CA2225861A1

CA2225861A1 - (aminostyryl)pyridinium compounds for radiolabelling cell membranes

Info

Publication number: CA2225861A1
Application number: CA002225861A
Authority: CA
Inventors: Carol Lambert; Ronnie C. Mease; John G. Mcafee
Original assignee: Individual
Current assignee: Research Corp Technologies Inc
Priority date: 1995-07-06
Filing date: 1995-07-06
Publication date: 1997-01-23
Also published as: WO1997002246A1

Abstract

A compound is provided of formula (I) wherein n is 4-16, Det is an organic group comprising a radioisotope or capable of chelating a radioisotope, and Z-is one equivalent of a biologically acceptable anion, which compounds are useful to radiolabel cellular membranes, as of hematopoietic cells.

Description

W O 97/02246 PCT~US95/08460 5 (AMINOSTYRYL)PYRIDINIUM COMPOUNDS FOR RADIOLABELL~G
C~ELL MEMBRANES
Ba~.uund of the Invention Lipophilic fluorescent membrane perm~nent dyes have been used for over 15 years to measure membrane potential in both resting and activated 10 neutrophils and lymphocytes in vitro. The lipophilic r.~lel~t~ n-oxine, ~ n-tropolone, and 99mTc-~PAO (h~Y~metllylpropylene amine oxime) are used clinically to label mixed leukocytes for detection of focal infl~tnm~tc1ry lesions.
Leukocytes labeled in vitro with 99mTc-HMPAO or the lllIn-chel~tes are still themost widely accepted means of im~ing infl~mm~tion See, E. LanKo, Scand. J.
15 Gastroent.. 29 Supp, 203~ 11 (1994). Because of improved i_age resolution, reduced radiation dose, low cost, and widespread clinical availability, 99mTc-~ HMPAO is generally preferred for ~l~tecting acute abdf-min~l.c sepsis,infl~mm~tory bowel disease (I13D), soft tissue sepsis and o~Leo~yelitis. This is in spite of the fact that 99mTc-E~PAO is less stable both in vitro and in vivo than20 either of the IllIn radioph~rm~ce~l~icals, and that with 99mTc-E~PAO, there is diffuse abdominal r~iio~ctivity~ gall bladder uptake, and renal excretion of an unidentified polar species cont~inin~ 99mTc as early as three hours post injection.
Because of this 99mTc leakage from labelled cells, 99mTc-~aPAO is of no use in assessing urinarv tract infections. In addition, 99mTc-E~PAO leukocytes have 25 lower absolute uptake in abscesses and lower target to background ratios thancells labeled with lllIn-oxine. See, J.G. McAfee et al., Eur. J. Nucl. Med.. 13,353 (1987).
In general, lllIn-leukocytes are preferable for im~ging chronic infection, renal sepsis, fevers of unknown origin, and intraabdominal abscesses in 30 communication with the bowel lumen (A.M. Peters et al., J. Nucl. Med. ~, 65 (1992)). Quantification of whole body retention of radioactivity with lllIn-labeled granulocytes and of fecal excretion of radioactivity with ll'In-labeled neutrophils have been used as methods by which to quantify IBD and Crohn's Disease. S.H.
SavelyllluUIl et al., Gastroent.. 85, 1333 (1983). This quantification is not W O 97/02246 PCT~US95/08460 generally possible using 99mTc-HMPAO unless early SPECT im~ging is p~lr~ .ed using the procedure of Weldon (Scand. J. Ent.~ 29 Supp 203 61 (1994)).
To ~limin~te the leukocyte halvt;~Lil~g step required when 99mTc-HMPAO or "IIn-chelates are used for whole blood labeling, invPstiE~tor.q have 5 ~L~ Led a variety of directly injected in vivo agents for infl~mm~til~n im~in~ However, these techniques, which have employed labeled antibodies and liposomes, have serious drawbacks, incllltling bone marrow uptake of the radiolabel, and low target tissue uptake.
Therefore, a need exists for improved agents to label m~mm~ n 10 cells, such as blood cells, associated with infl~mm~finn, infection, m~ n~ncies, and related pathologies.

Summarv of the Invention The present invention provides compounds which can effectively 15 radiolabel cellular membranes, methods of using them, and int~netli~tP.~ for the p~ Lion thereof. Preferred compounds of the invention are (aminostyryl)pyridinium salts of formula (I):

Det--N~3 ~3N(CIlH2n+l )2 Z = (I) wherein Det is an organic group comprising a cletect~hle radioisotope, n is 4-16, preferably 6-10, and Z~ is one equivalent of a biologically acceptable cation, e.g., (I) is the pyri~linillm salt of an inorganic or organic acid which does not interfere with the ability of the compound of formula (I) to penetrate and label the 25 membranes of target cells and is not toxic to the cells.
Preferably, Det is -CH2-CH=CH-X wherein X is a radioisotope of iodine, i.e., 123I, '2sI, or '31I; or Det is a chelating group comprising one equivalent CA 0222=,861 1997-12-29 W O 97/02246 PCTrUS95/08460 of a metallic radioisotope such as lllIn or 99mTc, ~~.hel~ted by a polycarboxylic acid.
The compounds of formula (I) are ~ r~l~bly employed in vifro, in combination with a ph~rm~centically acceptable carrier or vehicle, to label 5 pop~ tion~ of m~mm~ n cells, such as blood cells, inrlll-lin~ mixed leukocytes or lymphocytes. When introduced into a m~mm~ n host, such as a human patient or ~nimal, the labelled cells such as the leukocytes or lymphocytes, localize at a site of infl~mm~tinn, infection, m~ n~ncy, or the like, t_us enabling the im~;in~; of said site, for ~ nostic purposes or to enable the effective 10 ~al~gel.l1g of therapeutic agents.
Useful intermediates for the p~al~lion of the compounds of formula (I) wherein Det is *I-CH=CH-CH2- are col.lp.,unds of formula (II):

(n-bu~)3Sn--CH=CH--CH2--~N(CnH2n+l )2 Z
(II) wherein n is 4-16, preferably 6-10, and Z~ is one equivalent of a cation.
15 Intermediates of formula (I) wherein Det is a chel~ting moiety which does notcomprise a radiolabel, but which is capable of chelating a radioactive metal, also are an embodiment of the invention.
For example, compounds (I) and (II~ are the pyric~inillm salts of an inorganic or organic acids, i.e., Z is halide, sulfate, carbonate, phosphate, 20 bicarbonate, acetate, citrate, L~, m~le~., malate, propionate, and the like.
Using l25I- and l3lI-labeled compounds of formula (I), canine mixed leukocytes and m~nnml~lear cells were labeled in high yield (80-90%). Canine mixed leukocytes labeled with one compound of the invention, l25I- or l3'I-~, show a higher degree of loc~ tinn in a sodium urate in~ c.ecl abscess in the dog25 model than do ~llIn-oxine labeled mixed leukocytes.
Thus, the compounds of formula (I) can exhibit one or more of the following utilities:

