Long emission wavelength chemiluminescent compounds and their use in test assays

An assay method incorporating at least two different chemiluminescent compounds for detection and/or quantitation of at least two substances in a test sample is described. The synthesis of chemiluminescent reagents or conjugates for use in such methods as well as kits incorporating such reagents are also disclosed. The assays have particular application in the field of clinical diagnostics.

BACKGROUND OF THE INVENTION 
1. Technical Field 
The present invention relates to a new class of chemiluminescent, aromatic 
ring-fused acridinium compounds (AFAC) which emit green or yellow light 
upon simple chemical treatments. This invention also relates to conjugates 
formed from AFAC and binding partners, e.g. biological molecules, and test 
assays utilizing the conjugates. Furthermore the invention relates to test 
assays in which the detection and/or quantitation of two or more 
substances or analytes in a test sample can be carried out simultaneously 
due to the discernable and non-interfering light emission characteristics 
of two or more chemiluminescent conjugates. 
2. Cross-Reference 
The following application, filed concurrently herewith, describes a 
luminometer for detecting emission spectra of at least two 
chemiluminescent compounds in a test sample and is identified as U.S. Ser. 
No. 08/035,341. The disclosure of said application is commonly assigned 
and incorporated herein by reference. 
3. Technical Review 
Chemiluminescent compounds that emit light with separated wavelength maxima 
and minimally overlapping but correctable emission spectra can be very 
useful in analytical assays, particularly in industrial assays, and in 
clinical diagnostic assays for multi-substance, e.g. multi-analyte, 
determinations. Such compounds can be used to tag or label binding 
partners, e.g. biological molecules, such as antigens, antibodies, and 
nucleic acids to form conjugates or tracers that are capable of producing 
mutually non-interfering, or minimally over-lapping light emission signals 
or spectra, that allow the simultaneous detection and/or quantitation of 
multiple substances in a test sample. For example, simultaneous 
determinations of serum levels of luteinizing hormone (LH) and follicle 
stimulating hormone (FSH) from one patient sample is possible and is 
demonstrated below, because two chemiluminescent compounds having light 
emission spectra which span about 100-250 nm for a spectral region with 
signal intensity above 5% of peak height, but differing in their emission 
maxima so that the signals are discernable. In one example the emission 
maxim of two chemiluminescent signals differ by about 60 nm and preferably 
by 80 nm or more for labeling or tagging anti-LH and anti-FSH. A further 
example where simultaneous determinations is possible according to the 
methods described herein, is in the assay of amplified nucleic acid 
sequences, e.g. oncogenes associated with malignant transformation. See 
EP-A-0 481 704 (priority app. U.S. Ser. No. 598,269 (Oct. 16, 1990) and 
references cited therein) which is commonly assigned and incorporated 
herein by reference. In such assays, the inclusion of a parallel, internal 
reference material for a known, different target sequence in the same 
working vessel or reaction medium as a positive control is recognized as 
important to assay performance, for example, to safeguard false negative 
results. In other assay formats, the inclusion of a known control 
substance will also serve to assess assay performance. 
The economical benefit and the experimental necessity of determining and/or 
quantitating two or more substances, e.g. analytes, in a test sample were 
the two main underlying motives in the development of a multiple-tracer 
assay system of the present invention. It was further envisioned that an 
ideal multiple-tracer system would emit multiple-wavelength signals under 
identical chemical conditions. It was recognized that it would be less 
desirable and more cumbersome to combine two chemiluminescent tracers in a 
multiple-analyte assay system that required two different sets of signal 
generating mechanisms, conditions and timings as would be in the case of 
utilizing two different classes of chemiluminescent compounds such as 
acridinium compounds pairing with luminol series or with stable 
dioxetanes, which involve the use of other chemicals or enzymes to 
generate the signal. Furthermore the two or more different 
chemiluminescent compounds or conjugates must have emission efficiency 
differing by not more than one order of magnitude. 
A still further fundamental requirement was the adequate stability of the 
chemiluminescent compounds in aqueous media or environment, which will 
withstand the shipping conditions for commercial products when placed in 
kit form. 
Applicant has achieved these goals by developing stable chemiluminescent 
analogues within the same general class that exhibit bathochromic shifts 
in their emission maxima, and emit light with comparable efficiency under 
identical chemical treatments. 
Chemiluminescent acridinium compounds which are shown herein to emit blue 
light upon treatment with hydrogen peroxide and metal hydroxide have been 
well documented. U.S. Pat. Nos. 4,745,181, 4,918,192, 5,110,932, U.S. Ser. 
No. 07/826,186, U.S. Pat. No. 5,227,489, and U.S. Ser. No. 07/871,601, 
U.S. Pat. No. 5,241,070, describe stable polysubstituted-aryl acridinium 
esters; all of which are commonly assigned and incorporated herein by 
reference. Such acridinium compounds shall be referred to generally herein 
as "reference acridinium esters" or "acridinium esters". Such compounds as 
indicated include an acridinium ring system and, depending on the use of 
such compounds, further include an appropriate functional group(s), e.g. 
for attaching the label to a substance to form conjugates for use in a 
test assay, including the assays of the present invention. 
Batmanghelich, et al, EP-A-0 478 626 (priority GB 2233450A (Jun. 24, 
1989)), described the use of acridinium compounds of varying light 
emission, i.e. fast and slow durations to prepare different tracer 
conjugates, to achieve a "substantially simultaneous" quantitation of two 
or more different analytes. This approach, however, has several major 
drawbacks. First, one of the acridinium esters includes 
electron-withdrawing substituents on the phenolic moiety in order to 
achieve very short duration of light emission, i.e. complete emission or 
emission maxima in one second. This type of compound, e.g. a 
ortho-dihalogenated aryl acridinium ester, however, may suffer from lack 
of stability in aqueous environment. Second, light emission kinetics must 
be carefully examined to permit accurate correction in order to 
distinguish the light emission contributed individually by the two tracers 
during the overlapping period of light emission. The described method 
relies on the measurement of photons emitted in two separate time windows 
for sequential integration of light intensity. Unless the light emission 
overlap is relatively small, such correction could be a potential source 
for poor assay precision, particularly for detection of two analytes 
having widely different concentrations. The requirement for smaller light 
emission overlap would in turn demand the availability of a pair of 
chemiluminescent compounds, one having very short and the other very long 
duration of light emission; and would lead to compounds which either have 
stability problems or render the dual-analyte assay unpractical due to 
excessively prolonged signal-collection time. 
Where the signal collection time is extended, an advantage of performing 
two assays in a single test sample would be lost. It is noted that in one 
automated analyzer using chemiluminescent detection and/or quantitation, a 
first test result is reported at fifteen (15) minutes and thereafter at 
every twenty (20) seconds during operation, see EP-A-0 502 638 (priority 
U.S. Ser. No. 665,196 (Mar. 4, 1991, abandoned)) which is commonly 
assigned and incorporated herein by reference. 
Batmanghelich et al also described an acridinium compound of different 
light emission spectra to prepare different tracer conjugates. The 
approach they used was to extend the electronic conjugation of the 
acridinium nucleus to obtain 3-(4-carboxybutadienyl)-acridinium ester 
(compound 2b) with bathochromic shift of about 80 nm in the emission 
maximum as compared to the parent acridinium ester (compound 2a). 
