EP0650372A1 - Compounds for cancer imaging and therapy - Google Patents

Abstract

The present invention relates to compounds having affinity for certain cancer cells, e.g. lung carcinomas, colon carcinomas, renal carcinomas, malignant melanomas, gliomas, neuroblastomas and pheochromocytomas. The compounds 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 compounds are of general formula (I) wherein X is a radionuclide; R2 is -N(R3)2 or a 5 to 6 member nitrogen containing heterocyclic ring, optionally substituted with at least one alkyl group; the remaining variables are defined on page 5 of the disclosure. Methods are provided for diagnostic imaging and for the detection and treatment of tumors containing the cancer cells described above.

Description

COMPOUNDS FOR CANCER IMAGING AND THERAPY

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 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 carcinomas and renal 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 carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) .

SCLC is a neuroendocrine tumor that secretes several peptide growth factors including

SHEET 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. 5 NSCLC accounts for about 75% of all lung cancer cases and encompasses a variety carcinomas including adenocarcinomaε, large cell carcinomas and squamous cell carcinomas. NSCLC tumors secrete transforming growth factor-alpha (TGF-σ) to stimulate _Q 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.

20 Whereas the prognosis for superficial spreading melanoms can be quite good, there is a much poorer prognosis for nodular melanomas in which distant metastases frequently form.

Many lives could be saved if lung carcinomas,

25 melanomas, gliomas, neuroblastomas, pheochromocytomas, colon carcinomas and renal carcinomas 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 n treat such cancers since these procedures can cause disfiguration or disablement. Accordingly an outstanding need exists for highly selective and non-

35

SUBSTITUTESH -\ invasive procedures permitting early detection and treatment of cancer.

A variety of radiopharmaceuticals have been evaluated for diagnostic imaging. For example, t- 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. a^. have disclosed [ 123I and 125I]N- (diethylaminoethyl)4-iodobenzamide (i.e. IDAB) for _Q 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. United States Patent No. 4,279,887 to Baldwin et al. , United States Patent No. 5,154,913 to De Paulis et al. and Murphy et a]^. (1990 J. Med. Chem. 33:171-178)

2o disclose radioiodonated benzamide compounds for use in imaging the brain only, e.g. ¹²³I-N-β-phenethyl-o- iodobenzamide or (S)-N-[ ( l-ethyl-2-pyrrolidinyl)methyl]- 2-hydroxy-3-iodo-6-methoxybenzamide (IBZM). However, the structure and utility of the compounds disclosed by c Baldwin et cil . , De Paulis et. a_l. and Murphy et al_^. is distinct from those provided herein.

The present invention provides compounds which bind with high specificity and affinity to the cell surface of cancer cells. These compounds bind, for

__n 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

35 •, immortalized neuroblastoma and glioma cell lines (Walker et al. 1990 Pharmacol. Reviews 2: 355-400; and Villner et al.. 1992 in Multiple Sigma and PCP Receptor Liqands: Mechanisms for Neuromodulation and Neuroprotection? tr Ka enka 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. 0 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, π- 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 0 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. 5

CH₂)_j—CZ—NR₃—(CH2)_y—R₂ I

5

SUBSTITUTESHEET 2 wherein :

X is a radionuclide; Z is =0 or two -H; each R_χ is independently H, halo, lower alkyl π or lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring; 0 ^R ₂ ^is —^N(^R ₃)₂ ^or a 5 to 6 me bered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent; each R₃ is independently hydrogen or lower alkyl; 2 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. 2o 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 n vitro detection of a cancer

35 cell in a mammalian tissue sample which includes contacting a mammalian tissue sample with an iri 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.

wherein Z, R_a, R_b, R , q, j are as described above;

Q is a radionuclide, halide or an activating group;

R 4, is — (*R3,)'„2 or an N-linked 5 to 6 membered nitrogen containing heterocyclic ring which can have at

SUB T 2 least one alkyl substituent, wherein each R₃ is independently lower alkyl or hydrogen;

R₅ is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl t- substituent; m is an integer from 2 to 6; n is an integer from 3 to 6. Such preferred compounds can also be used in the methods of the present invention. 0 Compositions and kits containing the present compounds are also provided herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS:

Fig. 1 illustrates the log molar amount of nonradioactive IPAB needed to competitively inhibit binding of radioactive IPAB to malignant melanoma cells. The K_± obtained from these data was 6.8 nM.

Fig. 2A provides a scintigraphic image obtained at 6 hrs. after a nude mouse bearing a human o malignant melanoma tumor received [¹³¹I]PAB. The arrow indicates the implanted tumor.

Fig. 2B provides a scintigraphic image obtained at 24 hrs. after a nude mouse bearing a human malignant melanoma tumor received [¹³¹I]PAB. The arrow _c indicates the implanted tumor.

Fig. 3A provides a scintigraphic image obtained at 6 hrs. after a nude mouse bearing a human malignant melanoma tumor received [ I]DAB. The arrow indicates the implanted tumor. Q Fig. 3B provides a scintigraphic image obtained at 24 hrs. after a nude mouse bearing a human

5

SUBSTITUTE SHEET malignant melanoma tumor received [131,I]DAB. The arrow indicates the implanted tumor.

Fig. 4 provides a scintigraphic image obtained at 30 hrs. after a nude mouse bearing a human lung adenocarcinoma tumor received [ I]PAB. The arrow indicates the implanted tumor.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides novel compounds

10 and methods for detecting and treating certain types of cancer, e.g. neuroblastomas, gliomas, pheochromocytomas, melanomas, colon, renal and lung 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

20 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 of any one of formulae I, II, III or IV:

30

35

SUBSTITUTESHEET V

wherein:

X is a radionuclide;

Q is a radionuclide, halide or an activating group;

Z is =0 or two -H; each R_j is independently H, halo, lower alkyl or lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₂ is —^N(^R ₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent; each R is independently hydrogen or lower alkyl;

R 4„ is —N(R3,)',Z or an N-linked 5 to 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent, wherein each R₃ is independently lower alkyl or hydrogen;

R₅ is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent; 0 j and y are independently an integer from 0 to 6; q is an integer from 0 to 2; m is an integer from 2 to 6; n is an integer from 3 to 6; and _t- 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 o 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 r 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 0 method for in vitro detection of a cancer cell in a mammalian tissue sample which includes contacting a mammalian tissue sample with an iri vitro diagnostic

5

SUBSTITUTESHEET 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 γ-emitting radionuclides which can be detected by radioimaging procedures, e.g. by scintigraphic imaging. Such γ-emitting radionuclides emit radiation which is sufficently penetrating to be detected through tissues. Moreover, for diagnostic imaging preferred radionuclides do not emit a particle, e.g. an a or β particle. Preferred X and Q groups for diagnostic imaging include

123 I_, 124I_, 125_τI, 131_τI, 1BF_, 76-B,r and, 77_Br. M.,ore pref_eerred_J X_T and Q groups for diagnostic imaging include ³I, I and F. 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 β-emitting or an σ-emitting radionuclides. However, as contemplated herein, any cytotoxin which exhibits a localized cell killing activity can be used in place of a X or Q radionuclide. The preferred X and Q groups for treating cancers include ¹³¹I, ²¹¹At, ²¹²Pb, ²¹²Bi, ⁷⁶Br, ⁷⁷Br and the like. However, compounds for treating cancer more preferably have X or Q as ¹³¹I.

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

SUBSTITUTE SHEET 2 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 -,_Q electrophilic aromatic substitution. Preferred activating groups include tributyl-tin, trimethylsilyl, t-butyldimethylsilyl, iodide and the like.