W O 97/02246 PCT~US95/08460 a. Mixed leukocytes labeled with the radioiodinated compounds can be reinjected into the donor for the detection of either acute or chronic sites of infl~mm7~tinn/infection by ~ nnstic im~ging techniques.
b. Autologous lymphocytes labeled with the radioiodinated 5 compounds can be used for in vivo lymphocyte tracking and clinical im~gin~ of lymphatic m~lign~ncies.
c. The present compounds can replace lllIn-oxine as the plt;rel,~d agent with which to label cultured lymphocytes for im~ging met~et~tic m~l~nc)m~
prior to and after adoptive immllnotherapy.
d. Any isolated cell population can be radiolabeled using the present compounds for in vitro testing or tracking in vivo.
e. Autologous lymphocytes labeled with the present co...p~ullds can be used to detect the lymphocytic infiltration of the pancreas which occurs prior to and during the early stages of Type I Diabetes Mellitus. Diagnostic im~gin~
15 with the labeled lymphocytes could be used to assess the effectiveness of dirrele~-l drugs for treating Type I Diabetes during the early stages of the disease.
f. Autologous lymphocytes radiolabeled with the present co...pou.-ds can be used to detect lymphocyte loc~li7~tion in transplanted organs as a means of early detection of the host's rejection of the transplanted tissue.
More specifically, mixed leukocytes labeled with 99mTc- cont~ining compounds of formula (I) can be used to image sites of acute infl~mm~tion.
Nonspecific abdominal and renal uptake typical of 99mTc-~PAO should be reduced using 99mTc ~hP.l~te~l by a compound of formula (I). Mixed leukocytes labeled with ~I~In-, l3lI-, or '23I- cont~ininp: compounds of formula (I) can be used to image sites of chronic infl~mm~tion. Ultimately, autologous lymphocytes labelled in accord with the present method could be used for in vivo lymphocyte tracking and clinical im~ging of lymphatic m~ ;n~ncies. The present compounds may replace lllIn-oxine as the preferred agent with which to label cultured lymphocytes for im~ging metastatic melanoma prior to adoptive immunotherapy W O 97J02246 PCT~US95/08460 Detailed Description of the Invention To prepare compounds of f~ (I), wherein Det is I-CH=CH-CH2-, the (dialkylamino)~ylyl~y~idines, cl~ n~ted Di-X-ASP wherein X is the length of the alkyl chain, can be synth~si7ed from an aminobenzene as shown in S Figure 1, by the general method of A. ~s~ner et al., J. Or~;. Chem.~ 49, 2546 (1 984).
Det is an organic group bound to, or which can bind to, or chelate a radioisotope. The radioisotope can be covalently bound to the organic group, as when Det is CH2CH=CH-X and X is radioactive iodine, or Det can comprise a 10 metal ~hel~tin~ functionality, optionally chelated to a metallic radioisotope.
Preferred among these ~ h~l~ting compounds or ~Irhel~tors~ are such molecules as EDTA, DTPA or DCTA or analogs or homologs thereof, or the compound of the formula:

f~ ~R3 sH2cooM
N~

This formula depicts a cyclohexane-based metal chelator which may be attached to the pyridinium ring N through positions 4 or 5, or through alkyl group R3 and which carries from 1 to 4 metal or nonmetal cations, monovalent cations, or the alkaline earth metals. Thus, with metals of oxidationstate +1, each individual cyclohexane-based molecule may carry up to 4 metal 20 cations (where both R3 groups are CH2COOM). As is more likely, with higher oxidation states, the number of metals will decrease to 2 or even 1 per cyclohexane skeleton. The cyclohexane functionality admits of varying stereochemistry, and the aforementioned formula is not int.o-ntled to limit the molecule to any specific stereochemistry. In particular, both amino functionalities 25 may be either cis or trans to each other.
A preferred cyclohexane ring-cont~ining chelator is of the formula:

W O 97/02246 PCT~US95/08460 ~N(R )CH2C(O)NH(CH2)NHC(O)CH2 N(CH2C02 )2 S wherein R3 is (Cl-C4)alkyl or CH2CO2- and M is one equivalent of a cationic metallic radioisotope, such as 'I'In or 99mTc.
The cyclohexane may be unsubstituted (except for the two nitrogen functionalities) or may be substit~lte(l~ especially at the 4-position, with a h~d~ y or acylated hy~ y group, such as with a lower acyl substit~ltinn For purposes of this invention, other cyclohexane-based analogs such as alkyl deliv~lives (e.g., lower alkyl) or substitution products, wherein the deliv~ a~ion or substitution do not interfere with the linking of the cyclohPx~n~
~keleton to N, with the ~h~l~tin~ ability (affinity, geometry, etc.) of the individual c~h~l~ting moieties, are equivalent to those actually shown. Substitl~tion~ which 15 are equivalent for the purposes of this invention are hydroxy, acyl, halogen, amino, and the like.
Any metal capable of being detected in a diagnostic procedure in vivo or in vi27o can be employed as M in the Det moieties. Particularly, any radioactive metal ion capable of producing a diagnostic result in a human or 20 ~nimal body or in an in vitro diagnostic assay may be used in the practice of the present invention. Suitable ions include the following: Antimony-124, Antimony-125, Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, C~lmillm-109, C~millm-llSm, Calcium-45, Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-56, Cobalt-57, 25 Cobalt-58, Cobalt-60, Erbium-169, Eulupiulil-152, Gadolinium-153, Gold-195, Gold-l99, ~fnillm-175, ~fnillm-175-181, Indium-111, Iridium-192, Iron-55, Iron-59, Krypton-85, Lead-210, Manganese-54, Mercury-197, Mercury-203, :

