Methods for cancer imaging and therapy using benzamine compounds

The present invention relates to a class of compounds having affinity for certain cancer cells, e.g. lung carcinomas, colon carcinomas, renal carcinomas, prostate carcinomas, breast carcinomas, malignant melanomas, gliomas, neuroblastomas and pheochromocytomas. The compounds of the present invention can also bind with high specificity to cell surface sigma receptors and can therefore be used for diagnostic imaging of any tissue having an abundance of cells with sigma receptors. The present invention provides such compounds as agents for diagnostic imaging and for detecting and treating tumors containing the cancer cells described above.

FIELD OF THE INVENTION 
The present invention relates to a class of compounds having particular 
affinity for a specific cell surface receptor prevalent on certain cancer 
cells, e.g. lung carcinomas, malignant melanomas, gliomas, neuroblastomas, 
pheochromocytomas, colon carcinomas, renal carcinomas, breast carcinomas, 
prostate carcinomas and the like. In particular the present invention 
provides such compounds as agents for detecting and treating tumors, 
particularly tumors having cancer cells which possess a cell surface sigma 
receptor. 
BACKGROUND OF THE INVENTION 
Lung carcinomas, malignant melanomas, gliomas, neuroblastomas, 
pheochromocytomas, colon, renal, prostate and breast carcinomas are 
aggressive forms of cancer, the early detection and treatment of which are 
of paramount importance. If left undetected or untreated for several years 
or even months the median survival time of patients having these types of 
cancers is dramatically reduced. 
Of these cancers, lung cancer has lead to the highest number of fatalities. 
In 1992 alone, lung cancer caused about 165,000 deaths within the United 
States. Two major types of lung carcinomas are responsible for most of 
these deaths: small cell lung carcinomas (SCLC) and non-small cell lung 
carcinoma (NSCLC). 
SCLC is a neuroendocrine tumor that secretes several peptide growth factors 
including bombesin/gastrin releasing peptide (BN/GRP). SCLC is responsive 
to chemotherapy and radiation therapy, but relapse occurs frequently, and 
the median survival time is only about one year. 
NSCLC accounts for about 75% of all lung cancer cases and encompasses a 
variety carcinomas including adenocarcinomas, large cell carcinomas and 
squamous cell carcinomas. NSCLC tumors secrete transforming growth 
factor-alpha (TGF-.alpha.) to stimulate cancer cell proliferation. NSCLC 
is generally treated with chemotherapy and surgical resection. However the 
median survival time for patients with NSCLC is only about 5 years. 
Melanomas are among the most serious manifestations of skin cancer and lead 
to a greater number of fatalities than any other form of skin cancer. 
Melanomas can metastasize through the lymphatic system to regional nodes 
and then via the blood to secondary sites on the skin or in the liver, 
lungs and brain. Whereas the prognosis for superficial spreading melanomas 
can be quite good, there is a much poorer prognosis for nodular melanomas 
in which distant metastases frequently form. 
Breast cancer is a major cause of death for women, and estrogen receptors 
have been reported to play a major role in the development and growth of 
breast tumors. Deprivation of estrogen is one of the clinically effective 
methods for the treatment of breast cancer patients. Several growth 
factors such as insulin-like growth factor I (IGF-I), transforming growth 
factors (TGF-.alpha. and -.beta.), epidermal growth factor (EGF), and 
platelet-derived growth factors have been shown to be involved in the 
growth and progression of human breast cancer cells. Some growth factors 
such as TGF-.beta. act as inhibitors of tumor growth. Despite the 
development of numerous antiestrogen and other drugs, the clinical utility 
of antiestrogen is limited due to resistance by the tumor cells. 
Many lives could be saved if lung carcinomas, melanomas, gliomas, 
neuroblastomas, pheochromocytomas, colon, prostate and renal carcinomas 
and breast tumors were detected and treated at an early stage. Moreover 
many patients are reluctant to undergo radical surgical or broad spectrum 
chemotherapy procedures which are frequently used to treat such cancers 
since these procedures can cause disfiguration or disablement. 
Current techniques diagnose breast cancer by first identifying suspect 
tumors by single plane or 2D mammography screening. A biopsy is then 
required to differentiate tumors from other lesions. In the United States 
alone, 21 million mammographies are performed each year; 700,000 suspect 
tumors are biopsied and 182,000 women are diagnosed with breast cancer. 
This suggests that 400,000-500,000 women are subject to unnecessary biopsy 
each year. 
Accordingly an outstanding need exists for highly selective and 
non-invasive procedures permitting early detection and treatment of 
cancer. 
A variety of radiopharmaceuticals have been evaluated for diagnostic 
imaging. For example, Michelot, J. M. et al. (1991 J. Nucl. Med. 
32:1573-1580; Meyniel G. et al. (1990 C. R. Acad. Sci. Paris 311(1):13-18; 
and French Patent Publication No. 2,642,972 by Morean et al. have disclose 
.sup.123 I and .sup.125 I!N-(diethylaminoethyl)4-iodobenzamide (i.e. 
IDAB) for imaging malignant melanoma in humans. Unfortunately, the 
synthesis of IDAB is problematic and, more significantly, IDAB is taken up 
in high concentrations by non-melanoma cells in the liver and lung. 
Accordingly, IDAB does not have optimal specificity for melanoma cells and 
its uptake by non-tumor cells undermines its utility for routine screening 
of cancer. 
U.S. Pat. No. 4,279,887 to Baldwin et al., U.S. Pat. No. 5,154,913 to De 
Paulis et al. and Murphy et al. (1990 J. Med. Chem. 33:171-178) disclose 
radioiodonated benzamide compounds for use in imaging the brain only, e.g. 
.sup.123 I-N-.beta.-phenethyl-o-iodobenzamide or 
(S)-N-(1-ethyl-2-pyrrolidinyl)methyl!-2-hydroxy-3-iodo-6-methoxybenzamide 
(IBZM). However, the structure and utility of the compounds disclosed by 
Baldwin et al., De Paulis et al. and Murphy et al. is distinct from those 
provide herein. 
The present invention provides compounds which bind with high specificity 
and affinity to the cell surface of cancer cells. These compounds bind, 
for example, to receptors on the cancer cell surface. One such receptor is 
a sigma receptor. Sigma receptors are known to be present on neural 
tissues and certain immortalized neuroblastoma and glioma cell lines 
(Walker et al. 1990 Pharmacol. Reviews 42: 355-400; and Villner et al. 
1992 in Multiple Sigma and PCP Receptor Ligands: Mechanisms for 
Neuromodulation and Neuroprotection? Kamenka et al., eds. NPP Books, pp 
341-353). However, it has been surprisingly found by the present inventors 
that sigma receptors are prevalent on some types of cancer cells, e.g. 
neuroblastoma, melanoma, glioma, pheochromocytoma, colon, renal and lung 
carcinoma cells. Recently, John et al. have found that MCF-7 breast tumor 
cells express sigma receptors. (1994 J. Med. Chem. 37: 1737-1739). 
Therefore the compounds of the present invention are useful for detecting 
and treating tumors, e.g. those containing cells with sigma receptors. 
The present compounds are also useful for diagnostic imaging any tissue 
having a sigma receptor, e.g., a neural tissue such as the brain or spinal 
cord. 
SUMMARY OF THE INVENTION 
The present invention provides a method for diagnosing a mammal for the 
presence of a mammalian tumor which includes administering to a mammal a 
diagnostic imaging amount of a compound of the present invention, and 
detecting binding of the compound to a tumor in the mammal. The compounds 
of the present invention are of the general formula I. 
##STR1## 
wherein: 
X is a radionuclide; 
Z is .dbd.O or two --H; 
each R.sub.1 is independently H, halo, lower alkyl or lower alkoxy; 
R.sub.a and R.sub.b are independently H, halo, lower alkyl, lower alkoxy or 
R.sub.a and R.sub.b together with the carbon atoms to which they are 
attached form a cycloalkenyl or heterocyclic ring; 
R.sub.2 is --N(R.sub.3).sub.2 or a 5 to 7 membered nitrogen containing 
heterocyclic ring which is unsubstituted or substituted with at least one 
alkyl or substituted or unsubstituted arylalkyl substituent; 
each R.sub.3 is independently hydrogen or lower alkyl; 
j and y each are independently an integer from 0 to 6; 
q is an integer from 0 to 2; and with the proviso that the compound is not 
an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide. 
The present invention also provides a method for treating a mammalian tumor 
which includes administering to a mammal a composition including a 
tumor-inhibiting amount of a compound of formula I. 
The present invention further provides a method for diagnostic imaging of a 
mammalian tissue which has cell surface sigma receptors which includes 
administering to a mammal a diagnostic imaging amount of a compound of the 
present invention and detecting an image of a tissue having an abundance 
of cells with sigma receptors. 
A further aspect of the present invention provides a method for in vitro 
detection of a cancer cell in a mammalian tissue sample which includes 
contacting a mammalian tissue sample with an in vitro diagnostic imaging 
amount of a compound of formula I for a time and under conditions 
sufficient for binding of the compound to the cancer cell and detecting 
such binding. 
Another aspect of the present invention provides a preferred compound of 
formula I, e.g. a compound of any one of formulae II, III or IV. 
##STR2## 
wherein Z, R.sub.a, R.sub.b, R.sub.1, q, j are as described above; 
Q is a radionuclide, halide or an activating group; 
R.sub.4 is --N(R.sub.3).sub.2 or an N-linked 5 to 7 membered nitrogen 
containing heterocyclic ring which can have at least one alkyl or 
substituted or unsubstituted arylalkyl substituent, wherein each R.sub.3 
is independently lower alkyl or hydrogen; 
R.sub.5 is a 5 to 7 membered nitrogen containing heterocyclic ring which 
can have at least one alkyl or substituted or unsubstituted arylalkyl 
substituent; 
m is an integer from 0 to 6; 
n is an integer from 3 to 6. Such preferred compounds can also be used in 
the method of the present invention. 
Compositions and kits containing the present compounds are also provided 
herein.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides novel compounds and methods for detecting 
and treating certain types of cancer, e.g. neuroblastomas, gliomas, 
pheochromocytomas, melanomas, colon, renal, prostate, lung and breast 
carcinomas. The compounds of the present invention bind to a cell surface 
sigma receptor and exhibit exquisite cell specificity and affinity for the 
above cancerous cells and for cells having sigma receptors. 
In one embodiment the present invention is directed to a method for 
detecting a mammalian tumor which includes administering to a mammal a 
diagnostic imaging amount of a compound of the present invention, and 
observing retention of the compound in a tissue of the mammal; wherein the 
compound is any one of formulae I, II, III or IV; 
##STR3## 
wherein: 
X is a radionuclide; 
Q is a radionuclide, halide or an activating group; 
Z is .dbd.O or two --H; 
each R.sub.1 is independently H, halo, lower alkyl or lower alkoxy; 
R.sub.a and R.sub.b are independently H, halo, lower alkyl, lower alkoxy or 
R.sub.a and R.sub.b together with the carbon atoms to which they are 
attached form a cycloalkenyl or heterocyclic ring; 
R.sub.2 is --N(R.sub.3).sub.2 or a 5 to 7 membered nitrogen containing 
heterocyclic ring which is unsubstituted or substituted with at least one 
alkyl or substituted or unsubstituted arylalkyl substituent; 
each R.sub.3 is independently hydrogen or lower alkyl; 
R.sub.4 is --N(R.sub.3).sub.2 or an N-linked 5 to 7 membered nitrogen 
containing heterocyclic ring which can have at least one alkyl 
substituent, wherein each R.sub.3 is independently lower alkyl or 
hydrogen; 
R.sub.5 is a 5 to 7 membered nitrogen containing heterocyclic ring which 
can have at least one alkyl or substituted or unsubstituted arylalkyl 
substituent; 
j and y are independently an integer from 0 to 6; 
q is an integer from 0 to 2; 
m is an integer from 0 to 6; 
n is an integer from 3 to 6; and with the proviso that the compound is not 
an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide. 
The present invention also provides a method for treating a mammalian tumor 
which includes administering to a mammal a composition including a 
tumor-inhibiting amount of a compound of formula I, II, III or IV. 
The present invention further provides a method for diagnostic imaging of a 
mammalian tissue which has cell surface sigma receptors which includes 
administering to a mammal a diagnostic imaging amount of a compound of the 
present invention and detecting an image of a tissue having an abundance 
of cells with sigma receptors. 
The present invention further provides a method for in vitro detection of a 
cancer cell in a mammalian tissue sample which includes contacting a 
mammalian tissue sample with an in vitro diagnostic imaging amount of a 
compound of formula I for a time and under conditions sufficient for 
binding of the compound to the cancer cell and detecting such binding. 
When used for diagnostic imaging X or Q as a radionuclide is used. Moreover 
X or Q radionuclide groups which are preferably used for diagnostic 
imaging are .UPSILON.-emitting radionuclides which can be detected by 
radioimaging procedures, e.g. by scintigraphic imaging. Such 
.UPSILON.-emitting radionuclides emit radiation which is sufficiently 
penetrating to be detected through tissues. Moreover, for diagnostic 
imaging preferred radionuclides do not emit a particle, e.g. an .alpha. or 
.beta. particle. Preferred X and Q groups for diagnostic imaging include 
.sup.123 I .sup.124 I .sup.125 I .sup.131 I .sup.18 F .sup.76 Br, .sup.77 
Br, .sup.99m Tc and .sup.111 In. .sup.123 I is especially preferred for 
diagnostic imaging. 
When used for therapeutic purposes X or Q as a radionuclide is used. 
Preferably X and Q radionuclides employed for therapy are .beta.-emitting 
or an .alpha.-emitting radionuclides. However, as contemplated herein, any 
cytotoxin which exhibits a localized cell killing activity can be used in 
place of an X or Q radionuclide. The preferred X and Q groups for treating 
cancers include .sup.131 I, .sup.211 At, .sup.212 Pb, .sup.212 Bi, .sup.76 
Br, .sup.77 Br and the like. However, compounds for treating cancer more 
preferably have X or Q as .sup.131 I. 
As provided herein Q is a radionuclide, a halide or an activating group. 
Compounds having Q as a halide or an activating group are provided as 
non-radioactive compounds of the present invention which can be readily 
converted into the corresponding radioactive compound. Since the utility 
of a radioactive compound relates to the specific activity of such a 
radioactive compound, it is often preferred to add the radionuclide just 
before use. Accordingly, compounds having Q as halide or as an activating 
group are provided, for example, in a form useful for storage or 
transport. 
When Q is a halide, such a halide is preferably Br or I. 
As provided herein an activating group is a group which is easily displaced 
by a radionuclide via electrophilic aromatic substitution. Preferred 
activating groups include tributyl-tin, trimethylsilyl, 
t-butyldimethylsilyl, iodide and the like. 
According to the present invention Z is .dbd.O or two hydrogen atom 
substituents. Since the --CZ-- moiety is adjacent to an amine, when Z is 
.dbd.O an amide (--CO--NH--) is formed. When Z is two hydrogen atoms a 
methylene (--CH.sub.2 --) is formed. Therefore compounds of the present 
invention can be amide or alkylamino compounds, e.g. compounds of formula 
I can have one of the following side chains: 
EQU --(CH.sub.2)--CO--NR.sub.3 --(CH2).sub.y --R.sub.2 or 
EQU --(CH.sub.2).sub.j --CH.sub.2 --NR.sub.3 --CH2).sub.y --R.sub.2. 
In a preferred embodiment Z is .dbd.O, i.e., the --CZ-- group forms a 
carbonyl. When --CZ--NR.sub.3 -- is --CH.sub.2 --NR.sub.3 --, the R.sub.3 
is preferably an alkyl. 
The term lower alkyl, when used singly or in combination, refers to alkyl 
groups containing one to six carbon atoms. Lower alkyls may be straight 
chain or branched and include such groups as methyl, ethyl, propyl, 
isopropyl, butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, 
neopentyl, hexyl and the like. The preferred alkyl groups contain one to 
four carbon atoms. 
As used herein, a lower alkylene, singly or in combination with other 
groups, contains up to six carbon atoms in the main chain and a total of 
10 carbon atoms if the alkylene is branched. Lower alkylene groups include 
methylene, ethylene, propylene, isopropylene, butylene, t-butylene, 
sec-butylene, isobutylene, amylene, isoamylene, pentylene, isopentylene, 
hexylene and the like. The preferred lower alkylene groups contain one to 
four carbon atoms. 
The term cycloalkenyl refers to a partially saturated cyclic structure, 
i.e., a ring, having 3-7 ring carbon atoms which can have one or two 
unsaturations. Since the cycloalkenyl groups of the present invention are 
fused to a phenyl moiety such cycloalkenyls are partially unsaturated. The 
subject cycloalkenyls groups include such groups as cyclopropenyl, 
cyclobutenyl, cyclopentenyl, cyclohexenyl and cycloheptenyl rings. 
As used herein, lower alkoxy refers to a lower alkyl group attached to the 
main chain via an oxygen atom. 
Halo refers to a halogen, especially bromine, iodine, chlorine and 
fluorine. As used herein a halo group is a commonly available, 
non-radioactive halogen isotope. Preferred halo groups include iodide, 
chloride, bromide and the like. 
Aryl refers to a compound whose molecules have ring structures 
characteristic of benzene, naphthalene, phenanthrene, etc. These compounds 
have either the six-carbon ring of benzene or the condensed six-carbon 
rings of other aromatic derivatives. These compounds may be unsubstituted 
or substituted with halogens, alkoxy or alkyl groups. A preferred aryl 
group is phenyl, C.sub.6 H.sub.5. 
The term arylalkane refers to a compound containing both aliphatic and aryl 
structures. A preferred arylalkane is benzyl, CH.sub.2 C.sub.6 H.sub.5. 
These compounds may be unsubstituted or substituted with halogens, alkoxy 
or alkyl groups. 
As employed herein, a heterocyclic ring means a saturated, partially 
saturated or aromatic heterocyclic ring having at least one nitrogen or 
oxygen ring atom. As is known to the skilled artisan a saturated 
heterocyclic ring has no double bonds. As used herein a partially 
saturated heterocyclic ring can have at least one double bond. 
