthio" includes straight and branched chain radicals of the type (Cl-C7 alkyl) -S-wherein Cl-C7 alkyl is defined as before. When R
is alkylthio, it is preferably methylthio.

lZ~6~

The terms "C1-C7 alkylsulfinyl" and "C1-C7 alkylsulfonyll' designate radicals of the formulas (Cl-C7 alkyl) -SO-and (C1-C7 alkYl) -52 ' respectjvely, wherein C1-C7 alkyl is defined as before.
When R is alkylsulfinyl or alkylsulfonyl, methylsulfinyl and methylsulfonyl are preferred.
When R is -CH=NOR " ', it is preferably -CH=NOH
or CH=NOCH3. When R is -CONR'R'', it is preferably -CONH2 or -CON(CH3)2-In formulas (I) through (IV) hereinabove, y is preferably1; n. m, p or q is preferably one; and R is preferably located in the 3-position in structures (a), (b), (i) or (ii) and in the 4-position in structures (c) or (iii). R is preferably -CH=NOR''' or -CONR'R'' wherein R', R'' and R''' are as broadly defined here-inabove. Most preferably, R is -CONH2 or CH=NCCH3.
The expression "residue of a chelating agent capable of chelating with a metallic radionuclide, said chelating agent having at least one primary, secondary or tertiary amino functional group, said functional group being not essential for the complexing properties of said chelating agent, said residue being characterized by the absence of at least one of said primary, secGndary or tertiary amino functional groups from the chelating agent" is believed to be self-explanatory. By way of example, if a chelating agent having a primary amino function which is non-essential in terms of chelating ability can be represented by the structural formula 126~ 4 I--I1H2 ~
then-the corresponding residue could be depicted as in formulas (I) through (IY), the ring nitrogen atoms S in structures (a) through (c) and (i) through (iv) are thus located in the same position relative to the rest of the chelate structure as was the nitrogen atom of the original amino function. As a specific example, in the case of a chelating agent have the structure NH NH
O=C C=O

S S
H H

the corresponding residue would be NH NH
O=C C=O

S S
H H

.

126~

and the corresponding redox system-containing chelating agent precursor of formula (I) would have the structure CH2 - CHCH2 ~ N 3 ~ sx-t NH NH
O = C C = O Y

S S
H H

wherein y=l and -N ~ , s, X~and t are defined as with S formula (I). Similarly, when the chelating agent has the structure H3C \ IH2 ~H ~ CH3 then the corresponding residue is CH CH

H3C \ IH2 ~H ~ CH3 H3C SH HS \ CH3 ~26~;4 and the corresponding redox system-containing chelating agent precursor of formula (I) would have the structure NH NCH2CH2 ~ N ~ ~ sX t H3C \ 1 2 1 ~ CH3 y H3C SH HS \ CH3 wherein y=l and the other structural variables are defined as with formula (1).
As another example, when the chelating agent has the structure H3C \ / NH-C-NHCH3 NH2-CH=C~
~ C=N \
H3C NH-~C-NHCH3 or 1266~
^23-\ N-(CH2) ~ C=N-NH-C-NHR
C=N-NH-IC-NHR

wherein Rl, R2, R3 and R4 are each H or Cl-C3 alkyl and n' is an integer of O to 3, then the corresponding residue is H3C \ ~ NH-C-NHCH3 -CH=C
\ C=N \

or ( C H 2 ) n ~3 jS

C=N-NH-C-NHR

1266~64 respectively; and the corresponding redox system-containing chelating agent precursor of formula ~I) would have the structure _ _ S
H3C \ / NH-C-N~CH3 SX t C N+

Y s or ~ C ~ - (CH~) ~ S
C=N-NH-C-NHR

Y
respectively, wherein y=l and s, X and t are as defined with formula (I) and Rl, R2 and n' are as defined immediately aboYe.
It will be apparent from the foregoing that the exact structure of the amino function in the chelating agents is immaterial insofar as concerns the structure of the instant derivatives of formulas (I) through (IV), for in formulas (I) through (IV) the entire amino .. . .
.
, 12~6~

function in the parent chelating agents has been replaced with a dihydropyridine/pyridinium salt redox system. Thus, virtually any chelating agent capable of complexing with a radionuclide and having at least one primary, secondary or tertiary amine functional group which is non-essential in terms of chelating properties can provide the chelating a~ents residue' ,~ in the instant derivatives. Many illustrative such amine groups will be apparent to those skilled in the art;
most commonly, however, the chelating agent's func-tional group which is to be replaced with the redox system is simply an -NH2 group. And such amino gro`up can be readily introduced into the structure of a known chelating agent not already comprising same and then replaced by the instant redox system to give the desired derivatives, as described in more detail hereinbelow.
lt too will be appreciated that the radical re-presented by 2B - h~

in formulas (II) and (III) must enable the complex of formula (III) to penetrate the BBB and must also be capablè of being oxidized in vivo to the corresponding quaternary structure. The ionic entity which re-sults from such in vivo oxidation is prevented fromefflux from the brain, while elimination from the general circulation is accelerated. In contradis-tinction to the drug-carrier entities disclosed, for example, in Science, Vol. 214, December 18, 1981, 1266l3~

pp. 1370-1372, however, there is no readily metaboli-cally cleavable bond between drug and quaternary portions;
the active species delivered in the present case is the formula (IV) quaternary itself.
It will also be appreciated that a compound of formula (III) may be administered as the free base or in the form of a non-toxic pharmaceutically ac-ceptable salt thereof, i.e. a salt which can be re-presented by the formula 14 ~ ~ H ~
~o "`-- L ~r wherein M,'~ ,~. -N ~ , y and HX are defined as before;
and that, regardless of the actual form in which the compound is administered, it will be converted in vivo to a quaternary salt of formula (IV), the anion X being present in vivo. It is not necessary that the anion be introduced as part of the compound ad-ministered. Indeed, even when the compound of formula (III) is used in its salt form, the anion of the formula (IY) compound is not necessarily the same as that present in the formula (III) compound. Indeed, the exact identity of the anionic portion of the compound of formula (IY) is immaterial to the in vivo trans-formation of (III) to (IY).
Insofar as concerns the expression "said func-tional group being not essential for the complexing properties of said chelating agent", it will be ap-parent that this expression is intended to mean that any primary, secondary or tertiary amino functional group in a chelating agent which can be replaced with the instant redox system without destroying the chelating agent's ability to complex with the radionuclide is considered herein to be not essential for complexing properties. On the other hand, re-placement of an amino functional group which would lead to a redox system-containing structure which would be incapable of complexing with a radionuclide is not within the ambit of this invention.
In accord with the present invention, the sus-tained delivery of a radionuclide to the brain in sufficient concentrations for radioimaging can be effected with much lower concentrations in the peripheral circulation and other tissues. The present invention of course will allow such imaging of any other organs or glands in which sufficient radio-activity accumulates. Thus, for example, it is expect-ed that the quaternary form (IV) which is locked in the brain will be locked in the testes as well.
The novel radionuclide delivery system of this invention begins with the preparation of the novel redox system-containing chelating agent precursors of formula (1). The preparation of those precursors will be tailored to the particular chelating portion and redox portion to be combined, as well as to the presence or absence of other reactive functional groups (amino, mercapto, carboxyl, hydroxy) in either the chelating or redox portion. Typically, if such other t reactive groups are present, they are found in the chelating portion. In any event, when such groups are present and it is desired to protect them, a step that introduces appropriate protecting groups can be incorporated at a suitable stage of the synthetic pathway. Protective groups are well known in the art and include t-butoxycarbonyl for amino groups, N-methyleneacetamido for mercaptans, and N-hydroxy-succinimidyl for carboxyl groups. Acyl or carbonate groups are typically utilized to protect alcohol hydroxyls. When carbonate protecting groups are desired, the step of introducing the protecting groups will involve reacting the alcohol with a halocarbonate of the type ROCOCl or ROCOBr (formed by reaction of ROH with COC12 or COBR2), R typically being lower alkyl. For acyl protecting groups, the alcoholic hydroxyl is reacted with an acyl halide R'Cl or R'Br, R' being -COCH3 or -COC(CH3)3. Yet other reaction schemes and reactants will be readily apparent to those skilled in the art as will the appropriate means for removing such protective groups after they have achieved their function and are no longer needed.
In forming the precursors of formula ~1), at least one primary, secondary or tertiary amino functional group in a chelating agent will be replaced with -N
the hydrophilic. ionic pyridinium salt form of the dihydropyridine = pyridinium salt redox system.
It will be appreciated that by -N ~ there is intended any non-toxic redox moiety of structure (a), (b) or (c) hereinabove comprising, containing or including the pyridinium nucleus, whether or not a part of any larger basic nucleus, and whether substituted or un-1;~668~

substituted, the only criterion therefor being capacity for chemical reduction to the corresponding dihydro-pyridine form -N ~ , B~B-penetration of -N ~ and in vivo oxidation of -N ~ back to the quaternary pyridinium salt redox moiety -N ~ .
As aforesaid, the ionic pyridinium salt radio-pharmaceutical/redox entity of formula (IV) which results from in vivo oxidation of the dihydropyridine form (III) is prevented from efflux from the brain, while elimination from the general circulation is accelerated. Radioimaging of the radionuclide present in the "locked in" formula (IV) quaternary allows observation of the distribution of the localized radio-nuclide for diagnosis of pathological conditions, abnormalities, etc.
The following synthetic schemes illustrate various approaches to the preparation of the redox system-con-taining chelating agent precursors of formula (I), to the corresponding redox system-containing chelating agents of formula (II) and to the corresponding redox system-con.aining radiopharmaceuticals of formula (lII). Also shown are the corresponding "locked in"
quaternaries of formula (IV) formed by in vivo oxidatiG~
of the formula (III) chelates, said formula (IV) quater-naries being the primary localized materials whoseradionuclide content is imaged by radiation detection means.

~26~36~

PART A: SCHEME I

~C~ S2C12 O H

CN~NOH)CCOOC"H5 H2/PtO2 Cl-H3N CH2CHCOOC~H5 liC I ~C2H50H> HH3 C I

4~ Pyrldlns J~H CH'H
H3C~SkCH3 l NH3 ~CONH2 H3~ ~C~C63 \ LIAIH4 NaBH4 \~
~ , ~CONH2 ~_~CH2NH2 3~ Ca3 H3t~~ a33 , 1~66~6'~

PARI B, verslon 1: SCHEME 1, con't.
CH2HH2 , ,CH=NOCH3 ~NH NH ~CH~OCH3 H3C_lC _CH3 1 trlethyl3~lne NH qH
H3C--ISH ~~ CH3 ~ H3C--C C 'CH3 ~ZIncke reosent) Tc-99m Pertechnetote ond /
reouclng ogent, e.g. "~ reductlon, e.g No2S204, ~n ~051~ / ~Ith Ho2S204 medlum / In boslc medlum CH=NOCH3 H3~S ~Ic~ - s~ 3 < reouclng osent ~' ~r 12 ~ CH=NOCH3 3~H SH

I n vlvo 11 oxldotlon H3C>CS~ 7~H3 X-Cl'.~N~
CH=NûCH3 ~orm 'lockeo In~ broln lZ~;6~

SClll~ I con t.