W O 97102246 PCTrUS95/08460 Molybdenum-99, Neodymium-147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185 + 191, p~ m-103, Pl~timlm-l95m~ Praseody liu...-143, Promethium-147, Prot~ctinillm-233, Radium-226, Rh~.nillm-186, Rubidium-86, P~llthenillm_103, p~llth~nillm-106, Sc~n(~illm-44, Sc~n(lillm-46, Sf~l~nillm-75,5 Silver-llOm, Silver-lll, Sodium-22, Sl~ iu111-85, Sllull~iu11l-89, Sliu~ -90, Sulfur-35, T~nt~ m-182, Te~hn~tillm-99m~ Tellllrillm-125, Tellurium-132, Th~llillm-204, Thorium-228, Thorium-232, Th~lli-lm-170, Tin-l 13, Tit~nillm~4, Tungsten-185, V~n~illm48, V~n~lillm-49, Ytterbium-169, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, and Zirconium-95.
Autologous human lymphocytes can be labeled for in vivo tracking and for im~ging lymph nodes in normal hllm~n~ for im~Eing lymphatic m~lign~noies, for im~ing tumor-involved lymph nodes and staging Hodgkin's disease, for im~ging sites of chronic infl~mm~ti~n, and for the ~i~gnc~ of acutekidney-graft rejection. Cultured lymphocytes labeled with t_e present 15 compounds, inc~ ing interleukin-2 (IL-2) activated autologous peripheral blood lymphocytes (PBLs), tumor-activated killer lymphocytes (TAKs), lymphokine activated killer cells (LAKs), and tumor infiltrating lymphocytes (TILs) can be used to image tumors.
Lymphocytes are extremely sensitive to radiation damage, and 20 radiotoxicity resulting from nuclear ~ccllml~l~tion of IllIn results in decreased lymphocyte proliferative capacity and severe chromosomal aberration. It is well documented that radiotoxicity generally decreases as the distance of a nuclide from the cell nucleus increases. According to various studies, r~ tinn damage from Auger-electron emitters such as IllIn can be reduced 85-fold if the nuclide is 25 confmed to the cytoplasm rather than the nucleus, and reduced 120-fold if thenuclide is restricted to the cell membrane. Thus, the present compounds are expected to be particularly useful to label lymphocytes, as they are membrane-restricted.

W O 97/02246 PCTrUS9S/08460 Alkylation of Di-X-ASP with E-l-tributylst~nnyl-3-to~yl~l"pal,e (1), prepared by tosylation of the known 1-tributylstannyl-1-propen-3-01 (2) ~.E.
Jung et al., Tet. Lett.. ~, 3851 (1982)) gave compounds 3a-d ~Figure 2).
R~ .ting 3a-d with l2sI or l3~I, with or without carrier iodide, in acet -nitrile/water S cnnt~ining 1-10 ~lL 2/1 30% hydrogen peroxide/glacial acetic acid for 10 mimlt.~.,s followed by HPLC purification (Spherisorb phenyl colu_n, 6 mM sodium acetate in methanol) gave pure 4a-d. Yields of radioiodinated 4a-d ranged from 47-80%
for carrier-added reactions giving specific activities from 250 Ci/mmol to 12000Ci/mmol. Compound 4d was prepared in theoretical specific activity in a 50%
10 yield with NCA l2sI.
Labeling of mixed leukocytes (harvested from 20 mT~ canine blood) with ,4a-d was pelrol...ed using five different procedures: 1) labeling in Diluent C (a commercial non-ionic cell labeling media developed by Zynaxis Co. that is not approved for human use) after two saline washes of the cell pellet; 2) labeling 15 in saline after two saline washes of the cell pellet; 3) labeling in saline with no saline washes of the cell pellet; 4 and 5) labeling in ether 10% or 100% platelet poor plasma (PPP). Leukocyte labeling results are sllmm~n7:ed in Table 1.
Compounds 4a and 4b labeled leukocytes in high yield using either Diluent C, or saline with or without washes of the cell pellet. The introduction of plasma into 20 the labeling media reduced labeling yields. Longer chain compounds 4c and 4d only labeled leukocytes in useful yields using Diluent C, W O 97/02246 PCTrUS95/08460 ~o ~t U~
a ¢
~ \ ' CO ' ~ \ ' I

, _ ~ ~O t ~O 00 .

~ O\ ~ ~ ~

t-- ~ ~ o\

~ c ~, " E a~ ", f;

o .;

W O 97/02246 PCT~US95/08460 Peripheral blood lymphocytes (PBLs) from dogs and rats as well as rat splenic lymphocytes were labeled in high yields with either 4a or 4b (Table 2). Viability of labeled lymphocytes was >90% both before and after radiolabeling (Trypan Blue ~xclll~ion test).

W O 97/02246 PCT~US95/08460 o ~

~1 , I , ~D
~1 t_ ¢
_ ~ m m ~~ O
O

~a~ -E a ~ a a 1 ~

C

,, 0 O ~ ~
a _I

W O 97/02246 PCTrUS95/08460 Mixed leukocytes labeled wi~ 4b and l'lIn-oxine labeled mixed leukocytes were compared in dogs having a sodium urate-in~lced infl~mm~tion in the left stifle joint. Selected results are s~lmm~rized in Table 3.

CA 0222586l l997-l2-29 W O 97/02246 PCTrUS95/08460 G g ~~ ~\ ~\ ~\ ~\ ~\ ~\
O ~ a>
O~

o ~ O ~ U~
a ~ ~ ,s~
a O ~ s~
'~ a> ~ ~
a~
X ~ 5 ~ a ~ ~ ~ \ ~ O\ O\ O\ O\ O\ O\ O\ O\ O\
. _ . _, ,,~ s:4 ~ ~ ~ o o o ~ o o o ~ o . ~ ~ ~ o o O O o o O o o o o o o 0 aU~ ~
V ~ V C,) V V ~ O V
~o a~
a~

3 a~
,_ o ~ O O O O O O O O O O
o ~ a~
.
~o a~ a~

oo ~ ~ ~

a~ Z

~. ~ o CA 0222586l l997-l2-29 W O 97/02246 PCTrUS95/08460 s:
-C~

o X
o ~
~ X

C~ ~

C) ~
~ ~>

o o i3 ~ o --o ~ ~) o o o ~ o C~
o o ~ o .