Extension of electronic conjugation of the acridinium nucleus does not 
necessarily lead to major bathochromic shift in the emission maximum which 
is practically needed to construct a dual-analyte immunoassays and a 
possible reduction in emission efficiency. No teaching was made of 
benzacridinium chemiluminescent compounds or conjugates for use in such 
assays. 
McCapra et al, EP-A-0 322 926 (priority U.S. Ser. Nos. 140,040 (Dec. 31, 
1987), abandoned, and 291,843 (Dec. 29, 1988), abandoned), suggested the 
"chemiluminescent moiety" consisting of heterocyclic ring or ring system 
with ester, amide linkages attached to one of the carbon atoms on the ring 
or ring system. This chemiluminescent compound was said to include 
benz[a]acridinium, benz[b]acridinium, and benz[c]acridinium but the 
synthesis and structure of these compounds or conjugates was not 
described. Neither emission wavelength maxima, nor light emission 
efficiency of these structures were predicted; nor were the use of at 
least two chemiluminescent compounds or conjugates in an assay method, nor 
the utility of such compounds when used in assays based on their emission 
spectra. 
The nomenclature of benz[a]acridinium and benz[b]acridinium utilized in 
this disclosure is based on Rule 21.5 of Definitive Rules for Nomenclature 
of Organic Chemistry, Ed. International Union of Pure and Applied 
Chemistry in the 1957 REPORT OF THE COMMISSION ON THE NOMENCLATURE OF 
ORGANIC CHEMISTRY. 
According to the example given on Benz[a]anthracene, the compound arising 
from fusing benzene ring to the peripheral sides of the acridnium nucleus 
(structure below) should therefore be named according to whether side a, 
b, or c of the acridinium nucleus is fused with the benzene ring. 
##STR1## 
The following abbreviations are utilized in the disclosure: 
______________________________________ 
1. ABAC: angular benz[a]acridinium compound 
2. AFAC: aromatic ring fused acridinium compound 
3. EtO: ethoxy 
4. DMAE: dimethyl acridinium ester 
5. DIPAE: diisopropyl acridinium ester 
6. LBAC: linear benz[b]acridinium compound 
7. LEAC: longer emission acridinium compound 
8. LEAE: longer emission acridinium ester 
9. MeO: methoxy 
10. NSE: N-sulfoethyl 
11. NSP: N-sulfopropyl 
12. PCT: percent cross talk 
13. PMP: paramagnetic part icles 
14. QAE: quaternary ammonium ethoxy 
15. RLU: relative light units 
______________________________________ 
SUMMARY OF THE INVENTION 
A method is described for detection and/or quantitation of at least two 
substances in a test sample comprising simultaneously detecting the 
emission signals of at least two chemiluminescent conjugates; each 
chemiluminescent conjugate being associated with a substance sought to be 
detected and/or quantitated in the test sample. The emission signals of 
each of the chemiluminescent conjugates are discernable by their spectral 
emissions, so that the substances may be detected and/or quantitated. 
A chemiluminescent compound for use in the assays of the present invention 
is described in the formula: 
##STR2## 
where W is carbon; alternatively, C.sub.7, W, C.sub.9 or C.sub.10 can be 
replaced with --N.dbd.; or W can be omitted and C.sub.7 connected to 
C.sub.9, and C.sub.7, C.sub.9 or C.sub.10 can be replaced with --O--, 
--S--, --NH-- or --NR--; Y is a branched or straight chained alkyl 
containing optionally up to 20 carbon atoms, halogenated or unhalogenated, 
or a polysubstituted aryl moiety of the formula: 
##STR3## 
R.sub.1 is an alkyl, alkenyl, alkynyl or aralkyl containing optionally up 
to 20 heteroatoms; 
R.sub.2, R.sub.3, R.sub.9 and R.sub.10 are identical or different groups 
selected from hydrogen, substituted or unsubstituted aryl (ArR or Ar), 
halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --OR, 
--SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R; 
R.sub.2 includes a single or multiple substituent at C.sub.1-4 ; 
R.sub.2 can also be a fused aromatic ring with or without heteroatoms; 
R.sub.3 includes a single or multiple substituent at C.sub.7, W, C.sub.9 or 
C.sub.10 ; 
A-- is a counter ion including CH.sub.3 SO.sub.4.sup.-, FSO.sub.3.sup.-, 
CF.sub.3 SO.sub.4.sup.-, C.sub.4 F.sub.9 SO.sub.3.sup.- , CH.sub.3 C.sub.6 
H.sub.4 SO.sub.3.sup.- and halide; 
X is a heteroatom including nitrogen, oxygen or sulfur, such that when X is 
oxygen or sulfur Z is omitted, when X is nitrogen then Z is --SO.sub.2 
--Y' and Y' is equal to Y and where the substituents to Y and Y' do not 
have to be the same; 
R.sub.4 and R.sub.8 are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol, 
amido, 
R.sub.5 and R.sub.7 are any of R.sub.3, R.sub.9 and R.sub.10 defined above; 
R.sub.6 =--R.sub.11 --R.sub.12, 
where R.sub.11 is not required but optionally can be branched or 
straight-chained alkyl, substituted or unsubstituted aryl or aralkyl 
containing optionally up to 20 heteroatoms, 
and R.sub.12 is a leaving group or an electrophilic functional group 
attached with a leaving group or --Q--R--Nu, --Q--R(I).sub.n Nu, --Q--Nu, 
--R--Nu or --Nu, n is a number of at least 1, Nu is a nucleophilic group, 
Q is a functional linkage, I is an ionic or ionizable group; 
R.sub.5 and R.sub.6, and R.sub.6 and R.sub.7 are interchangeable; and 
R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally up to 
20 heteroatoms. 
A chemiluminescent compound or conjugate is characterized in that upon 
chemical treatment the compound or conjugate emits a blue-green, green, 
yellow, orange and red-orange light having a discernable emission spectra 
peak or maximum. In one embodiment, i.e. compound, the emission maxima is 
greater than or equal to 480 nm and in a preferred embodiment greater than 
or equal to 515 nm. 
An amplification method is described for target sequences, including one or 
more nucleic acid sequences, in a test sample comprising providing a test 
sample suspected of containing one or more target sequences, adding an 
internal reference to said test sample, amplifying the target sequences, 
providing at least two chemiluminescent conjugates, each chemiluminescent 
conjugate being associated with target sequences and the internal 
reference, and simultaneously detecting and/or quantitating amplified 
target sequences and the internal reference by emissions of the 
chemiluminescent conjugates. 
Accordingly, it is a primary object of the invention to provide a method 
for the simultaneous detection and/or quantitation of at least two 
substances in a test sample by use of at least two different 
chemiluminescent compounds or conjugates each having discernable emission 
spectra. 
Another object of the invention is to provide an assay method for the 
simultaneous detection and/or quantitation of an analyte and an internal 
standard or control in a single test medium or transfer tube. 
Still another object of the invention is to increase the efficiency of 
automated analyzers by providing for the simultaneous performance of two 
assays on a test sample in a single reaction medium or transfer tube. 