PAR~ B verS!ol) 2:
tH2NH2 tH2NH2 ~ H3C ~ cll5 NH NH ~ _N N
H3C ~CH2 CH~CH3 ~ CH3-t-C~13~ H3C ¦ CH2 H3C SH SH CH3 H3C C1'3 H3C CH3 CH=NOCH3 triethYlomine H3C ~ ~ ~CH3 80 ~ 9 ~ethonol ~ C ~ ~N IN ~C\
(zincke H3C ¦ CH2 CH2 ¦ CH3 reogent) S - C C - S
H3C C~ 3 H3~ C1~3 lCIo I (1) HgC12

(2) H2S
~ '_ < CH=NOCH3 conlinue os in ~CH~N~) Cl~
13 version I NH NH
I
H3C CH2 'cH~cH3 H C' ' 5H~CH3 lHEME ~, con't.
PART B. verslon 3 CH2NH2 H C ~CH2NH2 NH NH 3 ~ N N CH
t (CH~)~CO > C I I ~C-- 3 C C,H~CH3 S~C CIH~ l`CH3 H C--g S\CH H3C CH3 H3C CH3 8 8o ~\, tONH2 l~ trIethYICr~jne~ H3C~ N~CH2 N~CONH2 ~ N02 methonol ,C I I C ~ 3 ~$ H3C ¦ CI H2 ICH2 ¦ CH3 ~2 69 H3C CH3 H3C CH3 Zincke reosent) lOb ¦ (1) HgC12 ~2) H25 ~CH2 N;~ C 1 NH NH CON~I2

3 \C C,H~ CH3 H3C ~ gH gH ~ CH3 lOC
Tc-99m Dertechnetate ond /
~ reductlon, e 9. ~Ith reduclns ogent, e.g ~ NO S O In bos~c No35204~ In ~ ~ 3 2 4 ~CH2-H C)~N/ \N~ N~TC4/Oe9denCtln9 ~ I ~CH3 CH2N/=~ H3C ~SH 5H~CH3 120 \=( tONH2 llo In ~Ivo ootlon H3C TC ~ CH3 X

CH2~

.30 form ~locked In~ broln ~2668~

PART A

NH2 -C IH2NCH2CI HCC`OC2H5 reduct I on 1 (e'~i ~lth reduct I on e . 9. H2NCH2CHCH20H
CH~NûH)CCOOC2H5 ~Ith LIAIH4 NH2 H3C _~SyCH3 ~CH20H
ocL~H ~ o ' 16 CH CH

? H3C/ \g I ~ CH3 6H ~ CH20H L ~ iSOBr2 or PBr3 H3C ~ ICH2 C,H~ CH3 ~
H3C/S~ SH~CH3 ~_<CH2Br 180 ~ H CH
11 \ H3C ~ ~ CH3 LIAIH4 I PBr3 \ 18 ~ -COOC2H5 "~ ILIAIH4 3 ~ C CH--CH3 ~ ~CH2Br H3C - \S _ S CH3 NH H
H3~ H2CH3 Ig 1266~S'~

SCHEME 2. con't.
pARI B, verslon 1:
,~CONH2 ~H2Br ONH2 ~ /--<CH2-N~

H3C ~ ~ 2 CH ~f H3C 2 Cl~:H~/
H3C--~H SH CH3 H ~ C r Tc-99m Pertechnetote ono reduclng ogent ~
medl~/ rldhctlGn, e.g.
~ meo l um H3C Tr H N3TC04/reauclng ~;cH2-N/=~coNH2 ~_ S~ ~ S--fC 3 osent H H )=~
H3C ~ ~ CH < ~~
C-2 1'2 ~h 2.2 2 lln viYo lOxla~Ton H3C~ S ~TC~ s_~CH3 H3C~ tONH2 H2N~) X

23 ~>
form ~lockeo In~ broln ~Z~6864 SCHE~E 2 con t.

PARI B. verslon 2:

NH ~NHCH20H C ~ '' CH20H CH3 H3C CH2 ~ ~ CH3 ~ CH3-C-CH3 _~ H3C I lH2 CH2 ¦ CH3 H3C ~SH SH ~ CH3 H3C CH3 H3C CH3 18a lBb O~IN2 ~PBr3 H3C ~ t ~ ~3 3C ~ _ N c~CH2Br CH3 H C ~ C I I C ~ H C ¦ CH2 CH2 ¦ CH3 H3C CH3 H3C CH3 ~ CONH2 H3C CH3 H3C CH3 lOd ~ l9a ¦ ~1) HStl2 (2) H2S
~ ~ ~- CONH2 'H2N ~ Br~
~ `
NH tlH
H3C 'H2 C ~ 3 contlnue os In verslon 1 ~ 23 H3C ~ SH SH ~ CH3 lOe ~266~6~

SCI~ 3 O~ CO H2NCH2CH2A'H2 ~;3 ~CH33 H N~
H3 ?( S--S~CH3 18~CH2CNH2 1~2COI~'H2 H ~S--S~CH~3 1~A~ H4 ~NH NCH2CH2NH2 H33CC~I_5 ,~CH3 [~XSNO2 NH NCH2CH2NH2 02N ~ 270 ~ 2~7 - .

.
' ~- , stHEME 3. con't.

~ CH=NOCH3 ~ NH NCH~CH2N ~ Cl~
NH NCH2CH2NH2 HH3CC ~ ~ CCH3 HH3C ~ ~ CCHH33 ~ H=NOCH3 S 15 3 ~ Nl trlcthyloml~ S
02N ~ ~ 2 ~ N02 methanoJ 02N ~ ~ NO

(Zlncke reogent) / removol of Drotectlng ~ grouDs and reductlon Tc-99m Dertechnetote / e.s. ~Ith No2S204 ond reduclng oqent, / ~ In boslc medlum e.s. No2S204, In boslc medlu~ /
~ ~ H=NOCH3 Co~Dlex ~Ith NH NCH2CH2N
fYStem In ;edeucdedX NoTcO4~reduClns `CH2 CH2 H3 ~ H H~ CH3 ln ~Ivo 29 ~oxla~FTon Quoternory form of rodloDhormoceut ~locked ln~ braln iZ6~i864 NH NH
H2NCH2CHCH20H O'C C~O

/ C~ C1 40 ¦(1)HOHCt!3 / IBH4 CH2 ~HCH20H CH2 - CHCH2tl ~(2)CICH2COCI / NH INH. NH NH
~CH2- ~CHCH20H ,~ ~ ~C SOC I ~ 'C C=O
r ~ ~COSNO I f CH2 CH2 H2 CH2 ~ O I l~o o~c c=o 32; ~ 33 lSOBr2 or PBr3 34 1 NOI
~<CONH2 CH2-CHCH2-N r'`~ ~ CH -CHCH Br I I ~ Br~ 1 2 1 2 CH2 - CHCH21 NH INH ~ __C~CONH2 H NIH NH NH
H2 ~H2 ~ ~ ,H2 CIH~O2 =SH2 ~SH=2 34b \ NH3 ~ CONH2 / ~
~hH2-,CHCH2-N~ X- \~ \~/
o= C-O
,H2 1H~ CH2 - CHCH2_~
NH3 1 ~ I
O'C c~o 36tX ;Br_) CH2 CH2 360(X ~l ) O=CS tS=O
~ ~ 350 126~

- 4 0 -SCHE~E 4. con~t.

~COHH2 /=~CONH2 H2-çHcH2-N 0 ~ reductlon, e.s. ~Ith H2-ÇHCH2-~
H hH ~ X- Ho2S04 ln b~s c mealu~ H hH \~==;/
0= `~0 0~ ~-0 ~H2 ~H~ H2 ~H~
H 1H ~H
36 or 360 37 \ Tc-99m Pertechnetote ¦ NoTcO4/reduclng ond reduclns osent, e.s. osent \ N2S24~ In boslc \ me~lum ~ , 38 \~

In vlvo oxldotlon ~ , ~ ~ ONH2 C~H ~ X-form ~locke~ In~ braln .
. ~, . .. .

~266~S4 H2NCH2CHCOOC2H5 11) NoHC03 CH2 - CHCOOC2H5 NH2 12) ClCH2tOCI NH NH
0=C C~0 CH2 C, H2 ~l Cl ~ ~ ~~

CH2 - ItHcooc2H5 CH2 - CH-CH0 CHz - CHCONH2 NH NH NH NH
NH NH NH3 O=ICH C=0 NoCNBH3 0=C C=0 0=C C~0 ~ ~ 2 , 2 CH2 CH2 CH2 CH2 S S SH gH
SH SH 0=C C~0 4? ~ ~ 420 ¦ LIAIH4 41 ~ NH3 CH2 - CH-CH=NH2 RH HH
CH2--CHCH2NH2 CH2--CHCH2NH2 . I
NH NH NH NH CH, CH2 0~C C~0 an~/or CH2 CH2 SH SH
C~2 CH2 ~CH2 CH2 42 SH SH SH SH
NoBH4 4,3 44 ` , ..
`:
-- ' ` ' .

1266~36~

SCHErE 5, con't.
PART B. verslon 1:

HH HH ~CH~NOCH3 CH2--CHCH2N~ C I
CH2 ~N2 ~ NO2 CH2 C 2 (Zlncke reogent) Tc-99m Pertechnetote / reductl~n, e.g.
and reauclng ogent, ~ ~Ith H~2524 e.g. No2S204, In / In bosJc boslc medJum / medlum CH=NOCH3 ~ ~oTcO4/reduclng CH2 CHCH2N~=) O y U~<CH=~IOCH3 CH2 CH~

I In vlvo oxlaotlon CH=NOCH3 CH2N~ X-form ~locked In~ broln -i~6686 SCHErE 5. con't.
PART B. verslon 2:
,~CH=NOCH3 'H2--~CHCH2HH2 CH2--CHCH2N~> Cl-NH NH ~ , CH=NOCH3 NH NH
CH2 CH2 ~ ' ~ ,0~ ~ CH2 C~ H2 ,CH2 ~H2 ~$ , N02 ~sHH2 SHH2 (Zlncke reogent) Tc-99m Pertechnetate / reductlon, e.g and rNdUSIOng Isen ~ ~Ith H2524 baslc mRd ~ medlum ~=<CH=NOCH3 p NoTcO4~reduclng CH2--CHCH2N >
C S ~ Tc _ ~ ~ ogent CH2 CH2 y ~=~ CH=:lOCH3 CH2 CH2 52 tH2N~ SH SH

l!n vlvo oxldotlon T~c CH=NOCH3 CH2N~ X-form ~ocked In~ braln SCHEME 5, con't.

PART B, verslon 3:

NH NH ,, \ C--N N--C
CH2 CH2 ~ CH3-C-CH3 3 S ~ CH;~ CH2 ~ g 3 CH2 C~ H2 CH2 CH2 SH SH

, ~CH=NOCH3 440 ~ 9 trlethylr~mine~ CH2 - CHCH2N O ~ tl-(Zlnckemethonol H3C
reogent) \ C ~ N N - C - CH3 H3C I CH2 CH2 ¦ C1~3 HgC 1 2 ~,( 2 ) H2S
CH=NOCH3 53 contlnue os In verslon 2 CH2 - CHCH2N~ ~ Cl~
tlH NH
~CH2 ,CH2 SH SH

_ 4 5 _ 3LZ6~864 ~E 6 H2HtH2CHCH20Ht-buty] chloroformote t-BOC-NHCH2CHCH20H
NH2 NH-t-BOC

tl~ t-butyl llthlun ~¦~t2) ICH2CH20H

CH2--CHCH20C~12CH20H ~ t-BOC-NHCH2CHCH20CH2CH20H
NH2 NH2 NH-t-BOC

tl ) NoHC03 t2) CICH2COCl ~ (3) HoHC03 CH2--C~HCH20CH2CH20H CH2--CHCH20CH2CH20H
NH NH r--~ HH NH
O=C C-O~ CSHo o C
CH2 CH2 ~ CH2 CH2 Cl Cl S S

[~ 58 1 SOBr2 or PBr3 CH2 - CHCH20CH2CH2H ~ Br~ CH2 - CHCH20CH2CH2Br NH NH NH NH
O=C C'O COHH2 O=C C=O
CH2 CH2 ~ C~H2 C,H2 O=C C=O < O=C C=O

~0 59 lZ66~6~

SCHEnE 6, con't.