According to the present invention Z is =0 or two hydrogen atom substituents. Since the -CZ- moiety ■_jπ is adjacent to an amine, when Z is =0 an amide (-C0-NH-) is formed. When Z is two hydrogen atoms a methylene (-CH₂~) 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

20 side chains:

—(CH₂).—CO—-NR₃—(CH2)_y—R₂ or

—(CH₂) —CH₂— R₃—(CH2)_y—R₂.

In a preferred embodiment Z is =0, i.e. the -CZ- group forms a carbonyl. When -CZ-NR₃~ is -CH₂-NR₃-, the R₃ is

25 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,

30 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.

35

SUBSTITUTE SHEET 2 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 c include methylene, ethylene, propylene, isopropylene, butylene, t-butylene, sec-butylene, isobutylene, a ylene, isoamylene, pentylene, isopentylene, hexylene and the like. The preferred lower alkylene groups contain one to four carbon atoms. 0 ^Tne 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, r chloride, bromide and the like.

As employed herein, a heterocylic 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 has at least one double bond.

5

SUBSTITUTE SHEET 2 The present heterocyclic rings can have up to three heteroatoms and up to a total of six ring atoms. Accordingly heterocyclic rings of the present invention can have about 2 to about 5 ring carbon atoms, c 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 0 preferred that the heterocylic 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, o 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 5 morpholine.

As used herein R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring. When R_a and R_b 0 together form a cycloalkenyl or heterocylic ring, such a ring is fused to the phenyl.

5

SUBSTITUTE SHEET 2 Such a cycloalkenyl ring formed from R_a and R_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 c formed by R_a and R_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.

2o When a heterocyclic ring is formed by R_a and

R_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

■_jπ 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 ring carbon atoms. Thus, the total

2o 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

2c to 3 ring heteroatoms. The preferred ring heteroatoms are oxygen and nitrogen, especially oxygen. Preferred heterocyclic rings formed by R_a and R_b include dihydrofuranyl, dihydropyrrolyl, tetrahydropyridinyl and the like. _Q As provided herein each R_χ is independently H, halo, lower alkyl or lower alkoxy. In a preferred embodiment R_l is H, halo or lower alkoxy. More

5

SUBSTITUTE SHEET 14

- 16 -

preferred R₁ groups include H and halo. However, in one embodiment R_χ is preferably alkoxy.

The variable q is defined herein as an integer ranging from 0 to 2 which describes the number of R₁ groups on the phenyl moiety. Since the phenyl is also substituted with R_a, R_b, X (or Q) and a side chain amide or amine moiety, the maximal number of R_α groups is 2 (i.e. q can maximally be 2). When q is less than 2 some positions on the phenyl group are unsubstituted; in this case a hydrogen is present at the positions having no R₁ 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. X (or Q) and the amide or amine side chain moiety.

In a preferred embodiment the

group is selected from the following;

orBr

SUBSTITUTE SHEET 4

-17-

wherein R_χ is as described hereinabove and Q is a radionuclide (e.g. X), a halide or an activating group.

As described herein, R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent; wherein each R₃ is independently hydrogen or lower alkyl. Preferably R₃ is lower alkyl in the —N(R₃)₂ groups of the present invention. Preferred R₂ 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 R₆ lower alkyl. R₆ is preferably attached to the nitrogen of the piperidinyl, pyrrolidinyl or morpholinyl rings.

SUBSTITUTE SHEET η. In one embodiment R„2 can be R4, as defined herein. In another embodiment R„ 2 can be R_c5 as defined herein. In still another embodiment R₂ can be —^N(^R ₃)₂ as defined herein, t- As provided herein, R₄ is —N(R₃)₂ or an N- linked 5 to 6 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 -,0 chain through a nitrogen atom. R₄ is used in formula II to indicate a preference for attachment of the nitrogen present within the heterocyclic ring to the main chain. Preferred R₄ heterocyclic rings include rings of the formulae:

35

SUBST_ITUTESHEET 2 wherein R_g is hydrogen or lower alkyl and each i is independently an integer from 0 to 1. Preferred R₄ heterocyclic rings include N-piperidinyl, N- pyrrolidinyl, N-pyridine and the like.

5 In another embodiment preferred compounds have heterocyclic rings that are not attached via the ring nitrogen. R₅ is used in formula III to describe such compounds, wherein R₅ is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one 0 alkyl substituent. In a preferred embodiment R₅ is any one of the following:

wherein each i is independently an integer from 0 to 1 and R₆ is hydrogen or lower alkyl. More preferred R₅ heterocyclic rings include piperidinyl, pyrrolidinyl or _Q which are N-substituted with an R₆ lower alkyl, or pyridinyl which can have an R₆ lower alkyl. Such an R₆

5

SUBSTITUTE SHEET 2 lower alkyl is preferably methyl, ethyl, propyl or butyl.

The compounds of formula IV have an -N(R₃)₂ group which is hydrogen or lower alkyl. In a preferred c embodiment for -N(R₃), R₃ 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- 0 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-NR₃- and R₂ groups in the -CZ-NR₃—(CH2) —R₂ moiety of formula I. Preferably y is 1 to 3; more preferably y is 1 to 2.

Like y, the variable m defines the length of 0 the alkylene chain separating the -CZ-NR₃~ and the R₅ group in the -CZ-NR₃—(CH2)_m—R₅ moiety of formula III. The variable m is an integer ranging from 2 to 6. However, m is preferably 2 to 4 and more preferably 2 to 3. r The length of the alkylene chain separating the -CZ-NH- and the —N(R₃)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 _Q include the following.

5

SUBSTITUTE SHEET

35

SUBSTITUTE SHEET

10

20

30

35

SUBSTITUTE SHEET

R.

SUBSTITUTE SHEET 2 The various combinations and permutations of the Markush groups of X, Q, Z, R_a, R_b, R , R₂, R₃, R₄ and R₅ described herein are contemplated to be within the scope of the present invention. Moreover, the present c- 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_a, R_b, R , R₂, R₃, R₄ and R₅

20 and the various combinations thereof. Thus, for example, the present invention contemplates that R₂ may be one or more of the substituents listed hereinabove or any and all of the substituents of N(R,)„, R„ and R_c.

The present compounds can bind to a specific

25 cell receptor prevalent on certain types of cancer cells. Such cancer cells include lung carcinoma, colon carcinoma, renal carcinoma, melanoma, glioma, pheochromocytoma, neuroblastoma and related cells. An example of the cell receptor to which the present _Q 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 p_t- 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

__n the subject compounds.

For example, as demonstrated by the present inventors, a malignant melanoma cell line binds the

35

SUBSTITUTE SHEET 2 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 0 (e.g. melanocyte secreting hormone peptide), 5- hydroxytryptamine-1 (e.g. mianserin) , 5-hydroxy- tryptamine-la (e.g. NAN-190), 5-hydroxytryptamine-lc (e.g. ketanserine) , 5-hydroxytryptamine-2 (e.g. ketanserine and mianserin) and 5-hydroxytryptamine-3 c (e.g. 3-tropanyl-dichloroben) 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 o 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 5 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, melanoma, pheochromocytoma, glioma, _n neuroblastoma and the like. For example, several lung carcinoma cell types have demonstrated binding affinity for the present compounds including an adenocarcinoma, a

5

SUBSTITUTE SHEET 2 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 c 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.