A further object of the invention is to provide methods for synthesis of 
chemiluminescent compounds and intermediate products which may be used to 
synthesize such chemiluminescent compounds. 
An object of the invention is to provide chemiluminescent, aromatic 
ring-fused acridinium compounds (AFAC) that emit green or yellow light. 
Another object of the invention is to provide chemiluminescent, aromatic 
ring-fused acridinium compounds (AFAC) that emit green or yellow light 
with wavelength maxima or peaks greater than or equal to 515 nm. 
Still another object of the invention is to provide a simultaneous dual 
chemiluminescent label assay. 
A further object of the invention is to provide hydrophilic AFAC which 
carries one or more ionic and/or ionizable groups with or without, 
additionally, the reactive functional groups useful for forming covalent 
linkage with other micro- or macromolecules or encapsulation inside 
liposomes. 
An object of this invention is to provide AFAC conjugates formed between 
AFAC directly or indirectly with binding partners, e.g. biological 
molecules. 
Another object of this invention is to provide test assays involving the 
use of acridinium ester and AFAC conjugates. 
Still another object of the invention is to provide multianalyte assays in 
which the determination of two or more analytes or substances or 
combination thereof present in the sample as a mixture, can be carried out 
simultaneously in the same reaction medium or transfer tube due to the 
mutually non-interfering, or minimally overlapping but correctable light 
signals produced by the same chemical treatments of two or more different 
chemiluminescent tracers or compounds. 
A further object of the present invention is to provide test kits for 
performing dual chemiluminescent label test assays. 
An object of the present invention is to provide test kits having two or 
more chemiluminescent reagents for simultaneously assaying at least two 
substances in a test sample. 
Another object of the invention is to provide intermediate compounds to be 
utilized in the synthesis of labels for use in analytical assays. 
Still another object of the present invention is to provide 
chemiluminescent compounds having light emission spectra which span about 
100-250 nm, for a spectral region with signal intensity above 5% of peak 
height. 
These and other objects in view, as will be apparent to those skilled in 
the art, the invention resides in the combination of elements set forth in 
the specification and covered by the claims appended hereto.

DESCRIPTION OF PREFERRED EMBODIMENT 
The AFAC of present invention comprise linear benz[b]acridinium (LBAC), 
furanoacridinium, thiophenoacridinium, pyrroacridinium compounds and 
pyridoacridinium compounds. By virtue of the specific position of the 
aromatic ring fused to the acridinium nucleus LBAC was unexpectedly found 
to generate a chemiluminescent emission signal with much greater 
bathochromic shift than the angular benz[a]acridinium compounds (ABAC), 
and to the corresponding "reference acridinium esters", i.e. DMAE-Bz, 
DIPAE-Bz and 3-MeO-DMAE-Bz, see FIG. 1. 
The general structure of AFAC is represented by Formula I: 
##STR4## 
The general structures of linear benz[b]acridinium compounds and the 
isomeric, furano-, thiopheno-, pyrro- and pyrido- acridinium compounds are 
shown by the Formulas II, IIIA-III C, and IV A-IV D respectively. 
##STR5## 
One subclass of the AFAC contains reactive functional group(s), in addition 
to the fundamental chemiluminescent compound with properties described 
above, to enable formation, i.e., by covalent linkage, with binding 
partners, and particularly with biological molecules, to produce 
conjugates useful as non-isotopic tracers in binding assays or as a key 
integral part of a multianalyte assay system. Another subclass of the AFAC 
contains one or more ionic and/or ionizable groups which enhance the 
solubility of the compounds in aqueous media and/or allow them to be 
encapsulated inside liposomes with low leakage. Such hydrophilic LBAC can 
also be modified to carry additional reactive functional groups to allow 
forming conjugates with other micro or macromolecules. 
Preferred LBAC, Furanoacridinium compounds, Thiophenoacridinium compounds, 
Pyrroacridinium compounds and Pyridoacridinium compounds having the 
above-mentioned characteristics and being suitable for above-described 
utilities include chemiluminescent compounds represented by the above 
Formulas I, II, IIIA-III C, and IV A-IV D respectively, where: 
W is a carbon, Formula I becomes the LBAC class of chemiluminescent 
compounds as represented by Formula II; or W can be omitted and C7 
connected to C9 and one of C7, C9 or C10 can be replaced with --O--, 
--S--, --NH--, or --NR-- to form a 5-membered aromatic ring fused linearly 
to the acridinium nucleus as shown in Formula III A-C; or C7, W, C9, or 
C10 can be replaced with --N.dbd. to form a 6-membered pyrido ring fused 
linearly to the acridinium nucleus as shown in Formula IV A-D; 
R.sub.1 is an alkyl, alkenyl, alkynyl, or aralkyl containing optionally up 
to 20 heteroatoms, preferably nitrogen, oxygen, halogen, phosphorus or 
sulfur. 
R.sub.2, R.sub.3, R.sub.9 and R.sub.10 are identical or different groups 
selected from hydrogen, substituted or unsubstituted aryl (ArR or Ar), 
halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --CO.sub.2 H, --SCN, 
--OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R. 
R is alkyl, alkenyl, alkynyl, aryl, or aralkyl, containing optionally up to 
20 heteroatoms. 
R.sub.2 includes a single or multiple substituents) at C.sub.1-4. R.sub.3 
includes a single or multiple substituent(s) at C.sub.7, W, C.sub.9 or 
C.sub.10. 
R.sub.2 can be also a fused aromatic ring with or without heteroatoms. 
A-- is a counter ion including CH.sub.3 SO.sub.4 --, FSO.sub.3 --, CF.sub.3 
SO.sub.4 --, C.sub.4 F.sub.9 SO.sub.3 --, CH.sub.3 C.sub.6 H.sub.4 
SO.sub.3 --, and halide. 
X is a heteroatom including nitrogen, oxygen, or sulfur, when X is oxygen 
or sulfur Z is omitted, when X is nitrogen then Z is --SO.sub.2 --Y'. 
Y is a branched or straight chained alkyl containing optionally up to 20 
carbon atoms, halogenated or unhalogenated, or a polysubstituted aryl 
moiety of Formula V: 
##STR6## 
Y' is equal to Y, and the substituents to Y and Y' do not have to be the 
same. 
R.sub.4, and R.sub.8 are alkyl, alkenyl, alkynyl, alkoxyl, alkylthio, 
amido, groups positioned to ensure better stability of AFAC in aqueous 
media or environment. The stability of AFAC rendering them suitable for 
commercialization is attributed to the steric effect, electronic effect, 
or a combination thereof resulting from the presence of these two groups. 
R.sub.5 and R.sub.7 are as recited for R.sub.3, R.sub.9, and R.sub.10 in 
Formula I. For conjugating AFAC to biological molecules, R.sub.6 can be a 
leaving group or an electrophilic functional group attached with a leaving 
group, or functional groups which can be readily converted into such 
reactive groups, directly attached or connected via a spacer to the ring. 
A "spacer" is defined as branched or straight-chained alkyl, substituted 
or unsubstituted aryl or aralkyl, optionally containing up to 0-20 
heteroatams. Examples of such functional groups include: 
##STR7## 
--N.dbd.C.dbd.S, --N.dbd.C.dbd.O, --N.sub.2 +U--, --N.sub.3, --COOH, --U, 
or --SO.sub.2 U, where U is a halide. 