CO~H2 CONH2 CH2 - CHcH2ocH2cH2N ~ Br CH2 - CHCH2OCH2CH2N ~ Br O-C c o O-C C~O
,CH2 ICH2CH2 CH~

O~t C~0 Tc-99m Pertechnetote / reductlon, e.g.
ond rNedUS189 a~ent ~ ~Ith N2524 boslc med ~ ~ ,ledbauslc 4 ogent CH2 - 1C-~H20~H2CH2 y ~ CNH2 ,CH2 ~CH2 CH20CH~CH2N ~ SH SH

xl ~ on < CONH2 tH20CH2CH2N;~) X-fonm ~racked In~ 3raln lZ66~6~

t-butyl chloroformote t-BOC
HOOCCH2HHCH2CH2SH ~ HOOCCH2NCH2CH2SH

~

H2NCH2CH2NHc O t-BOCH~NCH2CH2NH2 r~ NHcocH2N~H2cH2sH
HSCH~CH2CHNHCCH2NCH2CH2SH ~ S 1 O t-BOC

68 ~ ~ trlethylomlne~ ~ NCH2CH2NHC o t BOC
~ ~ NO methonol tONH~ HSCH2CH2CH~HCCH2NCH~CH2SH

~2 (Zlncke reogent) 69 ~ HCI

~NCH2CH2NHC o CONH2 HscH2cll2cHNHccH2NHcH2cH2sH

1266~64 SCHEME 7~ con't, ~NCH2CH2NHC o CONH2 HscH2cH2cHNHccH2NHcH2cH2sH

\ Tc-99m Pertechnetote reductlon, e.g. \ ond reduclna agent, e.g ~Ith H2524 \ HO2s2o4~ Tn boslc In boslc medlum ~ edlum NoTcO4/reducIng ~===\ o ogent ) ~It~ reduced form of ~ NCH2CH2NHC ~l redox system CUNH2 HscH2cH2cHNHccH2NHcH2cH2sH

72 In vlvo oxlaGtlon Ouoternory form of rodlooharmaCeutlcol ~locked In~ broln ~26686~

HOCH NHCOCH
HSCH-CNHCH2COOH 2 3 > SCH-CNHCH2COOH

NH
C-o 1 ~NH2/DCC
~ H3 Cl H3 CH2 o C-O < H2NCH2CH2NH2 SCH-,CNHCH2CNH

C~O C=O

CH=NOCH3 Cl H3 78 ~ ~ SCH,CNHCH2CNHCHCH2CH25H
~Y trlethylanlne~ CH20 C~O CH=NOCH
~ethonol I I ~ 3 2 Cl- C,'HO NHCH2CH2H~
NO2 CH3 C 1 ~
(Zlncke reogent) 79 126686'~

- 5 O-SCH'~E 8, ~on't.

reductlon, e.g. Hl th Ho2S204 In t~oslc C, H3 edlu~ Cl H3 SCHCNIIC112CHHCHCH2CH25H SCH~CHHCH2~CNHCHCH2CH25H
gH20 C~O ¢ ~tH~OCH3 gH20 C-O ~=~tH'NOCH3 C~O NHCH2CH2N~,) C-O NHCH2CH
CH3 Cl CH3 Tc-99m Dertechnetat~\ /NoTcO4/reduclng ona reauclng ogent, \ / osent e.s. No2S204, Jn t~oslc \
medlum CcrPlex Hlth technetlum, redox system In reduced form ~n vlvo ~ oxlaotlon Ouoternory form of roaloDhormoceut Icol ~locked In~ t~roln ~26~86~

PART A: SCHEtlE 9 H N~COOH NCI H2N~COOC2H5 (3,4-dl~Jnlnobenzolt otld) ~4 ~1 )NoHC03 (2)tlCH2COCI

~CS CH2 CN~OOc2 H5 ~ C 1 CH2CNH~O~C2H5 86 CCH2SC~> HH6CH~CI

¦(1 )NH3 HSCH2CNI~ CONH2 oC~CH2SH

or ~ fla8H4~ocetlc otld HscH2cH2NH ~ CH2HH2 1~66~3~,jL~

SCHE~E 9, c~n't.
PART B, ver510n 1:

HSCH2CH2~11~CH2NH2 ~H~HOc#3 CH~lOCH3 >J '~,~ Cl trlethYlrmlne~ HSCH2CH2~1H~H2-~
KHCH2CH2SH N2 NHtH2CH25H

(Z~ncke reQgent) Tt-99m Pertechnetate / rcductlon, e.s.
and reduclng Osent,/ ~Ith H2524 e.g. Ha2524~ / In boslc medlu~
In boslc ~edlr/ , ~5--~ ~ 5~ HscH2cH2NH~ ~CH=NOCH3 Tt4/rO9eUntng NHCH2CH2SH
~ < 90 CH2-N~
H=NOCH3 n a~lT

~S~s;s~
X~
CH2~
92 H~OCH3 form ~]ocked In~ broln ~266864 SCH'~,E 9, con't.
PART B, verslon 2: CH3 HSCH2CH2N~CH2NH2 ~ CH3-C-CH3--3 CH2CH2N~H2NH2 88 NHCH,CH2SH C-S 880 S--C ~ C H3 CH=NOC H3 880 ~ 9 trlethylr~mlne ~ CH2CH2N ~ O ~ CH2N` ~ Cl~
(Zlncke CH30H y reogent) , C-S

¦~1) HgC12 ~2) H2S
,CH=NOCH3 92 contJnue os In verslon 1 HSCH2CH2NH ~ CH2N ~ Cl~
NHCn Cl' Sll .

- 5 4 - 1~66864 112H~COOH ClCH20CI Clcll2~cltlH~ooH
HH2 83 NH~CHLCI

tetrobutyl~mnonlum SOC1 borohyarlde < tlC 25 ~O)--ClCH2tHH~H2oH N~2CI

o ¦~CSNO

R ~ ~\ . SOtl2 or Polymer ~--~ n ~SCH2CNH~C CC14 > ~CSCH2CHH<~CH2cl ~,CH2So~ 97 lHoCH2S~~

SHeNotH3 HStH2CN:~ ~CI HStH2CN~

rJH,oCH2SH C~CH2SH

~ H-NOCH3 Note ~ , ,0, _~CH
CH-NûCH3 N CCH2SC
~ N~ 8 ~ ~

, -.

.~66864 SCHE~IE 10. con't.

reauct I on, e. 9. H=NOCH
t~lth N2524 r C 3 ~_~CH~HOCH3 In boslC medlu~ HscH2tH~cH2A/=\) HSCH2CN~ tH2~
Cl NH~CH2SH
NHoCH2SH \ 100 / NoTcO4/reduclng Tc-3~ .,er;ecl:netote ~ / osent ona reauclns ogent, e.s.
No2S204, In boslt mealun~
o 5 ~C~5 ~0 G~

101 ~
`CH~NOCH3 ¦In vlvo oxlaotlon ~s ~s~
~ S
~ X~
CH2-N~
laZ H=HOCH3 form ~lockeo In~ broln ~zf~

SCHE~1E 11 (1 )methyl 11 thlum 02N~ CH20H (2 ) I CH2CH20H ~ 02N~CH20CH2CH20H
N2 NO, (3 ,4-d I nl t ro~en2yl Gl cohol ) lQ3 lQ4 ¦reductlon, e.g.
lSn/HCI
( 1 ) ~laHCO~
n (2) CICH~COCI
ClcH2cN~cH2ocH2cH2oH ~ (3) NoHC03 2~CH20CH2CH20H
H~CCli2CI

¦<~1SNO

Phosphorus o n trl~ronlo~
<~CSCH2Clll~{H20CH2CH20H ~0 C" ~H20CH2tH2Br ~bCH25&~) NH~CH~S

~_~COhH2 ~ H~,~

HSCH2CNI~CH20CH2CH2~ No2C03 ~-CSC112CHI~CH~OCH,CH
NH~CH2SH Br~ NHnCH25 llo lQs 5 7 ~126&i~

SCHEVE 11. con't.

HSCH2CNI~ CH20CH2CH21~j tlH~CH25H Br-\ Tc-99m Dertechnetote hlth N2S24 ~ ~ d reduclng ogent, In boslc medlum ¦ ~ boslc medlum \~
NoTcO /reduclng ~ ~
~0~ /r~ ~ tONH24 ogent > ~ s ~ ~ 5 HSCH2CNli~ CH20CH2CH2~ ~ ~0 I~H~CH25H ~) 1'1 ~ ONH2 CH20CH2CH2N~=~

In vlvo /
oxraa ~

0~s~s~

~CONH2 CH20CH2CH2N~) X

form ']ocked In' broln - 5 8 - iZf~;~86~

dl-t-butyl dlcor~onote H2N ~O~CH20H ~ ~ t-BOC-HH ~ CH20H
NH2 114 HH-t-BOC

(3,4-dl~nlnoclenZYI olcohol I (1) butyl 11 thlun DreDored for ex~DDle bY
~,tK Sn/HClJ - 9 ~ (2~ ICH2CH20H

H2R ~CH20CH2cH2oH < -t-BOC-NH ~ CH~ocH2cH2oH

NH2 105 NH-t-BOC
(I ) NoHC03 (2) CICH2COCI ~ o ~ (3) HoHC03 ~ CSHa CICH COHH~CH2ocH2cH2oH~ ~cscH2cHH~cH2ocH2cH2oH
NH,CCH2C I 106 HH,C, CH2SC

1~07 ¦ SOCI2 or polymer ~ound trlDhenYI-~ DhosDhlne/ccl4 ~> ~
L ` ~ CH20CH2cH2H~ Cl- ~ CSCH2CNH ~ CH20CH2CH2CI
HH,CCH2SC~) CONH;~ NHaCH2S

- 5 9- ~2~i686~

SCHEME 12. Con~ t .

<~ CSCH2CNH ~ CH20CH2CH2N~ C 1- HSCH~CNH ~ CH2CCH2CH2t~
NH8CH2S,t,~ CONH~ NHCCH2SH COHH2 11~ 119 Tc-99m Pertechnetote reductlon, e.g. ¦
ond reduclng osent, t(lt~ N2524~
e.s. N2524~ In In boslc medlum boslc medlun ~ , ,, N~C04/r~dUCIn9 0 S-- ~ S~ HSCH2CNH ~ CH20CH2CH2N~=~

NH,C, CH2SH CO 2 \=~

l?l ln ~
on o ~ - s - ~c~s~
o ~ CO!'H2 CH20CH2tH2N~ X

form~locked In~ broln ~Z66~364

- 6 0 -SCHE~E 1~

~ dl(tert-butyl) dlcnrbonote A
H2N~CH2HH2 ~ H2H ~CH2NH-t-BOC

(3,4-dlomlnobenzylomlne) 124 12,3 CSHa ~ ~

~tSCH2CHH~CH2NH-t-BOC CltH2tNH ~ CH2HH-t-BOC
NHCCH2SC~ HHCCH;~CI

trlfluoroacetlc acld tH-NOtH3 CSCH2Ctl ~ ~H=NOCH3 N NHCCH2SC=O
CSCH2CNH ~ CH2NH2 ~ H2 trlethylomlne > ~ 98c NH,CCH2Sb ~ N2 IZlncke reogent) reductlon, e.g. ~lth 127 9 ~ Na2S204 In baslc mealum 102 contlnue as In Scheme 10 100 1~6i~i4 ~ hus, Schemes 1 (version 1), 5 (verslons 1 and 2) and 9 (version 1) above ~llustrate typical converslon of a carboxylic acid ester group to the corresponding amide (-CONH2); reduction of the amide function to the corresponding amine (-CH2NH2); replacement of the -NH2 group with the desired quaternary function -N
utilizing a Zincke reagent; and reduction of the resultant quaternary of formula (I) to the correspond-ing dihydro of formula (II), or conversion of (I) directly to the formula (III) radiopharmaceutical.
Variations of this type of reaction sequence are shown in versions 2 and 3 of Scheme 1, version 3 of Scheme S
and version 2 of Scheme 9, in which a protecting group is introduced prior to reaction with the Zincke reagent and then removed prior to reduction of the quaternary function. In the case of the chelating agents shown in these schemes, reaction with acetone protects both the secondary amino and thiol functions by formation of thiazolidine structures so that those functions do not interfere in the reaction with the Zincke reagent.
Subsequently, the secondary amino and mercapto groups are regenerated by reacting the protected intermediate with mercuric chloride in an organic solvent ~uch as methanol. conveniently at room temperature, and then decomposing the resulting complex with hydrogen sul-fide. See, for example British Patent Specification No. 585,250, which utilizes such a procedure for the production of esters of penicillamine.
Schemes 2 (version 1), 4 and 10 above illustrate typital conversion of an alcohol (-CH20H), which may be obtained from the corresponding carboxylic acid ester, to the corresponding halide (-CH2Cl or -CH2Br);