The present heterocyclic rings can have up to three heteroatoms and up to a 
total of seven ring atoms. Accordingly, heterocyclic rings of the present 
invention can have about 2 to about 6 ring carbon atoms. Preferably, a 
heterocyclic ring has only one nitrogen or one oxygen heteroatom, or one 
nitrogen atom and one oxygen heteroatom. Heterocyclic rings can also have 
a mixture of nitrogen or oxygen heteroatoms, e.g. morpholine with one 
oxygen and one nitrogen. It is preferred that the heterocyclic ring 
contain one or two ring heteroatoms, most preferred is one ring nitrogen 
or oxygen heteroatom. 
Heterocyclic rings of the present invention are monocyclic; such monocyclic 
rings can be fused to a phenyl ring to form a bicyclic ring. 
Representative partially saturated and heteroaromatic heterocyclic rings 
include furan, pyran, oxazine, isoxazine, pyrrole, pyrazole, pyridine, 
pyrazine, triazole, tetrazole, triazine, pyrimidine, pyridazine, furazan 
and the like. Preferred heteroaromatic groups include pyridine and the 
like. 
Representative saturated heterocyclic rings include tetrahydrofuran, 
pyrazolidine, imadazolidine, pyrrolidine, azetidine, piperidine, 
piperazine and morpholine. Preferred heterocyclic rings include piperidine 
and the like. 
As used herein R.sub.a and R.sub.b are independently H, halo, lower alkyl, 
lower alkoxy or R.sub.a and R.sub.b together with the carbon atoms to 
which they are attached form a cycloalkenyl or heterocyclic ring. When 
R.sub.a and R.sub.b together form a cycloalkenyl or heterocyclic ring, 
such a ring is fused to the phenyl. 
Such cycloalkenyl ring formed from R.sub.a and R.sub.b has only one 
unsaturation in the cycloalkenyl ring and that unsaturation is contributed 
by the phenyl ring to which the cycloalkenyl is fused. While a 
cycloalkenyl formed by R.sub.a and R.sub.b can be a 5 or 6 membered ring, 
such rings are preferably 5-membered rings., e.g. cyclopentenyl. Examples 
of the fused cycloalkenyl-phenyl ring include indanyl and 
tetrahydronaphthyl, e.g., 5,6,7,8-tetrahydronaphthyl, and the like. 
When a heterocyclic ring is formed by R.sub.a and R.sub.b, the heterocyclic 
ring preferably has one nitrogen or oxygen heteroatom and 5 or 6 ring 
atoms. As used herein, heterocyclic is as defined hereinabove. The 
heterocyclic ring contains at least two ring carbon atoms when the 
heterocyclic ring is a 5-membered ring, and the number of ring carbon 
atoms present can range from 2-4 carbon ring atoms. When the heterocyclic 
ring is a 6-membered ring, the number of ring carbon atoms can range from 
2-5 carbon atoms. Thus, the total number of ring carbon atoms will range 
from 6-8 ring carbon atoms when the phenyl ring is fused to a 5-membered 
heterocyclic ring and 6-9 ring carbon atoms when the phenyl ring is fused 
to a 6-membered heterocyclic ring. The heterocyclic ring can contain up to 
3 ring heteroatoms. The preferred ring heteroatoms are oxygen and 
nitrogen, especially oxygen. Preferred heterocyclic rings formed by 
R.sub.a and R.sub.b include dihydrofuranyl, dihydropyrrolyl, 
tetrahydropyridinyl and the like. 
As provided herein each R.sub.1 is independently H, halo, lower alkyl or 
lower alkoxy. In a preferred embodiment R.sub.1 is H, halo or lower 
alkoxy. More preferred R.sub.1 groups include H and halo. However, in one 
embodiment R.sub.1 is preferably alkoxy. 
The variable q is defined herein as an integer ranging from 0 to 2 which 
describes the number of R.sub.1 groups on the phenyl moiety. Since the 
phenyl is also substituted with R.sub.a, R.sub.b, X (or Q) and a side 
chain amide or amine moiety, the maximal number or R.sub.1 groups is 2 
(i.e. q can maximally be 2). When q is less then 2 some positions on the 
phenyl group are unsubstituted; in this case a hydrogen is present at the 
positions having no R.sub.1 group. Preferred values for q are 0 to 1. An 
especially preferred value for q is 0, i.e. the phenyl has hydrogen at all 
positions except those occupied by R.sub.a, R.sub.b, X (or Q) and the 
amide or amine side chain moiety. 
##STR4## 
group is selected from the following: 
##STR5## 
wherein R.sub.1 is as described hereinabove and Q is a radionuclide (e.g. 
X), a halide or an activating group. 
As described herein, R.sub.2 is --N(R.sub.3).sub.2 or a 5 to 7 membered 
nitrogen containing heterocyclic ring which is unsubstituted or 
substituted with at least one alkyl or substituted or unsubstituted 
arylalkyl substituent; wherein each R.sub.3 is independently hydrogen or 
lower alkyl. Preferably R.sub.3 is lower alkyl in the --N(R.sub.3).sub.2 
groups of the present invention. Preferred R.sub.2 heterocyclic rings 
include N-piperidinyl, N-pyrrolidinyl, N-pyridinyl, N-morpholinyl, 
N-pyrrolyl, piperidinyl, pyrrolidinyl, pyridinyl, morpholinyl or pyrrolyl, 
which can be substituted with an R.sub.6 lower alkyl or substituted or 
unsubstituted arylalkyl. R.sub.6 is preferably attached to the nitrogen of 
the piperidinyl, pyrrolidinyl or morpholinyl rings. The arylalkyl compound 
may be unsubstituted or substituted with halogens, alkoxy or alkyl groups. 
In one embodiment R.sub.2 can be R.sub.4 as defined herein. In another 
embodiment R.sub.2 can be R.sub.5 as defined herein. In still another 
embodiment R.sub.2 can be --N(R.sub.3).sub.2 as defined herein. 
As provided herein, R.sub.4 is --N(R.sub.3).sub.2 or an N-linked 5 to 7 
membered nitrogen containing heterocyclic ring which can have at least one 
alkyl substituent. As defined herein N-linked means that the nitrogen 
containing heterocyclic ring is attached to the main chain through a 
nitrogen atom. R.sub.4 is used in formula II to indicate a preference for 
attachment of the nitrogen present within the heterocyclic ring to the 
main chain. Preferred R.sub.4 heterocyclic rings include rings of the 
formulae: 
##STR6## 
wherein R.sub.6 is hydrogen or lower alkyl and each i is independently an 
integer from 1 to 3. Preferred R.sub.4 heterocyclic rings include 
N-piperidinyl, N-pyrrolidinyl, N-pyridine and the like. 
In another embodiment preferred compounds have heterocyclic rings that are 
not attached via the ring nitrogen. R.sub.5 is used in formula III to 
describe such compounds, wherein R.sub.5 is a 5 to 7 membered nitrogen 
containing heterocyclic ring which can have at least one alkyl or 
substituted or unsubstituted arylalkyl substituent. In a preferred 
embodiment R.sub.5 is any one of the following: 
##STR7## 
wherein each i is independently an integer from 1 to 3 and R.sub.6 is 
hydrogen or lower alkyl or substituted or unsubstituted arylalkyl. More 
preferred R.sub.5 heterocyclic rings include piperidinyl, pyrrolidinyl or 
pyridinyl which are N-substituted with an R.sub.6 lower alkyl or 
substituted or unsubstituted arylalkyl. Such an R.sub.6 lower alkyl is 
preferably methyl, ethyl, propyl or butyl. Such an R.sub.6 arylalkyl is 
preferably benzyl. The arylalkyl compound may be unsubstituted or 
substituted with halogens, alkoxy or alkyl groups. 
The compounds of formula IV have an --N(R.sub.3).sub.2 group which is 
hydrogen or lower alkyl. In a preferred embodiment for --N(R.sub.3), 
R.sub.3 is lower alkyl, e.g. methyl, ethyl, propyl or butyl. 
The variable j, as used herein, refers to an integer ranging from 0 to 6 
which defines the length of the alkylene chain separating the phenyl and 
--CZ-- moieties of the present compounds. Preferably, j is an integer from 
0 to 3. More preferably, j is an integer from 0 to 2. For compounds where 
--CZ-- is --CO--, j is preferably 0. 
As defined herein y is 0 to 6. The variable y defines the length of the 
alkylene chain separating the CZ--NR.sub.3 --and R.sub.2 groups in the 
--CZ--NR.sub.3 --(CH2).sub.y --R.sub.2 moiety of formula I. Preferably y 
is 0 to 3; more preferably y is 0 to 2. 
Like y, the variable m defines the length of the alkylene chain separating 
the --CZ--NR.sub.3 -- and the R.sub.4 (or R.sub.5) group in the 
--CZ--NR.sub.3 --(CH2).sub.m --R.sub.4 (or R.sub.5) moiety of formula II 
or III. The variable m is an integer ranging from 0 to 6. However, m is 
preferably to 4 and more preferably 0 to 3. 
The length of the alkylene chain separating the --CZ--NH-- and the 
--N(R.sub.3).sub.2 moieties in formula IV is described herein by n. The 
variable n is an integer ranging from 3 to 6. In a preferred embodiment n 
is 3. 
Preferred compounds of the present invention include the following: 
##STR8## 
The various combinations and permutations of the Markush groups of X, Q, Z, 
R.sub.a, R.sub.b, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 described 
herein are contemplated to be within the scope of the present invention. 
Moreover, the present invention also encompasses compounds and 
compositions which contain less than all of the elements in the Markush 
grouping. Thus, the present compounds and compositions contain one or more 
elements of each of the Markush groupings in X, Q, Z, R.sub.a, R.sub.b, 
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 and the various 
combinations thereof. Thus, for example, the present invention 
contemplates that R.sub.2 may be one or more of the substituents listed 
hereinabove or any and all of the substituents of N(R.sub.3).sub.2, 
R.sub.4 and R.sub.5. 
The present compounds can bind to a specific cell receptor prevalent on 
certain types of cancer cells. Such cancer cells include lung carcinoma, 
colon carcinoma, renal carcinoma, melanoma, glioma, pheochromocytoma, 
neuroblastoma, prostate carcinomas, breast carcinomas and related cells. 
An example of the cell receptor to which the present compounds bind is a 
cell surface sigma receptor. 
The binding characteristics of the present compounds were determined by 
observing whether binding was inhibited by known sigma receptor 
antagonists. Many antagonists are known which have demonstrated binding 
specificities for a given cell surface receptor. Such antagonists can be 
tested as competitive inhibitors for cellular binding by compounds of the 
present invention. If a given antagonist is a competitive inhibitor the 
receptor to which the antagonist binds must also bind the subject 
compounds. 
For example, as demonstrated by the present inventors, a malignant melanoma 
cell line binds the present compounds with strong specificity and 
affinity. Only antagonists which bind to the same site as the present 
compounds can inhibit binding of the subject compounds. Antagonists which 
can be tested include antagonists specific for cell receptors such as 
sigma (e.g. using SE2466-2), sigma-1 (e.g. fluphenazine at low 
concentrations), sigma-2 (e.g. fluphenazine at high concentrations), 
dopamine-1 (e.g. SCH23390), dopamine-2 (e.g. raclopride), melanocyte 
secreting hormone receptor (e.g. melanocyte secreting hormone peptide), 
5-hydroxytryptamine-1 (e.g. mianserin), 5-hydroxytryptamine-1a (e.g. 
NAN-190), 5-hydroxytryptamine-1c (e.g. ketanserine), 5-hydroxytryptamine-2 
(e.g. ketanserine and mianserin) and 5-hydroxytryptamine-3 (e.g. 
3-tropanyldichloroben) cell receptors and the like. 
As provided herein, antagonists with demonstrated binding specificity for 
cell surface sigma receptors (e.g. fluphenazine) can act as competitive 
binding inhibitors for compounds of the present invention. In contrast, 
antagonists that do not bind to cell surface sigma receptors cannot 
inhibit binding of the present compounds to melanoma cells. Therefore, the 
present compounds can bind to cell surface sigma receptors. 
Cell types which have sigma receptors include normal neural tissues (e.g., 
brain, spinal cord and the like) as well as lung carcinoma, colon 
carcinoma, renal carcinoma, breast and prostate carcinoma, melanoma, 
pheochromocytoma, glioma, neuroblastoma, all other neural tumors and the 
like. For example, several lung carcinoma cell types have demonstrated 
binding affinity for the present compounds including an adenocarcinoma, a 
squamous carcinoma and large cell lung carcinoma cells. In a further 
example metastatic malignant melanoma cells have demonstrated high 
affinity and specificity for the present compounds. In a preferred 
embodiment the present compounds are used to detect and treat melanomas 
and non-small cell lung carcinoma (NSCLC). Such NSCLC cancers include lung 
adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma 
and the like. 
Breast cancer cells, particularly MCF-7, T47-D and MDA-MB231 tumor cells, 
have also demonstrated binding affinity for the present compounds. In a 
preferred embodiment, the present compounds are used to detect and treat 
breast cancer. In another preferred embodiment, the present compounds are 
used to detect breast cancer in women with dense breasts. 
According to the present invention a method for detecting a mammalian tumor 
or a tissue containing cell surface sigma receptor includes administering 
to a mammal a composition including a diagnostic imaging amount of at 
least one of the present compounds. Such a diagnostic imaging amount is a 
dosage of at least one of the subject compounds which permits sufficient 
tumor or tissue localization of the compound to allow detection of the 
tumor or tissue. This dosage can range from about 1 .mu.g to about 1 g of 
the compound per liter which can be administered in doses of about 1 ng/kg 
body weight to about 10 .mu.g/kg body weight. Preferred dosages of the 
present compounds are in the range of about 10 ng to about 2 .mu.g/kg for 
diagnostic imaging. Moreover, for diagnostic imaging the amount of 
radioactivity administered should be considered. Preferably about 0.1 
millicuries (mCi) to about 20 mCi of radioactive compound is administered. 
As described herein a tumor or tissue labeled with one or more of the 
present compounds can be detected using a radiation detector, e.g. a 
.gamma.-radiation detector. One such procedure utilizes scintigraphy. 
Tomographic imaging procedures such as single photon emission computed 
tomography (SPECT) or positron emission tomography (PET) can also be used 
to improve visualization. 
In another embodiment the present invention is directed to a method for 
treating a mammalian tumor which includes administering to a mammal a 
composition including a tumor-inhibiting amount of at least one compound 
of the present invention. Such a tumor-inhibiting amount is an amount of 
at least one of the subject compounds which permits sufficient tumor 
localization of the compound to diminish tumor growth or size. As provided 
herein tumor growth or size can be monitored by any known diagnostic 
imaging procedure, e.g. by using the present methods. This dosage can 
range from about 0.1 mmole/kg body weight to about 500 mmole/kg body 
weight. A preferred dosage is about 5 to about 50 mmole/kg body weight. 
The amount of radioactivity administered can vary depending on the type of 
radionuclide. However, with this in mind the amount of radioactivity which 
is administered can vary from about 1 mCi to about 800 mCi. Preferably, 
about 10 mCi to about 600 mCi is administered. 
Moreover when considering a dosage for diagnostic imaging or therapy, the 
specific activity of the radioactive compound should be taken into 
consideration. Such a specific activity is preferably very high, e.g. for 
.sup.123 I-labeled compounds the specific activity should be at least 
about 1,000 Ci/mM to about 220,000 Ci/mM. More preferably the specific 
activity for .sup.123 I-labeled compounds is, e.g. about 10,000 Ci/mM to 
about 220,000 Ci/mM. 
In another embodiment the present invention provides a method for in vitro 
detection of a cancer cell in a mammalian tissue sample which includes 
contacting a mammalian tissue sample with an in vitro diagnostic imaging 
amount of a compound of any one of formulae I, II, III or IV for a time 
and under conditions sufficient for binding of the compound to a cell 
surface sigma receptor on the cancer cell and detecting such binding. 
Samples can be collected by procedures known to the skilled artisan, e.g. 
by collecting a tissue biopsy or a body fluid, by aspirating for tracheal 
or pulmonary samples and the like. 
As used herein any mammalian tissue can be tested in vitro. Preferred 
tissues for in vitro testing include lung, bronchial, lymph, skin, brain, 
liver, prostate, breast, any tumor of neural origin and the like. Samples 
can be sectioned, e.g. with a microtome, to facilitate microscopic 
examination and observation of bound compound. Samples can also be fixed 
with an appropriate fixative either before or after incubation with one of 
the present compounds to improve the histological quality of sample 
tissues. 
Conditions sufficient for binding of the compound to a cell surface sigma 
receptor on the cancer cell include standard tissue culture conditions, 
i.e. samples can be cultured in vitro and incubated with one of the 
present compounds in physiological media. Such conditions are well known 
to the skilled artisan. Alternatively, samples can be fixed and then 
incubated with a compound of the present invention in an isotonic or 
physiological buffer. 
An amount of at least one of the present compounds for in vitro detection 
of a cancer cell can range from about 1 ng/l to about 1000 .mu.g/l. A 
preferred amount is about 1 .mu.g/l to about 100 .mu.g/l. 
When the present compounds are used for in vitro diagnosis of cancer X or Q 
as a radionuclide is used. Preferable X and Q radionuclides for in vitro 
diagnosis of cancer include .sup.125 I, .sup.18 F, --.sup.35 S-alkyl, 
--.sup.35 SO.sub.3, --.sup.35 SO.sub.4, --.sup.14 COOH, --.sup.14 
CH.sub.3, .sup.3 H and the like. 
For detection, of cellular binding of one of the present compounds, samples 
can be incubated in the presence of a selected compound, then washed and 
counted in a standard scintillation counter. Alternatively, samples can be 
dipped in photoemulsion and the signal detected under light microscopy 
after several days, as exposed silver grains. 