Alternatively, R.sub.6 can be a protected or unprotected nucleophilic 
functional group directly attached or connected via a spacer to Y. Thus 
R.sub.6 =--Q--R--Nu, or --Q--R(I)n--Nu, --Q--Nu, --R--Nu, or --Nu, where R 
is defined as above. 
Q is a functional linkage arising from the covalent coupling between two 
functional groups each of which resides originally as substituents on Y 
and R or Nu, respectively. The introduction of Q in the construct of 
R.sub.6 represents a modular concept which allows the attachment of R--Nu, 
R(I)n--Nu or Nu directly to a preformed AFAC. Examples of Q include: 
--C(O)--, --C(O)NH--, --NHC(O)--, --NHC(O)O--, --NH--, --O--, --S--, 
--NHC(O)NH--, --NHC(S)NH--, --C(.dbd.N+H.sub.2)NH--, --SO.sub.2 --, 
--SO.sub.3 --, 
(I)n is an ionic or ionizable group including but not limited to quaternary 
ammonium, 
--COOH, --SO.sub.3 H, --SO.sub.4 H, --PO.sub.3 H.sub.2, and --PO.sub.4 
H.sub.2, where n is a number of at least 1. 
The presence of the ionic or ionizable group(s) will enhance the 
hydrophilicity of AFAC and compatibility for its usage in aqueous media. 
The choice and positioning of such ionic or ionizable groups have the 
advantage of enhancing the binding of the biological molecule/AFAC 
conjugate to the corresponding binding partners of said biological 
molecule. Nu is a nucleophilic group on the compound that will facilitate 
conjugation of compound with biological molecules which may lack 
nucleophilic group for coupling, but may have electrophilic group or its 
readily converted precursor. Examples of the protected nucleophilic 
functional groups or groupings include: 
t-Butyloxycarbonylamino and 3-(2-pyridinyldithio)propionyl (PDP). 
##STR8## 
t-Butyloxycarbonyl (t-Boc) is the protective group on amino which can be 
removed by acid, e.g. trifluoroacetic acid, treatment. The S-2-pyridinyl 
group in PDP is a protective moiety which can be removed to generate free 
--SH group upon treatment with dithiothreotol (DTT) at suitable pH. The 
usage of these protective groups of --NH.sub.2 and --SH nucleophilic 
groups is known to those skilled in the arts of organic chemistry. 
Examples of the unprotected nucleophilic functional groups include: amino, 
thiol, hydroxyl, active methylene adjacent to strong electron-withdrawing 
group, organic metallic moieties. Examples of such a nucleophilic R.sub.6 
grouping, its conjugation to biological molecules, and the conjugate 
utilities have been disclosed in EP-A-0 361 817 (priority U.S. Ser. No. 
249,620 (Sep. 26, 1988), abandoned) which is commonly assigned and 
incorporated herein by reference. 
To provide more hydrophilic compounds that can be encapsulated inside 
liposomes for the purpose of constructing signal-enhancing lumisomes, 
R.sub.2, R.sub.3 or R.sub.6 can be strongly ionizable groups directly 
attached or more suitably connected via spacer to the aromatic rings. 
Examples of strongly ionizable groups include: phosphate, phosphonate, 
sulfate, and sulfonate. Examples of such a R.sub.6 grouping, the 
incorporation of the hydrophilic chemiluminescent molecules into liposomes 
and the utility of the resulting lumisomes have been disclosed in EP-A-0 
361 817 (priority U.S. Ser. No. 226,639 (Aug. 1, 1988), abandoned) which 
is commonly assigned and incorporated herein by reference. Similarly, to 
provide hydrophilic AFAC that can be conjugated with biological molecules 
directly, R.sub.2 and/or R.sub.3 can be ionic or strongly ionizable groups 
directly attached or more suitably connected via spacer to the 
aromatic-ring fused acridinium nucleus, and R.sub.6 can be reactive 
functional group-containing side chain as recited above. The positions of 
R.sub.2, R.sub.3, R.sub.5 and R.sub.6, and R.sub.6 and R.sub.7 
substituents in all AFAC are interchangeable. 
One of the possible precursors to AFAC should be AFAC with the R.sub.6 
substituent being hydrogen or R, with R defined as above. 
When X is nitrogen, Y can be a branched or straight chained alkyl of 1 to 
20 carbon atoms or a moiety equal to Formula V above with all the possible 
substituents as recited, and Z is represented by the following Formula VI: 
EQU Z=--SO2--Y' (Formula VI) 
where Y' is a branched or straight chained alkyl of 1 to 20 carbon atoms, 
halogenated or unhalogenated, or a moiety equal to the Formula V shown 
above with all the possible substitutents as recited. The substituents to 
both Y and Y' do not necessarily have to be the same. 
A preferred aromatic ring-fused acridinium compounds (AFAC) should be as 
described above. More preferentially, they are the LBAC series with the 
following substituents: R.sub.1 is a methyl, sulfopropyl or sulfoethyl 
group; R.sub.9, R.sub.10 are hydrogen, methoxy or halogen; R.sub.2 is a 
hydrogen, 2-MeO, 2-quarternaryammoniumalkoxy, 3-MeO, 3-EtO, 
3-quarternaryammoniumalkoxy, or 3-carboxyalkyloxy group; R.sub.3, R.sub.5 
and R.sub.7 are hydrogen; when X is oxygen or sulfur, R.sub.4 and R.sub.8 
are methyl, ethyl, isopropyl groups; R.sub.6 is one of the following 
groups attached to the 4-position of Formula V, carboxylate, 
N-succinimidyloxycarbonyl, benzyloxycarbonyl, N-aminoalkylcarbamoyl, 
Sulfomethylcarbamoyl, 
N-[N-(2-amino-3-S-(3'-sulfopropyl)-thiopropionyl)-2-aminoethyl]carbamoyl, 
N-7-(1,3-disulfonaphthalenyl)carbamoyl, 
N-[1-carboxyl-2-(3-sulfopropylthio)ethyl]carbamoyl, 
N-(2-sulfonyloxyethyl)carbamoyl, N-(2-phosphonoethyl)carbamoyl, 
N-(2-phosphonoxyethyl)carbamoyl and alkoxyiminoethyl; when X is nitrogen, 
Y represents Formula V with R.sub.5 and R.sub.7 being hydrogen and R.sub.6 
being carboxylate, N-succinimidyloxycarbonyl, 
N-succinimidyloxycarbonylalkyl, benzyloxycarbonyl, or 
N-aminoalkylcarbamoyl; Z represents Formula VI with Y' being an alkyl or 
phenyl. 