~Z~6~36~

reaction of the halo der~vatiYe wlth the appropriate pyridine derivat~ve H-N ~ to afford the desired formula (I) quaternary, and reduction to the corres-ponding formula (II) dihydro or conversion directly to the corresponding formula (III~ radiopharmaceutical.
Schemes 4 and I0 also illustrate removal of a protect-ing group immediately after formation of the quater-nary, while version 2 of Scheme 2 illustrates intro-duction and removal of the thiazolidine protecting I0 group discussed above with respect to versions 2 and 3 of Scheme I, etc.
In Scheme 3 above, there is shown a typical method for introducting a longer alkylene chain between an atom which is involved in forming the chelate structure IS and a pendant NH2 group which is to be replaced with the quaternary structure. As depicted in this scheme, a secondary amino yroup `NH is reacted with a haloalka-mide, e.g. BrCH2CONH2, replacing the hydrogen of the `NH w1th -CH2C0NH2. Reduction of the amide affords the correspond~ng `NCH2CH2NH2 compound. That amine can then be reacted with a Zincke reagent to replace the -NH2 with N ~ , followed by reduction as in the other schemes; preferably, however, any free thiol groups are protected prior to reaction with the Zincke reagent.
Schemes 6, 11 and 12 illustrate yet other methods for lengthening the alkylene chain, the chain here being interrupted by one or more oxygen atoms. Thus, a -CH2OH group is typically converted to the corresponding lithium salt and then reacted with an iodoalkanol, e.g. ICH2CH20H, to convert the -CH20-Li~ group to a -CH2OCH2CH2OH group. [Obviously, the chain could be lengthened by utilizing a longer-chain iodoalkanol, or by repeating the two steps just described (in which case additional inServening oxygen atoms would be -63- lZ668~

introduced.)] The -CH20CH2CH20H group is then converted to the corresponding -CH20CH2CH2Br or -CH20CH2CH2Cl, which is then reacted with the selected pyridine derivative H-N ~ to form the desired quaternary salt. In the schemes shown, a protecting group is removed immediately after quaternization to afford the formula (I~ quaternary, which is subsequently reduced as in the other schemes.
In Schemes 7, 8 and 13 above, replacement of an -NH2 group with the corresponding quaternary -N ~
is shown, utilizing a Zincke reagent. Where appropriate, quaternary formation i5 followed by removal of protecting groups, as in Schemes 7 and 13. The resultant formula (I) quaternary is then reduced as shown in the other schemes.
Many of the earliest steps in the reaction schemes depicted above parallel reactions described in Frit2berg U.S. Patent No. 4,444,690. See, for example, the conversion of 14 to 15 in Scheme 2; the conversion of 16 to 32 to 33 in Scheme 4; the conversion of 15 to 40 to 41 in Scheme 5; the conversion of 56 to ~7 to 58 and the conversion of 60 to 61 in Sc'heme 6;
and so on.
Similar schemes can be shown for the preparation of the other derivatives of this invention. The steps of introducing and removing protecting groups are only included when necessary. Also, the order of steps may be altered; in particular, quaternization may occur earlier in the reaction scheme, depending of course on the particular compounds involved. Other reaction schemes, reactants, solvents, reaction con-ditions, etc. will be readily apparent to those skilled 66~

in the art. Also, insofar as concerns the quaternary derivativeS, when an anion different from that obtained is desired, the anion in the quaternary salt may be subjected to anion exchange via an anion exchange S resin or, more conveniently, by use of the method of Kaminski et al, Tetrahedron, VQ1. 34, PP. 2857-2859 (1978). According to the Kaminski et al method, a methanolic solution of an HX acid will react with a quaternary ammonium halide to produce the methyl halide and the corresponding quaternary X salt.
The processes exemplified by Schemes 1, 3, 5,

7, 8, 9 and 13 include the step of reacting a compound containing an -NH2 group with a Zincke reagent. The Zincke reaction can be used to derive the instant derivatives in which ; ~ is the residue of a primary amine, or their protected counterparts, directly from the corresponding primary amine/parent chelating agent.
However, if it j5 desired to prepare the instant derivatives in which ~ ~ is the residue of a secondary or tertiary amine, or théir protected counterparts, via the Zincke r'eaction, then one will not use the parent secondary or tertiary amine chelating agent as the starting material but would instead use the corresponding primary amine as the starting material.
2~ Alternatively, a compound of the formula / I Hal J
wherein Hal is chloro or bromo and ' ~ is the residue of the chelating agent as defined hereinbefore or its protected counterpart can be reacted with a pyridine derivative of the formula 1~6B6'~

H H
~, ~orH~, (R)n (R)p tR)~I

wherein R, n,p and q are defined with formula (1), e.g. nicotinamide, isonicotinamide, picolinamide, 3-quinolinecarboxamide, 4-isoquinolinecarboxamide or the corresponding oximes in which a -CH=NOCH3 group is present in place of the -CONH2 group of nicotinamide etc. See, for example, Schemes 2, 4, 6, 10, 11 and 12. The starting pyridine derivatives are readily available or can be prepared in known manner, e.g.
3-quinolinecarboxamide can be prepared by treating the corresponding acid with ammonia.
When a Zincke reagent is utilized in the reaction sequence, such reagent can be prepared by reacting 1-chloro-2,4-dinitrobenzene with a compound of the formula or ~ , ~R)n (R)p ~R)a wherein R, n, p and q are defined as with formula (I), to afford the corresponding Zincke reagent of the formula 6~36~

R~

C~- l Cl- Cl-~2 ~ ~2 ~ ~' N02 respect;vely. Thus, for example, the specific Z;ncke reagent depicted in Scheme 7 can be prepared by re-acting nicotinamide with l-chloro-2,4-dinitrobenzene.
See also Zincke et al, Annalen, 1904, 333, 296; Lettré
et al, Annalen, 1953, 579, 123; Keijzer et al, Heterocycles, Vol. 16, No. 10, 1981, 1687. Preferred Zincke reagents are those in which n, p or q is one and R is -CONH2 or -CH=NOCH3 and is located in the 3-position of the pyridinium or quinolinium structure or in the 4-position of the isoquinolinium structure. Typically, the Zincke reagent is reacted with the primary amine, which may be very conveniently employed in the form of its acid addition salt, in the presence of a suitable base, e.g. triethylamine, in an appropriate organic solvent, e.g. methanol, to afford the desired quaternary salt.
When a starting material of the formula ~ Hûl wherein~ ~and Hal are defined above is utilized to prepare the quaternary salt, said starting material can be prepared from the corresponding alcohol, e.g.
by methods such as those depicted in Schemes 2, 4, 6, 10, 11 or 12.

-67- 1'~6~

Reduction of the quaternary salt of formula (I) to the corresponding dihydro derivative of formula (II) can be conducted at a temperature from about -10C
to room temperature, for a period of tim~ from about lO minutes to 2 hours, conveniently at atmospheric pressure. Typically, a large excess of reducing agent is employed, e.g., a 1:5 molar ratio of reducing agent to starting compound of formula (I). The process is conducted in the presence of a suitable reducing agent, preferably an alkali metal dithionite such as sodium dithionite or an alkali metal bGrohydride such as sodium borohydride or lithium aluminum boro-hydride, in a suitable solvent. Sodium dithionite reductjOn is conveniently carried out in an aqueous solution; the dihydro product of formula (II) is usually insoluble in water and thus can be readily separated from the reaction medium. In the case of sodium boro-hydride reduction, an organic reaction medium is employed, e.g., a lower alkanol such as methanol, an aqueous alkanol or other protic solvent. More typically, however, the quaternary of formula (I) is reduced in the same reaction mixture as the reduction of the radionuclide (pre-ferably technetiùm) to an appropriate oxidation state, affording the formula (III) radiopharmaceutical in one step from the formula (I) quaternary. Further details of the one-step reduction are given hereinbelow.
It will be apparent from the foregoing that a wide variety of derivatives of formulas (I) through (IV) can be obtained in accord with this invent10n.
In a particularly preferred embodiment of this invention, however, there are provided novel chelating agent precursors of the formula 1~6t~6'~
-6~-SH HS
Rl-t ,~R2 s~x t (1 Rl \HN C-R2 wherein each Rl is independently selected from the group consisting of H and Cl-C7 alkyl, or an R1 can be combined with the adjacent ~C-Rl such that ,C

represents~C=O; each R2 is independently selected from the group consisting of H and Cl-C7 alkyl, or an R2 can be combined with the adjacent,C-R2 such ~ /R2 ~ ~
that ,C\ represents C=O; HN NH is a radical of the formula H H
H~7CH or R3 R3 R3 ol k-N~) olk-N~
wherein each R3 is independently selected from the group consisting of H and Cl-C7 alkyl, alk is a straight or branched lower alkylene group (Cl-C8) which additionally may contain 1, 2 or 3 nonadjacent oxygen atoms in the chain, and -N ~ is as defined with formula (I) hereinabove; X~ and t are as defined with formula (I); and s' is a number which when multiplied by t is equal to one. Preferably, the salts of formula (la) have the partial structure - 6 9- ~266~'}

~SH HS\ H3C /S ~ ~CH3 / Sll HS\
H2C I H3C l C ~ Cl;; H2C tH2 O~C /C=O ~ H2~ ~ CH2 or H2C ~CH2 HN~NH \HH~NH \H~ NH

or are position isomers and/or homologs of the first two partial structures shown. It is also preferred that when HN NH
HN~,~,NH ` /~
C i S R3 R3 R3 o1k-N~

then each R3 is preferably H and alk is preferably a Cl-C6 alkylene group, or a Cl-C6 alkylene group interrupted by an oxygen atom in the chain; and that when HN~ ~NH
HN~NH j S

ol k-N~

then alk is preferably a Cl-C6 alkylene group, or a Cl-C6 alkylene group interrupted by an oxygen atom in the chain. Preferred values for -N ~ in formula (~a) are as given in conjunction with formula (I) hereinabove.
Corresponding to the preferred novel chelating agent precursors of formula (Ia) are the preferred novel chelating agents of the formula ' ' ~ ' ' .: ' , _ 7 o ~;~t;6~

- SHHS
R~ /\ 'R2 Rl- C, , `R2 111 DH

wherein Rl and R2 are as defined with formula (Ia) and HN NH is a radical of the formula a ~H "Ç HI~HH
R3 R3 R3 alk-N~ olk-~10 wherein R3 and alk are as defined with formula (Ia) and -N ~ is as defined with formula (II) herein-above. Preferred compounds of formula ~IIa) are the dihydro derivatives corresponding to the pre-ferred compounds of formula (Ia).
Likewise preferred are the novel radippharma-ceuticals in which a formula (IIa) compound is chelated with a radioactive metal, especially with technetium. Especially preferred radiopharmaceuticals have the formula o 1 ~ ~ R2 wherein R1 and R2 are as defined with formula (Ia) and N N is a radical of the formula DH

- 7 1 - 126~

R~ R3 R3 ~Ik-N~) or wherein R3 and alk are as defined with formula (Ia) and -N ~ is as defined with formula (Il) herein-above; and the corresponding quaternaries, especially those of technetium, "locked in" the brain, which have the formula o R~R2 s 'X t ~ IV~

wherein Rl, R2, s', X and t are as d~fined with formula (Ia) and N ~ N is a radical of the formula `N N
1 0 R3 R3 R3 ~1 k-N~) or ~ ) ~

wherein R3 and alk are as defined with formula (Ia) and N ~ is as defined with formula (I) hereinabove.
The preferred comp1exes of formulas (IIIa) and (IVa) are those which correspond to the preferred derivatives Of formulas (Ia) and (Ila).
In order to further illustrate the present in-vention and the advantages thereof, the following specific examples are given, it being understood that same are intended only as illustrative and in no wise limitative.

:~2t;6~6 EXAMPLE

To a stirred solution containing 115.6 9 (1.6 mol) Df isobutyraldehyde 1 in 184 9 of carbon tetra-chloride are added dropwise, at 40-50C, 10~ 9 (0.8 5 mol) of 97% sulfur monochloride. The addition is carried out during a 2.5 hour period, under a nitrogen atmosphere, with occasional cooling. The solution is maintained at 30-45C, with stirring, for an ad-ditional 48 hour period, under a current of nitrogen, 10 to remove the hydrogen chloride liberated. The solution is distilled under vacuum to give 72 9 of the desired 3,4-dithia-2,2,5,5-tetramethylhexane-1,6-dione, i.e.
Compound 2 of Scheme 1. lH NMR(CDC13) C 9.1(s,2-CH0), 1.4[s,12,-C(CH3)2-]-To 10 9 (0.07 mol) of ethyl cyanoglyoxylate-2-oxime 3 are added 125 mL of absolute ethanol, 15 9 of hydrogen chloride gas and 1 9 of platinum oxide. The mixture is hydrogenated using a Parr-hydrogenation 20 apparatus. Hydrogen uptake is complete in 3 hours.
The product is removed by filtration and taken up in 75 mL of hot 95X ethanol. The ethanol solution is filtered. The filtrate is then cooled and the crystalline product which separates on standing is 25 removed by filtration. There is thus obtained ethyl 2,3-(diammonium)propionate dichloride, i.e. Compound 4 of Scheme 1. Yield 5 9 (35~), melting point 164-166C
(lit. 164.5-165C); lH NMR(D20) ~ 4.5(m,3,-NCHC0-, -OCH2CH3), 3.5(m,2,-NCH2CH-), 1.3(t,3,-OCH2CH3).