Compounds of the present invention can be prepared by any procedure 
available to the skilled artisan using protecting groups, leaving groups, 
activating groups and the like as needed. Starting compounds can be chosen 
which have the desired R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and 
R.sub.6 groups at the requisite positions. Alternatively, a leaving group 
may be used in place of the desired R.sub.1, R.sub.2, R.sub.3, R.sub.4, 
R.sub.5 or R.sub.6 group, and the appropriate group may replace the 
leaving group in a later synthetic step. Another alternative is to employ 
a protecting group on a reactive group which may be present on starting 
materials, e.g., an amine or similar reactive group on the chosen starting 
material. The use of leaving or protecting groups prevents undesirable 
side reactions from occurring, while permitting desired reactions to take 
place. 
As is generally known in the art, and for the purposes of the present 
invention, a leaving group is defined as a group which is readily broken 
away from its union with a carbon atom. These groups are readily 
recognizable by one skilled in the art. Suitable leaving groups are 
generally electron attracting groups, either because of their 
electronegativity or because they have an inductive effect, and may 
include groups such as halides, N.sub.3, HO-Aryl, or HSO.sub.3 -Aryl 
groups, and the like. For example, a leaving group can be present at the 
position of X or Q on a starting material for the present compounds; such 
a leaving group is preferably a halide, e.g. Br or I. 
A protecting group is covalently bound to a reactive group to render the 
reactive group unreactive while allowing desired reactions to take place. 
To be useful, a protecting group must in addition be easily removed 
without chemically altering the remainder of the molecule, and must 
regenerate the correct structure of the reactive group. Examples of 
protecting groups effective with, for example, primary and secondary amino 
groups include acetyl, carbobenzoxy (cleaved by acid hydrolysis), benzyl 
(cleaved by catalytic hydrogenation), tertbutoxycarbonyl (cleaved by mild 
acid treatment) and 9-fluorenylmethoxycarbonyl (cleaved by secondary 
amines). A comprehensive review of useful protecting groups is provided in 
Greene, 1981 Protective Groups in Organic Synthesis (John Wiley & Sons, 
New York). 
As provided herein an activating group is a group which is easily displaced 
by a radionuclide via electrophilic aromatic substitution. The activating 
group is used to facilitate substitution of a radionuclide onto the 
present compounds. Activating groups contemplated by the present invention 
include tributyl-tin, trimethylsilyl, t-butyldimethylsilyl, iodide and the 
like. 
The present compounds can be prepared from readily available starting 
materials, for example, by amidation of a substituted phenylalkyl acid or 
a substituted benzoic acid with an appropriate amine. Such a reaction 
yields a compound of any one of formulae I to IV. 
In an exemplary procedure for synthesis of a benzamide compound of formula 
I, a substituted phenylalkyl acid or substituted benzoic acid can be used 
as a starting material. For example, a substituted phenylalkyl acid or 
substituted benzoic acid (V) having a leaving group (Y) at the desired X 
(or Q) position can be amidated in the presence of a halogenating reagent 
with an amine of formula VI, as depicted below. 
##STR9## 
wherein Y is a leaving group and R.sub.1, R.sub.a, R.sub.b, q, j, y and 
R.sub.2 are as described hereinabove. Preferably Y is a halo group, e.g. 
Cl, Br or I. More preferred Y groups are Br in a meta position and I in a 
para position relative to the carboxyl group, when the X or Q is to be 
placed in such a respective meta or para position. 
Halogenating reagents for the above described reaction include those which 
can convert the carboxylate to an acid halide, e.g. thionyl halide such as 
SOCl.sub.2, PCl.sub.5, PCl.sub.3 and the like. A preferred halogenating 
reagent is SOCl.sub.2 in the presence of dimethylformamide. 
To facilitate formation of such an acid halide, the reaction can be heated 
to reflux temperatures. A preferred solvent for this reaction is a 
nonpolar volatile solvent, e.g. chloroform. The acid chloride so formed is 
sufficiently stable to be isolated, for example, by evaporation of 
solvent. After conversion of V to the acid halide, the amine (e.g. VI) can 
be condensed with the halide in the presence of a base such as 
triethylamine. The solvent for this reaction is also preferably a nonpolar 
solvent, e.g. chloroform. 
The skilled artisan can readily modify the reactions described above to 
generate a compound of any one of formulae I, II, III or IV. For example, 
to produce a compound of formula II, an amine of the formula NH.sub.2 
--(CH.sub.2).sub.m --R.sub.4 can be used in place of the compound of 
formula VI. Similarly, to produce a compound of formula III or IV, an 
amine of the formula NH.sub.2 --(CH.sub.2).sub.m --R.sub.5 or NH.sub.2 
--(CH.sub.2).sub.n --N(R.sub.3).sub.2, respectively, can be used in place 
of VI. 
When Z is .dbd.O, the leaving group (Y) can be directly replaced to produce 
a compound of any one of formulae I, II, III or IV. When Z is two --H, the 
carbonyl of the amide moiety formed by the above condensation must be 
converted into a methylene. To convert the --CO--NH-- to a --CH.sub.2 
--NH-- a reducing agent can be used, e.g. boron hydride, sodium 
borohydrate, lithium aluminum hydride and the like. More preferred 
reducing agents are boron hydride (BH.sub.3) or lithium aluminum hydride 
(LAH) in the presence of tetrahydrofuran (THF). For example, the carbonyl 
of a compound of formula VII can be converted to a methylene by the 
following reaction: 
##STR10## 
When the R.sub.3 of --CZ--NR.sub.3 -- is lower alkyl, such a lower alkyl is 
added, e.g. by alkylation, after condensation of the acid halide and the 
amine and after conversion of the amide (--CO--NH--) to the alkylamine 
(--CH.sub.2 --NH--). Alkylation can be done by any available procedure, 
e.g., using an alkyl halide with a sodium salt in dimethylformamide or 
ethanol. For example, an alkyl halide (e.g. iodomethane) can be reacted 
with a compound of formula VIII in the presence of sodium bicarbonate or 
sodium carbonate using dimethylformamide as solvent. 
If a compound of any one of formulae II, III or IV is desired, a Q group 
can replace the Y leaving group, e.g. on VII or VIII. As provided herein Q 
is a radionuclide, a halide or an activating group. When Q is a halide a 
starting material having the desired halide at the position of Q can be 
utilized, e.g. V can be bromophenyl alkyl acid, iodophenyl alkyl acid, 
iodobenzoic acid, and the like. An activating group can be placed at the 
position of Y by available procedures to generate a compound of any one of 
formulae II, III or IV, wherein Q is the activating group. The activating 
group (Q) can in turn be readily replaced by a radionuclide (i.e. X) to 
generate compounds of formulae I, II, III or IV, wherein X is the desired 
radionuclide. 
For example, activation can be achieved using palladium catalyzed 
stannylation with bis(tributyltin), as depicted below. 
##STR11## 
In this case Q is tributyltin (Bu.sub.3 Sn). This reaction is effective 
whether Z is .dbd.O or two --H. 
When using t-butyldimethylsilyl chloride (TBDMSCl) or trimethylsilyl 
chloride with N-butyl lithium or t-butyl lithium, a protecting group 
(R.sub.7) is first placed on the --CZ--NR.sub.3 -- amine, if R.sub.3 is 
hydrogen. When the R.sub.3 of the --CZ--NR.sub.3 is lower alkyl, no such 
protecting group is needed. Protecting groups used for a --CZ--NR.sub.3 
--amine can be any protecting group for a secondary amine, e.g. 
carbobenzoxy (i.e. CBz, cleaved by acid hydrolysis), benzyl (cleaved by 
catalytic hydrogenation), tert-butoxycarbonyl (i.e. t-BOC, cleaved by mild 
acid treatment) and the like. The silylation reaction can be then be 
performed as depicted below, e.g. using an amine protected compound of 
formula VIII. 
##STR12## 
In this case Q is trimethylsilane (Me.sub.3 Si). The conditions used for 
this reaction include low temperature (e.g. -78.degree. C.) and a polar 
solvent (e.g. tetrahydrofuran). 
The R.sub.7 group can be removed by standard techniques, e.g. when R.sub.7 
is CBz or t-BOC acid hydrolysis can remove R.sub.7 and restore the 
secondary amine (--NH--). Silylation is preferred for compounds wherein Z 
is two --H. 
The radioactively labeled compounds of the present invention can be 
produced with high specific activity and high yield by reacting a 
radioisotope (e.g. .sup.123 I, .sup.125 I or I) with an activated 
intermediate (e.g. a compound of formula IX or X) in the presence of an 
oxidizing agent. Any oxidizing reagent which can convert the negatively 
charged radionuclide to a positively charged radionuclide can be used. 
Preferred oxidizing reagents include iodogen beads, peroxides such as 
peracetic acid, hydrogen peroxide and the like, as well as 
N-chloro-4-toluene-sulfonamide (i.e. chloramine-T). A more preferred 
oxidizing reagent is chloramine-T. An acid, e.g. HCl, can also be added. 
An example of a reaction where the radionuclide replaces the activating 
group is depicted below using, e.g. a compound of formula IX. 
##STR13## 
When R.sub.a and R.sub.b together with the carbon atoms to which they are 
attached form a cycloalkenyl or heterocyclic ring the desired cycloalkenyl 
or heterocyclic ring can be in place on the starting material. For 
example, the R.sub.a and R.sub.b of formula V together with the carbon 
atoms to which they are attached can form the desired cycloalkenyl or 
heterocyclic ring. 
As is recognized by the skilled artisan, the above procedures can be 
modified for making the present compounds to include other known and 
commonly available procedures. The procedures provided herein are intended 
to be illustrative and are not exhaustive; therefore the illustrated 
procedures should not be viewed as limiting the invention in any way. 
Another embodiment of the present invention provides a compartmentalized 
kit for detection of a mammalian tumor which includes a first container 
adapted to contain at least one of the compounds of the present invention. 
A further embodiment of the present invention provides a compartmentalized 
kit for treating a mammalian tumor which includes a first container 
adapted to contain at least one of the compounds of the present invention. 
Compounds of the present invention which are provided in a kit for 
detecting or treating a mammalian tumor can have any one of formulae I, 
II, III, IV, VII, VIII, IX or X. However more preferred compounds for the 
present kits are of any one of formulae II, III, IV, VII, VIII or IX. 
Especially preferred compounds of the present invention which placed in 
kits include compounds of formula II or IX. 
Compounds provided in the present kits preferably have a Q rather than an X 
group and such a Q group is preferably an activating group. Activating 
groups present on compounds provided in the subject kits include 
tributyl-tin, trimethylsilyl or t-butyldimethylsily. Tributyl-tin is an 
especially preferred activating group for compounds provided in the 
present kits. 
The kits of the present invention can be adapted to contain another 
container having a reagent for replacing an activating group with a 
radionuclide. For example, such a reagent can be an oxidizing reagent, 
e.g. chloramine-T. 
Preferred radiolabeled compounds of the present invention include .sup.125 
I-(2-piperidinylaminoethyl)-4-iodobenzamide (.sup.125 I!PAB), .sup.125 
I-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-.sup.125 I!BP), .sup.125 
I-(N-benzylpiperidin-4-yl)-3-iodobenzamide (3-.sup.125 I!BP), .sup.125 
I-(N-benzylpiperidin-4-yl)-2-iodobenzamide (2-.sup.125 I!BP) and 
N-4-.sup.125 I-iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethylamine 
(4-.sup.125 I!PEMP). 
Especially preferred radiolabeled compounds include .sup.125 I!PAB, 
2-.sup.125 I!BP, 4-.sup.125 I!BP and 4-.sup.125 I!PEMP. 
In a further embodiment, the kits of the present invention can be adapted 
to contain another container having a material for separating unattached 
radionuclide from the radiolabeled compounds of the present invention 
having an attached X group. Such a material can be any chromatographic 
material including a thin layer chromatography plate, a molecular 
exclusion resin, a silica gel, a reverse phase resin and the like. For 
convenience, such resins can also be provided in the form of a prepacked 
column. 
The present compounds can be administered to a mammal as a pharmaceutical 
composition. Such pharmaceutical compositions contain a diagnostic imaging 
or an anti-tumor amount of at least one of the present compounds together 
with a pharmaceutically acceptable carrier. 
The compositions can be administered by well-known routes including oral, 
intravenous, intramuscular, intranasal, intradermal, subcutaneous, 
parenteral, enteral, topical and the like. Depending on the route of 
administration, the pharmaceutical composition may require protective 
coatings. 
The subject compounds may be incorporated into a cream, solution or 
suspension for topical administration. 
The pharmaceutical forms suitable for injection include sterile aqueous 
solutions or dispersions and sterile powders for the extemporaneous 
preparation of sterile injectable solutions or dispersions. In all cases 
the ultimate solution form must be sterile and fluid. Typical carriers 
include a solvent or dispersion medium containing, for example, water, 
buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyol 
(glycerol, propylene glycol, polyethylene glycol and the like), suitable 
mixtures thereof, surfactants or vegetable oils. Sterilization can be 
accomplished by any art recognized technique, including but not limited 
to, addition of antibacterial of antifungal agents, for example, paraben, 
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Further, 
isotonic agents, such as sugars or sodium chloride may be incorporated in 
the subject compositions. 
Production of sterile injectable solutions containing at least one of the 
present compounds is accomplished by incorporating these compounds in the 
required amount in the appropriate solvent with various ingredients 
enumerated above, as required, followed by sterilization, preferably 
filter sterilization. To obtain a sterile powder, the above solutions are 
vacuum-dried or freeze-dried as necessary. 
When the present compounds are administered orally, the pharmaceutical 
compositions containing an effective dosage of the compound, can also 
contain an inert diluent, an assimilable edible carrier and the like. 
orally administered compositions can be provided in hard or soft shell 
gelatin capsules, tablets, elixirs, suspensions, syrups and the like. 
The subject compounds are thus prepared for convenient and effective 
administration in pharmaceutically effective amounts with a suitable 
pharmaceutically acceptable carrier in a dosage which permits diagnostic 
imaging or cancer cell death. These amounts are preferably about 1 .mu.g 
to about 1 g of the compound per liter and are administered in doses of 
about 1 ng/kg body weight to about 10 .mu.g/kg body weight, or from about 
0.1 mmole/kg body weight to about 500 mmole/kg body weight. Preferred 
compositions provide effective dosages of the present compounds in the 
range of about 10 ng to about 2 .mu.g/kg for diagnostics and preferably 
about 5 to about 50 mmole/kg body weight for therapy. 
Moreover when considering a dosage for diagnostic imaging of therapy, the 
specific activity of the radioactive compound should be taken into 
consideration. Such a specific activity is preferably very high, e.g. for 
.sup.123 I-labeled compounds the specific activity should be at least 
about 1,000 Ci/mM to about 240,000 Ci/mM. More preferably the specific 
activity for .sup.123 I-labeled compounds is, e.g. about 10,000 Ci/mM to 
about 220,000 Ci/mM. 
As used herein, a pharmaceutically acceptable carrier includes solvents, 
dispersion media, coatings, antibacterial and antifungal agents, isotonic 
agents, and the like which are physiologically acceptable. The use of such 
media and agents are well-known in the art. 
The following Examples further illustrate the invention. 
EXAMPLE 1 
Synthesis of .sup.125 I-2-(PiperidinylAminoethyl)-4-IodoBenzamide 
Materials and Methods 
Melting points were determined with a Fisher-Johns apparatus. .sup.1 H and 
.sup.13 C NMR spectra were recorded on a Brucker 300 AM spectrometer. 
Unless noted, chemical shifts were expressed as ppm using 
tetramethylsilane as an internal standard. The thin layer chromatography 
(TLC) system consisted of Analtech uniplate silica gel GF plates (250 
microns, 10.times.20 cm), using CHCl.sub.3 /MeOH:80/20 as solvent. 
Radioactive spots were scanned and recorded by a Bioscan 300 imaging 
scanner equipped with automatic plate reader. Mass spectra (chemical 
ionization) were recorded on Finnigan 1015 mass spectrometer. Na.sup.131 I 
was obtained from duPont NEN and Na.sup.125 I was obtained from Bristol 
Meyers Squibb. Elemental analyses were performed by Galbraith Laboratory 
of Knoxville, Tenn. 
Preparation of (2-piperidinylaminoethyl)-4-bromobenzamide (A) 
A round bottom flask was charged with 4-bromobenzoic acid (2.0 g, 9.95 
mmol) in chloroform (150 mL). To the solution was added thionyl chloride 
(3 mL) in chloroform (10 mL), 2-3 drops of dimethylformamide (DMF). The 
slurry was heated at reflux for 3 hr. while monitoring the reaction 
through a bubbler. A clear solution of 4-bromobenzoyl chloride was 
obtained, the volatiles were removed and a light yellow oil was obtained 
which solidified upon cooling. 
The 4-bromobenzoyl chloride was dissolved in chloroform (30 mL) and added 
to a flask containing 1-(2-aminoethyl)-piperidine (1.29 g, 10 mmol) in 
chloroform (20 mL) and triethylamine (10 mL). The mixture was stirred at 
room temperature for 1 hr. and the volatiles were removed in vacuo. The 
resulting slurry was washed with 2% sodium bicarbonate (2.times.50 mL). 
The organics were dissolved in CHCl.sub.3 (100 mL), separated from aqueous 
layer and dried over anhydrous Na.sub.2 SO.sub.4. The solvent was removed 
to give a colorless solid (A, 2.7 g, yield, 87%). Rf (CHCl.sub.3 
/MeOH:90/10) 0.45. .sup.1 H R (.delta. ppm): 1.46 (t, 2H, CH.sub.2); 1.54 
(broad m, 4H, CH.sub.2); 2.43 (broad s, 4H, NCH.sub.2); 2.52-2.56 (t, 2H, 
NCH.sub.2); 2.68 (m, 2H, NCH.sub.2); 3.49-3.53 (dt, 2H, NCH.sub.2); 7.21 
(bs, 1H, NH); 7.52-7.55 (m, 2H, arom); 7.65-7.68 (m, 2H, arom). 