Intermediate compounds which may be utilized to synthesize the 
chemiluminescent compounds of the present invention include: 
An intermediate of the formula: 
##STR9## 
where w is carbon; 
alternatively, C.sub.7, W, C.sub.9 or C.sub.10 can be replaced with 
--N.dbd.; 
or W can be omitted and C.sub.7 connected to C.sub.9, and C.sub.7, C.sub.9 
or C.sub.10 can be replaced with --O--, --S--, --NH-- or --NR--; 
R.sub.2, R.sub.3, R.sub.9 and R.sub.10 are identical or different groups 
selected from hydrogen, substituted or unsubstituted aryl (ArR or Ar), 
halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --R, 
--OR, --SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R; 
R.sub.2 includes a single or multiple substituent at C.sub.1-4 ; 
R.sub.3 includes a single or multiple substituent at C.sub.7, W, C.sub.9 
and C.sub.10 ; 
R.sub.2 can also be a fused aromatic ring with or without heteroatoms; and 
R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally up to 
20 heteroatoms. 
An intermediate of the formula: 
##STR10## 
where W is carbon; 
alternatively, C.sub.7, W, C.sub.9 or C.sub.10 can be replaced with 
--N.dbd.; 
or W can be omitted and C.sub.7 connected to C.sub.9, and C.sub.7, C.sub.9 
or C.sub.10 can be replaced with --O--, --S--, --NH-- or --NR--; 
Y is a branched or straight chained alkyl containing optionally up to 20 
carbon atoms, halogenated or unhalogenated, or a polysubstituted aryl 
moiety of the formula: 
##STR11## 
R.sub.2, R.sub.3, R.sub.9 and R.sub.10 are identical or different groups 
selected from hydrogen, substituted or unsubstituted aryl (ArR or Ar), 
halide, amino, hydroxyl, nitro, sulfonate, --R, --CN, --COOH, --SCN, --OR, 
--SR, --SSR, --C(O)R, --C(O)OR, --C(O)NHR, or --NHC(O)R; 
R.sub.2 includes a single or multiple substituent at C.sub.1-4 ; 
R.sub.3 includes a single or multiple substituent at C.sub.7, W, C.sub.9 or 
C.sub.10 ; 
R.sub.2 can also be a fused aromatic ring with or without heteroatoms; 
X is a heteroatom including nitrogen, oxygen or sulfur, such that when X is 
oxygen or sulfur Z is omitted, when X is nitrogen then Z is --SO.sub.2 
--Y', Y' is equal to Y and the substituents to Y and Y' do not have to be 
the same; 
R.sub.4 and R.sub.8 are alkyl, alkenyl, alkynyl, alkoxyl, alkylthiol, 
amido; 
R.sub.5 and R.sub.7 are any of R.sub.3, R.sub.9 and R.sub.10 defined above; 
R.sub.6 =--R.sub.11 --R.sub.12, 
where R.sub.11 is not required but optionally can be branched or 
straight-chained alkyl, substituted or unsubstituted aryl or aralkyl 
containing optionally up to 20 heteroatoms; 
and R.sub.12 is a leaving group or an electrophilic functional group 
attached with a leaving group or --Q--R--Nu, --Q--R(I).sub.n Nu, --Q--Nu, 
--R--Nu or Nu where n is a number of at least 1, Nu is a nucleophilic 
group, Q is a functional linkage, I is an ionic or ionizable group; 
R.sub.5 and R.sub.6, and R.sub.6 and R.sub.7 are interchangeable; and 
R is alkyl, alkenyl, alkynyl, aryl or aralkyl containing optionally up to 
20 heteroatoms. 
The following examples describe the synthesis of the preferred compounds 
and intermediates of the present invention, the structures of which are 
shown in FIG. 1. The examples are intended to illustrate and not to limit 
the invention and may be used as a guide by those skilled in the art to 
synthesize compounds having alternate substitutents than those shown in 
the examples. 
EXAMPLE 1 
Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (LEAE-Bz) 
3-Anilino-2-naphthoic Acid 
A mixture of 3-hydroxy-2-naphthoic acid (Aldrich cat. #H4600-7) (376 g, 2.0 
mol) and aniline (376 ml, 4.1 mol) was heated at 170.degree. C. with 
stirring under nitrogen for 16 hours. The resulting mixture, when hot, was 
poured into 1 N HCl (2.5 ml), heated to 100.degree. C., and stirred at 
this temperature for 5 minutes. The mixture, when hot, was filtrated and 
the solid was washed with 0.2 N HCl (600 ml). The wet material was boiled 
and mechanically stirred with 0.5 N sodium carbonate solution (6.0 ml) for 
10 minutes, cooled and filtered. The reddish filtrate was treated dropwise 
with 5 N HCl with stirring to .sup..about. pH 7. The resulting yellow 
precipitate was collected, washed with small amount of water and 
crystallized from ethanol (400 ml) to give 3-anilino-2-naphthoic acid (36 
g, 7%). Rf 0.6 (silica gel, EM Art. 5715, 20% methanol/chloroform). MS 
(EI): m/z 264 (M). 
12-Chloro-benz[b]acridine 
A mixture of 3-anilino-2-naphthoic acid (10.0 g, 37.98 mmol) and 
phosphorous oxychloride (35.4 ml, 379.8 mmol) was refluxed at 150.degree. 
C. under nitrogen with stirring for 2 hours. The resulting purple mixture 
was cooled and evaporated under reduced pressure to dryness. The content 
was added with stirring to a mixture of chloroform/ice/conc. ammonium 
hydroxide (200 ml/200 g/200 ml). The chloroform layer was separated and 
dried over calcium chloride. Removal of the solvent under reduced pressure 
gave 12-chlorobenz[b]acridine (9.4 g, 95%). Rf 0.8 (silica gel, 
hexane/ethyl acetate 2:1). MS (EI): m/z 263 (M). 
12-Cyano-benz[b]acridine 
A mixture of 12-chloro-benz[b]acridine (2.3 g, 8.68 mmol), potassium 
cyanide (620 mg, 9.55 mmol) and copper(I) cyanide (391 mg, 4.43 mmol) in 
anhydrous methanol (16 ml) was bubbled with nitrogen for 1 minute and then 
kept in a sealed tube. The mixture was heated at 160.degree. C. with 
stirring for 4.5 hours and cooled. The red-brown mixture was evaporated 
and the residue was flash-chromatographed (W.C. still et al: J. Org. 
Chem., 43, 2923, (1978)) on a silica column (Baker silica gel, Cat# 
7024-1) packed with hexane and eluted with 10% ethyl acetate-hexane, 
yielding red 12-cyano-benz[b]acridine (1.54 g, 70%). Rf 0.7 (silica gel, 
hexane/ethyl acetate 2:1). MS (FAB, Thioglycerol Matrix): m/z 255 (M+1). 
Benz[b]acridine-12-carboxylic Acid Hydrochloride 
A mixture of 12-cyano-benz[b]acridine (557 mg, 2.19 mmol) and 
tetrabutylammonium bromide (71 mg, 0.22 mmol) in 50% sulfuric acid (v/v, 
50 ml) was heated at 160.degree.-170.degree. C. under nitrogen with 
stirring for 44 hours and cooled. The resulting mixture was poured into 
ice-water (500 ml); the purple precipitate was collected and washed with 
water. The wet material was dissolved with warming in 2 N NaOH (100 ml) 
and then filtered. The filtrate was acidified in an ice-water bath with 
concentrated HCl to pH 3-4, giving purple benz[b]acridine-12-carboxylic 
acid hydrochloride (510 mg, 75%). Rf 0.4 (silica gel, 
chloroform/methanol/water 65:25:4). MS (FAB, Thioglycerol Matrix): m/z 274 
(M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl Benz[b]acridine-12-carboxylate 
A suspension of benz[b]acridine-12-carboxylic acid hydrochloride (370 mg, 
1.2 mmol) in anhydrous pyridine (50 ml) was warmed at 60.degree. C. for 5 
minutes. The slightly cloudy solution was then cooled to 0.degree. C. and 
treated with p-toluenesulfonyl chloride (388 mg, 2.33 mmol) at 0.degree. 