~;~66~

Procedure 1 To 1.0 g (5 mmol) of the bisaldehyde 2 is added dropwise a solution of 1.0 9 (S mmol) of the ester 4 and 0.9 mL of pyridine in 30 mL of methano~ at 0C
while under a nitrogen atmosphere. The addition takes place during a 10 minute period. The solution is then allowed to stand for 1 hour, after which time 10 mL of water is added. The solution turns turbid and warms to 26C. The solution is stirred for an additional 20 minute period, after which time the white precipitate which forms settles out of solution.
The precipitate is removed by filtration and then is taken up in chloroform. The chloroform solution is dried over sodium sulfate. Removal of the solvent and trituration of the residue with petroleum ether gives white plate-like crystals of the desired product, 5,8-diaza-1,2-dithia-6-ethoxycarbonyl-3,3,10,10-tetra-methylcyclodeca-4,8-diene, i.e. Compound S of Scheme 1, in 53% yield (1 9), melting point 98-99C. IR (thin film) 3450, 1740, 1650 cm 1; 1H NMR(CDCl3) 6.9(m,2,c-N=CH-), 3.0-4.6(m,5,-OCH2CH3, -NCH2CH-N-), 1.5[m,15,2 ,C(CH3)2, -OCH2CH3].

Procedure II

Z5 To 1.0 9 (S mmol) of the bisaldehyde 2 in 10 mL
of methanol is added dropwise l.Q g (S mmol) of the ester 4 and 1 9 (12 mmol) of sodium bicarbonate in 20 mL of a 50:50 by volume mixture of methanol and 1~66~36~

water. The mixture 1s stirred at 0C for 10 minutes, after which time 10 mL of water is added. The resultant mixture is maintained at room temperature.
wlth stirring, for 2 hours. Water is added until the white precipitate which forms separates out of solu-tion. The precipitate is removed by filtration and taken up in chloroform. Removal of the solvent by rotary evaporation affords 0.4 9 (21~ yield) of Com-pound 5, having a melting point and lH NMR spectrum identical to the product of Procedure I.
Procedure III
A solution of 8 9 of the ester 4 and 7 mL of pyridine in 200 mL of methanol is added dropwise over a two hour period to a solution of 8 9 of bisaldehyde 2 in 25 mL of methanol. The reaction mixture is cooled in an ice bath after the addition for 1 hour, then is allowed to remain at room temperature for 1 hour. The reaction mixture is then placed in a freezer (-20C) overnight. The solution is concentrated to one-third volume, water is added and the aqueous solution is extracted with cloroform. The chloroform extract is washed with saturated aqueous sodium chloride solution and dried over magneslum sulfate. Removal of the solvent leaves a viscous mass, which is dissolved in 20 mL of hexane. The hexane solution is cooled ln an acetone/dry ice bath until a white powder separates.
The product is removed by filtration and taken up in chloroform. The chloroform solution is concentrated.
~hite crystals of Compound 5 are formed on standing.
Yield 7 9, melting point 95-96C. NMR and IR as in Procedure I.

lZ~

Procedure I:
A solution of 5 9 of the ester 5 in 20 mL of tetrahydro~uran and 20 mL of aqueous ammonia is stirred at room temperature for 2 hours. after which time it is allowed to stand at room temperature for 24 hours.
Removal of solvent leaves a white powder which is removed by filtration. The product, 6-carbamoyl-5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene, l.e. Compound 6 of Scheme 1, is crystallizedfrom a mixture of isopropanol and water. Yield 4 9 (88X), melting point 181-183C. IR (KBr) 3300, 3100, 1650 cm 1; lH NMR (CDC13) ~ 7.0(m,2,-HC=N-), 6.4(~road band, 2, -CON~ ), 3.8-4.6[m,3,-NCH2-CH(N-)CO-], 1.5, 1.4[s, 12, _C(CH3)2]-Procedure 11 A solution of S g of the ester 5 in 20 mL of tetrahydrofuran, 20 mL of ethanol and 20 mL of aqueous ammonia (28X) was stirred at room temperature for 16 hours. Removal of the solvent leaves Compound 6 as a white powder, which crystallizes from toluene as white plates. Yield 4 9, melting point 193-194C. IR and NMR as ln Procedure 1.

lZ6~

EXAMPLE S

To 3.7 9 of the amide 6 in 25 mL of 95% ethanol is added 2 9 of sodium borohydride. The mixture is stirred at room temperature for 2 hours, then is heated at reflux for 2 hours. The solution is thereafter concentrated _ vacuo and water is added to precipitate the product. The white crystalline product is removed by filtration. Recrystallization from a mixture of isopropanol and water affords 6-carbamoyl-5,8-diaza-101,2-dithia-3,3,10,10-tetramethylcyclodecane, i.e.
Compound 7 of Scheme 1, as fine white needles melting at 138-139C. Yield 3 9. 1H NMR(CDCL3) ~ 2.3-4.0[m,7, -NCH2CH-N-, 2-NCH2-C(CH3)-S-], 1.8(broad band, 2, -CONH2), 1.3[m,14, C(CH3)2, -CNH-CH2-].

A solution of 1.8 9 of the amide 7 in 50 mL of dry tetrahydrofuran is added dropwise to a slurry of 1 9 of lithium aluminum hydride in 100 mL of dry tetrahydrofuran. The addition takes place over a 30 minute period. The mixture is then heated at the reflux temperature for 20 hours. At the end of that time, the reaction mixture is first cooled and then quenched with saturated Na-K tartrate solution. The aqueous phase is extracted with chloroform. The CODl-bined organic phase is then dried over sodium sulfate.Removal of the solvent by rotary evaporation affords, as a viscous oil, 5-aminomethyl-4,7-diaza-2,9-dimethyl-decane-2,9-dithiol, i.e. Compound 8 of Scheme 1; lH NMR
(CDC13) ~2.8rm,9,-NCH2CH-C(CH2)NH-, 2-NCH2-C(CH3)2S-], l.S[m,14,`C(~H3)2. -SH].

~ 6 Methoxyamine hydrochloride (5 9; 0.06 mol) is dis-solved in SO mL of methanol and the solution is neutral-ized to pH 6 with 1 M methanolic KOH. The resultant S mixture is filtered and to the filtered solution is added 3.2 9 (0.03 mol) of 3-pyridinecarboxaldehyde.
That mixture is heated at reflux for 4 hours. The methanol is evaporated and the solid is crystallized from a mixture of ethanol and water. There is thus obtained 0-methyl-3-pyridinealdoxime having the structural formula [~ CH~NOCH3 A mixture of 2.7 9 (22 mmol) of 0-methyl-3-pyridine-aldoxime and 7 9 (33 mmol) of 1-chloro-2,4-dinitrobenzene is maintained on a water bath for 1 hour while beins stirred to a red homogenous mixture. The resultant mixture is dissolved in 35 mL of methanol, treated with charcoal and filtered. The filtrate is treated successively with two 100 mL portions of ether. The ether mixture is stirred and the product is removed by filtration. Obtained in this manner is 3-[(methoxy-imino)methyl]-1-(2,4-dinitrophenyl)pyridinium chloride, i.e. the Zincke reagent identified as Compound 9 in Schemes 1, 3, S, 8, 9 and 13.

12~

A solution of 1.6 9 (5 mmol) of the Zincke reagent 9 in 2 mL of methanol is added dropwise to 2.65 9 (10 mmol) of the amine 8 in 2 mL of methanol. The reaction mixture is heated at reflux for 2 hours, after which time ether is added to precipitate the product.
Alternatively, amine 8 may be utilized as its hydro-bromide salt and the reaction may be conducted in the presence of triethylamine (5-10 mmol). There is thus obtained 1-{~2',3'-bis-{N-~(2''-mercapto-2''-methyl)propyl]amino}propyl}}-3-~(methoxyimino)-methyl]pyridinium chloride, i.e. Compound 10 of Scheme 1.

To 3.15 9 of the dialdehyde 2 is added 4.0 9 of ethylenediamine, with stirring and cooling, over a period of 10 minutes. The thick mass which results is stirred for an additional one minute period, then allowed to stand for 1 hour at room temperature and subsequently cooled for 16 hours in a freezer (-20C).
The solid is removed by filtration and washed with 500 mL of water. The white product is then taken up in chloroform and the chloroform solution is dried over sodium sulfate. Removal of the chloroform gives 2.5 9 of 5,8-diaza-1,2-dithia-3,3,10,10--tetra-methylcyclodeca-4,8-diene, i.e. Compound 24 of Scheme 3, as a white crystalline product, melting at 168-170C (lit. 162-164C, 163-166C). lH NMR(CDC13) ~ 6.9(s,2,-HC=N-), 4.2,3.0(doublet of doub1et, 2, 2-CH2-CH2), 1.40[S,6,-C(CH3)2-3. Anal. Calcd. for C10 C18N2S2: C, 52.13; H, 7.88; N, 12.16; 5, 27.83.
Found: C, 52.20; H, 7.90; N, 12.14; S, 27.74.

1i~6~

EXAMPLE lo A solution of 0.5 9 of 24 and 0.3 9 of sodium borohydride in 23 mL of ethanol is stirred at room temperature for 1 hour, then is heated at the reflux temperature for 20 minutes. Then, 10 mL of water are added and the mixture is heated for an additional 10 minutes.
The solvent is partially removed by rotary evaporation and the residue is extracted three times with 10 mL
portions of chloroform. The chloroform extract is dried over sodium sulfate and the solvent is removed by rotary evaporation. The resultant liquid solidifies on cooling. Flash chromatography (eluent hexanes/di-chloromethane/isopropanol 5 :1:1 by volume) gives 5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane, i.e. Compound 25 of Scheme 3, as a solid, melting at 52-53C. 1H NMR(CDC13) ~ 3-2.1(m,10 ring protons), 1.1,1.2(s,6 CH3, CH3).

N-(t-butoxycarbonyl), N-(2-mercaptoethyl)glycyl homocysteine thiolactone 6? is prepared as described in Examples 1 and Z of Byrne et al U.S. Patent No.
4,434,151, and is dissolved (1.0 gram; 3 millimoles) in 25 milliliters of tetrahydrofuran (THF). The resulting solution is then cooled to about 0C and ethylenediamine (1.8 grams; 30 millimoles) is added to form a new solution. The resulting new solution is maintained for about one hour. The vo1atile componentS
of the solution are thereafter removed with a rotary evaporator. n-~utanol (about 10 milliliters) is added lZ66~6~

to the "dried" solution components and the liquid components of the resulting composition are again removed by rotary evaporation. The last step is re-peated until the vapors remaining in the evaporation S vessel do not cause a moistened pH-indicator paper to indicate a basic pH valuè, thereby also indicating that the ethylendiamine has been substantially removed and that the N-(t-butoxycarbonyl), N-(2-mercaptoethyl)-glycyl N'-(2-aminoethyl)homocysteinamide, i.e. Compound 68 of Scheme 7, so obtained is substantially pure.
-A mixture of 8 9 (66 mmol) of nicotinamide and209 (99 mmol) of 1-chloro-2,4-dinitrobenzene is maintained on a water bath for one hour, with stirring. The lS red homogenous mixture which results i5 dissolved in 100 mL of methanol and decolorized with charcoal.
The filtrate is then treated with 100 mL of ether and the yellow product which separates is removed by filtration and washed with S00 mL of ether. The highly hygroscopic product, 1-(2,4-dinitrophenyl)-3-carbamoylpyridinium chloride, is the Zincke reagent 69, employed for example in Scheme 7 and version 3 of Scheme 1. lH NMR (D20) ~ 8.5-lO.O(m,7,ArH,Py-H).

, 126f~B~

The procedure of Example 8 is substantially re-peated, except that an equivalent quantity of N-tt-butoxycarbonyl), N-(2-mercaptoethyl)glycyl N'-(2-aminoethyl)homocysteine (68) is used in place of the amine 8 and an equivalent quantity of 1-(2.4-dinitro-phenyl)-3-carbamoylpyridinium chloride (69) is used in place of the Zincke reagent 9. Obtained in this manner is Compound 70 of Scheme 7.

Compound 70 (0.002 mol) is dissolved with stirring in absolute ethanol (50 milliliters) and cooled to about 0C in an ice-water bath. HCl gas is bubbled through the stirred solution for 15 minutes, and the solution is thereafter stirred for an additional 15 minutes. Diethyl ether (200 mi11iliters).is thereafter added to the solution to precipitate the salt. The precipitate is filtered and washed with diethyl ether and the solid is then dried in vacuo to provide the corresponding de-protected quaternary. Compound 71 of Scheme 7.