Preparation of (2-piperidinylaminoethyl)4-iodobenzamide, (B) 
This was prepared using a procedure like that described above for A but 
using 4-iodobenzoic acid as starting material. A white solid (B) was 
obtained in 89% yield. .sup.1 H R (.delta. ppm): 1.43-1.45 (broad m, 2H, 
NCH.sub.2); 1.53-1.60 (broad m, 4H, NCH.sub.2); 2.41 (broad m, 4H, 
NCH.sub.2); 2.50-2.54 (t, 2H, J=7.8 Hz, NCH.sub.2); 3.44-3.48 (dt, 2H, 
NCH.sub.2); 7.02 (bs, 1H, NH); 7.47-7.49 (m, 2H, arom.); 7.73-7.76 (m, 2H, 
arom.). m.p. 114-115 C. Anal C.sub.14 H.sub.19 N.sub.2 Ol calcd. C, 46.91; 
H, 5.31; N, 7.82, found C, 46.91; H, 5.42; N, 7.68. 
Preparation of (2-piperidinylaminoethyl)4-tributyltinbenzamide, (C) 
A flame dried flask was charged with 4-bromobenzamide (1.0 g, 3.21 mmol) in 
triethylamine (40 mL). Tetrakis (triphenylphosphine) palladium (370 mg, 
0.321 mmol), and bistributyltin (2.4 g, 3.80 mmol) were added, and the 
mixture was refluxed under nitrogen for 12 hr. The mixture was cooled, 
solvents decanted from the black residue, and the volatiles were removed 
in vacuo. The resulting black oil was passed through a silica gel column 
with elution with CHCl.sub.3 (100 mL), followed by elution with CHCl.sub.3 
/MeOH: 90/10. The desired fractions, as characterized by thin layer 
chromatography, were pooled and solvent was evaporated to give an oil (C, 
0.4 g, 56%). m/e=523 (M.sup.+ +H) (100%); 233 (M.sup.+ -SnBu.sub.3) (40%). 
.sup.1 H NMR (.delta. ppm): 0.82-0.93 (m, 16H, Bu.sub.3 and CH.sub.2); 
1.01-1.05 (m, 4H, Bu.sub.3); 1.22-1.37 (m, 8H, Bu.sub.3); 1.45-1.67 (m, 
8H, piperidinyl ring); 2.49-2.51 (t, 2H, NCH.sub.2 piperdinyl ring); 
2.60-2.63 (t, 2H, J=6 Hz, NCH.sub.2); 3.53-3.58 (dt, 2H, J=5.34 Hz, 
NCH.sub.2); 7.30-7.41 (bs, 1H, NH); 7.49-7.78 (m, 4H, arom). .sup.13 C R 
(.differential., ppm): 9.60, 13.58, 25.743, 27.30, 29.00, 36.09, 54.29, 
57.15, 126.03, 128.39, 132.00, 136.54, 167.69. 
Radiolabeling of n-tributyltin PAB (C) with I-125 to Yield .sup.125 
I(2-piperidinylaminoethyl)4-iodobenzamide (D) 
To 100 .mu.L of an ethanolic solution of 
(2-piperidinylaminoethyl)4-tributyltinbenzamide (1 mg/ml), was added a 
solution of .sup.125 I! sodium iodide (1.5 mCi, 3 .mu.L) in 0.1N NaOH, 
followed by the addition of 0.05N HCl (50 .mu.L) to adjust the pH of the 
solution to pH 4.5-6. Fifty .mu.L of a freshly prepared solution of 
N-chloro-4-toluenesulfonamide sodium monohydrate chloramine-T (1 mg/ml) 
was added to the above mixture. The contents were stirred for 10-15 
minutes at room temperature and 100 .mu.L of a solution of sodium 
metabisulfite (200 mg/ml) were added. The reaction mixture was neutralized 
with a saturated solution of NaHCO.sub.3 (0.2 mL). 0.4 mL of normal saline 
was added and the organics were extracted in CHCl.sub.3 (1.0 mL) after 
vortexing 30 seconds. The chloroform layer was evaporated in a stream of 
nitrogen. The radioactivity of the aqueous layer and the organic residue 
was counted. The total recovered radioactivity in the residue ranged from 
74 to 89% (n=6). The residue (D) was dissolved in 90% ethanol, and 10% 
0.01M phosphate buffer (400 .mu.L). A portion of D was spotted on a TLC-SG 
plate along with a sample of nonradioactive 
(2-piperidinylaminoethyl)4-iodobenzamide (B, as above). The TLC-SG plates 
were developed with CHCl.sub.3 /MeOH: 90/10 (Rf=0.45). Another portion of 
D was injected into a Gilson HPLC fitted with a Waters Z-module radial 
compression separation system containing a micro BondaPak C-18 reverse 
phase column equipped with Rheodyne 4125 injector (0.5 mL loop). The 
retention time for D (.sup.125 I-PAB) using isocratic elution with 90/10 
EtOH/0.1M phosphate buffer (pH=6.7) at a flow rate of 1 mL/min. was 8.5 
min., a value identical to that of non-radioactive 
(2-piperidinylaminoethyl)4-iodobenzamide. 
Radiolabeling of n-tributyltin PAB (C) with .sup.131 I Yield .sup.131 I 
(2-piperidinylaminoethyl)4-iodobenzamide (E) 
The same protocol as described above for .sup.125 
I(2-piperidinylaminoethyl)4-iodobenzamide (D) was used except that the 
amount of 0.05N HCl added to adjust pH between 4.5-6 was different due to 
different concentration of aqueous sodium hydroxide solution in which 
Na.sup.131 I was commercially supplied. The workup of and the purification 
of .sup.131 I(2-piperidinylaminoethyl)4-iodobenzamide (E) was identical to 
.sup.125 I(2-piperidinylaminoethyl)4-iodobenzamide (D) above. 
The reactions described hereinabove are depicted in Reaction Scheme I. 
##STR14## 
EXAMPLE 2 
Synthesis of 
5-iodo-(N,N-diethylaminoethyl)-2,3-dihydrobenzofuran-7-carboxamide 
Materials and Methods 
Melting points were determined with a Fisher-Johns apparatus. .sup.1 H and 
.sup.13 C R spectra were recorded on a Brucker 300 AM spectrometer. Unless 
noted, chemical shifts were expressed as ppm using tetramethylsilane as an 
internal standard. The thin layer chromatography (TLC) system consisted of 
Analtech uniplate silica gel GF plates (250 microns, 10.times.20 cm), 
using CHCl.sub.3 /MeOH:80/20 as solvent. Radioactive spots were scanned 
and recorded by a Bioscan 300 imaging scanner equipped with automatic late 
reader. Mass spectra (chemical ionization) were recorded on Finnigan 1015 
mass spectrometer. Na .sup.131 I was obtained from duPont NEN and 
Na.sup.125 I was obtained from Bristol Meyers Squibb. Elemental analyses 
were performed by Galbraith Laboratory of Knoxville, Tenn. 
Synthesis of 5,7-dibromo-2,3-dihydrobenzofuran (F) 
To a solution 2,3-dihydrobenzofuran (25 g, 0.21 mol) in chloroform (100 mL) 
was added dropwise at 0.degree. C., a solution of bromine (67 g, 0.42 mol) 
with stirring. The reaction mixture was stirred overnight at room 
temperature. The excess bromine was destroyed by addition of a saturated 
solution of sodium thiosulfate (30 ml). The organic layer was separated 
from the inorganic layer and washed with 2% sodium bicarbonate (2.times.50 
ml), then dried over anhydrous sodium sulfate. The volatiles were removed 
in vacuo to provide a light yellow oil (51 g, 87%). .sup.1 H NMR 
(CDCl.sub.3) .delta. ppm: 3.21-3.27 (t, J=9 Hz, 2H, CH.sub.2): 4.57-4.63 
(t, J=9 Hz, 2H, OCH.sub.2): 7.14-7.15 (t, 1H, arom.): 7.32-7.33 (t, 1H, 
arom.) 
Synthesis of 5-bromo-7-carboxy-2,3-dihydrobenzofuran (G) 
To the above dibromo compound (15 g, 53.9 mmol) was added anhydrous 
tetrahydrofuran (50 ml). The solution was cooled at -78.degree. C. under 
nitrogen atmosphere. A solution of n-butyl-lithium (2.0M. 27 ml) was added 
to the mixture dropwise. The mixture turned light yellow brown. After 5 
minute of stirring at -78.degree. C., carbon dioxide was bubbled through 
the mixture, giving a straw yellow color to the mixture. The mixture was 
then warmed up to room temperature and stirred for 30 minutes. A dirty 
white color solid was obtained upon filtration (7.0 g, 53%). .sup.1 H NMR 
(d.sup.6 -DMSO) .delta. ppm: 2.85-2.95 (t, J=9 Hz, 2H, CH.sub.2 : 
4.35-4.45 (t, J=9 Hz, 2H, OCH.sub.2): 7.1 (m, 1H, arom): 7.4 (m, 1H, 
arom). .sup.13 C NMR (d.sup.6 -DMSO and CDCl.sub.3) .delta. ppm: 27.88, 
71.78, 110.51, 114.17, 131.02, 131.25, 159.09, 164.87. Anal., C.sub.9 
H.sub.7 BrO.sub.3 calcd. C, 44.44; H, 2.88; found C, 44.52; H, 2.97. 
Synthesis of 5-bromo-(N, 
N'-diethylaminoethyl)-2,3-dihydrobenzofuran-7-carboxamide (H) 
A round bottom flask was charged with bromocarboxylic acid (1.79 g, 7.36 
mmol) and chloroform (50 ml). The slurry was stirred and thionyl chloride 
(2.0 ml) in chloroform (8 ml) was added to the slurry along with 2 drops 
of DMF. The mixture was refluxed for 90 min to give a clear solution. The 
volatiles were removed in vacuo to give yellow solid. This acid chloride 
was used without further purification for the condensation with amine. To 
another flask containing N,N-diethylethylenediamine (0.82 g, 6.99 mmol) 
and triethylamine (15 ml) and CHCl.sub.3 (30 ml) was added a solution of 
the above acid chloride in CHCl.sub.3 (15 ml). The mixture was stirred for 
3 hours. The volatiles were removed, the residue was washed with water (50 
ml) and the organics were dissolved in CHCl.sub.3 (75 ml). The organic 
layer was separated, dried over anhyd. Na.sub.2 SO.sub.4, and the 
volatiles removed again to give a light yellow color oil. The oil was 
purified by passage through a silica gel column and elution with 
CHCl.sub.3 /MeOH:90/10. The fractions containing the desired compound were 
pooled together, and the volatiles were removed to give the carboxamide 
(1.9 g, 80%). TLC silica gel Rf (0.7) CHCl.sub.3 /MeOH:90/10. The 
hydrochloride salt was made with an ethanolic solution of hydrogen 
chloride gas upon trituration with anhydrous ether. .sup.1 H NMR 
(CDCl.sub.3) .delta. ppm: 0.96-1.00 (t, J=7 Hz, 6H, NCH.sub.2 CH.sub.3): 
2.46-2.53 (q, J=7 Hz, 4H, NCH.sub.2 CH.sub.3): 2.55-2.59 (t, J=7 Hz, 4H 
NCH.sub.2):3.17-3.22 (t, J=8 Hz, 2 H, CH.sub.2): 3.40-3.44 (m, 2H, 
NCH.sub.2): 4.63-4.69 (t, J=9 Hz, 2H, OCH.sub.2): 7.30 (m 1H. arom): 
7.961-7.968 (m, 1H, arom). Anal. C.sub.15 H.sub.21 BrN.sub.2 O.sub.2.2HCl, 
Calcd. C,47.68; H,5.82; N,7.41; found C,47.38; H,5.80; N,7.35. 
Synthesis of 
5-tributyltin-(N,N'-diethylaminoethyl)-2,3-dihydrobenzofuran-7-carboxamide 
(J) 
A round bottom flask was charged with 5-bromocarboxamide (1.0 g, 2.93 
mmol), bis(tributyltin) (2.4 g, 4.1 mmol), palladium tetrakis 
(triphenylphosphine) (0.35 g, 0.29 mmol) and triethylamine (55 ml). The 
mixture was refluxed for 3 hours. The volatiles were removed in vacuo and 
the residue was dissolved in CHCl.sub.3. This solution was loaded onto a 
silica gel column and eluted first with CHCl.sub.3 (100 ml) and then with 
CHCl.sub.3 /MeOH:90/10 whereby a light brown band was collected. The 
volatiles were removed in vacuo to give an oil (1.3 g). The TLC showed a 
slightly impure compound. The oil was passed through a short silica gel 
column again and eluted with CHCl.sub.3 /MeOH:90/10 to give 0.9 g pure tin 
compound. TLC (silica gel) Rf=0.45 (CHCl.sub.3 /MeOH:90/10). .sup.1 H NMR 
(CDCl.sub.3) .delta. ppm: 0.83-1.60 (m, 33, H, nBu.sub.3 and NCH.sub.2 
CH.sub.3); 2.51-2.59 (q, J=7 Hz, 4H, NCH.sub.2 CH.sub.3); 2.60-2.66 (t, 
2H, CH.sub.2); 3.18-3.23 (t, J=8 Hz, CH.sub.2); 3.44-3.50 (q, J=6 Hz, 2H, 
CH.sub.2); 4.64-4.70 (t, J=9 Hz, 2H, OCH.sub.2); 7.32 (m, 1H, arom); 7.97 
(m, 1H, arom). .sup.13 C(CDCl.sub.3) (.delta. ppm): 9.67, 12.01, 13.60, 
16.45, 26.97, 27.30, 27.82, 29.03, 37.58, 47.06, 51.72, 71.74, 115.87, 
127.26, 132.65, 135.53, 136.76, 158.04, 164.92. 
Synthesis of 
5-iodo-(N,N-diethylamino-ethyl)-2,3-dihydrobenzofuran-7-carboxamide (K) 
Tributyltincarboxamide (300 mg) and iodine (0.8 g) were stirred together in 
CHCl.sub.3 at room temperature for 48 hours. The mixture was quenched with 
a saturated solution of sodium thiosulfate. The organic layer was 
separated, dried and the volatiles were removed in vacuo to give a 
colorless oil. The oil was passed through the silica gel column and eluted 
with CHCl.sub.3 /MeOH:95/5. The first few fractions contained tributyltin 
iodide and were discarded. The later fractions provided the desired iodo 
(0.2 g, 95%) compound. TLC silica gel Rf=0.3 (CHCl.sub.3 /MeOH:90/10). 
.sup.1 H NMR (CDCl.sub.3): 1.34-1.39 (t, J=8 Hz, 6H, NCH.sub.2 CH.sub.3); 
3.14-3.33 (overlapping multiplet and triplet, 8H); 3.85-3.91 (q, J=6 Hz, 
2H, NCH.sub.2); 4.71-4.76 (t, J=9 Hz, 2H, OCH.sub.2); 7.54 (m, 1H, arom); 
8.02 (m, 1H, arom). 
Synthesis of 
5-bromo-1-(2-aminoethylpiperidinyl)-2,3-dihydrobenzofuran-7-carboxamide 
(L) 
A round bottom flask was charged with bromocarboxylic acid (2.0 g, 8.23 
mmol) and chloroform (50 ml). The slurry was stirred and thionyl chloride 
(4.0 ml) in chloroform (10 ml) was added to the slurry along with 2-3 
drops of dimethylformamide. The mixture was refluxed for 60 min to give a 
clear solution. The volatiles were removed in vacuo to give a yellow 
solid. The acid chloride was used without further purification for the 
condensation with amine. To another flask containing 
1-(2-aminoethyl)piperidene (1.1 g, 8.58 mmol), triethylamine (15 ml) and 
CHCl.sub.3 (40 ml) was added a solution of the above acid chloride in 
CHCl.sub.3 (20 ml). The mixture was stirred for 3 hours at room 
temperature. The volatiles were removed and the residue was taken up in 
CHCl.sub.3 (100 ml) and washed with water (2.times.50 ml). The organic 
layer was separated, dried over anhyd Na.sub.2 SO.sub.4, and the volatiles 
removed in vacuo to give a light yellow oil. The oil was purified by 
passage through a silica gel column when elution with CHCl.sub.3 
/MeOH:90/10. The desired fractions were combined and the volatiles were 
evaporated to give light yellow oil (2.4 g, 83%). Rf (TLC silica gel 
CHCl.sub.3 /MeOH:90/10)=0.7. .sup.1 H NMR (.delta. ppm): 1.42-1.60 (m, 6H, 
piperidinyl CH.sub.2 's); 2.42 (bs, 4H, piperidinyl NCH.sub.2); 2.48-2.52 
(t, J=6 Hz, 2H, CH.sub.2); 3.20-3.26 (t, J=9 Hz, 2H, NCH2); 3.48-3.54 (m, 
2H, NHCH.sub.2); 4.68-4.73 (t, J=9 Hz, 2H, OCH.sub.2); 7.33-7.35 (m, 1H, 
arom); 7.98-7.99 (m, 1H, arom); 8.05 (bt, 1H, NH). 
Synthesis of 
5-n-tributyltin-1-(2-aminoethylpiperidinyl)-2-3-dihydrobenzofuran-7-carbox 
amide (M) 
5-bromo-carboxamide (2.0 g, 5.63 mmol), bis(tributyltin) (3.3 g, 5.7 mmol), 
and palladium tetrakis (triphenylphosphine) (0.33 g, 0.28 mmol) were 
refluxed overnight (15 hrs.) in triethylamine (100 ml). The black residue 
was separated from the solvent. The volatiles were removed and the yellow 
residue was passed through a silica gel column and eluted first with 
CHCl.sub.3 (150 ml) and then with CHCl.sub.3 /MeOH:90/10. The fractions 
containing the desired compound were combined together and the volatiles 
were removed to give a light yellow viscous oil (1.4 g). .sup.1 H NMR 
(.delta. ppm): 0.82-0.87 (t, J=7 Hz, 9H, nBu.sub.3); 0.98-1.59 (m, 25 H, 
nBu.sub.3 ; and piperidinyl CH.sub.2); 2.48 (bm, 4H, piperidinyl 
NCH.sub.2); 2.49-2.54 (t, J=7 Hz, 2H, CH.sub.2); 3.21-3.26 (t, J=9 Hz, 2H, 
CH.sub.2); 3.51-3.55 (m, 2H, NCH.sub.2); 4.63-4.69 (t, J=9 Hz, 2H, 
OCH.sub.2); 7.34 (m, 1 H, arom); 7.95 (m, 1H, arom). 