: ~ ~ ~ 3 ~

.. ~ o o ..
In W O 97/02246 PCT~US95/08460 (~ m m~ camera images of the neck, chest, abdomen, pelvis, right and left legs were obtained at 15 mimltes, 1 hour, 2 hours, and 3 hours for Experiment 3 and at 3 hours, 24 hours, and 48 hours for ~ iment 4. No lung sequestration was observed with l3lI-4b labeled leukocytes at 15 minlltes 5 indicating that 4b and the labeling procedures do not activate leukocytes. At early time points (between 1 hour and 3 hours) images obtained with both the 4b and IIIIn-oxine were co~pal~ble, and with either radioph~ ce~ltical tbe abscess was visible at 3 hours.
In Experiment 3, the 3 hour abscess to contralateral knee and 10 abscess to ~ul~u~ding tissue ratios were 3.7 and 2.3 for l3lI4b and 2.7 and 2.0 for IIIIn-oxine. At 48 hours (Experiment 4), the abscess to contralateral knee ratio were 9.1 for l3lI-4b and 4.0 for IIIIn-oxine while the abscess to surrounding tissue ratio was 6.3 for l3lI-4b and 2.6 for lllIn-oxine. The 48 hour percent injected dose per organ estimated from region of interest ~ROI) mea~ul~ ents for15 the liver and abscess were 8.7% and 1.15% for l3lI-4b and 13.1% for lllIn-oxine.
The effects of a reduced volume of plasma on the cell labeling yield in saline were det~-rrnined by eV~ ting the labeling with l2sI-4b (in 1.5 ml saline) of mixed leukocytes, whose prelabeling cell pellets were washed with either 2 x 10 mL saline or 1 x 10 mL saline or not washed. Unwashed mixed 20 leukocytes in 1.5 mL 10% PPP in saline were also tested. Labeling yields were82%, 84%, 80%, and 83%, respectively. No evidence was found that residual plasma in unwashed cells reduced the labeling yield. Although 10% PPP reduced the yield somewhat, labeling yields are high enough to make this method practical. This ability to label leukocytes in the presence of as little as 10%
25 plasma protects cells from premature activation and damage.
Using l2sI-4b and '3lI-4b, mononuclear leukocytes harvested from 20 mT canine blood via the isolymph method (sodium diatrizoste and Ficol 400, d = 1.077) were labeled in 77% and 85% yields, respectively. Thus, the present compounds can label both lymphocytes and mixed leukocytes in high yields, and 30 that the latter can be used to image sites of focal infl~mm~tion. The higher CA 0222586l l997-l2-29 W O 97/02246 PCTrUS~5/~1C0 locali_ation of leukocytes labeled with l3lI~b col~ed to IllIn-oxine in inrlllced infl~mm~tnry sites is ~ci~nific~nt, since IllIn-oxine gives the highest absoluteinfl~mm~tory lesion uptake of all agents yet tested.
The increased loc~li7~tinn observed with the present couu~c,uuds S may be due to an increase in cell viability. Since it has been sllgg~ted that the lymphocyte colul)ol-ent of labeled mixed leukocytes increases the sensitivity ofthese mixtures for ~let~ctin~ chronic infections over the se~iLiviLy obtained with pure granulocytes, this improved loc~li7~tinn may also be due to an increased contribution by labeled lymphocytes spared the r~ tinn damage they would 10 receive from lllIn-oxine. Thus, the present compounds may be a~pl~,pliate fortagging lymphocytes in in vivo tracking studies. These results also suggest thatmixed leukocytes labeled with the present compounds may image chronic infectious foci with low neutrophilic e~nld~tinn better than other agents.

15 Synthesis of Bifunctional Chelatin~ A~ents for Preparin~ lllIn-coordinated Membrane Permeant ASP Compounds Two routes by which to attach bifunctional rhel~ting agents ~BCAs) for lllIn to ASP dyes, were developed.
ALkylation of Di-10-ASP with 3-bromopropylisothiocyanate gave 10. which allows ~ chment of BCAs conf~ining a free amine group. Cyclohexyl EDTA
20 (CDTA) ligand was used to prepare a lllIn-binding BCA with a free amine or with an alkylating group for direct addition to Di-10-ASP. CDTA makes it possible to monofunctionalize (i.e., add a linking group to a single carboxylateof) the ligand. See, R.C. Mease et al., U.S. Patent No. 5,021,571.

R~}N~cH2)ncH3 9 R = ~ NH2 10 R =~--NCS

CA 0222586l l997-l2-29 W O 97/02246 PCTrUS95/08460 Triester 12, shown in Figure 3, was made by two routes. In the first, ethylenedi~mine was ~l-v~l~d to the mono-t-Boc d~liv~Live and reacted with CDTAMA to give 13 (Figure 3). Methyl iodide ~t~rific~tion gave 14 and was followed by selective hydrolysis to 12 (overall yield 15%). Compu~L,d 11 was also collv~ d to 12 by r~ xi/-~ the ammonium salt of 11 in a solution of methanol, chloroform, and sulfuric acid over 3A molecular sieves (50% yield).
10 ~ompound 12 reacted with the NHS ester of several a-h~lo~cetic acids to give 15. Reaction of 15 (X = I) with Di-10-ASP, and 11 with 10 gave Di-10-ASP-CDTA conjugates 16 and 17, where the metal-binding carboxylates are m~ked as esters. Mild nucleophilic methods of deesterification will yield 18 and 19.
The invention will be further described by reference to the~5 following detailed examples.

W O 97/02246 PCT~US95/08460 Example 1 a f c d N~N--(CH2CH2(CnH2~CH~2 a b c d Synthesis of 4-[2-14-(N,N-dibutylamino)phenyl]ethenyl]pyridine (Di-4-ASP);
S Preparation of 4-12-14-(N,N-dihe~cylamino)phenyl]ethenyllpyridine (Di-6-ASP); Preparation of 4-[2-14-(N,N-diocblamino)phenyl]ethenyl]pyridine (Di-8-ASP); Preparation of 4-12-[4-(N,N-didecylamino)phenyllethenyl]pyridine (Di-10-ASP): These compounds were prepared following the procedure of A.
Hassner et al., J. Or~. Chem.. 49. 2546 (1984). lH NMR data (300 MHZ, 10 CDCl3) for these compounds is listed below.
Di-10-ASP: H" ~8.48 (d, J=5.4Hz, 2H); Hb o7.27 (d, J=5.4Hz, 2H); Hc o6.58 (d,J=7.2Hz, 2H); Hd ~7.37 (d, J=7.2Hz, 2H); He or Hf ~7.18 (d, J=15.8Hz, lH);
He or Hf ~6.72 (d, J=15.8Hz, lH); Hg ~3.28 (app.t, J=7.1Hz, 4H); Hh ~1.58 (br s, 4H); Hi ~0.88 (app.t, J=7.1Hz, 6H); Hj ~1.27 (br s, 28H).
Di-8-ASP: H" ~8.48 (d, 2H); Hb ~7.27 (d, 2H); H~ ~6.58 (d, 2H); Hd 7.37 (d, 2H); He or Hf o7.18 (d, IH); He or Hf o6.72 (d, lH); Hg â3.2g (app.t, 4H);
Hh ~ 1.58 (br s, 4E~; Hi oO.88 (app.t, 6H); H, ol .27 (br s, 20H).
Di-6-ASP: H, ~8.48 (d, 2H); Hb ~7.27 (d, 2H); Hc ~6.58 (d, 2H); Hd ~7.37 (d, 2H); He or Hf o7.18 (d, lH); He or Hf ~6.72 (d, lH); Hg ~3.28 (app.t, 4H);
20 Hh ~1.58 (br s, 4H); Hi ~0.88 (app.t, 6H); Hj ~1.27 (br s, 12H).
Di-4-ASP: H" o8.48 (d, 2H); Hb ~7.27 (d, 2H); Hc ~6.58 (d, 2H); Hd ~7-37 (d, 2H); He or Hf ~7.18 (d, lH); He or Hf ~6.72 (d, lH); Hg ~3.26 (app.t, 4H);
Hh ol.55 (br m, 4H); Hi o0.95 (app.t, 6H); Hj ~1.33 (br m, 4H).