N-~2-(S-acetamidomethyl)mercaptopropionyl)glycyl homocysteine thiolactone (Compound 77 of Scheme 8), prepared as described in Examples 7 and 9 of 8yrne et al U.S. Patent No. 4,434,151, is suspended ~1.0 gram; 3 millimoles) in 25 milliliters of THF. The ' ,, , :
.
::
- :

~2~i6~364 resulting suspension is cooled to a temperature of about 0C in an ice-water bath, and ethylendiamine (1.8 grams; 30 millimoles) is added to form a new solution. N-[2-(acetamidomethyl)mercaptopropionyl]-glycyl N'-(2-aminoethyl)homocysteinamide, i.e. Com-pound 78 of Scheme 8, is thereafter obtained in a manner substantially similar to that described in Example 11 for the analogous compound.

The procedure of Example 8 is substantially re-peated, except that an equivalent quantity of N-~2-(acetamidomethyl)mercaptopropionyl]glycyl N'-(2-amino-ethyl)homocysteinamide (78) is used in place of the amine 8. Obtained in this manner is Compound 79 of Scheme 8.

1-{{2',3'-bis-{N-[(2''-mercapto-2''-methyl)propyl]-amino~propyl}}-3-[(methoxyimino)methyl]pyridinium chloride, i.e. Compound 10 (0.17 mmol), is dissolved in 1.0 mL of absolute ethanol and 1.0 mL of lN NaOH.
A 1.0 mL generator eluant of 99mTc04- (5 to 50 milli-Curies) in saline is added. Then, 0.5 mL of dithionite solution, prepared by dissolving 336 mg of Na2S204 per mL of 1.0 NaOH, is added and the mixture heated sufficiently to reduce both the technetium and the pyridinium salt and to form the comp7ex between the dihydropyridine-containing 7igand and the oxotechnate-99m ion. The complex so prepared, i.e. Complex 12 ~Z6~6'~

of Scheme 1, is buffered by the addition of 1.0 mL
of lN NaCl and 4.0 mL of 0.1 mL of NaH2P04, pH 4.5 buffer.

The general procedure of Example 17 can be repeated to convert Compound 20 to Complex 22; Compound 28 to Complex 30; Compound 36 to Complex 38; Compound 46 to Complex 48; Compound 50 to Complex 52; Compound 61 to Complex 63; Compound 71 to Complex 73; Compound 79 to Complex 81; Compound 89 to Complex 91; Compound 99 to Complex 101; Compound 110 to Complex 112;
Compound 118 to Complex 121; and so forth.

To a slurry of 11 9 of lithium aluminum hydride in 300 mL of dry tetrahydrofuran is added dropwise, over a 2 hour period and under an argon atmosphere, 13 9 of the amide 6 in lS0 mL of dry tetrahydrofuran. After the addition is complete, the reaction mixture is heat-ed at reflux for 30 hours, then quenched with satur-ated Na-K tartrate solution. Treatment with 3N hydro-chloric acid and then wlth saturated sodium carbonate solution, followed by filtration and extraction of the filtrate with dichloromethane affords an organic solu-tion which is dried over magnesium sulfate. Removal of the solvent affords the desired amine, Compound 8 of Scheme 1, as a viscous oil.
A sample of the free amine thus obtained is dis-solved in diethyl ether and hydrogen chloride gas is 126~;S6~

added. The white powder which separates ls removed by flltration and purified from ethanol/water to give the corresponding hydrochloride salt melting at 225-228C.
lH NMR (D20) ~ 3.3-4.2(m,9H,HCl,NH2CH2, -HCl NHC~ ), 1.5[m,12H,C(CH3)2]. Anal. Calcd. for C11H30C13N3S2-H20: C,33.63; H,8.21; N,10.69; C1,27.07; S,16.32.
Found: C,33.93; H,7.94; N,10.60; Cl,27.05; S,16.25.

A mixture of 1 9 of the amine 8, 75 mL of acetone and a catalytic amount of p-toluenesulfonic acid is heated at reflux for 24 hours. The solvent is removed by rotary evaporation and the residue is taken up in chloroform and treated successively with saturated aqueous sodium bicarbonate solution, aqueous sodium hydroxide solution (10~) and saturated aqueous sodium chlorlde solutlon. The solutlon is dried over magnes-lum sulfate. Removal of the solvent leaves a viscous mass. Thin layer chromatography (CHC13/methanol, 2:1) lndlcates two major components having Rf values of 0.13 and 0.73. The component with the lower Rf value shows a posltlve nlnhydrin test, confirming that it is the deslred prlmary amlne 8a, whlle the component with the hlgher Rf value is negative. lH NMR of the Rf 0.73 component (CDC13): ~ 2.9, 2.5, 1.3-l.S. lH NMR of the Rf 0.13 component (CDC13): ~ 3.0, 2.8, 2.3, 1.2-1.7.
Obtained in this manner is the desired bisthiazolidine primary amine, Compound 8a of Scheme 1.

, -, , lZ6~3S'~

Reaction of the bisthiazolidine primary amine 8a with the Zincke reagent 9 according to the procedure of Example 8 affords the corresponding bisthiazolidine quaternary, ;.e. Compound lOa of Scheme 1, which can then be de-protected, e.g. by reaction with mercuric chloride, followed by treatment with hydrogen sulfide, to give the unprotected quaternary, Compound 10 of Scheme 1.

Reaction of the bisthiazolidine primary amine 8a with the Zincke reagent 69 according to the procedure of Example 8 affords the corresponding bi sthiazolidine quaternary, i.e. Compound lOb of Scheme 1; removal of lS the protecting groups, e.g. by successive treatments with HgC12 and H2S, affords the corresponding unprotected quaternary, i.e. Compound lOc of Scheme 1.

A solution of 7 9 (3 mmol) of the ester 5 (prepared, for example, as described in Example 3) in S0 mL of dry tetrahydrofuran is added dropwise over a period of 1 hour to 1.8 9 (47 mmol) of lithium aluminum hydride in 200 mL of dry tetrahydrofuran. The mixture is heated at reflux for 16 hours, after which time the reaction is quenched with K-Na tartrate solution. The 1;~6~

organic phase is dried over sodium sulfate. Removal of the solvent leaves a yellow viscous mass. Yield 4 9 (65~) of the desired alcohol, Compound 18a of Stheme 2. lH NMR (CDC13) ~ 2.2-2.8, 3.5, 2.3, 1.5.

.
A solution of 2 9 of PBr3 is added at 0C to 1 9 of the alcohol 18a. The mixture is heated at reflux for 30 minutes, then treated with saturated aqueous sodium bicarbonate solution and extracted with chloro-form. The chloroform extract is dried over sodiumsulfate. Removal of the solvent in vacuo left the corresponding bromo compound, i.e. Compound 19 of Scheme 2, as a clear viscous mass.

Following the general procedure of Example 20, but substituting an equivalent quantity of the alcohol 18a in place of the amine 8, affords the bisthiazolidine alcohol, Compound 18b of Scheme 2.

Reaction of the bisthiazolidine alcohol 18b with PBr3 according to the procedure of Example 24 affords the corresponding bromo compound, i.e. Compound l9a of Scheme 2.

1266l3~;4 A solution of 17 mL of 2N lithium borohydride in tetrahydrofuran is added to 300 mL of dry tetrahydro-furan under an argon atmosphere. To that solution are added 10 9 (0.035 mol) of the ester 40 in 100 mL of dry tetrahydrofuran. The resultant cloudy solution is heated at reflux for 1.5 hours. The reaction is quenched with water and the organic phase 1s washed with saturated aqueous sodium chloride solution and dried o~er magnesium sulfate. Removal of the solvent leaves, as a white powder which is very soluble in water, the corresponding primary alcohol, Compound 32 of Scheme 4. Yield 2 9 (24~); melting point 85-90C;
lH NMR tacetone-d6) ~ 7-8, 4.15, 3.3-4Ø

To 1 9 of the alcohol 32 ln 40 mL of dry ethanol is added a solution of sodium thiobenzoate prepared from 0.2 9 of sodium in 10 mL of ethanol and 1.26 9 of th10benzoic acid in 5 mL of ethanol. The reaction mixture is stirred at room temperature for 10 minutes, then is heated at 45C for an additional 10 minutes.
The mixture becomes very thick and difficult to stir and a yellow product separates. The product, Compound 33 of Scheme 4, is removed by filtration and washed with water. Yield 1.2 9, melting point 151-152C; lH
NMR (DMS0-d6/acetone-d6) ~ 7.4-8.3, 3.85, 3.1-3.6.

-88- 12~8~4 To 50 mL of dichloromethane are added 2 9 (4.5 mmol) of the alcohol 33 and 0.35 9 (4.5 mmol~ of dry pyridine. The solut~on is cooled and 0.8 9 (6.8 mmol) of thionyl chloride in 5 mL of dichloromethane ~s added dropwise over a ten minute period. The solution is allowed to stir overnight at room temperature. Then, an add~t~onal 50 mL of dichloromethane is added and the solution is washed successively w~th 2N hydrochloric ac~d, saturated sodium b~carbonate solution and water. Drying over magnesium sulfate and removal of the solvent left a yellow solid having an Rf (CH2C12/acetone) of 0.47. Yield 1.7 9 (81.6~) of the chloro derivative, Compound 34a of Scheme 4, which melts at 129-131C.
Chelating agent precursors. chelating agents and radiopharmaceuticals within the purview of the present invention can also be prepared based on the bifunctional chelating agents of Yokoyama et al U.S. Patent No.
4,287,362. Thus, for example, Yokoyama et al's chelating agents of the formula Rl S
HOOC-C - C=N-NH-C-NH-R4 R3-C=N-NH-C-NH-R4 S

wherein R1, R2, R3 and R4 are each H or Cl-C3 alkyl can be first converted to the corresponding esters (e.g. replacing -COOH with -COOC2H5), which can then 1~66~

be reduced to the corresponding alcohols (replacing -CDOC2H5 with -CH20H), which can then be converted to the corresponding -CH2Br or -CH2Cl derivatives.
which can in turn be reacted with the selected pyridine compound of the formula H-N ~ , thus replacing the halogen atom to afford the desired quaternary salt of formula (I) herein. Other process variations will be apparent from the many reaction schemes depicted hereinabove.
Another bifunctional chelating agent which can be readily converted to the redox system-containing chelating agent precursors, chelating agents and radio-pharmaceuticals of this invention is a compound of the formula ~H3 /NH-C-NHCH3 C=N S
/

NH2-CH=C
/ C=N \ S

which is also known as amino DTS and which is de-scribed in the literature, e.g. in Jap. J. Nucl. Med.
19, 610 (1982). Amino DTS can be readily converted to the derivatives of the present invention by reacting it with a Zincke reagent of the formula g o ~266~

whereln -NJ is as defined with formula (I) herein-above to afford the corresponding precursor of formula (1), which can then be utilized as generally described herein to prepare the corresponding compound of formula (Il) and radiopharmaceutlcals of formulas (Ill) and (IV). See, for example, Scheme 14 below.
Yet another group of known chelating agents which is particularly well-suited for conversion to the redox system-containing chelating agent precursors, chelatiny agents and radiopharmaceuticals of the present inven-tion can be represented by the formula ~ N- ~ CH2 ) n '4~3 S
R4 5=N_NH,C_hHR2 wherein R1, R2, R3 and R4 are each H or C1-C3 alkyl and n' is an integer of 0 to 3. See, for example, Yokoyama et al U.S. Patent No. 4,511,550 and Australian Patent No. 533,722. An especially preferred chelating agent encompassed by this group is known as amino-PTS, or AEPM, and has the structure H2N-CH2CH2 ~ C=N-NH-C-HHCH3 Ç=N-NH-,C-NHCH3 - 9l -Amino-PTS tan ~e converted to the derlvatives of the present inventlon via a Zincke reagent, as described supra in connection with amino-DTS. See, for example, Scheme lS below. The exact structure of the resultant technetium complex 136 has not been determined; it is possible that the C=N and C=S bonds are also reduced during one of the reduction steps. One possible structure for 136 is as follows:

~NH-CH-rlHCH3 ~=~N-CH2CH2 ~ ~TC6~
CH N ~ ~,5 I0 (A similar structure could be depicted for complex 131 of Scheme 14.) An alternate route to derivatives of amino PTS, amino DTS and the like is depicted in Schemes 16 and 17 below. This route can begin by utilizing known, com-mercially available pyrylium salts, e.g. Compound 138, to convert the primary amino group of amino PTS. amino DTS or the like into a pyridinium intermediate. The resultant pyridinium intermediate (e.g. 140 or 143) can then undergo nucleophilic displacement to afford the corresponding halo compound (e.g. 141 or 144). The halo derivative can then be reacted with the selected pyridine compound of the formula H-N~ , to afford the corresponding quaternary salt of formula (I) herein, which can be converted to the instant derivatives of formulas (II), (III) and (IV) as already described hereinabove.