The reactions described hereinabove are depicted in Reaction Scheme II. 
##STR15## 
EXAMPLE 3 
Synthesis of .sup.125 I(N-Benzylpiperidin-4-yl)-4-iodobenzamide 4-.sup.125 
!BP (N) 
Materials and Methods 
Melting points were determined with a Fisher-Johns apparatus. .sup.1 H and 
.sup.13 C NMR spectra were recorded on Brucker 300 AM spectrometer. Unless 
noted, chemical shifts were expressed as ppm using CDCl.sub.3 as an 
internal standard. All chemicals were obtained form the Aldrich chemical 
Company, Milwaukee, Wis. The thin layer chromatography (TLC) system 
consisted of Analtech uniplate silica gel GF plates (250 microns, 
10.times.20 cm), using CHCl.sub.3 /MeOH:90/10 as solvent. Radioactive 
spots were scanned and recorded by a Packard 7220/21 radiochromatogram. 
Mass spectra (chemical ionization) were recorded on Finnigan 1015 mass 
spectrometer. Na.sup.125 I was obtained from Amersham, Arlington Heights, 
Ill. 
Preparation of (N-benzylpiperidin-4-yl)-4-iodobenzamide 
A round bottom flask was charged with 4-iodobenzoic acid (3.0 g, 12.1 mmol) 
in chloroform (100 mL). To the solution was added thionyl chloride (5.0 
mL) in chloroform (10 mL) and 2-3 drops of dimethylformamide (DMF). The 
slurry was heated at reflux for 2 hours, while monitoring the reaction 
through an oil bubbler. A clear solution of 4-iodobenzoyl chloride was 
obtained. The volatiles were removed and a colorless oil was obtained 
which solidified upon cooling. 
The 4-iodobenzoyl chloride was dissolved in chloroform (40 mL) and added to 
a flask containing 4-amino-1-benzyl-piperidine (2.29 g, 12.1 mmol) in 
chloroform (75 mL) and triethylamine (20 mL). The mixture was stirred at 
room temperature overnight and the volatiles were removed in vacuo. The 
resulting slurry was washed with water (100 mL). The organics were 
dissolved in CHCl.sub.3 (100 mL), separated from the aqueous layer and 
dried over anhydrous Na.sub.2 SO.sub.4. The solvent was removed to give a 
solid (yield, 86%), m.p. 206.degree. C. m/e=420 (M.sup.+). .sup.1 H R 
(.delta. ppm): 1.47-1.60 (m, 2H, CH.sub.2); 1.97-2.11 (m, 2H, CH.sub.2); 
2.15-2.19 (m, 2H, CH.sub.2); 2.81-2.85 (m, 2H, CH.sub.2); 3.49 (s, 2H, 
benzyl CH.sub.2); 3.91-3.97 (m, 1H, CH); 5.98-6.00 (d, 1H, NH); 7.22-7.30 
(m, 5H arom); 7.43-7.45 (d, 2H, J=8 Hz, para substituted arom); 7.73-7.76 
(d, 2 H, J=8 Hz, para substituted arom). .sup.13 C NMR (.delta. ppm): 
32.23, 47.19, 52.25, 63.01, 127.14, 128.27, 128.49, 129.14, 134.18, 
137.75, 138.14, 166.02) 
Preparation of (N-benzylpiperidin-4-yl)-4-bromobenzamide 
(N-benzylpiperidin-4-yl)-4-bromobenzamide was prepared by a procedure like 
that described for N-benzylpiperidin-4-yl-4-iodobenzamide, but using 
4-bromobenzoic acid as starting material. 
Preparation of (N-benzylpiperidin-4-yl)-4-tri-n-butylstannylbenzamide 
A flame dried flask was charged with 
(N-benzylpiperidin-4-yl)-4-bromobenzamide (1.0 g, 2.68 mmol) in 
triethylamine (100 mL). Tetrakis (triphenylphosphine) palladium (300 mg, 
0.27 mmol), and bistributyltin (1.8 g, 3.10 mmol) were added, and the 
mixture was refluxed under nitrogen for 12 hours. The mixture was cooled 
and the volatiles were removed in vacuo. The resulting black oil was 
passed through a silica gel column with elution with CHCl.sub.3 (75 mL), 
followed by elution with CHCl.sub.3 MeOH: 95/5. The desired fractions, as 
characterized by thin layer chromatography, were pooled and solvent was 
evaporated to give an oil (57% yield). m/e=585 (M+1).sup.+ 1 H NMR 
(.delta. ppm): 0.82-1.99 (m, 31H, typical nBu.sub.3 and CH.sub.2); 
1.99-2.10 (m, 2H, CH.sub.2); 2.24-2.31 (m, 2H, NCH.sub.2); 2.92-2.97 (m, 
2H, NCH.sub.2); 3.61 (s, 2H benzylic CH.sub.2); 4.01-4.10 (m, 1H, CH); 
6.18-6.21 (d, 1H, NH); 7.24-7.67 (m, 9H, arom). .sup.13 C NMR (.delta., 
ppm): 9.61, 27.27, 28.99, 31.58, 46.42, 52.16, 62.68, 125.84, 127.63, 
128.40, 128.55, 129.52, 131.94, 131.21, 136.55, 167.19. 
Radiochemical Synthesis of .sup.125 
I(-N-benzylpiperidin-4-yl)-4-iodobenzamide 
To 100 .mu.L of a solution of 
(N-benzylpiperidin-4-yl)-4-tributyltinbenzamide (1 mg/ml) in ethanol was 
added a solution of Na.sup.125 I (0.5-1.0 mCi, 3-5 .mu.L) in 0.1N NaOH, 
followed by the addition of 0.05N HCl (50-100 .mu.L) to adjust the pH of 
the solution to pH 4.0-5.0. One hundred .mu.L of a freshly prepared 
solution of N-chloro-4-toluenesulfonamide sodium monohydrate 
(chloramine-T) (1 mg/ml) was added to the above mixture. The contents were 
incubated for 15 minutes at room temperature and 200 .mu.L of a solution 
of sodium metabisulfite (10 mg/ml) were added and incubated for 5 minutes. 
The reaction mixture was neutralized with a saturated solution of 
NaHCO.sub.3 (500 .mu.L). 0.4 mL of normal saline was added and the 
organics were extracted in CHCl.sub.3 (1.0 mL) after vortexing 30 seconds. 
The chloroform layer was evaporated in a stream of air. The residue was 
dissolved in methanol (400 .mu.L), and injected into a Gilson HPLC fitted 
with a Waters Z-module radial compression separation system containing a 
micro BondaPak C-18 reverse phase column equipped with Rheodyne 4125 
injector (0.5 mL loop). The fractions containing the desired compounds 
were pooled together and co-spotted on TLC along with authentic "cold" 
4-IBP and developed in CHCl.sub.3 /MeOH:90/10. The Rf of "cold" 4-IBP and 
4-.sup.125 I!BP was found to be 0.85 in the above solvent system. 
The reactions described hereinabove are depicted in Reaction Scheme III: 
##STR16## 
EXAMPLE 4 
Preparation of .sup.125 I-(N-Benzylpiperidin-4-yl)-3-iodobenzamide 
.sup.125 I-(N-Benzylpiperidin-4-yl)-3-iodobenzamide, 3-.sup.125 I!BP, was 
prepared by a procedure like that described above in Example 3, but using 
3-iodobenzoic acid as starting material. 
Unradiolabeled 3-IBP was prepared in 91% yield, m.p. 151-152.degree. C. 
m/e=420 (M.sup.+). .sup.1 H NMR (.delta. ppm): 1.58-1.62 (m, 2H, 
CH.sub.2); 1.97-2.01 (m, 2H, CH.sub.2); 2.14-2.22 (m, 2H, NCH.sub.2); 
2.85-2.89 (m, 2H, NCH.sub.2); 3.53 (s, 2H, benzylic CH.sub.2); 3.90-3.96 
(m, 1H, CH); 5.98-6.01 (d, 1H, NH); 7.11-7.16 (m, 1H, arom); 7.24-7.32 (m, 
5H, arom); 7.65-8.05 (m, 3H, arom). .sup.13 C NMR (.delta. ppm): 32.04, 
47.16, 52.20, 62.89, 126.06, 127.28, 128.32, 129.23, 130.22, 135.94, 
136.78, 137.67, 140.28, 165.28. 
EXAMPLE 5 
Preparation of .sup.125 I-(N-benzylpiperidin-4-yl)-2-iodobenzamide 
.sup.125 I-(N-benzylpiperidin-4-yl)-2-iodobenzamide, 2-.sup.125 I!BP, was 
prepared by a procedure like that described above in Example 3, but using 
2-iodobenzoic acid as starting material. 
Unradiolabeled 2-IBP was prepared in 90% yield, m.p. 145-146.degree. C. 
m/e=420 (M.sup.+). .sup.1 H NMR (.delta. ppm): 1.53-1.57 (m, 2H, 
CH.sub.2); 1.99-21.0 (m, 2H, CH.sub.2); 2.14-2.17 (m, 2H, NCH.sub.2); 
2.82-2.85 (m, 2H, NCH.sub.2); 3.50 (s, 2H, benzylic CH.sub.2); 3.91-3.97 
(m, 1H, CH); 6.02-6.10 (d, 1H, NH); 7.11-7.15 (m, 1H, arom); 7.23-7.29 (m, 
5H, arom); 7.65-7.79 (m, 2H, arom); 8.04 (m, 1H, arom). .sup.13 C 
(.delta., ppm): 32.13, 47.26, 52.20, 62.93, 126.04, 127.06, 128.20, 
129.07, 130.15, 135.89, 136.76, 138.13, 140.18, 163.26. 
EXAMPLE 6 
In Vitro Competitive Binding of Radioactive and Nonradioactive 
(2-PiperidinylAminoethyl)-4-IodoBenzamide 
Competitive binding studies indicate that compounds of the present 
invention bind malignant melanoma cells with very high affinity. 
Materials and Methods 
A2058 cells, derived from a brain metastasis of human malignant melanoma 
(Todaro et al. 1980 Proc. Natl. Acad. Sci. USA 77:5258) were obtained from 
the National Institutes of Health. These cells were grown in DMEM2 medium 
(Dulbecco's modification of Eagle's medium, EMEM) supplemented with 10% 
fetal bovine serum and 0.03% L-glutamine. 
(2-Piperidinylaminoethyl)4-iodobenzamide (i.e. IPAB, B) and .sup.125 
L(2-piperidinylaminoethyl)4-iodobenzamide (D) was synthesized as described 
in Example 1. 
In Vitro Cell Binding Assay 
A2058 cells, grown as described above, were harvested with calcium and 
magnesium free phosphate buffer (0.1M) containing 0.02% EDTA. Cells were 
washed twice with ice-cold RPMI 1640 medium (Gibco) without glutamine and 
resuspended in the same medium. Carrier-free .sup.125 I!PAB (0.1 ml) was 
added to eight aliquots of 0.1 ml test A2058 cells (1.5.times.10.sup.6 
cells in suspension). To observe competitive binding by non-radioactive 
IPAB, varying concentrations of non-radioactive IPAB were added in a 
volume of 0.1 ml. Cells were incubated at 37.degree. C. for 5 hr. after 
addition of radioactive and nonradioactive IPAB. 
After incubation, cells were collected by centrifugation for 5 min and 
washed twice with RPMI 1640 medium. The radioactivity bound to cells was 
counted using a Packard Autogamma 5650 scintillation counter. 
Data were analyzed with an INPLOT.RTM. iterative, non-linear least square 
curve fitting program. 
Results 
FIG. 1 illustrates that IPAB binds to human malignant melanoma cells with 
high affinity. In particular, FIG. 1 shows the amount of nonradioactive 
IPAB needed to competitively inhibit binding of radioactive IPAB. Binding 
of 50% of the radioactive IPAB was competitively inhibited by as little as 
6.8 nM (i.e. Ki is 6.8 nM). These data indicate that IPAB binding is so 
highly selective and stable that the interaction of IPAB with human 
malignant melanoma cells likely occurs by IPAB binding to a specific cell 
receptor. 
EXAMPLE 7 
In Vitro Binding Competition Between Pharmacological Antagonists and 
(2-PiperidinylAminoethyl)-4-IodoBenzamide 
Competitive binding studies indicate that compounds of the present 
invention bind cell surface sigma receptors on malignant melanoma cells. 
Materials and Methods 
A2058 cells, derived from a brain metastasis of human malignant melanoma 
(Todaro et al. 1980 Proc. Natl. Acad. Sci. USA 77:5258) are obtained from 
the National Institutes of Health. These cells are grown in DMEM2 medium 
(Dulbecco's modification of Eagle's medium, EMEM) supplemented with 10% 
fetal bovine serum and 0.03% L-glutamine. 
(2-Piperidinylaminoethyl)4-iodobenzamide (i.e. IPAB, B) and .sup.125 
I(2-piperidinylaminoethyl)4-iodobenzamide (D) is synthesized as described 
in Example 1. 
Pharmacological antagonists and the corresponding receptors which are 
tested include SE2466-2 (i.e. sigma receptor antagonist), fluphenazine 
(sigma-1 at low concentrations and sigma-2 at high concentrations), 
SCH23390 (dopamine-1), raclopride (dopamine-2), melanocyte secreting 
hormone peptide (melanocyte secreting hormone receptor), mianserin 
(5-hydroxytryptamine-1 receptor), NAN-190 (5-hydroxy-tryptamine-1a 
receptor), ketanserine (5-hydroxytryptamine-1c receptor), ketanserine and 
mianserin (5-hydroxytryptamine-2 receptor) and 3-tropanyldichloroben 
(5-hydroxytryptamine-3 receptor). 
In Vitro Cell Binding Assay 
A2058 cells, grown as described above, are harvested with calcium and 
magnesium free phosphate buffer (0.1M) containing 0.02% EDTA. Cells are 
washed twice with ice-cold RPMI 1640 medium (Gibco) without glutamine and 
resuspended in the same medium. Carrier-free .sup.125 I!PAB (0.1 ml) is 
added to eight aliquots of 0.1 ml test A2058 cells (1.5.times.10.sup.6 
cells in suspension). To observe competitive binding by pharmacological 
antagonists, varying concentrations of the antagonists are then added in a 
volume of 0.1 ml. Cells are incubated at 37.degree. C. for 5 hr. after 
addition of an antagonist and the radioactive IPAB. 
After incubation, cells are collected by centrifugation for 5 min and 
washed twice with RPMI 1640 medium. The radioactivity bound to cells is 
counted using a Packard Autogamma 5650 scintillation counter. 
Data can be analyzed with an INPLOT.RTM. iterative, non-linear least square 
curve fitting program. 
Results 
Antagonists with demonstrated binding specificity for cell surface sigma 
receptors (e.g. fluphenazine) can act as competitive binding inhibitors of 
IPAB binding to malignant melanoma cells. In contrast, antagonists that do 
not bind to cell surface sigma receptors cannot inhibit binding of 
radioactive IPAB to melanoma cells. Such data indicate that the present 
compounds bind to cell surface sigma receptors. 
EXAMPLE 8 
Binding Competition Between Pharmacological Antagonist and 
(2-PiperidinylAminoethyl)-4-IodoBenzamide 
Materials and Methods 
(2-Piperidinylaminoethyl)4-iodobenzamide (i.e. IPAB, B) was synthesized as 
described in Example 1. 
A sigma-1 binding assay was performed in guinea pig brain membranes and rat 
C6 glioma cells (purchased from American Tissue and Cell Collection, 
Rockville, Md.) in the presence of a sigma-1 selective ligand, .sub.3 
H!-(+)-pentazocine. 
A sigma-2 binding assay was performed in rat liver membranes in the 
presence of a sigma-2 selective ligand, .sup.3 H!DTG, in the presence of 
dextrallorphan to mask sigma-1 sites. 
Membrane Preparation 
A plasma membrane-mitochondrial (P2) membrane fraction was prepared from 
frozen guinea pig brains (Pel-Freeze, Rogers, Ak.), minus cerebellum. The 
brain tissue was thawed slowly before homogenization. A crude P2 membrane 
fraction was also prepared from the livers of rat Sprague-Dawley rats 
(150-220 g, Taconic Farms) liver. The animals were decapitated and their 
livers were minced and homogenized. The tissue homogenization was carried 
out at 4.degree. C. in ml/g tissue weight of 10 mM Tris-HCl/0.32M sucrose, 
pH=7.4 using 10 motor-driven strokes in a Potter-Elvehjem Teflon glass 
homogenizer. The crude homogenate was centrifuged for 10 min at 100 g and 
the crude nuclear (P1) pellet was discarded. Supernatants were centrifuged 
at 31000 g for 15 min to yield a plasma membrane-mitochondrial pellet 
(P2). This pellet was resuspended in 3 ml/g in 10 mM tris-HCl, pH 7.4 and 
used for binding studies. Protein concentrations were determined by the 
method of Lowry. 
Various concentrations of the IPAB ranging from 0.5-1000 nM were incubated 
with guinea pig brain membranes (300-500 microgram protein) in the 
presence of 3 nm .sup.3 H!-(+)-pentazocine (specific activity 52 Ci/mmol) 
in 0.5 ml of 50 mM Tris-HCl for 60 min at 37.degree. C. The amount of 
non-specific binding was determined by the addition of 10 mM Tris-HCl, pH 
8.0 followed by rapid filtration through glass filters using a Brandel 
Cell harvester (Gaithersburg, Md.). Filters were washed twice with 
ice-cold buffer. Prior to use, filters were soaked in 0.5% 
polyethyleneimine for about 30 min at 25.degree. C. Similarly rats liver 
membranes (sigma-2) or C6 glioma cell homogenates were incubated with 3 nM 
.sup.3 H!DTG (39.4 Ci/mmol) in the presence of 1 micromolar cold 
dextrallorphan and various concentration of the unlabeled IPAB. The amount 
of non-specific binding was determined by incubation of membranes in the 
presence of 5 micromolar haloperidol. 