Example 2.

a c d e (n-Bu)3Sn~ 0~0 CH3 d e Synthesis of ~1-tributylstannyl-1-propen~3-p-toluenesulfonate ~: E-1-5 tributylstannyl-1-propene-3-ol was prepared by the procedure of M.E. Jung et al., Tet. Lett.. 23, 3851 (1982). In a dry flask under nitrogen atmosphere, 0.500 g (1.44 mmol) of this material was dissolved in dry 1,2-dichloroethane, and 0.33 g(2.16 m mol) of 1,2,2,6,6-pPnt~methylpiperidine was added. p-Toluen~lllfonic anhydride (0.565 g, 1.73 mmol) was added and the reaction stirred at 50~C
10 overnight. At the end of this time, solvent was removed under reduced ~le~uleand the crude product redissolved in methylene chloride. Heptane was added and the volume reduced to give a white precipitate of pto.nt~m~ ylpi~P.ritlinillm tosylate. This solid was filtered and solvent was then removed under reduced ~les~ule from the solution cont~ining E-1-tributylst~nnyl-1-propene-3-p-15 toluenesulfonate. The product was chromatographed on silica gel with 5% ethylacetate/hexane to give 0.6321g (1.24 mmol, 86% yield) of product.
lH NMR (300 MHz, CDCl3) H" ~6.27 (dt, J=19.5,1.5Hz, lH); Hb ~5.88 (dt, J=19.5,5.4Hz, lH); Hc o4.52 (dd, J=5.4,1.5Hz, 2H); Hd o7.79 (d, J=7.9Hz, 2H);
Hc ~7.32 (d, J=7.9Hz, 2H); Hf (s, 3H); o1.43, o1.27, ~0.86 (m, butyl-Sn, 27H).
20 Elemental analysis: Calculated for C22H38S03Sn, C 52.71%, H 7.64%, S 6.40%;
Found C 52.83%, H 7.59%, S 6.47%.

CA 0222=7861 1997-12-29 W O 97/02246 PCT~US95/08460 Example 3.

k m a f c d nBu)3Sn~~ N~ /=\ h a e ~--N--(cH2cH2(cnH2n)cH3)2 5 Synthesis of 3-[4-12-[4-(N,N-didecylamino)phenyllethenyllpyridino]-E-l-tributylstannyl-propene (3d) (Bu3Sn-Di-10-ASP): In a dry flask under nitrogen atmosphere, Di-10-ASP (0.750 g, 1.57 mmol) was dissolved in 5 mL of 1,2-dichlorobenzene to give an amber solution. E-l-Tributylstannyl-l-propene-3-p-toluenesulfonate (0.867 g, 1 73 mmol) was added to the solution and the reaction10 was heated 2 days to give a deep red solution. Solvent was removed under reduced pressure at as low a temperature as possible to give the crude product as a thick red oil. The yield of the reaction was estimated by NMR to be at least 80%. The crude material was purified by HPLC prior to radioiodination.

Example 4.
Synthesis of 3-l4-l2-l4-(N,N-dibutylamino)phenyl]ethenyl]pyridinol-E-l-tributylstannyl-propene (3a) (Bu3Sn-Di-4-ASP); Preparation of 3-[4-l2-l4-(N,N-dihexylamino)phenyllethenyllpyridino]-E-l-tributylstannyl-propene (3b) 20 (Bu3Sn-Di-6-ASP); Preparation of 3-[4-12-l4-(N,N-dioctylamino)phenyl]ethenyllpyridino]-E-l-tributylstannyl-propene (_) (Bu3Sn-Di-8-ASP): These compounds were made and purified by the procedure described above for Bu3Sn-Di-10-ASP. lH NMR data (300 M:Hz, CDCl3 ) of the crude compounds is listed below.
25 Bu3Sn-Di-10-ASP: Ha ~8.56 (d, J=6.8Hz, 2H); Hb ~7.77 (d, J=6.8Hz, 2H~;
Hc ~6.57 (d, J=7.7Hz, 2H); Hd ~7.44 (d, J=7.7Hz, 2H); He or Hf ~7.49 (d, W O 97/02246 PCT~US95/08460 J=15.8Hz, lH); He or Hf ~6.71 (d, J=15.8Hz, lH); Hg ~3.27 (app.t, J=7.1Hz, 4H); ~h ~i ~j, butyl-Sn ol.58, ol.43, ol.26, oØ84 (br m ~legl~les higher than theoretical 65H); Hk o6.25 (m, lH); Hl ~5.97 (m, lH); Hm oS.00 (d, J=7.0Hz, 2H).
5 Bu3Sn-Di-8-ASP: Ha o8.53 (d, 2H); Hb ~7.75 (d, 2H); Hc o6.60 (d, 2H); Hd ~7.45 (d, 2H); He or Hf ~7.56 (d, lH); He or Hf ~6.76 (d, lEI); Hg ~3.32 (app.t, 4H); ~h>Hi,lI;, butyl-Sn ~1.58, ol.43, ol.26, ~Ø84 (br m integrates higher than theoretical 57H~; Hk ~6.28 (m, lH); Hl ~6.03 (m, lH); Hm oS.09 (d, 10 Bu3Sn-Di-6-ASP: Ha ~8.60 (d, 2H); Hb ~7.77 (d, 2H); Hc o6.60 (d, 2H); Hd ~7.45 (d, 2H), He or Hf ~7.55 (d, lH); He or Hf ~6.75 (d, lH); Hg ~3.30 (app.t, 4H); ~,Hi H;, butyl-Sn ol.58, ol.43, ol.26, oØ84 (br m integrates higher than theoretical 57H); Hk ~6.30 (m, lH); Hl ~6.06 (m, lE~); Hm ~5.15 (d, 2H).
Bu3Sn-Di-4-ASP: Ha ~8.50 (d, 2H); Hb ~7.76 (d, 2H); Hc ~6.60 (d, 2H), Hd ~7.44 (d, 2H); He or Hf o7.55 (d, lH); He or Hf o6.75 (d, lH); Hg o3.32 (app.t, 4H); Hh,Hi,Hj, butyl-Sn ~1.58, ~1.43, ~1.26, ~Ø84 (br m integrates higher than theoretical 41H); Hk ~6.31 (m, lH); Hl ~6.05 (m, lH); Hm o5.04 (d, 2H).
Example 5.