According to the present invention a method 0 ^ror 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 about 1 μg to about 1 g of the compound per liter which can be administered in doses of about 1 ng/kg body 0 weight to about 10 ug/kg body weight. Preferred dosages of the present compounds are in the range of about 10 ng to about 2 ug/kg for diagnostic imaging. Moreover, for diagnostic imaging the amount of radioactvity administered should be considered. Preferably about 0.1 r 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 γ-radiation detector. One such procedure utilizes scintigraphy. Tomographic imaging procedures such as single photon emission computed tomography (SPECT) or positron

5

SUBSTITUTE SHEET 6314

-27-

2 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 5 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 0 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 cr 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 o 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 5 the radioactive compound should be taken into consideration. Such a specific activity is preferably very high, e.g. for 123I-labeled compounds the specific activity should be at least about 1,000 Ci/ M to about 50,000 Ci/mM. More preferably the specific activity for ¹²³I-labeled compounds is, e.g. about 10,000 Ci/mM to 0 about 22,000 Ci/mM.

5

SUBSTITUTE SHEET 2 In another embodiment the present invention provides a method for jji vitro detection of a cancer cell in a mammalian tissue sample which includes contacting a mammalian tissue sample with an _in vitro c 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. 0 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 iji vitro testing include lung, bronchial, lymph, skin, brain, liver, any tissue of nervous origin and the like. Samples can be sectioned, e.g. with a microtome, to facilitate microscopic examination and observation of bound o 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 _t- compound to a cell surface sigma receptor on the cancer cell include standard tissue culture conditions, i.e. samples can be cultured ii vitro and incubated with one of the present compounds in physiological media. Such conditions are well known to the skilled artisan. 0 Alternatively, samples can be fixed and then incubated with a compound of the present invention in an isotonic or physiological buffer.

5

SUBSTITUTE SHEET 14

-29-

An amount of at least one of the present compounds for iji vitro detection of a cancer cell can range from about 1 ng/1 to about 1000 μg/1. A preferred amount is about 1 μg/1 to about 100 μg/1.

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 iri vitro diagnosis of cancer include ¹²⁵I, ¹⁸F, -³⁵S-alkyl, -³⁵S0₃, -³⁵S0₄, - COOH, -¹CH₃, ³H 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 , R₂, R₃, R₄, R₅ and R_g groups at the requisite positions. Alternatively, a leaving group may be used in place of the desired R , R₂, R₃, R₄, R₅ or R_g 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.

SUBSTITUTE SHEET 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 c 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, N₃, HO-Aryl, or HSO^-Aryl groups, and o 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 5 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 o 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) , tert-butoxycarbonyl (cleaved by mild c 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

5

SUBSTITUTESHEET 2 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, π iodide and the like.

The present compounds can be prepared from readily available starting materials, for example, by amidation of a substituted phenyl alkylcarboxylate or a substituted benzoic acid with an appropriate amine. 0 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 phenylcarboxyalkyl can be used as a starting material. c For example, a phenylcarboxyalkyl (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.

5

V VI

0

SUBSTITUTE SHEET 6314

-32 -

VII

wherein Y is a leaving group and R_χ, R_a, R_b q, j, y and 0 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. 5 Halogenating reagents for the above described reaction include those which can convert the carboxylate to an acid halide, e.g. thionyl halide such as S0C1₂, PC1₅, PC1₃ and the like. A preferred halogenating reagent is S0C1₂ in the presence of dimethylformamide. o ^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 acid halide in the presence of a base such as triethylamine. The solvent for this reaction is also preferably a nonpolar solvent, e.g. Q chloroform.

The skilled artisan can readily modify the reactions described above to generate a compound of any

5

SUBSTITUTE SHEET one of formulae I, II, III or IV. For example, to produce a compound of formula II, an amine of the formula NH,—CH,Z—R4„ can be used in p*"lace of the compound of formula VI. Similarly, to produce a compound of formula III or IV, an amine of the formula NH₂—(CH₂)_m—R₅ or NH₂—(CH₂)_π—N(R₃)₂, respectively, can be used in place of VI.

When Z is =0, 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 -CH₂-NH- a reducing agent can be used, e.g. boron hydride, sodium borohydrate, lithium aluminium hydride and the like, A preferred reducing agent is boron hydride (BH₃) 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.

—(CH2)_y—R₂

VII

SUBSTITUTE SHEET 4

-34-

rahydrofuran

VIII

When the R₃ of -CZ-NR..- 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 (—CH₂-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 carboxyalkyl, iodophenyl carboxyalkyl iodobenzoic acid, and the like. An activating group can be placed at the position of Y by available pocedures to generate a compound of any one of formulae

SUBSTITUTE SHEET 4

-35-

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.

(triphenylphosphine)₄ palladium + (tributyltin)₂ + triethylamine

In this case Q is tributyltin (Bu₃Sn) . This reaction is effective whether Z is =0 or two -H.

When using t-butyldimethylsilyl chloride (TBDMSC1) or trimethylsilyl chloride with N-butyl lithium or t-butyl lithium, a protecting group (R₇) is first placed on the -CZ-NR₃~ amine, if R₃ is hydrogen. When the R₃ of the -CZ-NR₃~ is lower alkyl, no such protecting group is needed. Protecting groups used for a -CZ-NR₃~ amine can be any protecting group for a secondary amine, e.g. carbobenzoxy (i.e. CBz, cleaved by

SUBSTITUTE SHEET 4

-36 -

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 then be performed as depicted below, e.g. using an amine protected compound of formula VIII.

(CH 2,)'y Villa

ilyl chloride lithium

)_y—R₂

In this case Q is trimethylsilane (Me₃Si). The conditions used for this reaction include low temperature (e.g. -78°C) and a polar solvent (e.g. tetrahydrofuran) .

The R_? group can be removed by standard techniques, e.g. when R_? is CBz or t-BOC acid hydrolysis can remove R₇ 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. ¹²³I, ¹²⁵I or ¹³¹I) with an activated intermediate (e.g. a

SUBSTITUTE SHEET 14

-37-

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. HC1, 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.

(CH₂) .—CO—NR₃- (CH2)_y—R₂ IX

radioisotope

+ oxidizing agent

NJ + acid

When R_a and R_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 and R of formula V

SUBSTITUTE SHEET 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 formuale II, III, IV,

VII, VIII or IX. Especially preferred compounds of the present invention which placed in kits include compounds of formula 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 presnt on compounds provided in the subject kits

SUBSTITUTE SHEET 2 include tributyl-tin, trimethylsilyl or t- butyldimethylsilyl. Tributyl-tin is an especially preferred activating group for compounds provided in the present kits. 5 The kits of the present invention can be adapted to contain another container having a reagent for replacing a activating group with a radionuclide. For example such a reagent can be an oxidizing reagent, e.g. chloramine-T.

20 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

2 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.

2o 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

25 carrier.

The compositions can be administered by well- known routes including oral, intravenous, intramuscular, intranasal, intrader al, subcutaneous, parenteral, enteral, topical and the like. Depending on the route

■ _Q of administration, the pharmaceutical composition may require protective coatings.

35

SUBSTITUTESHEET 2 The subject compounds may be incorporated into a cream, solution or suspension for topical administration.