When the assay was terminated, the membranes were filtered and the filtrate 
washed twice as above. The radioactivity was counted in Ecoscint (National 
Diagnostics, Manville, N.J.) after an overnight extraction of counts. 
The amount of IPAB required to inhibit binding of sigma-1 and sigma-2 
selective ligands by 50% (i.e. the IC.sub.50 values) was derived using the 
computerized iterative curve-fitting program, GraphPAD. K.sub.i values 
were calculated from the IC.sub.50 values using Cheng-Prusoff equation. 
______________________________________ 
Results 
The K.sub.i values for IPAB are shown in Table 1. 
Sigma-1 Sigma-2 Sigma-2 
Guinea Pig Brain 
Rat Liver 
C.sub.6 Glioma Cells 
______________________________________ 
0.89 nM 24.0 nM 130 nM 
______________________________________ 
These data demonstrate that IPAB binds to cell surface sigma receptors with 
very high affinity. 
EXAMPLE 9 
Inhibition Constants (Ki=nM) For Binding Affinities of IBP Isomers in 
Various Receptor Systems 
Materials and Methods 
(N-Benzylpiperidin-4-yl)-2-iodobenzamide (i.e. 2-IBP) was synthesized as 
described in Example 5. 4-IBP and 3-IBP were synthesized as described in 
Examples 3 and 4 respectively. 
The sigma-1 binding assay was performed in guinea pig brain membranes in 
the presence of a sigma-1 selective ligand, .sup.3 H!-(+)-pentazocine 
purchased from DuPont NEN, Boston Mass. 
The sigma-2 binding assay was performed in rat liver membranes in the 
presence of a sigma-2 selective ligand, .sup.3 H!DTG, in the presence of 
dextrallorphan to mask sigma-1 sites. .sup.3 H!DTG was purchased from 
DuPont NEN, Boston Mass. 
A similar procedure was used with each IBP compound but will be shown using 
the 2-IBP compound. 
Membrane Preparation 
A plasma membrane-mitochondrial (P2) membrane fraction was prepared from 
frozen guinea pig brains (Pel-Freeze, Rogers, Ak.), minus cerebellum. The 
brain tissue was thawed slowly before homogenization. A crude P2 membrane 
fraction was also prepared from the livers of Sprague-Dawley rats (150-220 
g, Taconic Farms). The animals were decapitated and their livers were 
minced and homogenized. The tissue homogenization was carried out at 
4.degree. C. in 10 mM tris-HCl/0.32M sucrose, pH=7.4 using 10 motor-driven 
strokes in a Potter-Elvehjem Teflon glass homogenizer. The crude 
homogenate was centrifuged for 10 min at 1000 g and the crude nuclear (P1) 
pellet was discarded. Supernatants were centrifuged at 31000.times.g for 
15 min to yield a plasma membrane-mitochondrial pellet (P2). This pellet 
was resuspended in 3 ml/g in 10 mM tris-HCl, pH 7.4 and used for binding 
studies. Protein concentrations were determined by the method of Lowry. 
Various concentrations of 2-IBP ranging from 10.sup.-4 to 10.sup.-12 M were 
incubated with guinea pig brain membranes (300-500 microgram protein) in 
the presence of 3 nM .sup.3 H!-(+)-pentazocine (specific activity 51.7 
Ci/mmol) in 0.5 ml of 50 mM tris-HCl for 120 min at 25.degree. C. The 
amount of non-specific binding was determined by the addition of 10 mM 
tris-HCl, pH 8.0 followed by rapid filtration through glass filters using 
a Brandel Cell harvester (Gaithersburg, Md.). Filters were washed twice 
with ice-cold 10 mM tris-HCL. Prior to use, filters were soaked in 0.5% 
polyethyleneimine for about 30 min at 25.degree. C. 
Similarly rats liver membranes (sigma-2) were incubated with 3 nM .sup.3 
H!DTG (39.4 Ci/mmol) in the presence of 1 .mu.M cold dextrallorphan and 
various concentration of the unlabeled IBP. The amount of non-specific 
binding was determined by incubation of membranes in the presence of 10 
micromolar haloperidol. 
When the assay was terminated, the membranes were filtered and the filtrate 
washed twice as above. The radioactivity was counted in Ecoscint (National 
Diagnostics, Manville, N.J.) after an overnight extraction of counts. 
The amount of IBP required to inhibit binding of sigma-1 and sigma-2 
selective ligands by 50% (i.e. the IC.sub.50 values) was derived using the 
computerized iterative curve-fitting program, GraphPAD. K.sub.i values 
were calculated from the IC.sub.50 values using Cheng-Prusoff equation. 
Results 
The K.sub.i values for IBP compounds are shown in Table 2: 
______________________________________ 
SIGMA-1 GUINEA PIG BRAIN 
SIGMA-2 RAT LIVER 
COMPD .sup.3 H!-(+)-PENT 
.sup.3 H!DTG + DEX 
______________________________________ 
4-IBP 1.70 .+-. 0.44 25.2 .+-. 1.28 
3-IBP 3.02 .+-. 1.06 84.6 .+-. 2.5 
2-IBP 1.64 .+-. 0.15 29.6 .+-. 0.49 
______________________________________ 
DOPAMINE D-2 MUSC RAT 
RAT BRAIN PCP RAT BRAIN 
BRAIN 
COMPD .sup.3 H!-(-)-SULPIRIDE 
.sup.3 H!TCP 
.sup.3 H!QNB 
______________________________________ 
4-IBP 382 .+-. 39 &gt;100 000 &gt;100 000 
3-IBP 24.8 .+-. 0.02 
&gt;100 000 &gt;100 000 
2-IBP 63.4 .+-. 10.8 
&gt;100 000 &gt;100 000 
______________________________________ 
These data demonstrate that all three IBP compounds bind sigma-1 sites on 
guinea pig brains with high affinity. The 2- and 4-IBP compounds also had 
moderate affinity for rat liver sigma-2 sites. The affinity of 3-IBP for 
sigma-2 sites was relatively low. 
The data in Table 2 show low affinity for dopamine, phenylcyclidine, PCP, 
and muscarinic receptors. 
EXAMPLE 10 
Biodistribution of .sup.125 I-(2-PiperidinylAminoethyl)-4-IodoBenzamide 
Biodistribution experiments were performed to assess the tumor-specificity 
of the present compounds. 
Materials and Methods 
A2058 tumor cells, derived from a brain metastasis of human malignant 
melanoma (Todaro et al. 1980 Proc. Natl. Acad. Sci. USA 77:5258) were 
obtained from the National Institutes of Health. 
Non-small cell lung carcinoma cell lines NCI-157, NCI-838 and NCI-1299 were 
obtained from the National Cancer Institute. The NCI-157 cell line is a 
squamous carcinoma cell line, while NCI-838 is an adenocarcinoma cell line 
and NCI-1299 is a large cell lung carcinoma cell. 
Tumor cells were grown in DMEM2 medium (Dulbecco's modification of Eagle's 
medium, EMEM) supplemented with 10% fetal bovine serum and 0.03% 
L-glutamine. 
.sup.125 I-N-(diethylaminoethyl)4-iodobenzamide (i.e..sup.125 I!DAB) was 
prepared as described in John et al. (1993 Nucl. Med. Biol. 20: 75-79). 
.sup.125 I(2-piperidinylaminoethyl)4-iodobenzamide (i.e. .sup.125 I!PAB) 
(D) was synthesized as described in Example 1. 
Animal Biodistribution Assays 
For in vivo studies, tumor cells were harvested using calcium and magnesium 
free PBS containing 0.02% EDTA. Suspension of 5.times.10.sup.6 cells 
(viability greater than 95%) in 0.2 mL of medium were inoculated 
subcutaneously in female Balb/c nu/nu mice. After about two weeks, solid 
tumors of about 1 cm in diameter appeared in approximately 85% of all 
inoculated mice. Mice with solid tumors having a diameter of about 1 cm 
were used for biodistribution studies. 
Balb/c nu/nu mice (17-22 g) were injected intravenously with 0.2 ml of a 
saline solution containing .sup.125 I!PAB (5-6 pCi). At 1, 6 and 24 hr. 
after injection, blood samples were collected by cardiac puncture and the 
mice were sacrificed immediately thereafter by cardiectomy while under 
halothane anesthesia. The organs of interest were subsequently excised, 
blotted with tissue paper, weighed, and the radioactivity was counted 
using a Packard automatic counter (autogamma 5650). The % injected dose/g 
(% ID/g) values were determined by comparison of tissue radioactivities 
with suitably diluted aliquots of the injected .sup.125 I!PAB dose 
divided by the weight of the organ. The values obtained were normalized to 
a mouse weighing 20 g. The differences between .sup.125 I!PAB and 
.sup.125 I!DAB were examined by Student's unpaired t tests. 
Results 
Tables 1-3 illustrate the biodistribution of .sup.125 I!PAB and .sup.125 
I!DAB in nude mice bearing human A2-058 melanoma xenografts in the flank 
at one, six, and twenty-four hours, respectively, after administration of 
the imaging agent. 
At one hr. post-injection (Table 1), the concentration of .sup.125 I!DAB 
(% injected dose/gm) was higher than the tumor concentration of .sup.125 
I!PAB in several tissues including non-tumorous liver, muscle, lung and 
heart tissues. Therefore, while .sup.125 I!DAB collected in the tumor at 
a marginally higher level than .sup.125 I!PAB, .sup.125 I!DAB was 
significantly less specific for the tumor site than .sup.125 I!PAB. 
By 6 hrs. after administering the diagnostic agents, mice receiving 
.sup.125 I!PAB had more of this diagnostic agent in the tumor than any 
other tissue. In contrast .sup.125 I!DAB was found at higher 
concentrations in the liver than in the tumor. Moreover, the concentration 
of .sup.125 I!DAB was significantly higher than that of .sup.125 I!PAB 
is non-cancerous blood, liver and intestinal tissues. 
By 24 hrs. mice receiving .sup.125 I!PAB had about four-fold more 
.sup.125 I!PAB in their tumors than in their livers. In contrast mice 
receiving .sup.125 I!DAB had only about half as much .sup.125 I!DAB in 
their tumors as their livers. These data indicate that high levels of 
.sup.125 I!DAB are non-specifically localized in the liver. These data 
also indicate that .sup.125 I!DAB has less tumor specificity than 
.sup.125 I!PAB. 
Moreover, the tumor concentration of .sup.125 I!PAB was almost twice as 
high as that of .sup.125 I!DAB indicating that IPAB binds to tumor cells 
with greater affinity and stability than IDAB. These data indicate that 
.sup.125 I!PAB is highly specific for malignant tumors which contain 
cells having sigma receptors. 
TABLE 1 
______________________________________ 
Biodistribution of N-(piperidinylaminoethyl)- 
4-iodo .sup.125 I!benzamide, .sup.125 I!PAB, and 
N-(diethylaminoethyl)4-iodo .sup.125 I!benzamide, 
.sup.125 IDAB, in nude mice xenografted with human 
melanotic melanoma % ID/g; mean (std. dev.), n = 61 
P Value for 
1 Hour .sup.125 I!PAB 
.sup.125 I!DAB 
Difference 
______________________________________ 
Blood 0.967(.168) 
1.03(.318) 
NS 
Liver 6.36(.770) 12.7(1.69) 
&lt;.001 
Spleen 3.11(.789) 3.46(.206) 
NS 
Kidney 3.82(.561) 4.63(.905) 
NS 
Bone 0.750(.0663) 
1.04(.476) 
NS 
Muscle 0.552(.0711) 
0.988(.125) 
&lt;.001 
Stomach 3.23(.697) 3.84(1.98) 
NS 
Intestine 10.64(.541) 
5.04(1.47) 
&lt;.001 
Thyroid 4.23(.594) 5.68(1.02) 
.013 
Lung 2.34(.277) 6.32(1.55) 
&lt;.001 
Heart 1.14(.207) 1.67(.210) 
.001 
Brain 0.895(.0887) 
1.04(.0855) 
.015 
Tumor 3.87(.470) 5.18(1.31) 
.044 
RATIO 
Tumor/Blood 4.16(1.09) 5.68(2.75) 
Tumor/Muscle 
7.16(1.50) 5.33(1.65) 
______________________________________ 
TABLE 2 
______________________________________ 
Biodistribution of N-(piperidinylaminoethyl)- 
4-iodo .sup.125 I!benzamide, .sup.125 I!PAB, and 
N-(diethylaminoethyl)4-iodo .sup.125 I!benzamide, 
.sup.125 I!DAB, in nude mice xenografted with human 
melanotic melanoma % ID/g; mean (std. dev.), n = 6! 
P Value for 
6 hour .sup.125 I!PAB 
.sup.125 I!DAB 
Difference 
______________________________________ 
Blood 0.208(.0542) 
1.03(.0.197) 
.001 
Liver 1.16(.212) 3.74(.427) &lt;.001 
Spleen 0.330(.105) 0.260(.0990) 
NS 
Kidney 0.483(.131) 0.435(.0909) 
NS 
Bone 0.115(.0236) 
0.100(.0297) 
NS 
Muscle 0.0983(.0306) 
0.0967(.0356) 
NS 
Stomach 0.757(.298) 0.475(.164) NS 
Intestine 2.46(1.18) 0.423(.0963) 
.002 
Thyroid 0.583(.203) 0.400(.124) NS 
Lung 0.387(.0568) 
0.458(.0993) 
NS 
Heart 0.167(.0372) 
0.150(.0329) 
NS 
Brain 0.1.22(.0331) 
0.132(.0204) 
NS 
Tumor 2.91(.463) 2.83(.388) NS 
RATIO 
Tumor/Blood 
14.9(5.07) 28.1(5.84) .002 
Tumor/Muscle 
32.5(12.0) 33.3(14.0) NS 
______________________________________ 
TABLE 3 
______________________________________ 
Biodistribution of N-(piperidinylaminoethyl)- 
4-iodo .sup.125 I!benzamide, .sup.125 I!PAB, and 
N-(diethylaminoethyl)4-iodo .sup.125 I!benzamide, 
.sup.125 I!DAB, in nude mice xenografted with human 
melanotic melanoma % ID/g; mean (std. dev.), n = 6! 
P Value for 
24 Hour .sup.125 I!PAB 
.sup.125 I!DAB 
Difference 
______________________________________ 
Blood 0.0617(.018) 
0.0350(.0084) 
.009 
Liver 0.263(.0216) 
1.12(.232) &lt;.001 
Spleen 0.0383(.015) 
0.0350(.023) 
NS 
Kidney 0.0850(.016) 
0.065(.0197) 
NS 
Bone 0.0133(.0052) 
0.0133(.0052) 
NS 
Muscle 0.0117(.0041) 
0.0150(.0084) 
NS 
Stomach 0.130(0.881) 
0.445(.386) NS 
Intestine 0.132(.0852) 
0.123(.0717) 
.NS 
Thyroid 0.100(.143) 0.0550(.0207) 
NS 
Lung 0.0717(.0075) 
0.0633(.0273) 
NS 
Heart 0.0283(.0075) 
0.0233(.0175) 
NS 
Brain 0.0067(.0052) 
0.0033(.0052) 
NS 
Tumor 1.028(.239) 0.553(.241) .006 
RATIO 
Tumor/Blood 
17.8(6.10) 15.5(4.69) NS 
Tumor/Muscle 
94.5(32.5) 39.7(9.61) .003 
______________________________________ 
EXAMPLE 11 
Biodistribution of .sup.125 I-(2-PiperidinylAminoethyl)-4-IodoBenzamide 
Biodistribution experiments were performed to assess the tumor-specificity 
of the present compounds. 
Materials and Methods 
Non-small cell lung carcinoma cell lines NCI-157, NCI-838 and NCI-1299 were 
obtained from the National Cancer Institute. The NCI-157 cell line is a 
squamous carcinoma cell line, while NCI-838 is an adenocarcinoma cell line 
and NCI-1299 is a large cell lung carcinoma cell. 
Tumor cells were grown in DMEM2 medium (Dulbecco's modification of Eagle's 
medium, EMEM) supplemented with 10% fetal bovine serum and 0.03% 
L-glutamine. 
.sup.125 I-N-(diethylaminoethyl)4-iodobenzamide (i.e. .sup.125 I!DAB) was 
prepared as described in John et al. (1993 Nucl. Med. Biol. 20: 75-79). 
.sup.125 I(2-piperidinylaminoethyl)4-iodobenzamide (i.e. .sup.125 I!PAB) 
(D) was synthesized as described in Example 1. 
Animal Biodistribution Assays 
For in vivo studies, tumor cells were harvested using calcium and magnesium 
free PBS containing 0,02% EDTA. Suspension of 5.times.10.sup.6 cells 
(viability greater than 95%) in 0.2 mL of medium were inoculated 
subcutaneously in female Balb/c nu/nu mice. After about two weeks, solid 
tumors of about 1 cm in diameter appeared in approximately 85% of all 
inoculated mice. Mice with solid tumors having a diameter of about 1 cm 
were used for biodistribution studies. 
Balb/c nu/nu mice (17-22 g) were injected intravenously with 0.2 ml of a 
saline solution containing .sup.125 I!PAB (5-6 .mu.Ci). At 1, 6 and 24 
hr. after injection, blood samples were collected by cardiac puncture and 
the mice were sacrificed immediately thereafter by cardiectomy while under 
halothane anesthesia. The organs of interest were subsequently excised, 
blotted with tissue paper, weighed, and the radioactivity was counted 
using a Packard automatic counter (autogamma 5650). The % injected dose/g 
(% ID/g) values were determined by comparison of tissue radioactivities 
with suitably diluted aliquots of the injected .sup.125 I!PAB dose 
divided by the weight of the organ. The values obtained were normalized to 
a mouse weighing 20 g. 
Results 
Tables 4-5 illustrate the biodistribution of .sup.125 I!DAB and .sup.125 
I!PAB, respectively, in nude mice bearing human squamous cell carcinoma 
xenografts in the flank at one, six, and twenty-four hours after 
administration of the imaging agent. 
By 24 hrs. mice receiving .sup.125 I!PAB had more .sup.125 I!PAB in their 
tumors than any other tissue. In contrast mice receiving .sup.125 I!DAB 
had about six-fold more .sup.125 I!DAB in their livers as their tumors. 