a b k m/=\ f c d N ~ ~N--(cH2cH2(cnH2n)cH3)2 a b c d 25 Synthesis of 3-[4-[2-[4-(N,N-didecylamino)phenyllethenyllpyridinol-E-l-iodo-propene (I-Di-10-ASP): Nonradioactive I-Di-10-ASP was prepared directly from Di-10-ASP by an alternative procedure and used as a standard against which to W O 97/02246 PCT~US95/08460 compare the physical properties of radioiodinated I-Di-10-ASP p~ aled from Bu3Sn-Di-10-ASP. Nonradioactive I-Di-10-ASP was char~c.t~ri7~d by NMR and the identification was confirmed by accurate mass mass spectral analysis.
lH NMR data (300 MHz, CDC13 ) Ha o8.74 (d, J=5.4Hz, 2H); Hb o7.62 (d, 5 J=5.4Hz, 2H); Hd+He or Hf o7.53-7.34 (m, 3H); Hk or Hl o6.96 (d, J=14.7Hz, lH); Hc+He or Hf+Hk or Hl o6.75-6.50 (m, 4H); Hm oS.13 (d, J=7.1Hz, 2H);
Hg o3.28 (app.t, J=7.1Hz, 4H); Hh ol.58 (br s, 4H); Hi oO.84 (app.t, J=7.1Hz, 6H); Hj ol.24 (br s, 28H).
High resolution mass spectrometry was performed on a Finnigan MAT 95 high 10 resolution mass spectrometer using a Cesium gun for Liquid SIMS (Secondary Ion Mass Spectrometry). Accurate mass analysis of nonradioactive I-Di-10-ASP
suggested an empirical formula of C36H56rN2 with a deviation of 0.8 mmu for ~ the cationic portion of the molecule. This is in agreement with the proposed structure.

Example 6.

H3C02C o a ~~ N N~ ~1 ~ N CO2CH3 0 a co2cH3 Synthesis of N-lmethyl(2-(iodoacetamido)ethyl)carbamide]-trans-1,2-rli~minocyclohexane N,N', N'-triacetic acid-trimethyl ester: N-~methyl(2-(iodo?~cet~mido)ethyl)carbamide]-trans-1,2-dt~minQcyclohexane N,N', N'-triaceticacid was prepared from cyclohexyl EDTA monoanhydride (R.C. Mease et al., 25 U.S. Patent 5,021,571) and ethylene diamine in a 94% yield as previously reported (R.C. Mease et al., U.S. Patent 5,334,729). This amine was converted to W O 97/02246 PCT~US95/08460 its hydrochloride salt by dissolving it in 100 mT 3M HCl, stirring for lh, then conce~ g it to a sticky solid. This solid was dried by the addition of 100 mL
meth~nnl followed by evaporation under reduced pres~ul~ to dryness. This dr,ving procedure was repeated three more times. The re~ulting powder was S placed in a 500 mT round bottom flask to which was added 200 mT methanol, 150 mL methylene rhloricle7 and 9 mL conce~ led H2SO4. The flask was equiped with a soxhlet filled with molecular sieves 3A and a reflux condenser.
The mixture was refluxed for 10 days, cooled and concentrated to a thick oil.
This oil was dissolved in 50 mL CH2Cl2 and added dropwise to a stirred mixture of 200 mL CH2Cl2 and 200 mL saturated NaHCO3 solution. Upon completion of the ~clrlition the CH2Cl2 layer was separated and the aqueous layer was extracted with 200 mL CH2Cl2. The methylene chloride portions were combined, dried over MgSO4, and concentrated to give 7.3 g (65% yield) of N-[methyl(2-aminoethyl)carbamide]-trans-1,2-(li~min- cyclohexane N,N',N'-triacetic acid-triethyl ester.
lH NMR (300 MHz, CDCl3) Ha (ol.15, m broad, 4H); Hb (~1.70, m, 2H); Hc (ol.95, m 2H); Hd (o2.50, m, lH) and Hd (~2.60, m, lH); He (~2.75, m, 2H);
Hg (o2.90, d, lH) and Hg (o3.18, d, lH); Hf (~3.45, m, 2H); ~3.70-3.40 (m, 15H, C02C_3, NCH2CO2); o8.45 (t, lH, C(O)N~).
N-Succinimidyl-iodoacetate was prepared by the method previously used to make N-succinimdyl-bromoacetate (R.C. Mease et al., U.S. Patent No.
5,089,663). Iodoacetic acid (2.0 g, 10.8 mmol) was dissolved in 100 mL dry CH2Cl2. To this was added 1.2 g (10.8 mmol) N-hyd~ y~uccinimi(lç, and 2.2 g (10.8 mmol) dicyclohexylcarbodiimide. This mixture was stirred for three days as room temperature under a nitrogen atmosphere. The by-product, dicyclohexylurea was filtered and the filtrate concentrated to give a yellow solid.
This was recryst~lli7ed from ethyl acetate to give 2.4 g (79%) of a solid (mp 144-147~C) H NMR (300 MHz, CDCl3) o2.85 (s, 2H), ~3.95 (s, 4H) W O 97/02246 PCT~US95/08460 N-Succir~imidyl-iodoace~te (1.52, 5.4 mmol) was dissolved in 50 rnL dry CHzCl2. To this was added dropwise a solution of 2.1g (4.9 rnrnol) N-[methyl(2-aminoethyl)carbamide]-trans-1,2-tli~minncyclohexane N, N', N'-triacetic-trimethyl ester in 50 mL dry CH2C12 under a nitrogen atmosphere at 5 room temperature. The reaction was stirred overnight then partitioned between 100 mL water and 100 mL CH2Cl2. The CH2C12 layer was collected, the aqueous layer extracted with 200 mL CH2C12, the organic layers combined, dried over MgSO4, filtered, and concentrated to a thick oil. This oil was purified by colurnn chromatography on silica gel using 15:1 CH2Cl2/methanol as eluate to give a 10 thick oil which solidified to yield 1.52 g (52%) of N-[methyl(2-(iodo~cet~mido)ethyl)carbamide]-trans-1,2-~ minocycloh~x~ne N,N', Nl-triacetic acid-trimethyl ester as a glassy solid.
'H N~ (300 MHz, CDC13) Ha (~1.15, m broad, 4H); Hb (~1.70, M, 2H); Hc (~1.95, m, 2EI); Hd (~2.50, m, 1H) and Hd (~2.60, m, 1H~; Hg (~2.90, d, lH) 15 and Hg (~3.15, d, lH); ~3.70-3.30 (m, 21,CO2CH3, NCH2CO2, C(O)NCH2CH2NC(O), NC(O)CH2I).