The pharmaceutical forms suitable for 5 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 0 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 c can be accomplished by any art recognized technique, including but not limited to, addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thi erosal, and the like. Further, isotonic agents, such as sugars or o 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 5 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. _Q When the present compounds are administered orally, the pharmaceutical compositions containing an effective dosage of the compound, can also contain an

5

SUBSTITUTE SHEET 2 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 o amounts are preferably about 1 μg to about 1 g of the compound per liter and are administered in doses of about 1 ng/kg body weight to about 10 ug/kg body weight, or from about 0.1 mmole/kg body weight to about 500 mmole/kg body weight. Preferred compositions provide 5 effective dosages of the present compounds in the range of about 10 ng to about 2 ug/kg for diagnostics and preferably about 5 to about 50 mmole/kg body weight for therapy.

Moreover when considering a dosage for o 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 ¹²³I-labeled compounds the specific activity should be at least about 1,000 Ci/mM to about π 50,000 Ci/mM. More preferably the specific activity

12₃ for I-labeled compounds is, e.g. about 10,000 Ci/mM to about 30,000 Ci/mM.

As used herein, a pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, Q 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.

5

SUBSTITUTE SHEET The following Examples further illustrate the invention.

SUBSTITUTE SHEET EXAMPLE 1

Synthesis of I- (2-PiperidinylAminoethyl)-4-IodoBenzamide

Materials and Methods

Melting points were determined with a Fisher- Johns apparatus. H and ¹³C 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 x 20 cm), usind CHCl₃/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. Na¹³¹I was obtained from duPont NEN

125 and Na I was obtained from Bristol Meyers Squibb. Elemental analyses were performed by Galbraith Laboratory of Knoxville, TN.

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.

SUBSTITUTESHEET 2 The 4-bromobenzoyl chloride was dissolved in chloroform (30 mL) and added to a flask containing l-(2- aminoethyl)-piperidine (1.29 g, 10 mmol) in chloroform (20 L) . Triethylamine (10 L) was added dropwise. The 5 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 x 50 mL) . The organics were dissolved in CHC1₃ (100 mL) , separated from aqueous layer and dried over anhydrous Na₂S0₄. The o solvent was removed to give a colorless solid (A, 2.7 g, yield, 87%). Rf (CHCl₃/MeOH: 90/10) 0.45. H R (d ppm): 1.46 (t, 2H, CH₂); 1.54 (broad m, 4H, CH₂) ; 2.43 (broad s, 4H, NCH₂); 2.52-2.56 (t, 2H, NCH₂) ; 2.68 (m, 2H, NCH₂); 3.49-3.53 (dt, 2H, NCH₂) ; 7.21 (bs, 1H, NH) ; 7.52- 7.55 (m, 2H, arom) ; 7.65-7.68 (m, 2H, arom) .

Prepartion of (2-piperidinylaminoethyl)4-iodobenzamide, (B)

This was prepared using a procedure like that o described above for A but using 4-iodobenzoic acid as starting material. A white solid (B) was obtained in

89% yield. H R (d ppm): 1.43-1.45 (broad m, 2H, NCH₂) ;

1.53-1.60 (broad , 4H, NCH₂); 2.41 (broad m, 4H, NCH₂) ;

2.50-2.54 (t, 2H, J=7.8Hz, NCH₂) ; 3.44-3.48 (dt, 2H, NCH₂); 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 C₁₄H_ιgN₂01 calcd. C, 46.91; H, 5.31; N, 7.82, found C, 46.91; H,

5.42; N, 7.68.

0

5

SUBSTITUTE SHEET 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 jLn 0 ^vacuo- ^τhe resulting black oil was passed through a silica gel column with elution with CHC1₃ (100 mL), followed by elution with CHCl₃/MeOH: 90/10. The desired fractions, as characterized by thin layer chromatography, were pooled and solvent was evaporated 5 to give an oil (C, 0.4 g, 56%). m/e = 523 (M^*+H)

(100%); 233 (M^*-SnBu₃) (40%). ^XH R (d ppm): 0.82-0.93 (m, 16H, Bu₃ and CH₂) ; 1.01-1.05 (m, 4H, Bu₃) ; 1.22-1.37 (m, 8H, Bu₃); 1.45-1.67 (m, 8H, piperidinyl ring); 2.49- 2.51 (t, 2H, NCH₂ piperdinyl ring); 2.60-2.63 (t, 2H, o J=6Hz, NCH₂); 3.53-3.58 (dt, 2H, J=5.34Hz, NCH₂) ; 7.30- 7.41 (bs, 1H, NH); 7.49-7.78 (m, 4H, arom). ¹³C R (d , 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 1-125 to Yield ¹²⁵I(2-piperidinylaminoethyl)4-iodobenzamide (D)

To 100 uL of an ethanolic solution of (2- piperidinylaminoethyl)4-tributyltinbenzamide (1 mg/ml),

125 was added a solution of [ I]sodium iodide (1.5 mCi,

3 uL) in 0.1 N NaOH, followed by the addition of 0.05 N 0

HC1 (50 uL) to adjust the pH of the solution to pH 4.5-

6. Fifty uL of a freshly prepared solution of N-chloro-

5

SUBSTITUTE SHEET 6314

-46 -

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 uL of a solution of sodium metabisulfite (200 mg/ml) were added. The reaction mixture was neutralized with a saturated solution of NaHC0₃ (0.2 mL) . 0.4 mL of normal saline was added and the organics were extracted in CHC1₃ (1.0 mL) after vortexing 30 seconds. The chloroform layer was evaporated in a stream of nitrogen. ^Tne 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% 0.01 M phosphate buffer (400 uL) . 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 CHCl₃/MeOH: 90/10 (Rf=0.45). Another portion of p 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 (¹²⁵I-PAB) using isocratic elution with 90/10 EtOH/O.lM 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 131, I Yield ¹³¹I ( 2-piperidinylaminoethyl ) 4-iodobenzamide (E)

The same protocol as described above for

I ( 2-piperidinylaminoethyl ) 4-iodobenzamide (D) was used

SUBSTITUTE SHEET except that the amount of 0.05 N HCl added to adjust pH between 4.5-6 was different due to different concentration of aqueous sodium hydroxide solution in which Na I was commercially supplied. The workup of and the purification of ¹³¹I(2-piperidinylaminoethyl)4- iodobenzamide (E) was identical to ¹²⁵I(2- piperidinylaminoethyl)4-iodobenzamide (D) above.

The reactions described hereinabove are depicted in Reaction Scheme I.

REACTION SCHEME I

la : X =< Br lb : X - I

79-94%

SUBSTITUTE SHEET 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. H and ¹³C 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 x 20 cm), using CHCl₃/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. Na¹³¹I was obtained from duPont NEN and Na I was obtained from Bristol Meyers Squibb. Elemental analyses were performed by Galbraith Laboratory of Knoxville, TN.

Synthesis of 5,7-dibromo-2,3-dihydrobenzofuran (F) To a solution 2,3-dihydrobenzofuran (25 g., 0.21 mol) in chloroform (100 L) was added dropwise at 0°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 X 50 ml), then dried over anhydrous sodium sulfate. The volatiles were removed in vacuo to provide a light

SUBSTITUTE SHEET yellow oil (51 g, 87%). H NMR (CDCL₃) d ppm: 3.21-3.27 (t, J = 9 Hz, 2H, CH₂): 4.57-4.63 (t, J = 9 Hz, 2 H, OCH₂): 7.14-7.15 (t, 1 H, arom.): 7.32-7.33 (t, 1 H, 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° C under nitrogen atmosphere.