These data indicate that high levels of .sup.125 I!DAB are 
non-specifically localized in the liver. These data also indicate that 
.sup.125 I!DAB has less tumor specificity than .sup.125 I!PAB. 
Moreover, the tumor concentration of .sup.125 I!PAB was more than 
three-fold higher than that of .sup.125 I!DAB at 24 hrs. post-injection 
indicating that IPAB binds to tumor cells with greater affinity and 
stability than IDAB. These data indicate that .sup.125 I!PAB is highly 
specific for lung carcinomas which contain cells having sigma receptors. 
TABLE 4 
______________________________________ 
Biodistribution of N-(diethylaminoethyl)- 
4-iodo .sup.125 I!benzamide, .sup.125 I!DAB, in nude mice 
xenografted with human squamous cell carcinoma 
% ID/g; mean (std. dev.), n = 6! 
Tissue 1 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 1.35(0.42) 0.301(0.04) 
0.027(0.00) 
Liver 13.77(0.72) 5.84(0.51) 1.25(0.10) 
Spleen 3.58(0.28) 0.51(0.08) 0.01(0.00) 
Kidney 7.64(0.35) 1.07(0.16) 0.05(0.00) 
Bone 1.85(0.22) 0.21(0.01) -- 
Stomach 5.41(0.53) 2.7(0.98) 0.15(0.02) 
Intestine 5.64(0.58) 1.34(0.27) 0.05(0.01) 
Thyroid 8.22(0.72) 1.48(0.55) 0.03(0.01) 
Lung 6.38(0.89) 1.07(0.14) 0.03(0.09) 
Heart 2.21(0.16) 0.38(0.11) 0.00(0.09) 
Brain 1.57(0.05) 0.23(0.05) 0.00(0.00) 
Tumor 5.13(0.74) 2.17(0.09) 0.18(0.02) 
______________________________________ 
TABLE 5 
______________________________________ 
Biodistribution of (piperidinylaminoethyl)- 
4-iodo .sup.125 I!benzamide, .sup.125 I!PAB, in nude mice 
xenografted with human squamous cell carcinoma 
% ID/g; mean (std. dev.), n = 6! 
Tissue 1 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 1.99(0.35) 0.51(0.03) 0.15(0.07) 
Liver 10.48(1.26) 3.47(0.23) 0.39(0.02) 
Spleen 3.97(0.18) 0.86(0.20) 0.05(0.00) 
Kidney 6.56(0.04) 1.61(0.28) 0.13(0.00) 
Bone 2.03(0.28) 0.56(0.18) 0.02(0.00) 
Stomach 6.57(1.31) 3.46(0.18) 0.20(0.03) 
Intestine 12.63(0.43) 11.41(0.52) 
0.38(0.09) 
Thyroid 7.84(0.91) 2.80(0.49) 0.09(0.01) 
Lung 4.91(0.25) 1.10(0.03) 0.08(0.00) 
Heart 2.25(0.07) 0.49(0.03) 0.03(0.00) 
Brain 1.80(0.17) 0.29(0.01) 0.01(0.00) 
Tumor 4.27(0.40) 3.27(0.33) 0.66(0.12) 
______________________________________ 
EXAMPLE 12 
Biodistribution of .sup.125 I-(N-Benzylpiperidin-4-yl)-4-IodoBenzamide 
Biodistribution experiments were performed to assess the clearance of 
.sup.125 I-(N-benzylpiperidin-4-yl)-4-IodoBenzamide, 4-.sup.125 I!BP. 
Materials and Methods 
.sup.125 I-(N-Benzylpiperidin-4-yl)-4-iodobenzamide, 4-.sup.125 I!BP, was 
prepared as described in Example 3. 
For in vivo studies, Wistar rats were used. 
Animal Biodistribution Assays 
Male Wistar rats (200-230 g), while under anesthesia, were injected 
intravenously with 0.1 ml of a saline solution containing 20% ethanol 
solution of 4-.sup.125 I!BP(4-5 pCi). At 1, 4, 6 and 24 hr. after 
injection, blood samples were collected by cardiac puncture and the mice 
were sacrificed immediately thereafter by cardiectomy while under 
anesthesia. The organs of interest were subsequently excised, blotted with 
tissue paper, weighed, and the radioactivity was counted using a Packard 
automatic counter (autogamma 5650). The % injected dose/g (% ID/g) values 
were determined by comparison of tissue radioactivities with suitably 
diluted aliquots of the injected 4-.sup.125 I!BP dose divided by the 
weight of the organ. 
A similar procedure was performed with 2-.sup.125 I!BP and 3-.sup.125 
I!BP but blood samples were only collected at 1, 6 and 24 hours. 
Results 
Table 6 illustrates the biodistribution of 4-.sup.125 I!BP in Wistar male 
rats at one, four, six and twenty-four hours after administration of the 
radiolabeled compound. 
The results in Table 6 show that 4-.sup.125 I!BP cleared quickly form the 
blood pool. After 24 hours, thyroid activity was minimal indicating that 
there is no in vivo dehalogenation. The results show that the compound 
crossed the blood brain barrier and is retained in the brain after 24 
hours. 
There is high uptake of 4-.sup.125 I!BP in the liver, lungs and kidneys. 
These data indicate that 4-.sup.125 I!BP is highly selective for organs 
containing cells that have sigma receptors. 
The results for 3-.sup.125 I!BP are shown in Table 8. These data show fast 
clearance from the blood, 0.75%ID/organ at one hour, but a high level in 
the liver. 
The results for 2-.sup.125 I!BP are shown in Table 7. These data show fast 
clearance from the blood and normal organs. 
The results for 3-.sup.125 I!BP are shown in Table 8. These data show fast 
clearance from the blood, 0.75%ID/organ at one hour, but a high level in 
the liver. 
TABLE 6 
______________________________________ 
4-.sup.125 I!-(N-Benzylpiperidin-4-yl)-4- 
iodobenzamide, 4-.sup.125 I!BP in Wistar Male Rats 
(% ID/g; each data point represent average of four rats) 
Tissue 1 hr. 4 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 0.04 .+-. 0.00 
0.02 .+-. 0.00 
0.04 .+-. 0.00 
0.02 .+-. 0.00 
Heart 0.75 .+-. 0.05 
0.74 .+-. 0.04 
0.75 .+-. 0.02 
0.65 .+-. 0.03 
Lung 5.05 .+-. 0.26 
5.22 .+-. 0.78 
4.85 .+-. 0.17 
3.70 .+-. 0.38 
Liver 4.77 .+-. 0.13 
7.76 .+-. 0.36 
7.20 .+-. 0.40 
5.37 .+-. 0.53 
Spleen 1.60 .+-. 0.10 
1.51 .+-. 0.19 
1.57 .+-. 0.12 
1.47 .+-. 0.28 
Kidney 2.10 .+-. 0.15 
2.05 .+-. 0.02 
2.10 .+-. 0.08 
1.54 .+-. 0.05 
Gonads 0.39 .+-. 0.03 
0.30 .+-. 0.02 
0.34 .+-. 0.01 
0.34 .+-. 0.01 
Muscle 0.24 .+-. 0.02 
0.20 .+-. 0.02 
0.12 .+-. 0.02 
0.12 .+-. 0.01 
Bone 0.24 .+-. 0.01 
0.24 .+-. 0.05 
0.44 .+-. 0.03 
0.47 .+-. 0.07 
Brain 1.24 .+-. 0.13 
1.29 .+-. 0.05 
1.23 .+-. 0.08 
1.24 .+-. 0.06 
Thyroid 0.07 .+-. 0.01 
0.12 .+-. 0.00 
0.23 .+-. 0.05 
0.29 .+-. 0.04 
Ratios: 
Brain/Blood 
33.00 64.50 35.07 54.89 
Heart/Blood 
134.78 260.80 138.57 164.22 
______________________________________ 
TABLE 7 
______________________________________ 
Biodistribution of .sup.125 I-(N-Benzylpiperidin- 
4-yl)-2-iodobenzamide, 2-.sup.125 I!BP 
in Sprague Dawley rats (% ID/whole organ) 
each data point represents an average of four rats. 
Tissue 1 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 2.06 .+-. 0.42 
1.30 .+-. 0.12 
0.50 .+-. 0.05 
Heart 0.17 .+-. 0.03 
0.05 .+-. 0.00 
0.01 .+-. 0.00 
Liver 22.36 .+-. 1.29 
6.07 .+-. 0.18 
0.59 .+-. 0.02 
Spleen 0.32 .+-. 0.07 
0.12 .+-. 0.01 
0.02 .+-. 0.00 
Kidney 1.68 .+-. 0.08 
0.39 .+-. 0.06 
0.05 .+-. 0.01 
Lung 1.06 .+-. 0.21 
0.21 .+-. 0.02 
0.04 .+-. 0.00 
Muscle 16.15 .+-. 2.13 
6.75 .+-. 0.32 
1.95 .+-. 0.11 
Brain 0.48 .+-. 0.12 
0.05 .+-. 0.01 
0.01 .+-. 0.00 
Thyroid 0.14 .+-. 0.04 
1.91 .+-. 0.23 
2.11 .+-. 0.35 
______________________________________ 
TABLE 8 
______________________________________ 
Biodistribution of .sup.125 I-(N-Benzylpiperidin- 
4-yl)-3-iodobenzamide, 3-.sup.125 I!BP 
in Sprague Dawley rats (% ID/whole organ) 
each data point represents an average of four rats. 
Tissue 1 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 0.75 .+-. 0.05 
0.55 .+-. 0.13 
0.52 .+-. 0.05 
Heart 0.15 .+-. 0.01 
0.07 .+-. 0.01 
0.15 .+-. 0.01 
Liver 13.35 .+-. 0.62 
9.06 .+-. 0.71 
6.65 .+-. 1.63 
Spleen 0.47 .+-. 0.02 
0.37 .+-. 0.05 
0.65 .+-. 0.08 
Kidney 3.49 .+-. 0.23 
2.25 .+-. 0.16 
1.75 .+-. 0.13 
Lung 0.82 .+-. 0.07 
0.43 .+-. 0.05 
1.05 .+-. 0.04 
Muscle 9.36 .+-. 0.58 
1.23 .+-. 0.49 
03.7 .+-. 0.44 
Brain 0.61 .+-. 0.04 
0.53 .+-. 0.26 
0.81 .+-. 0.07 
Thyroid 0.13 .+-. 0.02 
0.53 .+-. 0.26 
3.43 .+-. 0.21 
______________________________________ 
EXAMPLE 13 
Saturation Binding of 4-.sup.125 I!BP in MCF-7 Breast Cancer Cells 
Competitive binding studies were performed with compounds of the present 
invention to assess their affinity for sigma sites on human breast cancer 
cells. 
Materials and Methods 
MCF-7 cells, a line of human breast cancer cells, were obtained from the 
National Cancer Institute. These cells were cultured in serum supplemented 
medium (RPMI-1640) containing 10% heat inactivated fetal bovine serum 
(GIBCO) at 37.degree. C. The cells were adherent and split weekly in a 
1:20 ratio using trypsin/EDTA (GIBCO). The cells were then transferred to 
24 well plates and allowed to be adherent and confluent (about 0.5 million 
cells) or the cells were grown in T75 cell culture flasks and were 
detached when they were confluent using trypsin/EDTA with DMEM. 
I-(N-Benzylpiperidin-4-yl)-4-iodobenzamide, 4-.sup.125 I!BP, was prepared 
according to the description in Example 3. 
.sup.3 H!DTG was obtained from DuPont NEN Boston, Mass. 
Scatchard Analysis of Binding of .sup.3 H!DTG in MCF-7 Cell Membranes 
Crude membranes from MCF-7 cells were prepared by homogenization of cells 
(Potter-Elvehjem homogenizer with teflon pestle) in ice-cold 10 mM 
tris-HCl, pH 7.4 containing 0.32M sucrose at a density of 1.times.10.sup.7 
-10.sup.8 cells/ml. The homogenate was then centrifuged at 31,000.times.g 
for 15 min. at 4.degree. C. and the pellet resuspended in ice-cold 10 mM 
tris-HCl, pH 7.4 to a protein concentration of 15-20 mg/ml, as determined 
by method of Lowry with bovine serum albumin as standard. Binding assay 
with .sup.3 H!DTG was carried out under the conditions described in 
Example 9 for sigma-2 receptors except that a temperature of 37.degree. C. 
was used. Using 15 different concentrations ranging from 1-400 nM, .sup.3 
H!DTG was incubated in the presence of 1 .mu.M dextrallorphan. A 
combination of labeled and unlabeled ligand was used to achieve 
concentrations above 15 nM for .sup.3 H!DTG. 
In Vitro Scatchard Plot of 4-.sup.125 I!BP in MCF-7 Breast Cancer Cells 
The Scatchard analysis was carried out using cell suspension in culture 
tubes (13.times.100 mm). 4-.sup.125 I!BP was incubated with MCF-7 cells 
(10,000-20,000 cells) in 12 concentrations at 0.01 nM to 300 nM. A 
combination of labeled and unlabeled ligand was used. The non-specific 
binding was determined in the presence of 10.sup.-5 M 4-IBP. The cells 
were incubated for 1 hr. at 37.degree. C. in a CO.sub.2 incubator. The 
cells were filtered through a Brandel Cell Harvester on a Whatman Filter 1 
and washed twice with 3.0 mL of de-ionized water. The activity associated 
with the cells was counted in Beckman Gamma Counter (DP 5500). The data 
obtained were analyzed using the iterative curve-fitting program, BDATA 
(EMP Software, Baltimore, Md.). The amount of protein was determined using 
bicinchonic acid (BCA) protein assay reagent obtained from Pierce, 
Rockford, Ill. with bovine serum albumin as the standard. 
Results 
The Scatchard analyses for binding of .sup.3 H!DTG in MCF-7 cells are 
shown in FIG. 5. 
The Scatchard plot for 4-.sup.125 I!BP binding in MCF-7 cells is shown in 
FIG. 6. 
The results indicate that 4-.sup.125 I!BP exhibits saturable binding with 
Kd=26 nM and B=4000 fmol/mg protein. 
The results for .sup.3 H!DTG, a known sigma ligand, gave Kd of 38.2 nM and 
B.sub.max of 3867 fmol/mg protein. 
The results in Table 7 show the results of Scatchard analysis of .sup.3 
H!(+)pentazocine binding to sigma-1 sites and .sup.3 H!DTG binding to 
sigma-2 sites. 
TABLE 7 
______________________________________ 
Sigma-1 Sigma-2 
Cell Line (.sup.3 H!(+)-pentazocine 
(.sup.3 H!DTG + DEX) 
______________________________________ 
MCF-7 breast 
No specific binding 
K.sub.d = 24.54 .+-. 5.57 
adenocarcinoma 
T47D breast K.sub.d 1 = 6.62 .+-. 1.03 
K.sub.d = 19.95 .+-. 3.53 
ductal carcinoma 
B.sub.max 1 = 108 .+-. 64.6 
B.sub.max = 1221 .+-. 264 
K.sub.d 2 = 261 .+-. 41.48 
B.sub.max 2 = 1690 .+-. 164 
LNCaP.FGC prostate 
K.sub.d = 38.44 .+-. 17.78 
K.sub.d = 39.00 .+-. 0.40 
B.sub.max = 11.96 .+-. 490 
B.sub.max = 727 .+-. 5.67 
______________________________________ 
EXAMPLE 14 
Homologous and Heterologous Competition Binding Studies in MCF-7 Cells 
The affinity of compounds for sites labeled by 4-.sup.125 I!BP and 
2-.sup.125 I!BP in MCF-7 cells was determined by homologous and 
heterologous in vitro competitive binding assays in intact cells. 
Materials and Methods 
Haloperidol, a known non-selective sigma ligand was obtained from RBI, 
Boston, Mass. 
DTG, a known selective sigma ligand, was obtained from RBI, Boston Mass. 
4-.sup.125 I!BP was prepared according to the description in Example 3. 
2-.sup.125 I!BP was prepared according to the description in Example 5. 
In Vitro Affinity of 4-.sup.125 I!BP And 2-.sup.125 I!BP for MCF-7 Breast 
Cancer Cells Intact adherent cells were washed (2.times.1 mL) with 10 mM 
phosphate buffer (pH=7.2). The cells were incubated with DMEM and 
incubated with 4-.sup.125 I!BP and varying concentrations (10.sup.-4 to 
10.sup.-12 M) of 4-IBP keeping a total volume 1.0 mL constant in each 
well. The optimum pH for the binding was found to be between 7-7.5. Each 
data point represent an average of three values. The cells were incubated 
at 37.degree. C for 1 hr. and subsequently washed with phosphate buffer 
(10 mM; pH 7.2) (3.times.1 mL). The cells were then dissolved in 0.2N NaOH 
(1.0 mL) and the activity counted on a Beckman (DP 5500) Gamma Counter. 
The competition binding assay results are listed in Table 7. A similar 
procedure was repeated with 2-.sup.125 I!BP with MCF-7, MDA-MB 231 and 
T47D breast cancer cells. 
Results 
Homologous competition binding of radiolabeled 2- and 4-.sup.125 I!BP to 
MCF-7 cells showed high affinity binding. FIG. 8 shows homologous 
competition binding assays of 2-.sup.125 I!BP in MCF-7 breast cancer 
cells. FIGS. 9 and 10 show the homologous competition binding assays of 
2-.sup.125 I!BP in MDA-MB-231 and T47D breast cancer cells suggesting the 
high affinity binding is common to other commercially available cells 
line. 
Heterologous competition assays using DTG and haloperidol showed high 
affinity, concentration-dependent inhibition of specific binding. 
Competition assays for the binding of 4-.sup.125 I!BP and 2-.sup.125 
I!BP with haloperidol in MCF-7 breast tumor cells are shown in FIGS. 7 and 
8. 
The Ki for DTG and haloperidol were found to be 56.+-.15 and 4.6+0.9 nM, 
respectively, suggesting the labeling of sigma sites by 4-.sup.125 I!BP 
in MCF-7 cells. 