C. for 10 minutes and at room temperature for another 15 minutes to give 
the first reaction mixture. This reaction mixture was further treated with 
benzyl 2,6-dimethyl-4-hydroxybenzoate, see U.S. Pat. No. 4,745,181, (694 
mg, 2.71 mmol) to give the second reaction mixture, which was stirred at 
room temperature under nitrogen for 40 hours, and then evaporated under 
reduced pressure to dryness. The residue was flash-chromatographed on a 
silica column packed with hexane and eluted with 50% ether-hexane to give 
orange-red (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
benz[b]acridine-12-carboxylate (450 mg), 74%). Rf 0.6 (silica gel, 20% 
ethyl acetate/toluene). MS (FAB, Thioglycerol Matrix): m/z 512 (M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (LEAE-Bz) 
To a solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
benz[b]acridine-9-carboxylate (115 mg, 0.23 mmol) in anhydrous methylene 
chloride (5 ml) was added methyl fluorosulfonate (0,128 ml, 2.25 mmol). 
The solution was stirred at room temperature under nitrogen for 20 hours, 
and then treated with anhydrous ether (10 ml). The resulting precipitate 
was collected and washed with ether (100 ml), giving dark-brown 
(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-methyl-benz[b]acridinium-12-carboxylate fluorosulfonate (136 mg, 97%). 
MS (FAB, Thioglycerol Matrix): m/z 526 (M). 
EXAMPLE 2 
Preparation of (2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (LEAE-NHS) 
(4-Carboxy-2,6-dimethyl)phenyl Benz[b]acridine-12-carboxylate Hydrobromide 
A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
benz[b]acridine-12-carboxylate (198 mg, 0.39 mmol), prepared in Example 1, 
in 30% hydrogen bromide-acetic acid (5 ml) was stirred at 
55.degree.-60.degree. C. under nitrogen for 4 hours. The mixture was 
treated with anhydrous ether (20 ml); the precipitate was collected and 
washed with ether (100 ml) to give (4-carboxy-2,6-dimethyl)phenyl 
benz[b]acridine-12-carboxylate hydrobromide quantitatively. Rf 0.4 (silica 
gel, 5% methanol/chloroform). MS (EI): m/z 421 (M). 
(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
Benz[b]acridine-12-carboxylate 
To a solution of (4-carboxy-2,6-dimethyl)phenyl 
benz[b]acridine-12-carboxylate hydrobromide (108 mg, 0.22 mmol) in 
anhydrous N,N-dimethylformamide (5 ml) was added at 0.degree. C. 
dicyclohexylcarbodiimide (111 mg, 0.54 mmol). After stirring at this 
temperature for 30 minutes, N-hydroxysuccinimide (62 mg, 0.54 mmol) was 
added. The solution was stirred under nitrogen at 0.degree. C. for 10 
minutes and then at room temperature for 24 hours. The resulting mixture 
was treated with acetic acid (3 drops) and evaporated to dryness under 
reduced pressure. The residue was extracted with chloroform and the 
chloroform extract was evaporated under reduced pressure to dryness. The 
residue was flash-chromatographed on a silica column packed and eluted 
with ether to give (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
benz[b]acridine-12-carboxylate (20 mg, 18%). Rf 0.6 (silica gel, 20%). MS 
(El): m/z 518 (M). 
(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (LEAE-NHS) 
To a solution of (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
benz[b]acridine-12carboxylate (14 mg, 0.026 mmol) in anhydrous methylene 
chloride (1 ml) was added methyl fluorosulfonate (0.021 ml, 0.26 mmol). 
The resulting brown solution was stirred at room temperature under 
nitrogen for 20 hours, and then treated with anhydrous ether (1 ml). The 
precipitate was collected and washed with ether (40 ml), yielding 
(2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
5-methylbenz[b]acridinium-12-carboxylate-fluorosulfonate (11 mg, 65%). MS 
(FAB, Thioglycerol Matrix): m/z 533 (M) 
EXAMPLE 3 
Preparation of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (DIP-LEAE-Bz) 
3,5-Diisopropyl-4-hydroxybenzoic acid 
This acid was prepared according to the procedure of W. H. Meek et al. J. 
Chemical and Engineering Data, 14(3), 388, (1969). To a solution of 
2,6-diisopropylphenol (Aldrich cat. #D12660-8) (37.0 ml, 0.20 mol) in 
anhydrous N,N-dimethylacetamide (150 ml) was added sodium methoxide (16.2 
g, 0.30 mol). Carbon dioxide was passed through the mixture throughout the 
subsequent reaction period. The mixture was heated with stirring and the 
solvent was slowly distilled out during 2 hours of the period until the 
pot temperature reached to 180.degree. C.. The mixture was allowed to 
continue stirring at 180.degree. C. for another 1.5 hours and then cooled 
to 90.degree. C. The flow of carbon dioxide was discontinued and water 
(400 ml) was added. The mixture, after further cooled to room temperature, 
was washed with toluene (4.times.60 ml) and then treated with conc. 
hydrochloric acid in an ice-water bath to pH 3. The resulting mixture was 
extracted with diethyl ether (2.times.150 ml); the ether extract was 
washed with brine (100 ml) and dried over anhydrous magnesium sulfate. 
Removal of the solvent under reduced pressure gave 
3,5-diisopropyl-4-hydroxybenzoic acid (10.4 g, 23%). Rf 0.5 (silica gel, 
50% diethyl ether/hexane). MS (CI, CH.sub.4): m/z 223 (M+1). 
Benzyl 3,5-Diisopropyl-4-hydroxybenzoate 
To a solution of 3,5-diisopropyl-4-hydroxybenzoic acid (1,223 g, 5.50 mmol) 
in methanol (25 ml) was added potassium hydroxide (308 mg, 5.50 mmol) in 
water (5 ml). The resulting solution was stirred at room temperature under 
nitrogen for 1 hour, and then evaporated completely to dryness under 
reduced pressure. This potassium salt was dissolved in anhydrous 
acetonitrile (30 ml) and N,N-dimethylformamide (15 ml), and treated with 
dibenzo-18-crown-6 (198 mg, 0.55 mmol). After 30 minutes of stirring at 
80.degree. C. under nitrogen, the solution was further treated with benzyl 
bromide (0,712 ml, 6.05 mmol). The stirring was continued at 80.degree. C. 
under nitrogen for 4 hours. The resulting mixture, after cooling, was 
filtrated. The filtrate was evaporated under reduced pressure to dryness. 
The residue was flash-chromatographed on a silica column packed with 
hexane and eluted with 20% ethyl acetate/hexane, yielding crystalline 
benzyl, 3,5-diisopropyl-4-hydroxybenzoate (1.35 g, 79%). Rf 0.6 (silica 
gel, 20% ethyl acetate/toluene). MS(EI). m/z 312 (M). 