A solution of n-buthyl-lithium (2.0 M. 27 ml) was added to the mixture dropwise. The mixture turned light yellow brown. After 5 minute of stirring at -78 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 filteration (7.0 g, 53%). ^αH NMR (d⁶-DMSO) 2.85-2.95

(t, J = 9 Hz, 2H, CH,: 4.35-4.45 (t, J = 9 Hz, 2H, OCH :

7.1 (m, 1H, arom): 7.4 (m, 1H, arom). 1³C NMR (d⁶-DMSO and CDC1₃): 27.88, 71.78, 110.51, 114.17, 131.02, 131.25, 159.09, 164.87. Anal., C_gH₇Br0₃ calcd. C, 44.44; H, 2.88; found C, 44.52; H, 2.97.

Synthesis of 5-bromo-(N, N'-diethylamino- ethyl)-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

SUBSTITUTE SHEET 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 triethyla ine (15 ml) and CHC1₃ (30 ml) was added a solution of the above acid chloride in CHC1₃ (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 CHC1₃ (75 ml). The organic layer was separated, dried over anhyd. Na₂S0₄, 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 CHCl₃/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) CHCl₃/MeOH: 90/10. The hydrochloride salt was made with an ethanolic solution of hydrogen chloride gas upon trituration with anhydrous ether. H NMR (CDC1₃) d ppm: 0.96-1.00 (t, J = 7 Hz, 6 H, NCH₂CH₃) : 2.46-2.53 (q, J = 7 Hz, 4 H, NCH₂CH₃) : 2.55-2.59 (t, J = 7 Hz, 4 H NCH₂) : 3.17-3.22 (t, J = 8 Hz, 2 H, CH₂) : 3.40-3.44 (m, 2 H, NCH₂): 4.63-4.69 (t, J = 9 Hz, 2 H, OCH₂) : 7.30 ( 1 H. arom): 7.961-7.968 (m, 1 H, arom). Anal. C₁₅H₂₁BrN₂0₂.2HCl, 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' -diethylamino- ethyl) -2,3-dihydrobenzofuran-7-carboxamide (J) :

A round bottom flask was charged with 5-bromo- carboxamide (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

SUBSTITUTE SHEET was refluxed for 3 hours. The volatiles were removed in vacuo and the residue was dissolved in CHC1₃. This solution was loaded onto a silica gel column and eluted first with CHC1₃ (100 ml) and then with CHCl₃/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 CHCl₃/MeOH: 90/10 to give 0.9 g pure tin compound. TLC (silica gel) Rf=0.45 (CHCl₃/MeOH: 90/10). ^αH NMR

(CDC1₃): 0.83-1.60 (m, 33 H, nBu₃ and NCH₂CH₃); 2.51-2.59 (q, J = 7 Hz, 4 H, NCH₂CH₃); 2.60-2.66 (t, 2 H, CH₂); 3.18-3.23 (t, J = 8 Hz, CH₂); 3.44-3.50 (q, J = 6 Hz, 2 H, CH₂); 4.64-4.70 (t, J = 9 Hz, 2 H, OCH₂) ; 7.32 ( , 1 H, arom); 7.97 (m, 1 H, arom). ¹³C (CDC1₃) ( d 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 CHCl₃ 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

CHCl₃/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 (CHCl₃/MeOH:90/10) .

SUBSTITUTE SHEET H NMR (CDC1₃) : 1.34-1.39 (t, J = 8 Hz, 6 H, NCH₂CH₃) ; 3.14-3.33 (overlapping multiplet and triplet, 8 H); 3.85-3.91 (q, J = 6 Hz, 2 H, NCH₂) ; 4.71-4.76 (t, J = 9 Hz, 2 H, OCH₂); 7.54 (m, 1 H, arom); 8.02 ( , 1 H, arom) .

Synthesis of 5-bromo-l-(2-aminoethyl- piperidinyl)-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 l-(2-aminoethyl)piperidene (1.1 g, 8.58 mmol), triethylamine (15 ml) and CHC1₃ (40 ml) was added a solution of the above acid chloride in CHC1₃ (20 ml).

The mixture was stirred for 3 hours at room temperature.

The volatiles were removed and the residue was taken up in CHC1₃ (100 ml) and washed with water (2 x 50 ml).

The organic layer was separated, dried over anhyd Na₂S0₄, 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 CHCl₃/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 CHCl₃/MeOH:90/10) = 0.7. ^XH NMR ( d ppm): 1.42-1.60 (m, 6 H, piperidinyl CH₂'s); 2.42 (bs, 4

H, piperidinyl NCH ) ; 2.48-2.52 (t, J = 6 Hz, 2 H, CH₂);

SUBSTITUTE SHEET 3.20-3.26 (t, J = 9 Hz, 2 H, NCH2 ) ; 3.48-3.54 (m, 2 H, NHCH₂); 4.68-4.73 (t, J = 9 Hz, 2 H, OCH₂); 7.33-7.35 (m, 1 H, arom); 7.98-7.99 (m, 1 H, arom); 8.05 (bt, 1 H, NH) .

Synthesis of 5-n-tributyltin-l-(2-aminoethyl- piperidinyl ) -2-3-dihydrobenzofuran-7-carboxamide (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 CHC1₃ (150 ml) and then with CHCl₃/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) . ^αH NMR (d ppm): 0.82-0.87 (t, J = 7 Hz, 9 H, nBu₃); 0.98-1.59 (m, 25 H, nBu₃; and piperidinyl CH₂); 2.48 (bm, 4 H, piperidinyl NCH₂); 2.49- 2.54 (t, J = 7 Hz, 2 H, CH₂) ; 3.21-3.26 (t, J = 9 Hz, 2 H, CH₂); 3.51-3.55 (m, 2 H, NCH₂) ; 4.63-4.69 (t, J = 9 Hz, 2 H, OCH₂); 7.34 (m, 1 H, arom); 7.95 (m, 1 H, arom) .

The reactions described hereinabove are depicted in Reaction Scheme II.

SUBSTITUTE SHEET REACTION SCHEME II

SUBSTITUTE SHEET EXAMPLE 3

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 77J 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 I (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 (0.1 M) 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 [ 125IJPAB (0.1 ml) was added to eight aliquots of 0.1 ml test A2058 cells (1.5 X 10⁶ 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

SUBSTITUTE SHEET 37°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® 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. K_i 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.

SUBSTITUTE SHEET 2 EXAMPLE 4

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 o A2058 cells, derived from a brain metastasis of human malignant melanoma (Todaro et a^L. 1980 Proc. Natl. Acad. Sci . USA 2^: 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 125I (2-piperidinylaminoethyl)4-iodobenzamide (D) is synthesized as described in Example 1. o 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-la receptor), ketanserine (5- hydroxytryptamine-lc receptor), ketanserine and _Q mianserin (5-hydroxytryptamine-2 receptor) and 3- tropanyl-dichloroben (5-hydroxytryptamine-3 receptor).

5

SUBSTITUTE SHEET 1 In Vitro Cell Binding Assay

A2058 cells, grown as described above, are harvested with calcium and magnesium free phosphate buffer (0.1 M) 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 [ 125I]PAB (0.1 ml) is added to eight aliquots of 0.1 ml test A2058 cells (1.5 X 10⁶ cells in suspension). To observe competitive binding by pharmacological

20 antagonists, varying concentrations of the antagonists are then added in a volume of 0.1 ml. Cells are incubated at 37°C for 5 hr after addition of an antagonist and the radioactive IPAB.

After incubation, cells are collected by

25 centrifugation for 5 min and washed twice with RPMI 1640 medium. The radioactivity bound to cells is counted using a Packard Autσgamma 5650 scintillation counter.

Data can be analyzed with an INPLOT® iterative, non-linear least square curve fitting 0 program.