Table 8 shows inhibitor constants, Ki, for 4-.sup.125 I!BP binding in MCF-7 
breast cancer cells for various drugs. In addition, MCF-7 cells showed 
little or no specific binding of (+)-pentazocine suggesting the absence of 
sigma-1 receptors in this cell line. However in T47-D breast cells, the 
binding of .sup.3 H!DTG and .sup.3 H!-(+)-pentazocine suggest the 
presence of both sigma-1 and sigma-2 receptors. 
TABLE 8 
______________________________________ 
Ligands Ki 
______________________________________ 
Haloperidol 4.6 .+-. 0.9 
IBP 4.8 .+-. 2 
DTG 56 .+-. 15 
Spiperone 247 .+-. 37 
(.+-.) Verapamil 379 .+-. 75 
(-)-Pentazocine &gt;1000 
(+)-PPP &gt;1000 
(+)-Pentazocine 1479 .+-. 190 
(+)SKF 10,047 &gt;10000 
(-)SKF 10,047 &gt;10000 
______________________________________ 
EXAMPLE 15 
Diagnostic Imaging using .sup.125 
I-(2-PiperidinylAminoethyl)-4-IodoBenzamide 
These experiments illustrate the present diagnostic imaging procedures and 
the benefit of utilizing the present compounds in such procedures. 
Materials and Methods 
A2058 cells, derived from a brain metastasis of human malignant melanoma 
(Todaro et al. 1980 Proc. Natl. Acad. Sci. USA 77: 5258) were obtained 
from the National Institutes of Health. A human lung adenocarcinoma cell 
line, NCI-838, was obtained from the National Cancer Institute. These 
cells were grown in DMEM2 medium (Dulbecco's modification of Eagle's 
medium, EMEM) supplemented with 10% fetal bovine serum and 0.03% 
L-glutamine. .sup.131 I-N-(diethylaminoethyl)4-iodobenzamide (i.e. 
.sup.131 I!DAB) was prepared as described in John et al. (1993 Nucl. Med. 
Biol. 20: 75-79. 
.sup.131 I(2-piperidinylaminoethyl)4-iodobenzamide(D) was synthesized as 
described in Example 1. 
Nude Mice Imaging 
Balb/c nu/nu mice (17-22 g) bearing human melanoma or non-small cell lung 
carcinoma xenograft tumors were injected intravenously with 0.2 ml of 
saline solution containing .sup.131 I!PAB or .sup.131 I!DAB (150-200 
.mu.Ci). The animals were anesthetized with ketamine containing rompun 
before the imaging studies. The images were obtained using a scintigraphic 
camera with a pin-hole collimator at 6 and 24 hr. post injection. 
FIGS. 2 and 3 provide scintigrams of nude mice implanted with human 
melanoma xenografts and treated with .sup.131 I!PAB and .sup.131 I!DAB, 
respectively. 
At 6 and 24 hrs. post injection, .sup.125 I!PAB was detected only within 
the tumor (FIGS. 2A and 2B). In contrast, no .sup.131 I!DAB was detected 
in the tumor at either 6 or 24 hrs. after administration (FIGS. 3A and 
3B). Moreover, considerable uptake of .sup.131 I!DAB had occurred in the 
livers of mice receiving this agent by 6 and 24 hrs. post-administration 
(FIGS. 3A and 3B). Little or no .sup.131 I!PAB was observed in the liver 
at either 6 or 24 hours. post-administration. These data indicate IPAB is 
a significantly better diagnostic agent for tumor imaging than IDAB. 
FIG. 4 provides a scintigram of a nude mouse implanted with a human 
adenocarcinoma xenograft 30 hrs. after injection of .sup.131 I!PAB. These 
scintigraphic imaging studies easily visualized the implanted tumor at 
both 24 and 30 hrs. post-injection. 
EXAMPLE 16 
Synthesis of N-(4-.sup.125 
I!-Iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethylamine 
Preparation of N-methyl-2-(1-piperidinyl)ethylamine 
To an aqueous solution of (40%) of methylamine (235 ml, 3.0 mol) was added 
dropwise a solution of cholorethyl piperidine monohydrochloride (50 g, 
0.27 mol). The mixture was stirred overnight and then basified by addition 
of NaOH. A colorless oil separated from the aqueous solution. This oil was 
extracted (2.times.200 ml) with ether. The solvents were evaporated under 
low temperature and a clear oil was obtained. This oil was used without 
any further purification. 
Preparation of N-2-(4-bromophenyl) 
acetyl!-N-methyl-2-(1-piperidinyl)ethylamine 
To a solution of 4-bromophenylacetic acid (10 g, 46.5 mmol) in CHCl.sub.3 
(100 ml) was added thionyl chloride (8 ml) in CHCl.sub.3 (10 ml) and three 
drops of DMF. The mixture was reflux for one to two hours. The volatiles 
were removed in vacuo to give a light yellow oil. This acid chloride was 
dissolved in CHCl.sub.3 30 ml and added dropwise to another flask 
containing N-methyl-2-(1-piperidinyl)ethyl amine (6.1 g, 48 mmol) and 
triethylamine (25 ml) and CHCl.sub.3 (100 ml) at 0.degree. C. The reaction 
mixture was stirred overnight at room. The volatiles were removed, the 
residue dissolved in CHCl.sub.3 (150 ml) and washed with (2.times.100 ml) 
water and 2% NaHCO.sub.3 (50 ml). The organic layer was dried and the 
volatiles were removed to give a light yellow oil (15.0 g). The oil was 
passed through a silica gel column and eluted with CHCl.sub.3 /MeOH to 
give 13 g, (87%) of the desired pure compound. .sup.1 H NMR 1.30-1.54 (m, 
6H, piperidinyl CH.sub.2); 2.33-2.43 (m, 6H, NCH.sub.2); 2.92 (44%), 2.96 
(56%) (s, 3H, N-Me); 3.32-3.37 (46%), 3.45-3.49 (54%), (t, 2H, J=7.1 Hz, 
NCH.sub.2); 3.61 (56%), 3.67 (44%), (s, 2H, benzylic); 7.09-7.11 (d, 2H, 
arom); 7.38-7.41 (d, 2H, arom). 
Preparation of 
N-2-(4-bromophenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethylamine 
To a solution of the above amide (10 g, 29.4 mmol) in THF (200 ml) was 
added in small portions lithium aluminum hydride (LAH) (2.2 g, 2 fold 
excess). The mixture was heated under reflux for three hours and stirred 
overnight. The slurry was cooled and a saturated solution of sodium 
ammonium tartrate was added dropwise carefully. After all excess LAH had 
decomposed, CHCl.sub.3 (250 ml) was added. The organic layer was separated 
from the aqueous layer, dried over anhydrous Na.sub.2 SO.sub.4 and the 
volatiles were removed in vacuo to give a yellow oil. The oil was passed 
over a silica gel column and eluted with CHCl.sub.3 and then with 
CHCl.sub.3 /MeOH:90/10. The desired fractions were pooled together and the 
solvents evaporated to give 7.4 g of the desired product. .sup.1 H NMR 
1.4-1.5 (m, 2H, CH.sub.2); 1.7-1.8 (m, 4H, piperidinyl CH.sub.2); 2.35 (S, 
3H, NMe); 2.5-2.80 (m, 12H, NCH.sub.2); 7.09-7.11 (d, 2H, arom); 7.38-7.41 
(d, 2H, arom). 
Preparation of 
N-2-(4-n-tributylstannylphenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethyl 
amine 
To a solution of amine (3.0 g, 0.92 mmol) was added Pd(PPh.sub.3).sub.4 
(1.0 g, 0.092 mmol) and bistributyltin (5.3 g, 0.92 mmol) and triethyl 
amine (100 ml). The mixture was refluxed overnight. The solvents were 
removed in vacuo and the residue dissolved in CHCl.sub.3 (10 ml) and 
purified after passing through a silica gel column and eluting with 
CHCl.sub.3 (75 ml) and then CHCl.sub.3 /MeOH:90/10. The desired fractions 
were combined, the solvents were evaporated to give an oil (4.6 g). .sup.1 
H NMR: 0.8-1.1 (m, 12H, Bu.sub.3 and piperidinyl); 1.2-1.6 (m, 18H, 
nBu.sub.3); 2.3 (s, 3H, NMe); 2.4-2.8 (m, 15H, NCH.sub.2); 7.1-7.15 (d, 
2H, arom); 7.3-7.35 (d, 2H, arom). .sup.13 C NMR: 9.51, 13.65, 23.65, 
24.98, 27.37, 29.07, 33.45, 42.29, 54.67, 2.84, 59.66, 128.33, 128.42, 
136.51, 138.99, 139.84. m/e=537 (M+).sup.+ (30%); 521 (M-CH.sub.3) (35%). 
Preparation of 
N-2-(4-iodophenyl)ethyl!N-methyl-2-(1-piperidinyl)ethylamine 
A round bottom flask was charged with 
N-2-(4-n-tributylstannyl-phenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethyl 
amine (1.0 g, 1.86 mmol) and iodine (500 mg) and acetone (50 ml). The 
mixture was stirred at room temperature for 15 hours. An aqueous solution 
(25 ml) of sodium thiosulfate (15%) was added and the mixture was 
extracted in CHCl.sub.3. The organic layer was separated and dried and the 
solvent removed to give a light yellow oil. (M+1).sup.+ (100%). 
Preparation of N-(4-.sup.125 I!-Iodophenyl) 
ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine 
The radiolabeled compound was prepared according to the protocol used in 
Example 3 to prepare .sup.125 I(-N-benzylpiperidin-4-yl)-4-iodobenzamide, 
but using 
N-2-(4-n-tributylstannyl-phenyl)ethyl!-N-methyl-2-(1-piperidinyl)ethyl 
amine as starting material. The synthetic procedure is depicted below in 
Reaction Scheme IV. 
##STR17## 
EXAMPLE 17 
Biodistribution of N-(4-.sup.125 
I!-Iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 4-.sup.125 
I!PEMP 
Biodistribution experiments were performed to assess the clearance of 
N-(4-.sup.125 
I!-iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 4-.sup.125 
I!PEMP. The compound was prepared according to the procedure described in 
Example 16. For in vivo studies, Sprague Dawley rats were used. Animal 
biodistribution assays were performed according to the procedure described 
in Example 11. 
Results 
Table 9 illustrates the biodistribution of 4-.sup.125 I!PEMP in Sprague 
Dawley rats at one, six, and twenty-four hours after administration of the 
radiolabeled compound. 
The results in Table 9 show that 4-.sup.125 I!PEMP rapid clearance from 
the liver, lungs and kidneys. 
TABLE 9 
______________________________________ 
Biodistribution of N-(4-.sup.125 I!-iodophenyl) 
ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 
4-(.sup.125 I!PEMP in Sprague Dawley rats (% ID/whole organ) 
each data point represents an average of four rats. 
Tissue 1 hr. 6 hr. 24 hr. 
______________________________________ 
Blood 1.82 .+-. 0.20 
2.59 .+-. 0.32 
1.01 .+-. 0.12 
Heart 0.30 .+-. 0.02 
0.18 .+-. 0.00 
0.05 .+-. 0.00 
Liver 16.56 .+-. 2.19 
6.39 .+-. 0.48 
1.83 .+-. 0.16 
Spleen 2.52 .+-. 0.49 
0.96 .+-. 0.10 
0.28 .+-. 0.03 
Kidney 4.57 .+-. 0.88 
1.45 .+-. 0.03 
0.50 .+-. 0.02 
Lung 4.91 .+-. 0.23 
1.22 .+-. 0.12 
0.34 .+-. 0.03 
Muscle 15.60 .+-. 3.04 
11.73 .+-. 0.63 
5.55 .+-. 1.25 
Brain 1.79 .+-. 0.21 
0.57 .+-. 0.01 
0.23 .+-. 0.00 
Thyroid 0.19 .+-. 0.02 
0.20 .+-. 0.05 
0.58 .+-. 0.03 
Bone 0.23 .+-. 0.00 
0.28 .+-. 0.01 
0.17 .+-. 0.00 
______________________________________ 
EXAMPLE 18 
Binding Competition Between Pharmacological Antagonists and 
N-(4-I-iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 4-IPEMP 
Materials and Methods 
N-(4-I-iodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 4-IPEMP, 
and (bromoiodophenyl)ethyl!-N-methyl-2-(1-piperidinyl)-ethylamine, 
Br-PEMP, were synthesized as described in Example 16. Assays were prepared 
according to the procedure described in Example 9. 
Results 
The Ki values for 4-IPEMP and BR-PEMP are shown in Table 10: 
______________________________________ 
Sigma-1 Sigma-2 
Guinea Pig Brain 
Rat Liver 
.sup.3 H!-(+)-pentazocine 
.sup.3 H!DTG + DEX 
______________________________________ 
4-IPEMP 7.02 nM 52.65 nM 
Br-PEMP 6.27 nM 51.79 nM 
______________________________________ 
These data demonstrate that 4-IPEMP and its bromo precursor bind both 
sigma-1 and sigma-2 sites with high affinity. 
EXAMPLE 19 
Homologous and Heterologous Competition Binding Studies in MCF-7 Cells 
The affinity of compounds for sites labeled by 4-.sup.125 I!PEMP was 
determined by homologous and heterologous in vitro competitive binding 
assays in intact MCF-7 breast cancer cells and guinea pig brain membranes 
labeled by 4-.sup.125 I!PEMP. The assays were prepared according to the 
procedure described in Example 14. 
Results 
Heterologous competition assays using BD1008, a known sigma ligand show 
high affinity, concentration-dependent inhibition of specific binding. 
Competition assays for the binding of 4-.sup.125 I!PEMP with BD1008 in 
MCF-7 breast tumor cells are shown in FIG. 11. 
The Ki for BD1008, (+)pentazocine and haloperidol were found to be 5.6, 
35.9 and 36.5 nM, respectively, suggesting the labeling of sigma sites by 
4-.sup.125 I!PEMP in guinea pig brain membranes. 
EXAMPLE 20 
Ligan Binding Studies with Breast Tumor Biopsied Samples 
An approval was obtained by the Committee on Human Research, GWUMC for 
in-vitro binding studies of biopsied human tumors. 
A small piece of a breast tumor tissue (300 mg), surgically removed from a 
patient, was obtained from the Department of Pathology, GWUMC. 
Membrane Preparations 
A small piece of breast tumor tissue (200 mg) was suspended in 10 mL of 
tris-HCI buffer (50 mM, pH 9.0). The tissue was thoroughly homogenized on 
a Ultra-turrax polytron for a period of 5-10 minutes. The suspension was 
centrifuged on a Beckmann centrifuge (Model J 21B centrifuge) for 5 min at 
5000 rpm. The resulting pellet was washed with tris-HCI, (50 mM, pH 9.0) 
and centrifuged again for 20 min at 18000 rpm. The supernatant was 
discarded and the pellet was resuspended in 10 mL of tris-HCI (50 mM, pH 
9.0). The protein was determined using BCA protein assay reagent obtained 
from Pierce, Rockford, Ill. with BSA as a standard. 
Ligand Binding Studies with Breast Tumor Biopsied Samples 
The small portion (0.1 mL) of the membranes were aliquotted in tissue 
culture tubes and incubated with radio-iodinated ligands 4-.sup.125 I!BP 
and 2-.sup.125 I!BP (0.1 mL) and a varying amount of competing ligand 
(0.1 mL haloperidol). The contents were incubated in a waterbath for 1 hr 
at 37.degree. C. The assays were terminated by the addition of ice cold 
tris buffer (5 mL) and filtration through glass fiber filters using a cell 
harvester (Gaithersburg, Md.). The activity bound to membranes was then 
counted using Beckman Gamma Counter (DP 5500). The data obtained were 
analyzed using the iterative curve-fitting program, BDATA (EMF Software, 
Baltimore, Md.). A representative example is given in FIGS. 12 & 13. 
Results 
A high affinity binding of 2-.sup.125 I!BP with haldoperidol (Ki=6.3 nM) 
suggested the binding to sigma receptors present on the breast tissue 
membranes. Similarly, a high affinity dose dependent binding of 4.sup.125 
I!BP with haloperidol, a sigma ligand, was observed (Ki=3.8 nM). 
EXAMPLE 21 
Biodistribution of .sup.125 I-(N-benzylpiperidin-4-yl)-2-IodoBenzamide 
Biodistribution experiments are performed to assess the tumor-specificity 
of 2-.sup.125 I!BP. 
Materials and Methods 
Breast cancer cell lines MCF-7, T47D and MDA-MB-231 are obtained from ATCC, 
Rockwell, Md. 
Tumor cells are grown in DMEM2 medium (Dulbecco's modification of Eagle's 
medium, EMEM) supplemented with 10% fetal bovine serum and 0.03% 
L-glutamine. 
.sup.125 I-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide (i.e.2-.sup.125 
I!BP) is prepared as described in Example 5. 
Animal Biodistribution Assays 
For in vivo studies, tumor cells are harvested using calcium and magnesium 
free PBS containing 0.02% EDTA. Suspension of 5.times.10.sup.6 cells 
(viability greater than 95%) in 0.2 mL of medium are inoculated 
subcutaneously in female Balb/c nu/nu mice. In about two weeks, solid 
tumors of about 1 cm in diameter appear in approximately 85% of all 
inoculated mice. Mice with solid tumors having a diameter of about 1 cm 
are used for biodistribution studies. 
Balb/c nu/nu mice (17-22 g) are injected intravenously with 0.2 ml of a 
saline solution containing .sup.125 I!PB (5-6 .mu.Ci). At 1, 6 and 24 hr. 
after injection, blood samples are collected by cardiac puncture and the 
mice are sacrificed immediately thereafter by cardiectomy while under 
halothane anesthesia. The organs of interest are subsequently excised, 
blotted with tissue paper, weighed, and the radioactivity counted using a 
Packard automatic counter (autogamma 5650). The % injected dose/g (% ID/g) 
values are determined by comparison of tissue radioactivities with 
suitably diluted aliquots of the injected .sup.125 I!PB dose divided by 
the weight of the organ. The values obtained are normalized to a mouse 
weighing 20 g.