(4-Benzyloxycarbonyl-2,6-diisopropyl]phenyl Benz[b]acridine-12-carboxylate 
A suspension of benz[b]acridine-12-carboxylic acid hydrochloride from 
Example 1 (200 mg, 0.65 mmol) in anhydrous pyridine (30 ml) was warmed at 
60.degree. C. for 5 minutes. The slightly cloudy solution was then cooled 
to 0.degree. C. and treated with p-toluenesulfonyl chloride (247 mg, 1.29 
mmol). The solution was stirred at 0.degree. C. for 40 minutes and room 
temperature for another 10 minutes, and benzyl 2,6 
-diisopropyl-4-hydroxybenzoate (202 mg, 0.65 mmol) was added. This 
reaction mixture was allowed to continue stirring at room temperature 
under nitrogen for 20 hours, and then evaporated under reduced pressure to 
dryness. The residue was flash-chromatographed on a silica column packed 
with hexane and eluted with 25% ether-hexane to give orange 
(4-benzyloxycarbonyl-2,6-diisopropyl)phenyl benz[b]acridine-12-carboxylate 
(187 mg, 51%). Rf 0.6 (silica gel, 20% ethyl acetate/toluene). MS(CI, 
CH4): m/z 570 (M+3). 
(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate (DIP-LEAE-Bz) 
To a solution of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 
benz[b]acridine-12-carboxylate (50 mg, 0.088 mmol) in anhydrous methylene 
chloride (3 ml) was added methyl fluorosulfonate (0.072 ml, 0.088 mmol). 
The brown solution was stirred at room temperature under nitrogen for 24 
hours, and then treated with anhydrous ether (4 ml). The resulting 
precipitate was collected and washed with ether (10 ml), giving dark-brown 
(4-benzyloxycarbonyl-2,6-diisopropyl)phenyl 
5-Methyl-benz[b]acridinium-12-carboxylate fluorosulfonate (39 mg, 64%). MS 
(FAB, Thioglycerol Matrix): m/z 582 (M). 
EXAMPLE 4 
Preparation of 
N-(4-Methoxyphenyl-N-[3-(benzyloxycarbonyl)phenylsulfonyl]5-Methyl-benz[b] 
acridinium-12-carboxamide Fluorosulfonate (LEAC-Bz) 
3-[N-(4-Methoxyphenyl)sulfamido]benzoic acid 
To a solution of 3-(chlorosulfonyl)benzoic acid (Kodak cat. #1188655) (4.4 
g, 20.00 mmol) and triethylamine (2.78 ml, 20.00 mmol) in anhydrous 
methylene chloride (40 ml) was added at 0.degree. C. 4-anisidine (2.46 g, 
20.00 mmol). After 10 minutes of stirring at 0.degree. C., a large 
quantity of precipitate formed from the solution. The mixture was allowed 
to continue stirring at room temperature under nitrogen for 2 hours. After 
filtration, the collected off-white solid was washed with water (50 ml) 
and then with ether (50 ml), giving 
3-[N-(4-methoxyphenyl)sulfamido]benzoic acid (4.50 g, 74%). Rf 0.5 (silica 
gel, chloroform/methanol/water 65:25:4). MS(CI, CH4): m/z 308 (M+1). 
Benzyl 3-[N-(4-Methoxyphenyl)sulfamido]benzoate 
To a solution of 3-[N-(4-methoxyphenyl)sulfamido]benzoic acid (2.00 g, 6.52 
mmol) in N,N-dimethylformamide (20 ml) was added a solution of sodium 
hydroxide (260.6 mg, 6.52 mmol) in water (5 ml). The resulting solution 
was stirred at room temperature under nitrogen for 1 hour, and then 
evaporated completely to dryness under reduced pressure. This sodium salt 
was dissolved in anhydrous acetonitrile (40 ml) and N,N-dimethylformamide 
(20 mL), and treated with dibenzo-18-crown-6 (235 mg, 0.65 mmol). After 30 
minutes of stirring at 80.degree. C. under nitrogen, the solution was 
further treated with benzyl bromide (0.852 ml, 7.27 mmol). The stirring 
was continued at 80.degree. C. under nitrogen for 4 hours. The resulting 
mixture, after cooling, was filtrated. The white solid was washed with 
small amount of acetonitrile. The combined acetonitrile filtrate was 
evaporated under reduced pressure to dryness. The reside was 
flash-chromatographed on a silica column packed with hexane and eluted 
with 15% ethyl acetate/hexane, yielding crystalline benzyl 
3-[N-(4-methoxyphenyl)sulfamido]benzoate (2.00 g, 77%). Rf 0.5 (silica 
gel, 20% ethyl acetate/toluene). MS(CI, CH4): m/z 397 (M+1). 
N-(4-Methoxyphenyl)-N-[3-(benzyloxycarbonyl)phenylsulfonyl]Benz[b]acridine- 
12-carboxamide 
A suspension of benz[b]acridine-12-carboxylic acid hydrochloride (200 mg, 
0.65 mmol) in anhydrous pyridine (30 ml) was warmed at 60.degree. C. for 5 
minutes. The slightly cloudy solution was then cooled to 0.degree. C., and 
treated with p-toluenesulfonyl chloride (247 mg, 1.29 mmol). The solution 
was stirred at 0.degree. C. for 40 minutes and room temperature for 
another 10 minutes, and benzyl 3-[N-(4-methoxyphenyl)sulfamido]benzoate 
(257 mg, 0.65 mmol) was added. This reaction mixture was allowed to 
continue stirring at room temperature under nitrogen for 20 hours, and 
then was evaporated under reduced pressure to dryness. The residue was 
flash-chromatographed on a silica column packed with hexane and eluted 
with 25% hexane-ether to give orange 
N-(4-methoxyphenyl)-N-[3-(benzyloxycarbonyl)phenylsulfonyl]benz[b]acridine 
-12carboxamide (177 mg, 68%). Rf 0.4 (silica gel, 20% ethyl 
acetate/toluene). MS(CI, CH4): m/z 653 (M+1). 
N-(4-Methoxyphenyl)-N-[3-(benzyloxycarbonyl)phenylsulfonyl]5-Methyl-benz[b] 
acridinium-12-carboxamide Fluorosulfonate (LEAC-Bz) 
To a solution of 
N-(4-methoxyphenyl)-N-[3-(benzyloxycarbonyl)phenylsulfonyl]benz[b]acridini 
um-12-carboxamide (50 mg, 0.077 mmol) in anhydrous methylene chloride (3 
ml) was added methyl fluorosulfonate (0.062 ml, 0.77 mmol). The dark-brown 
solution was stirred at room temperature under nitrogen for 20 hours, and 
treated with anhydrous ether (10 ml). The resulting precipitate was 
collected and washed with ether (10 ml), giving blue-black 
N-(4-methoxyphenyl)-N-[3-(benzyloxycarbonyl)phenylsulfonyl]5-methyl-benz[b 
]acridinium-12-carboxamide fluorosulfonate (38 mg, 65%). MS (FAB, 
Thioglycerol Matrix): m/z 667 (M). 