~266~364 - 9 2 ^

~ CONH2 ~C~N ~ 3 ~ G2 (C2H5)3N > ~, ,C-N~

CH3 NH-~-NHCH3 Cl- CH3 N~-C-NHCH3

8 6,9 129 (Zlncke reogent~
lo~lno DTS, o llgand aescrlbed, for exa~P1e In JOD . I . NUC I . Med.
19, 610 (1982)1 ~
/ Tc-99m Pertechnetote reductlon, e.g / ond reduclng osent, e.g. ~Ith Ha2S O
/ No2S204, In boslc medlum I b 1 2 4i comPlex of 99mTcO CH3 Nl~-C-NHCH3 ~It reduced form CDNHj ~ , 131 NoTcO4~reducJng ~ H-CH=C

¦ In vlvo CH3 NH-C-NHCH3 oxldotlon S

Ouoternar form of rooloP~orrnoceutlcol ~locked In~ bro~n .

. . .
' : -126~;~6'~

SCHEME IS

H~N-cH2-cH2 ~-N-C-NCH3 'HCI ~pNH2 133 H3C g S H N NCol~

No' 5335172250 ona Austro~lon Potent N~2 69 ~Zlncke ~C2HS~3N~cH30H reosent) ~N-CH2CH2~ H ,S, H
Cl- H3C)= b s H
.34 / ~ S204, In Doslc / Tc-99m oertechnetote med~um k~Na2S204, Ynl609slac9emtdle 9 c~ H ,S, H
~N-CH2CH2 _~N-N-C-NCH3 reduce3 torm NTC4/roegdenlrs / 1,35 ~ N-N-C-NCH3 o~ redox system lJn vlvo oxlaotlon Ouoternary form of roaloDhonmoceutlcal ~locked ~n~ braln .

iz~

SCHEt ~ 16 ~f~
¢~ [~CNH~
H2N-CH2CH2~hl S H ~ 140 ~o~ N-N-C-NCH3 ~X

138 omlno PTS N-N-C-NCH3 (YokoyGmo et ol, U.S. N-g-C-~CH3 o pyryllum SQlt Patent No. 4 511 550) S

CONH2 ~ KBr H2NOC /--<
Br~ ~ I-CH2CH2 ~ H CS H N~ Br-cH2cH2~-h-c-NcH3 142 N-N-C-gCH3 )= H S H

\ reductlon, e.g. ~Ith / ~ 2S204, In bosic /Tc-99m Pertechnetote \ edium /ond reouclng ogent, e.g.
/ No2S204, In boslc medlum "~
~ CONH2 Comolex of 99mTcO ~ fi--~\ N-N-C-tlCH
~Ith reduced form NoTcO4/reduclng ( N-CH2CH2--(/
of redox sYstem < ogent \~ H S H

~ In vlvo oxldctlon Quoternory form of rodloDhGrmoceut Icol ~locked InU broln 1266~;4 9s ~o pyrylJum solt) (omlno DTS) H3C-h C-CH3 N N
H3C ~ ~ NH,C,NHCH3 HN NH
S=C C=S
Br-CH=C
HN NH
CsN
H3C ~ \ RHCNHCH3 H3C CH3 144 C0~H2 143 1 N ~

H2NOC H3C ~ " " NH,C,NHCH3 Br ~ -CH5C \ reductlon, e.g. wlth C=N N~S204, In boslc medlum ~3C ~ NH,C,NHCH3 ;45 S
Tc-99m Pertechnetote ond reduclng ogent, e.g.
No2S204, In boslc medlum 130 ~ , / NoTc04/reduclng ogent Complex of 93mTC
wlth reduced form ot redox system ~31 ~ L vlvo Oxldotlon Ouoternory form of rodloDhormaceut ~lockeo In~ btoln '': '''' .

lZ~ 6~

Fritzberg U.S. Patent No. 4,444,690 describes an interesting serieS of 2,3-bis(mercaptoalkanoamido)-alkanoic acid chelating agentS of the general formula ,R R' HS-CH-CO-NH-C-X
HS-CH-CO-NH-C-COOH
R R' wherein X is H or -COOH, and R and R' are H or lower alkyl, and water-soluble salts thereof, used to pre-pare the corresponding radiopharmaceuticals of the formula q ~ S ~

X R' R' C02H
wherein X iS H or -COOH, and R and R' are H or lower alkyl. The Fritzberg chelating agentS are-prepared from the corresponding 2,3-diaminoalkanoic acids by esterification With a lower alkanol Containing dry HCl, followed by treating the resultant alkyl eSter With a chloroalkanoyl chloride to form the bis(chloro-alkanoamide)ester, followed by treating that ester with ~ CSNa, followed by alkaline hydrolysis of the resultant 2,3-bis(benzoylmercaptoalkanoamido)-alkanoic acid ester to produce the 2,3-bis(mercapto-alkanoamido)alkanoic acid chelating agent. Preparation ~z~

of an analog from 3,4-diaminobenzoic acid is also disclosed by Fritzberg. Many of Fritzberg's synthetic steps can be adapted to produce the formula (I) derivatives of this invention in which, in place of the -COO~
group in Fritzberg's chel ~ ng agent, there is a -CH2N ~ X , -CH20CH2CH2N J X or like group, wherein N ~ and X~ are as define twith formula (I) hereinabove.
See, for example, Schemes 4, 5, 6 and 11 hereinabove.
Suitable nontoxic pharmaceutica7ly acceptable IO diluents or vehicles for use with the present complexes of formula (III) will be apparent to those skilled in this art. See, for example, Remington's Pharma-ceutical 5ciences, 4th Edition (1970). Obviously, the choice of suitable diluents or vehicles will depend upon the exact nature of the particular dosage form selected.
The dosage ranges for administration of the com-plexes according to this invention will vary with the size and species of the subject, the Dbjective for which the complex is administered, the particular dosage form employed, and the like, as discussed below. The quantity of given dosage form needed to deliver the desired dose of the radiopharma-ceutical, of course, depends upon the concentration Of the complex in any given pharmaceutical composi-tion/dosage form thereof and the radioactivity thereof.
3y way of example only, a 5-50 mg/kg dose of formula (III) radiopharmaceutical, injected into the tail vein or carotid vein of rats, due to the "lock in" mechanism will exhibit a very significant difference lZf~6i~

between brain and periphera1 levels of radioactivity, with consequent ready radioimaging of the brain;
imaging at approximately 60 to 90 minutes after ad-ministration will be most effective, since it will take advantage of this brain/peripheral differential.
The instant radiopharmaceuticals are generally administered intravenously. Sustained release ad-ministration, typically by slow intravenous infusion, will further enhance the site-specificity of the instant redox system. The rate of release of the formula (III) radiopharmaceutical from the sustained release system should be comparable to the rate of in vivo oxidation of the dihydro form (III) to the quaternary form (IY) in order to achieve the greatest degree of enhancement of specificity.
In a further aspect, the present invention also provides a process for the manufacture of a diagnostic agent for the visualization of an organ such as the brain. To that end, the blood-brain barrier penetrating form, formula (III), is admixed with an aqueous buffer medium having a p~ value of about 4 to about 8, pre-ferably of about 6.5 to about 7.5, in an effective radioimaging amount.
Preparation of the radiopharmaceutical can be carried out in the hospital or like location where the patient is found in order to minimize losses of radioactivity caused by the decay of radioactive metal. Inasmuch as the preparation for visualization is injectable, it must be sterile and pyrogen-free;
preferably, it is also isotonic. To this end, a so-called labeling kit car. be provided that permits a simple~ rapid and safe labeling of the solution to be lZ6f~

injected with the radioactive metal e.g., technetium-99m. Such kits are especially desirable when a short-lived radioisotope such as technetium-99m is used.
The kit includes a collecting vial for receiving and/or containing an aqueous medium in which the com-plexing reaction can be effected. Additionally. the kit includes the chelating agent of formula (II) or chelating agent precursor of formula (I) and a pharma-cologically acceptable reducing agent for reducing the radioactive element to an appropriate oxidation state for complexing with the chelating agent [and also for reducing the pyridinium moiety to the cor-responding dihydropyridine form, when a chelating agent precursor of formula (I) is present].
In the case of technetium-99m, the radioactive element is received from a radionuclide generator as an aqueous pertechnetate (Tc04) solution such as an eluate in isotonic saline, as is well-known in the art. The amount of Tc-99m required t~ produce a quantity of formula (III) radiopharmaceutical suf-ficient for diagnostic purposes is generally from 0.01 milliCurie (mCi) to about 500 mCi per mL of 99m-pertechnetate solution. The reducing agent for the pertechnetate can be a thiosulfate Or dithionite if the reducing reaction is to be carried out in a basic medium. or a tin (I~) salt such as SnC12 if the reducing reaction is to be carried out in an acid medium.
A kit for preparing an injectable radiopharma-ceutical, e.g., for complexing an organ-specific agent labeled with a radioactive metal, includes, in separate containers: (1) a biologically compatible, sterile 1266~

- 1 oo aqueous medium suitable for complex formation with a radioactive metal. (2) a dihydropyridine~~'pyridinium salt redox system-containing complexing agent of formula (I) or (II) compatible therewith, and (3) a pharma-ceutically acceptable reducing agent for the radio-active metal.
The dihydropyridine = pyridinium salt redox moiety may be present in the kit either in its oxidized or its reduced state, as desired. The reducing agent for the radioactive metal can be selected to reduce also the oxidized form of the redox moiety, if present, as the radioactive metal is reduced to form the complex preparatory to injection of the radiopharmaceutical into a test animal or a patient. In a preferred em-bodiment of this invention, a reducing agent capable of reducing both the oxidized form of the redox moiety and the radioactive metal is chosen and the chelating agent precursor of formula (I) is present in the kit.
In an especially preferred embodiment,the kit comprises, in separate containers (preferebly aspetically -and hermetically sealed vials of approximately 5-25 mL
volume), (1~ a biologically compatible, sterile aqueous medium, (2) a chelating agent precursor of formula (I), and (3) a pharmacologically acceptable re-ducing agent capable of reducing the chelating agent precursor of formula (I) to a chelating agent of formula (II) and also capable of reducing the radioactive metal to an oxidation state in which it is capable of complexing with the formula (II) chelating agent to form a radiopharmaceutical of formula (III). Most preferably, the reducing agent is sodium dithionite;

' .

~266~6~

also most preferably, the radioactive metal is technetium.
The dithionite reduction is preferably carried out in basic medium; this may be accomplished by providing that the aqueous medium (1) above is of basic pH, S or by adding an appropriate base (e.g. NaOH, Na2C03) when combining the kit components and the pertechnetate solution. As yet another alternative, the kit could comprise only two separate components: (1) the biologically compatible, sterile aqueous medium of essentially neutral pH containing the chelating agent precursor of formula (I); and (2) the reducing agent e.g. sodium dithionite or (2) the reducing agent together with ~he base, e.g. sodium dithionite and sodium carbonate.
Radioactive metal ions are typically not provided with the kit due to the relatively short half-lives of commonly utilized radionuclides. Rather, the radio-nuclide is provided separately as described ea~lier and admixed with the components of the kit shortly before use, as is known for other radiopharmaceutical delivery systems. In the case of technetium-99m, the pertechnetate solution and the basic aqueous medium may be first combined and then heated, e.g.
from 40 to 95C for 10 to 20 minutes, in the presence of the reducing agent, then cooled to about room temperature or below prior to addition of the formula (I) precursor. ln this instance, the technetium will be reduced prior to reduction of the quaternary moiety to the corresponding dihydro form,in which case a substantial portion of the quaternary salt (I) will likely chelate with the reduced technetium to form ~- .