Example 7.

k ~N-(cH2cH2(cnH2~)cH3~z Synthesis of N-[methyl(2-(Di-10-ASP-acetamido)ethyl)carbamidel-trans-1,2-diaminocyclohexane-N,N',N'-triacetic acid-trimethyl ester: Into a flask under a nitrogen atmosphere was added 20 mL dry 1,2-dichlorobenzene, 71.5 mg (0.15 25 mmol) Di-10-ASP, and 98 mg (0.165) N-[methyl(2-(iodoacetamido)ethyl~-carbamide]-trans-l,2-~ m inocyclohexane N,N', N'-triacetic acid-trimethyl ester.

W O 97/02246 PCT~US95/08460 The reaction was stirred and heated to 50~C under nitrogen for 14 days, then conce~ ed to dryness under reduced ples~ul~ to give a dark red colored thick oil. NMR analysis of the crude material showed about 90% collv~l~ion to product.
- S lH NMR (300 MHz, CDCl3) Ha (o8.75, d, 2H); Hb (~7.70, d,2H); He (o7.55, d, lE~; Hf (o6.75, d,lH); Hc (o7.45, d, 2H); Hd (o6.60, d, 2H~; Hk(oS.50, q, 2H);
o3.7-3.45 (m, 21H, Hp, Hq, Hr, and C02CH3; Hg (o3.30, m, 4H); Ho (o2.60, m, 2H) Hn (ol.95, m, 2H); Hm (ol.70, m, 2H); Hh (ol.55, m, 4H); Hj (ol.25, m, 28H); Hl (ol.10, m, 4H); Hi (oO.80, m, 6H).
Example 8. Radioiodination and cell labelin~.
A. Preparation of No-carrier-added 3-[4-[2-(N,N-didecylamino)phenyl]~ pyridino-~1-1125Il-iodo-propene:
Fifty micro liters of a conce~ Led solution of 3-[4-[2-(N,N-didecylamino)phenyl]~ y~ lyl]pyridino-E-l-tributylstannyl-propene was injected into an a 10 micron Spherisorb phenyl column (this colurnn must never have been exposed to an oxidant) and eluted with 5 mM
sodium acetate in methanol. The W absorbing fraction that eluted at 10-14 mimltç~ was collected (solution is a bright red color). A 1 mL
plastic microcentrifuge tube was charged with 130 ul of E~LC eluate fraction eluting at 10-14 min~lte~, the methanol evaporated under a stream of nitrogen and the residue reconstituted in 100 ul acetonitrile.
To this vial was added 10 ul water, 10 ul of a solution of Na'2sI (30 uCi), and 1 ul of a 2:1 mixture of 30% hydrogen peroxide/glacial acetic acid (200 ul 30% H202/100 ul glacial acetic acid). This was mixed with a micro pipet and allow to stand for 10 minllt~.c at room temperature.
The reaction was diluted with 200 ul 5 mM sodium acetate in methanol and injected on to another HPLC system equipped with an inline W
detector, a radioactive detector and a 10 micron Spherisorb column.
The column was eluted with 5 mM sodium acetate in methanol (1 CA 0222~86l l997-l2-29 W O 97/02246 PCTrUS~5/OY1CO

mL/min.). The radioactive peak that eluted at 22 mimlt~ was collected and this fraction was counted to give 15.9 uCi (53~/O). No W peak was observed at this ret~ntit)n time so the specific activity was estimated to be 2170 Ci/mmol. A sample of the product was mixed with authentic cold 3-[4-[2-(N,N-didecylamino)phenyl]ethyenyl]pyridino-E-l-iodo-propene and reinjected on to the ~'LC; the radioactive material and the W material co-eluted confirming the identitv of the radioactive product.

Example 9.

Preparation of carrier-added of 3-14-[2-(N,N-dihexylamino)phenyl]ell-y~--yl]pyridino-E~1-[~ -iodo-propene: Fifty micro liters of a concentrated solution of 3-[4-[2-(N,N-15 dihexylamino)phenyl]elllyellyl]pyridino-E-l-tributylstannyl-propene was injected into an a 10 micron Spherisorb phenyl column (this column must never have been exposed to an oxidant) and eluted with 5 mM sodium acetate in methanol.
The W absorbing fraction that eluted at 8-10 mimltes was collected (solution is a bright red color). A 1 mL plastic microcentrifuge tube was charged with 100 ul20 of HPLC eluate fraction eluting at 8-10 minlltes7 the methanol evaporated under a stream of nitrogen and the residue reconstituted in 100 ul acetonitrile. To thisvial was added 10 ul water, 10 ul of a solution of Na~3lI (1.18 mCi), 5 micro liters of a 0.28 mmol solution of nonradioactive sodium iodide (1.4 nanomole) inwater, and 10 ul of a 2:1 mixture of 30% hydrogen peroxide/glacial acetic acid 25 (200 ul 30% HzO2/lOO ul glacial acetic acid). This was mixed with a micro pipet and allowed to stand for 10 minutes at room temperature. The reaction was diluted with 60 ul 5 mM sodium acetate in methanol and injected on to another HPLC system equipped with an inline W detector, a radioactive detector, and a 10 micron Spherisorb column. The column was eluted with 5 mM sodium 30 acetate in methanol (1 mL/min.). The radioactive and W absorbing fraction that WO 97l02246 PCT/US95/08460 eluted at 16 mimlt~ was collected and this fraction was counted to give 858 uCi (73%). From the size of the W peak, the specific activity was estimated to be 300 Ci/mmol.

Example 10.

Radiolabeling mixed leukocytes with 3-14-[2-(N,N-dihexylamino)phenyl]~ yllpyridino-~1-ll3lI]-iodo-propene: A 1 mL
microcentrifuge tube was charged with 500 ul (600 uCi) of the HLPC eluate of 3-10 t4-[2-(N,N-dihexylamino)phenyl]~Lllyellyl]pyridino-E-1-[l3lI]-iodo-propene. This was evaporated to dryness under vacuum in a speed vac and reconstituted into 30 ul ethanol. Forty milliliters of canine blood was drawn into a 60 mT~ syringe cont~inin~ 6.5 mL acetate-citrate-dextrose solution. To this was added S.S mT.

