Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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:
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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
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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:
CH2COOM
f~ ~R3
sH2cooM
N~
R3
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:
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~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,
:
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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.
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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,
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~o
~t
U~
a
¢
~ \ ' CO ' ~ \ ' I
, _ ~ ~O t ~O 00
.
~ O\ ~ ~ ~
t-- ~ ~ o\
~ c ~, " E a~ ", f;
o .;
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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).
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11
o ~
~1 , I , ~D
~1
t_
¢
_ ~ m m
~~ O
O
~a~ -E a ~ a a 1 ~
C
,, 0
O ~ ~
a
_I
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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.
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13
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
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14
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
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(~ 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
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16
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
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17
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.
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18
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).
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19
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%.
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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,
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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
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22
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
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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)
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24
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
C02H
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.
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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
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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
CA 02225861 1997-12-29
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
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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
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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.