.

lZ~;6~64 the quaternary complex (IV) in the reaction mixture as an intermediate to the dihydro complex (III), rather than the quaternary salt (I) being first converted to the dihydro chelating agent (II ) and then to the S dihydro complex (III). Alternatively, if only minimal or no heating is done, the precursor may be present in the initial mixture made from the kit, and it is likely in this instance that the formula (I) quaternary will be first reduced to the formula (II) dihydro, which will then chelate with the reduced technetium to form the complex (III). If the mixture is mildly basic, e.g. pH 8 to 9, it may be administered as is, after the reduction and chelation have occurred to form the formula (III) radiopharmaceutical, or the pH may be adjusted to about 7. If the mixture is more strongly basic, e.g. pH 13, it is generally de-sirable to adjust the pH to a slightly alkaline or neutral value.
Whatever the exact configuration of the kit, it is preferable for it to contain excess chelating agent precursor (I) or chelating agent (II) with respect to the radionuclide to be complexed therewith, e.g.
a 1:2 molar excess. The reducing agent is present in a large excess with respect to the chelating agent precursor (I), e.g. 1:5 to 1:10. When the chelating agent ~1~) rather than the precursor (I) is present, then the reducing agent is preferably present in a slight excess with respect to the radionuclide.
To effect visualization, the diagnostic agent is administered to a patient, typically intravenouslY.
with or without further dilution by a carrier vehicle ~z~

such as physiological saline, phosphate-buffered saline, plasma, or the like. Generally, the unit dose to be administered has a radioactivity of about 0.01 milliCurie (mCi) to about lO0 milliCuries, preferably S about 1 mCi to about 20 mci. The solution to be in-jected into an adult patient per unit dosage is about 0.01 milliliter (mL) to about 1 milliliter.
After intravenous administration, imaging of the organ in vivo can take place after a few minutes.
If desired, imaging can also take place hours after the injection, depending upon the half-life of the radioactive material that has been introduced into the patient and upon the amount of such material intro-duced. Preferably, imaging takes place 60 to 90 minutes after intravenous administration.
Any conventional method of imaging for diagnostic purposes can be utilized when practicing the present invention.
In summary, then, in its broadest aspects the present invention can be seen to provide compositions of matter comprising: (1) the residue of a chelating agent having at least one primary, secondary or ter-tiary amino functional group, said functional group being not essential for the complexing properties of said chelating agent, said residue being charac-terized by the absence of at least one of said primary, secondary or tertiary amino functional groups, said chelating agent being either (a) capable of chelating with a metallic radionuclide or (b) chelated with a metallic radionuclide; and (2) a dihydropyridine =
pyridinium salt redox system, which in its oxidized form comprises a radical of the formula :'- , 12~

,~ . ~ or ~n ~R~D ~R)~
bl (c) wherein n, p, q and R are as defined with formula (I) hereinabove, and which in its reduced form comprises a radical of the formula ~3, ~ (R)~ or (R)n (R)p (R~m (I) (Il) (111) ~Iv) wherein n, p, q, m and R are as defined with formula (I1) hereinabove; said redox system being directly attached to said chelating agent residue, the ring nitrogen atom of said redox system occupying the same position relative to said chelating agent residue as the position occupied by said primary, secondary or tertiary amino functional group in sa.id che7ating agent.
While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substi-tutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims.

Claims (31)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A salt having the structural formula wherein is the residue of a chelating agent capable of chelating with a metallic radionuclide, said chelating agent having at least one primary, secondary or tertiary amino functional group, said functional group being not essential for the complexing properties of said chelating agent, said residue being characterized by the absence of at least one of said primary, secondary or tertiary amino functional groups; y is 1 or 2; is a radical of the formula wherein n is zero, one or two; p is zero, one or two, provided that when p is one or two, then each R in formula (b) can be located on either of the two fused rings; q is zero, one or two, provided that when q is one or two, then each R in formula (c) can be located on either of the two fused rings; and each R is in-dependently selected from the group consisting of halo, C1-C7 alkyl, C1-C7 alkoxy. C2-C8 alkoxy C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, C1-C7 alkylsulfinyl, C1-C7 alkylsulfonyl, -CH=NOR''' wherein R''' is H or C1-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl; X- is the anion of a pharmaceuti-cally acceptable organic or inorganic acid; t is the valence of the acid anion; and s is a number which when multiplied by t is equal to y.

2. A compound having the structural formula (II) or a non-toxic pharmaceutically acceptable salt thereof.
wherein is the residue of a chelating agent capable of chelating with a metallic radionuclide, said chelating agent having at least one primary, secondary or tertiary amino functional group. said functional group being not essential for the complexing properties of said chelating agent, said residue being characterized by the absence of at least one of said primary, secondary or tertiary amino functional groups; y is 1 or 2; and is a radical of the formula (I) (II) (III) (IV) wherein the dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system;
m is zero or one; n is zero, one or two; p is zero, one or two, provided that when p is one or two, then each R is formula (ii) can be located on either of the two fused rings; q is zero, one or two, provided that when q is one or two, then each R in formula (iii) can be located on either of the two fused rings;
and each R is independently selected from the group.
consisting of halo, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, C1-C7 alkylsulfinyl, C1-C7 alkyl-sulfonyl, -CH=NOR''' wherein R''' is H or C1-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl.

3. A chemical entity as defined by Claim 1, wherein y is 1.

4. A chemical entity as defined by Claim 3, wherein n, p, q or m is one.

5. A chemical entity as defined by Claim 4, wherein R us located in the 3 position of the pyridin-ium or dihydropyridine ring, in the 3 position of the quinolinium or dihydroquinoline ring system, or in the 4 position of the isoquinolinium or either of the dihy-droisoquinoline ring systems.

6. A chemical entity as defined by Claim 4, wherein R is -CH=NOR''' wherein R''' is H or C1-C7 alkyl.

7. A chemical entity as defined by Claim 4, wherein R is -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl.

8. A chemical entity as defined by Claim 5, wherein R is -CH=NOR''' wherein R''' is H or C1-C7 alkyl.

9. A chemical entity as defined by Claim 5, wherein R is -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl.

10. A salt as defined by Claim 1, having the structural formula wherein each R1 is independently selected from the group consisting of H and C1-C7 alkyl, or an R1 can be combined with the adjacent ?C-R1 such that represents ?C=O; each R2 is independently selected from the group consisting of H and C1-C7 alkyl, or an R2 can be combined with the adjacent ?C-R2 such that represent ?C=O; is a radical of the formula wherein each R3 is independently selected from the group consisting of H and C1-C7 alkyl, alk is a straight or branched lower alkylene group which additionally may contain 1, 2 or 3 nonadjacent oxygen atoms in the chain, and is as defined in Claim 1; X-and t are as defined in Claim 1, and s' is a number which when multiplied by t is equal to one.

11. A salt as defined by Claim 10, having the structural formula wherein s', X- and t are as defined in Claim 10.

12. A salt as defined by Claim 1, having the structural formula s'X-t (Ib) wherein , X- and t are as defined in Claim 1, s' is a number which when multiplied by t is equal to one, n' is an integer of 0 to 3, and R1 and R2 are each H or C1-C3 alkyl.

13. A salt as defined by Claim 12, having the structural formula s'X-t wherein -N? , s', X and t are as defined in Claim 1.

14. A salt as defined by Claim 1, having the structural formula ;

.

15. A compound as defined by Claim 2, having the structural formula (IIc)) wherein each R1 is independently selected from the group consisting of H and C1-C7 alkyl, or an R1 can be combined with the adjacent ?C-R1 such that represents ?C=O; each R2 is independently selected from the group consisting of H and C1-C7 alkyl, or an R2 can be combined with the adjacent ?C-R2 such that represents ?C=O; is a radical of the formula wherein each R3 is independently selected from the group consisting of H and C1-C7 alkyl, alk is a straight or branched lower alkylene group which additionally may contain 1, 2 or 3 nonadjacent oxygen atoms in the chain, and is as defined in Claim 2.

16. A compound as defined by Claim 15, having the structural formula wherein is as defined in Claim 15.

17. A compound as defined by Claim 2, having the structural formula (IIb) wherein is as defined in Claim 2, n' is an integer of 0 to 3 and R1 and R2 are each H or C1-C3 alkyl.

18. A compound as defined by Claim 17, having the structural formula wherein -N? is as defined in Claim 17.

19. A compound as defined by Claim 2, having the structural formula ;

;

.

20. A kit for preparing an injectable radio-pharmaceutical comprising, in separate containers:
(1) a compound of formula (II) as defined by Claim 2, (2) a pharmaceutically acceptable reducing agent capable of reducing a radioactive metal to an oxida-tion state in which said metal is capable of complexing with said compound of formula (II); and (3) a bio-logically compatible, sterile aqueous medium.

21. A kit for preparing an injectable radio-pharmaceutical comprising. in separate containers:
(1) a salt of formula (I) as defined by Claim 1;
(2) a pharmaceutically acceptable reducing agent capable of reducing said salt to the corresponding compound of the formula (II) wherein and y are as defined in Claim 1 and is a radical of the formula (I) (II) (III) (IV) wherein the dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system; m is zero or one; n is zero, one or two, p is zero, one or two, pro-vided that when p is one or two, then each R in formula (ii) can be located on either of the two fused rings; q is zero, one or two, provided that when q is one or two, then each R in formula (iii) can be located on either of the two fused rings;
and each R is independently selected from the group consisting of halo, C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, C1-C7 alkylsulfinyl, C1-C7 alkyl-sulfonyl, -CH=NOR''' wherein R''' is H or C1-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl; said reducing agent also being capable of reducing a radio-active metal to an oxidation state in which said metal is capable of complexing with said compound of formula (II); and (3) a biologically compatible, sterile aqueous medium.

22. A kit as claimed in Claim 21, wherein the radioactive metal which said reducing agent is capable of reducing is technetium.

23. A kit as claimed in Claim 21, wherein said reducing agent is sodium dithionite.

24. A kit as claimed in Claim 21, wherein said aqueous medium is of basic pH.

25. A kit for preparing an injectable radio-pharmaceutical comprising, in separate containers:
(1) a salt of formula (1) as defined by Claim 1, in a biologically compatible, sterile aqueous medium;
and (2) a pharmaceutically acceptable reducing agent capable of reducing said salt to the corresponding compound of the formula (III) wherein and y are as defined in Claim 1 and is a radical of the formula (I) (II) (III) (IV) wherein the dotted line in formula (i) indicates the presence of a double bond in either the 4 or 5 position of the dihydropyridine ring; the dotted line in formula (ii) indicates the presence of a double bond in either the 2 or 3 position of the dihydroquinoline ring system; m is zero or one; n is zero, one or two; p is zero, one or two, provided that when p is one or two, then each R in formula (ii) can be located on either of the two fused rings;
q is zero, one or two, provided that when q is one or two, then each R in formula (iii) can be located on either of the two fused rings; and each R is in-dependently selected from the group consisting of halo. C1-C7 alkyl, C1-C7 alkoxy, C2-C8 alkoxycarbonyl, C2-C8 alkanoyloxy, C1-C7 haloalkyl, C1-C7 alkylthio, C1-C7 alkylsulfinyl, C1-C7 alkylsulfonyl, -CH=NOR''' wherein R''' is H or C1-C7 alkyl, and -CONR'R'' wherein R' and R'', which can be the same or different, are each H or C1-C7 alkyl; said reducing agent also being capable of reducing a radioactive metal to an oxidation state in which said metal is capable of complexing with said compound of formula (II).

26. A kit as claimed in Claim 25, wherein the radioactive metal which said reducing agent is capable of reducing is technetium.

27. A kit as claimed in Claim 25, wherein said reducing agent is sodium dithionite.

28. A kit as claimed in Claim 25, wherein said aqueous medium is of approximately neutral pH.

29. A kit as claimed in Claim 25, wherein (2) contains a base in addition to said reducing agent.

30. A process for preparing a salt of formula (I) as defined by Claim 1, said process comprising:
(a) reacting a primary amine of the formula wherein is as defined in Claim 1, with a Zincke reagent of the formula wherein is as defined in Claim 1; or (b) reacting a compound of the formula wherein Hal is chloro or bromo and is as defined in Claim 1, with a compound of the formula wherein R, n, p and q are as defined in Claim 1;
(c) followed by, when desired, when the residue contains protecting groups, removal of said protecting groups from the resultant salt of formula (I);
(d) followed by, when desired, exchange of the anion Cl- or Br- in the resultant salt of formula (I) for another X- anion, X- being defined as in Claim 1.

31. A process for preparing a compound of formula (II) as defined by claim 2, said process comprising reducing a salt of formula (I) as defined by Claim 1.