2% methyl cellulose in saline. The con~e"L~ of the syringe were gently mixed by 15 several inversions of the syringe and the syringe placed vertically, needle end up, for 45 mimltt~.~ to allow the red blood cells to settle. The plasma was drawn off and placed into a S0 mL Falcon tube. The tube was centrifuged at 1100 rpm for 15 mimlt~s to pellet the leukocytes. The plasma was removed and the cells resuspended in 2 mL saline. In a 1 mL plastic microcentrifuge was placed O.S
20 mL saline. To this was added 20 ul (372 uCi) of the ethanolic solution of 3-[4-[2-(N,N-dihexylamino)phenyl]ethyenyl]pyridino-E-1-[l3'I]-iodo-propene and mixed.
The solution of 3-[4-[2-(N,N-dihexylamino)phenyl]ethyenyl]pyridino-E-1-[l3'I]-iodo-propene in saline was immediately added to the leukocytes and the cells were place vertically on a Nutator rocking ~pal~Lus and rocked gently for 20 25 minllt~.s at room temperature. The cells were then underlayered with 7.5 nnT of platelet poor plasma and cenkifuged at 1600 rpm for seven minut~ to pellet the cells. The supernatant was removed, the cells resuspended in 10 mL saline, and transferred to a new tube. The supernatant, cells, and labeling tube were counted for radioactivity. The cells were centrifuged again at 1600 rpm for 7 minllt~, the CA 0222=,861 1997-12-29 W O 97/02246 PCT~US95/08460 sUp~rn~t~nt removed, and the cells resuspended. After the second ce~lliÇugation,340 uCi (91%) of l3'I was bound to the cells.

Example 1 1.
5 Radiolabeling of rat splenic Iymphocytes ~ith 3-~4-[2-(N,N-dihexylamino)phenyll~ll.y~l~yllpyridino-~1-l~25Il-iodo-propene: A 400g Sprague-Dawley rat was sacrificed to remove the spleen (1.133 g). The spleen was perfused with phosphate buffered saline to remove the lymphocytes. The lymphocytes in 10 mL PBS were split into two equal portions and each portion 10 was layered onto 5 mL of Lympholyte-Rat (Cedar Lane Laboratories T imited, Ontario, Canada) in a separate 15 mL centrifuge tube. The tubes were c~ iruged ar 2700 rpm for 20 mimlt~s at room temperature. The lymphoctyes appeared as a layer at the int~rf~ce of the saline and Lympholyte-Rat layers. The saline layer was removed and discarded. The lymphocyte cont~ining layer from 15 each tube was removed and transferred to a single 50 rnL Falcon tube. To thistube was added 10 mT saline and the tube centrifuged for 7 mimlt~s at 1600 rpm.
The sUp~rn~t~nt was removed, the cells resuspended in 10 mT s~line, and centrifuged at 1600 rpm for 7 minutes. The supernatant was removed and the cells suspended in 1 mL saline. Using a hemocytometer, a total of 5 million 20 lymphocytes were counted. Trypan blue exclusion test showed that 12% of the cells were dead. To these cells was added 20 ul of an ethanolic solution of 3-[4-[2-(N,N-dihexylamino)phenyl]ethyenyl]pyridino-E- 1 -[l2 I]-iodo-propene ( 15 .6 uCi) dissolved in 500 ul of saline. The cells were place vertically on a Nutatorrocking appa~ s and rocked gently for 20 min~ltes at room temperature. The 25 cells were then underlayered with 7.5 mL of saline and centrifuged at 1600 rpm for seven minllte.s to pellet the cells. The supernatant ~as removed, the cells resuspended in 10 mL saline, and transferred to a new tube. The sup~rn~t~nt, cells, and labeling tube were counted for radioactivity. The cells were centrifuged again at 1600 rpm for 7 min~lt~, the supernatant removed, and the 30 cells resuspended. After the second centrifugation, 11.9uC~i (77%) of l3lI was CA 0222586l l997-l2-29 bound to the lymphocytes. After labeling, 12% of the cells were dead by the trypan blue e~rclll~ion test. Therefore, the labeling did not affect cell survival.
All publications, patents and patent documents are incorporated by reference herein, as though individually incollJol~led by reference. The invention 5 has been described with reference to various specific and ,o~rell~d embodiments and techniques. However, it should be understood that many variations and modifications may be made while r~m~inin~ within the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula (I):
wherein Det is an organic group comprising a detectable radioisotope. n is 4-16, and Z- is one equivalent of a biologically acceptable anion.

2. The compound of claim 1 wherein Det is -CH2-CH=CH-X wherein X is 123I, 125I, or 131I.

3. The compound of claim 1 wherein Det is a chelating group comprising a radioactive metal.

4. The compound of claim 3 wherein the radioactive metal is 111In or 99mTc.

5. The compound of claim 1 wherein Det is:

wherein M is one equivalent of a radioactive metal and R3 is (C1-C4)alkyl or CH2CO2-.

6. The compound of claim 5 wherein M is 111In or 99mTc.

7. The compound of claim 1 wherein n is 6-10.

8. A compound of formula (II):

wherein n is 4-16 and Z- is one equivalent of an anion.

9. The compound of claim 8 wherein n is 6-10.

10. A method for radiolabelling an isolated in vitro population of mammalian cells comprising contacting said population with an effective amount of a compound of formula (I):
wherein Det is an organic group comprising a detectable radioisotope, n is 4-16, and Z- is a biologically acceptable anion; so that the compound binds to the cell membranes.

11. The method of claim 10 wherein the cells are blood cells.

12. The method of claim 11 wherein the cells are leukocytes or lymphocytes.

13. An isolated in vitro population of mammalian cells comprising a membrane-bound compound of formula (1):

wherein Det is an organic group comprising a detectable radioisotope. n is 4-16, and Z- is one equivalent of a biologically acceptable anion.

14. The population of claim 13 wherein the cells are blood cells.

15. A method for detecting a locus of inflammation or infection in a mammal comprising introducing into the bloodstream of the mammal, leukocytes or lyphocytes comprising a membrane-bound compound of formula (I) wherein Det is an organic group comprising a detectable radioisotope. n is 4-16, and Z- is one equivalent of a biologically acceptable anion;
permitting the leukocytes or lymphocytes to localize at said locus; and detecting the presence of the compound at said locus.