Results Antagonists with demonstrated binding specificity for cell surface sigma receptors (e.g. 2 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 ■ Q present compounds bind to cell surface sigma receptors.

35

SUBSTITUTE SHEET 1 EXAMPLE 5

Binding Competition Between Pharmacological Antagonists and (2-PiperidinylAminoethyl)-4-IodoBenzamide

5

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 o 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, [₃H]-(+) -pentazocine.

A sigma-2 binding assay was performed in rat 5 liver membranes in the presence of a sigma-2 selective ligand, [³H]DTG, in the presence of dextrallorphan to mask sigma-1 sites.

Membrane Preparation: o ^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 5 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° C in ml/g tissue weight of 10 mM Tris-HCl/0.32 M 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 lOOOg and the crude nuclear (PI) pellet was discarded.

5

SUBSTITUTE SHEET 2 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 [ H]-(+) -pentazocine (specific activity 52 Ci/mmol)

20 ⁱⁿ 0-5 ml of 50 mM Tris-HCl for 60 min at 37°C. The amount of non-specific binding was determined by the addition of 10 M Tris-HCl, pH 8.0 followed by rapid filteration through glass filters using a Brandel Cell harvester (Gaithersburg, MD) . Filters were washed twice

25 with ice-cold buffer. Prior to use, filters were soaked in 0.5% polyethyleneimine for about 30 min at 25° C. Similarly rats liver membranes (sigma-2) or C6 glioma cell homogenates were incubated with 3 nM [ H]DTG (39.4 Ci/mmol) in the presence of 1 micromolar cold

2o 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

2 were filtered and the filtrate washed twice as above. The radioactivity was counted in Ecoscint (National Diagnostics, Manville, NJ) after an overnight extraction of counts.

The amount of IPAB required to inhibit binding

■^Q of sigma-1 and sigma-2 selective ligands by 50% (i.e. the IC₅₀ values) was derived using the computerized iterative curve-fitting program, GraphPAD. K_i values

35

SUBSTITUTE SHEET were calculated from the IC₅₀ values using Cheng-Prusoff equation.

Results The K₁ values for IPAB are shown in Table 1.

Sigma-1 Sigma-2 Sigma-2

.nea Pig Brain Rat Liver C_c Glioma C

0.89 nM 24.0 nM 130 nM

These data demonstrate that IPAB binds to cell surface sigma receptors with very high affinity.

SUBSTITUTE SHEET EXAMPLE 6

125 Biodistribution of

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 7_7: 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.

I-N-(diethylaminoethyl)4-iodobenzamide (i.e.

[ I]DAB) was prepared as described in John et a^. (1993

Nucl. Med. Biol. 2 : 75-79).

125 I(2-piperidinylaminoethyl)4-iodobenzamide

125

(i.e. [ I]PAB) (D) was synthesized as described in Example 1.

Animal Biodistribution Assays For jLn vivo studies, tumor cells were harvested using calcium and magnesium free PBS

SUBSTITUTE SHEET 2 containing 0,02% EDTA. Suspension of 5 x 10⁶ cells (viability greater than 95%) in 0.2 mL of medium were innoculated 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 innoculated 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 20 intravenously with 0.2 ml of a saline solution

125 containing [ I]PAB (5-6 μCi). At 1, 6 and 24 hr. after injection, blood samples were collected by cardiac puncture and the mice were sacrificed immediately thereafter by cardiecto y while under halothane

25 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

2o radioactivities with suitably diluted aliquots of the

125 injected [ I]PAB dose divided by the weight of the organ. The values obtained were normalized to a mouse

125 weighing 20 g. The differences between [ I]PAB and

[ 125I]DAB were examined by Student's unpaired t tests.

25

Results

Tables 1-3 illustrate the biodistribution of

[¹²⁵I]PAB and [¹²⁵I]DAB in nude mice bearing human A2058 melanoma xenografts in the flank at one, six, and ^ ^■ _Q twenty-four hours, respectively, after administration of the imaging agent.

35

SUBSTITUTE SHEET 2 At one hr. post-injection (Table 1), the concentration of [¹² I]DAB (% injected dose/gm) was higher than the tumor concentration of [¹² I]PAB in several tissues including non-tumorous liver, muscle, rr lung and heart tissues. Therefore, while [ 125I]DAB collected in the tumor at a marginally higher level than [¹⁵I]PAB, [¹²⁵I]DAB was significantly less specific for the tumor site than [¹²⁵I]PAB.

By 6 hrs. after administering the diagnostic 0 agents, mice receiving [ I]PAB had more of this diagnostic agent in the tumor than any other tissue. In contrast [ 125I]DAB was found at higher concentrati•ons i•n the liver than in the tumor. Moreover, the concentration of [¹²⁵I]DAB was significantly higher than 5 that of [ 125I]PAB in non-cancerous blood, liver and intestinal tissues.

125

By 24 hrs. mice receiving [ I]PAB had about four-fold more [¹²⁵I]PAB in their tumors than in their livers. In contrast mice receiving [ 125I]DAB had only o about half as much [¹²⁵I]DAB in their tumors as their livers. These data indicate that high levels of

[ IJDAB are non-specifically localized in the liver.

These data also indicate that [¹²⁵I]DAB has less tumor specificity than [¹²⁵I]PAB. 5 Moreover, the tumor concentration of [ I]PAB was almost twice as high as that of [ 125I]DAB indicating that IPAB binds to tumor cells with greater affinity and stability than IDAB. These data indicate that [ 125I]PAB is highly specific for malignant tumors which contain Q cells having sigma receptors.

5

SUBSTITUTE SHEET 14 -65-

Table 1

Biodistribution of N-(piperidinylaminoethyl)-

4-iodo [¹²⁵I]benzamide, [¹²⁵IJPAB, and

N-(diethylaminoethyl)4-iodo [ IJbenzamide,

[ IJDAB, in nude mice xenografted with human melanotic melanoma f% ID/g; mean (std. dev.), n=6]

P Value for 1 Hour [ ¹²⁵I]PAB [¹²⁵I]DAB Difference

SUBSTITUTE SHEET Table 2

Biodistribution of N-(piperidinylaminoethyl)-

4-iodo [ IJbenzamide, [ ²⁵IJPAB, and

N-(diethylaminoethyl)4-iodo [¹² I]benzamide,

[ I]DAB, in nude mice xenografted with human melanotic melanoma [% ID/q; mean (std. dev.), n=6]

SUBSTITUTE SHEET Table 3

Biodistribution of N-(piperidinylaminoethyl)-

4-iodo [¹²⁵I]benzamide, [¹²⁵I]PAB, and

N-(diethylaminoethyl)4-iodo [¹² I]benzamide,

[ I]DAB, in nude mice xenografted with human melanotic melanoma f% ID/q; mean (std. dev.), n=6T

SUBSTITUTE SHEET EXAMPLE 7

125 Biodistribution of

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.

125 I-N-(diethylaminoethyl)4-iodobenzamide (i.e. [ IJDAB) was prepared as described in John et ajL. (1993 Nucl. Med. Biol . _20: 75-79).

125 I(2-piperidinylaminoethyl)4-iodobenzamide (i.e. [¹²⁵I]PAB) (D) was synthesized as described in Example 1.

Animal Biodistribution Assays

For ij vivo studies, tumor cells were harvested using calcium and magnesium free PBS containing 0,02% EDTA. Suspension of 5 x 10 cells (viability greater than 95%) in 0.2 mL of medium were innoculated 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

SUBSTITUTE SHEET 2 innoculated 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 [¹²⁵I]PAB (5-6 μCi). 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 0 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 5 radioactivities with suitably diluted aliquots of the injected [¹²⁵I]PAB dose divided by the weight of the organ. The values obtained were normalized to a mouse weighing 20 g.

o Results

Tables 4-5 illustrate the biodistribution of [ IJDAB and [ IJPAB, respectively, in nude mice bearing human squamous cell carcinoma xenografts in the flank at one, six, and twenty-four hours after 5 administration of the imaging agent.

By 24 hrs. mice receiving [ 125IJPAB had more

125

[ IJPAB in their tumors than any other tissue. In

125 contrast mice receiving [ IJDAB had about six-fold more

[ 125IJDAB in their livers as their tumors. These data indicate that high levels of [ 125 Q IJDAB are non- specifically localized in the liver. These data also

5

SUBSTITUTE SHEET indicate that [¹²⁵IJDAB has less tumor specificity than [¹²⁵I]PAB.

Moreover, the tumor concentration of [ 125IJPAB was more than three-fold higher than that of [¹²⁵I]DAB at 24 hrs. post-injection indicating that IPAB binds to tumor cells with greater affinity and stability than IDAB. These data indicate that [¹²⁵IJPAB is highly specific for lung carcinomas which contain cells having sigma receptors.

SUBSTITUTE SHEET 14

-71-

Table 4

Biodistribution of N-(diethylaminoethyl)-

4-iodo [¹²⁵I]benzamide, [¹²⁵IJDAB, in nude mice xenografted with human s uamous cell carcinoma

SUBSTITUTE SHEET Table 5

Biodistribution of (piperidinylaminσoethyl)-

4-iσdo [ IJbenzamide, [ IJPAB, in nude mice xenografted with human squamous cell carcinoma

SUBSTITUTE SHEET /2 3 -73 -

EXAMPLE 8

125 Diagnostic Imaging Using I-(2-PiperidinylAminoethyl)-4-IodoBenzamide

5 These experiments illustrate the present diagnostic imaging procedures and the benefit of utilizing the present compounds in such procedures.

Materials and Methods 0 A2058 cells, derived from a brain metastasis of human malignant melanoma (Todaro et. al.. 1980 Proc. Natl. Acad. Sci. USA 7J_ : 5258) were obtained from the National Institutes of Health. A human lung adenocarcinoma cell line, NCI-838, was obtained from the 5 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.

¹¹I-N-(diethylaminoethyl)4-iodobenzamide (i.e. o [ IJDAB) was prepared as described in John et a_l. (1993 Nucl. Med. Biol. 2J): 75-79).

I(2-piperidinylaminoethyl)4-iodobenzamide (D) was synthesized as described in Example 1.

5 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 [¹³¹IJPAB or [^mIJDAB (150-200 μCi). _Q The animals were anesthetized with ketamine containing rompun before the imaging studies. The images were

5

SUBSTITUTESHEET - 74 -

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 [¹³¹IJPAB and [¹³¹I]DAB, respectively.

At 6 and 24 hrs. postinjection, [ IJPAB was detected only within the tumor (Figs. 2A and 2B) . In contrast, no [¹³¹IJDAB was detected in the tumor at either 6 or 24 hrs. after administration (Figs. 3A and 3B) . Moreover, considerable uptake of [ IJDAB had occurred in the livers of mice receiving this agent by 6 and 24 hrs. post-administration (Figs. 3A and 3B) . Little or no [¹³¹I]PAB was observed in the liver at either 6 or 24 hrs. 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 [¹³¹I]PAB. These scintographic imaging studies easily visualized the implanted tumor at both 24 and 30 hrs. post-injection.

SUBSTITUTE SHEET

Claims

WHAT IS CLAIMED:

1. A method for diagnosing a mammal for the presence of a mammalian tumor which comprises

administering to a mammal a diagnostic imaging amount of a compound of the formula:

wherein:

X is a radionuclide;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;

each R₃ is independently hydrogen or lower alkyl;

j and y are independently an integer from 0 to 6;

q is an integer from 0 to 2;

with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide; and detecting binding of said compound to a tumor in said mammal.

2. A method for treating a mammalian tumor which comprises administering to a mammal a composition comprising a tumor-inhibiting amount of a compound of the formula:

wherein:

X is a radionuclide;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;

each R₃ is independently hydrogen or lower alkyl;

j and y are independently an integer from 0 to 6;

q is an integer from 0 to 2; and

with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide.

3. The method of Claim 1 wherein X is a γ-emitting radionuclide.

4. The method of Claim 1 wherein X is ¹²³I,

¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁸F, ⁷⁶Br or ⁷⁷Br.

5. The method of Claim 1 wherein X is ¹²³I,

6. The method of Claim 2 wherein X is a ß-emitting or an α-emitting radionuclide.

7. The method of Claim 2 wherein X is ¹²³I,

²¹¹ At, ⁷⁶Br, ²¹²Pb, ²¹²Bi or ⁷⁷Br.

8. The method of Claim 2 wherein X is ¹²³I,

9. The method according to Claim 1 or 2 wherein Z is =0.

10. The method according to Claim 1 or 2 wherein Z is two -H.

11. The method according to Claim 1 or 2 wherein each R₁ is independently H, halo or lower alkyl

12. The method according to Claim 1 or 2 wherein each R₁ is H.

13. The method according to Claim 1 or 2 wherein q is 2.

14. The method of Claim 13 wherein R₁ is alkoxy.

15. The method according to Claim 1 or 2 wherein q is 1.

16. The method of Claim 15 wherein R₁ is alkoxy.

17. The method according to Claim 1 or 2 wherein q is 0.

18. The method according to Claim 1 or 2 wherein R_a and R_b are independently H, halo, lower alkyl or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring.

19. The method according to Claim 1 or 2

wherein R_a and R_b are independently H, halo, lower alkoxy or lower alkyl.

20. The method according to Claim 1 or 2 wherein R_a and R_b are independently H, halo, or lower

alkyl.

21. The method according to Claim 1 or 2 wherein R_a and R_b are independently H or halo.

22. The method according to Claim 1 or 2 wherein R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic

ring.

23. The method according to Claim 1 or 2 wherein

is:

,

,

,

, , , , .

or

24 The method of Claim 1 or 2 wherein j is

0 to 2

25. The method of Claim 1 or 2 wherein j

is 0.

26. The method of Claim 1 or 2 wherein y is

1 or 2

27. The method of Claim 1 or 2 wherein R₂ is —N(R₃)₂.

28. The method of Claim 27 wherein R₃ is

hydrogen.

29. The method of Claim 27 wherein R₃ is

lower alkyl.

30. The method of Claim 1 or 2 wherein R₂ is a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent.

31. The method of Claim 30 wherein said

heterocyclic ring is: ,

,

,

,

,

,

,

or

.

32. The method of Claim 30 wherein said heterocyclic ring is N-piperidinyl, N-pyrrolidinyl, N-pyridinyl, N-morpholinyl, piperidinyl, pyrrolidinyl, pyridinyl or morpholinyl which can be substituted with least one lower alkyl.

33. The method of Claim 30 wherein said heterocyclic ring is or piperidinyl or pyrrolidinyl which is N-substituted with lower alkyl.

34. The method of Claim 33 wherein said lower alkyl is methyl, ethyl, propyl or butyl.

35. The method of Claim 1 or 2 wherein said compound is:

_,

,

, ,

,

, ,

,

,

or

.

36. The method of Claim 1 or 2 wherein said tumor is a lung carcinoma, a colon carcinoma, a renal carcinoma, a melanoma, a glioma, a pheochromocytoma or a neuroblastoma.

37. The method of Claim 1 or 2 wherein said lung carcinoma is an adenocarcinoma, a squamous

carcinoma or a large cell lung carcinoma.

38. The method of Claim 1 or 2 wherein said tumor comprises cancer cells which have a cell surface sigma receptor.

39. The method of Claim 1 wherein the

dectection of the binding of said compound to a tumor is observed after about 6 to about 30 hours.

40. 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 amount of a compound of the formula:

wherein:

X is a radionuclide;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring; R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;

each R₃ is independently hydrogen or lower alkyl;

j and y are independently an integer from 0 to 6;

q is an integer from 0 to 2;

with the proviso that the compound is not a radioisotope of (N-diethylaminoethyl)-4-iodobenzamide;

for a time and under conditions sufficient for binding of said compound to a cancer cell and detecting said binding.

41. The method of Claim 40 wherein X is ¹²⁵I , ¹⁸F , ³⁵S-alkyl , ³⁵SO₃ , ³⁵SO₄ or ³H .

42 . The method of Claim 40 wherein said compound is :

_,

,

, ,

,

,

,

,

,

or

.

43. The method of Claim 40 wherein said cancer cell has a cell surface sigma receptor.

44. A compound of the formula:

wherein:

Q is a radionuclide, halide or an activating group;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₄ is -N(R₃)₂ or an N-linked 5 to 6 membered

nitrogen containing heterocyclic ring which can have at least one alkyl substituent, wherein each R₃ is

independently lower alkyl or hydrogen;

j is an integer from 0 to 6; and

q is an integer from 0 to 2.

45. A compound of the formula:

wherein: Q is a radionuclide, halide or an activating group;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₅ is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent;

m is an integer from 2 to 6;

j is an integer from 0 to 6; and

q is an integer from 0 to 2.

46. A compound of the formula:

wherein:

Q is a radionuclide, halide or an activating group;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring; each R₃ is independently lower alkyl or hydrogen;

n is an integer from 3 to 6;

j is an integer from 0 to 6; and

q is an integer from 0 to 4.

47. The compound of any one of Claims 44-46 wherein Q is a radionuclide.

48. The compound of Claim 47 wherein said radionuclide is ¹²³I, ¹²⁴ I, ¹²⁵I, ¹³¹I, ¹⁸F, ²¹¹At, ⁷⁶Br or ⁷⁷Br,

49. The compound of Claim 47 wherein said radionuclide is ¹²³I or ¹³¹I.

50. The compound of any one of Claims 44-46 wherein Q is an activating group.

51. The compound of Claim 50 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

52. The compound of any one of Claims 44-46 wherein Z is =0.

53. The compound of any one of Claims 44-46 wherein Z is two -H.

54. The compound of any one of Claims 44-46 wherein each R₁ is independently H, halo, lower alkyl.

55. The compound of any one of Claims 44-46 wherein each R₁ is alkoxy.

56. The compound of any one of Claims 44-46 wherein each R₁ is H.

57. The compound of any one of Claims 44-46 wherein q is 2.

58. The compound of Claim 57 wherein each R₂ is alkoxy.

59. The compound of any one of Claims 44-46 wherein q is 0.

60. The compound of Claim 44 wherein R₄ is —N(R₃)₂ and each R₃ is independently lower alkyl.

61. The compound of Claim 44 wherein R₄ is an

N-linked 5 to 6 membered nitrogen containing

heterocyclic ring which can have at least one alkyl substituent.

62. The compound of Claim 61 wherein said heterocyclic ring is

, ,

, or

.

63. The compound of Claim 51 wherein said heterocyclic ring is N-piperidinyl, N-pyrrolidinyl or N-pyridinyl.

64. The compound of Claim 45 wherein R₅ is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent.

65. The compound of Claim 64 wherein said heterocyclic ring is piperidinyl, pyrrolidinyl or pyridinyl which can have at least one alkyl substituent,

66. The compound of Claim 64 wherein said heterocyclic ring is:

, ,

, or

.

67. The compound of Claim 45 wherein m is 2.

68. The compound of Claim 46 wherein each R₃ is lower alkyl.

69. The compound of Claim 46 wherein each R₃ is hydrogen.

70. The compound of Claim 46 wherein n is 3.

71. A compound having any one of the formulae:

,

,

, ,

,

,

,

,

or

.

72. A pharmaceutical composition comprising a diagnostic imaging amount of the compound of any one of Claims 44-46 and 71, and a pharmaceutically acceptable carrier therefor.

73. A pharmaceutical composition comprising an anti-tumor amount of the compound of any one of Claims 44-46 and 71 and a pharmaceutically acceptable carrier therefor.

74. A compartmentalized kit for detection or treatment of a mammalian tumor comprising a container having at least one compound of the formula:

wherein:

Q is a radionuclide, halide or an activating group;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;

each R ₃ is independently hydrogen or lower alkyl; j and y are independently an integer from 0 to 6;

q is an integer from 0 to 2;

with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide.

75. The kit of Claim 74 wherein Q is an activating group.

76. The kit of Claim 75 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

77. The kit of Claim 74 which further

comprises another container having a reagent for

replacing said activating group with a radionuclide.

78. The kit of Claim 77 wherein said reagent is an oxidizing reagent.

79. The kit of Claim 78 wherein said oxidizing agent is chloramine-T.

80. The kit of Claim 74 which further

comprises a material for separating unattached

radionuclide from said compound.

81. The kit of Claim 80 wherein said material is a chromatographic material.

82. The kit of Claim 81 wherein said chromatographic material is a thin layer chromatography plate, a molecular exclusion resin or a reverse phase resin.

83. A kit for detection or treatment of a mammalian tumor which comprises a container having at least one of the compounds of any one of Claims 44-46 and 71.

84 The kit of Claim 83 wherein said compound has Q as an activating group.

85. The kit of Claim 84 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

86. The kit of Claim 83 which further

comprises another container having a reagent for

replacing said activating group with a radionuclide.

87. The kit of Claim 86 wherein said reagent is an oxidizing reagent.

88. The kit of Claim 87 wherein said oxidizing agent is chloramine-T.

89. A method for diagnostic imaging of a mammalian tissue which has cell surface sigma receptors which comprises administering to a mammal a diagnostic imaging amount of a compound of the formula:

wherein:

X is a radionuclide;

Z is =0 or two -H;

each R₁ is independently H, halo, lower alkyl, lower alkoxy;

R_a and R_b are independently H, halo, lower alkyl, lower alkoxy or R_a and R_b together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring; R₂ is —N(R₃)₂ or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;

each R₃ is independently hydrogen or lower alkyl,

j and y are independently an integer from 0 to 6;

q is an integer from 0 to 2;

with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide;

and detecting an image of a tissue having an abundance of cells with sigma receptors.

90. The method of Claim 89 wherein said tissue is a neural tissue.

91. The method of Claim 90 wherein said tissue is brain tissue.

92. The method of Claim 90 wherein X is a γ-emitting radionuclide.

93. The method of Claim 90 wherein X is ¹²³I,

¹²⁴ I, ¹²⁵I, ¹³¹I, ¹⁸F, ⁷⁶Br or ⁷⁷Br.

94. The method of Claim 90 wherein X is ¹²³I.

95. The method of Claim 90 wherein said compound is:

,

,

, ,

,

, ,

,

,

or .