EXAMPLE 5 
Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-Ethoxy-5-methyl-benz[b]acridinium-12 -carboxylate Fluorosulfonate 
(3-EtO-LEAE-Bz) 
Benzo[5,6]isatin 
Benzo[5,6]isatin was prepared according to the procedure of A. Etienne and 
A. Staehelin, Bull. Soc. Chim. France., 6, 743, (1954). 
3-Hydroxy-benz[b]acridine-12-carboxylic Acid 
A mixture of benzo[5,6]isatin (500 mg, 2.54 mmol) and potassium hydroxide 
(996 mg, 17.78 mmol) in water (2 ml) and n-butanol (2 ml) was heated with 
stirring to 100.degree. C. to give a homogeneous solution, followed by 
addition of resorcinol (1.95 g, 17.78 mmol). After the solution was 
further heated to 140.degree. C. and the solvents were slowly blown away 
with nitrogen during a 30-minute period, another 2 ml of water and 1 ml of 
n-butanol were added. The temperature of the solution was maintained at 
140.degree. C. while blowing of nitrogen continued for a total of 2 hours. 
The gummy mixture was cooled and dissolved in 100 ml of water; the 
solution was acidified with concentrated hydrochloric acid in an ice-water 
bath to pH 2. The resulting precipitate was collected, washed with water, 
and flash-chromatographed on a silica column packed with chloroform and 
eluted with 20% methanol-chloroform followed by chloroform-methanol-water 
(65:25:4) to give 3-hydroxy-benz[b]acridine-12-carboxylic acid (210 mg, 
29%). Rf 0.3 (silica gel, chloroform/methanol/water 65:25:4). MS (FAB, 
Thioglycerol Matrix): m/z 290 (M+1). 
Ethyl 3-Ethoxy-benz[b]acridine-12-carboxylate 
To a mixture of 3-hydroxy-benz[b]acridine-12-carboxylic acid (90 mg, 0.28 
mmol) and cesium carbonate (451 mg, 1.38 mmol) in methyl sulfoxide (3.5 
ml) was added bromoethane (207 ul, 2.77 mmol). The mixture was stirred 
under nitrogen at 25.degree. C. for 4 hours, and treated with water (10 
ml). The mixture was adjusted to pH 5 with 5% HCl; the resulting 
precipitate was collected and washed with water. The crude product was 
purified on a silica column packed with chloroform and eluted with 5% 
methanol-chloroform to give ethyl 
3-ethoxy-benz[b]acridinium-12-carboxylate (36 mg, 38%). Rf 0.5 (silica 
gel, diethyl ether/hexane 3:1). MS (FAB, Thioglycerol Matrix): m/z 346 
(M+1). 
3-Ethoxy-benz[b]acridinium-12-carboxylic Acid Hydrochloride 
A solution of ethyl 3-ethoxy-benz[b]acridinium-12-carboxylate (35 mg, 0.10 
mmol) in 16% sodium hydroxide (1 ml) and methanol (3 ml) was stirred under 
nitrogen at 25.degree. C. for 2 days and at 40.degree. C. for additional 4 
hours, and then evaporated to dryness under reduced pressure. The residue 
was suspended in water (20 ml) and adjusted to pH 3 in an ice-water bath 
with concentrated HCl. The precipitate was collected and washed with water 
to give 3-ethoxy-benz[b]acridine-12-carboxylic acid hydrochloride (30 mg, 
3%). Rf 0.8 (silica gel, chloroform/methanol/water 65:25:4). MS (CI, CH4): 
m/z 318 (M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-Ethoxy-benz[b]acridine-12-carboxylate 
A slightly-cloudy solution of 3-ethoxy-benz[b]acridine-12-carboxylic acid 
hydrochloride (19 mg, 0.054 mmol) in pyridine (1 ml) and 
N,N'-dimethylpropyleneurea (DMPU, 1.5 ml) was cooled at 0.degree. C. and 
p-toluenesulfonyl chloride (21 mg, 0.11 mmol) was added. After 20 minutes 
of stirring, benzyl 3,5-dimethyl-4-hydroxybenzoate (14 mg, 0.055 mmol) and 
N,N'-dimethylaminopyridine (1 mg) were added. The mixture was stirred at 
25.degree. C. under nitrogen for 20 hours and then evaporated under 
reduced pressure to dryness. The residue was treated with water (5 ml) and 
extracted with ether (5.times. 5 ml). The combined ether layer was washed 
with water (1.times.10 ml), brine (1.times.10 ml) and dried over magnesium 
sulfate. Removal of the solvent under reduced pressure gave a crude 
mixture, which was separated on a preparative-TLC plate (2 mm silica gel, 
EM Art. 5717) by developing with ether/hexane (3:2). The major orange band 
was collected and extracted with 10% methanol/ether. Removal of the 
solvents under reduced pressure gave orange 
(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-ethoxy-benz[b]acridine-12-carboxylate (5 mg, 17%). Rf 0.6 (silica gel, 
diethyl ether/hexane). MS (FAB, Thioglycerol Matrix): m/z 556 (M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-Ethoxy-5-methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate 
(3-EtO-LEAE-Bz) 
To a solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-ethoxy-benz[b]acridine-12-carboxylate (9 mg, 0.0162 mmol) in methylene 
chloride (1 ml) was added methyl fluorosulfonate (13.1, 0.162 mmol). The 
solution was stirred at 25.degree. C. under nitrogen for 36 hours and then 
treated with diethyl ether (10 ml). The precipitate was collected and 
washed with diethyl ether (20 ml), giving 
(4-benzyloxycarbonyl-2,6-dimethyl)phenyl-3-ethoxy-5-methylbenz[b]acridiniu 
m-12-carboxylate fluorosulfonate (3-EtO-LEAE-Bz, 7 mg, 65%) MS (CI, CH4): 
m/z 570 (M+2). 
EXAMPLE 6 
Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 3- (N, 
N-Diethyl-N-methyl-ammonium) 
ethoxy-5-methyl-benz[b]acridinium-12-carboxylate Difluorosulfonate 
(3-QAE-LEAE-Bz ) 
N,N-Diethylaminoethyl 
3-(N,N-Diethylamino)ethoxy-benz[b]acridine-12-carboxylate 
A solution of 3-hydroxy-benz[b]acridine-12-carboxylic acid (415 mg, 1.28 
mmol) in methyl sulfoxide (12 ml) was treated with cesium carbonate (5 g, 
15.4 mmol) at 25.degree. C. for 10 minutes and diethylamminoethyl bromide 
hydrobromide (1.5 g, 6.4 mmol) was added. The mixture was stirred at 
25.degree. C. under nitrogen for 4 hours and then quenched with water (100 
ml). The precipitate was collected and washed with water (50 ml). The 
crude product was separated on 4 preparative-TLC plates (2mm silica gel) 
by developing with 20% methanol/chloroform. The orange band was collected 
and extracted with 10% methanol/chloroform. Removal of the solvents under 
reduced pressure gave N,N-diethylaminoethyl 
3-(N,N-diethylamino)ethoxy-benz[b]acridine-12-carboxylate (73 mg, 12%). Rf 
0.6 (silica gel, 20% methanol/chloroform). MS (CI, CH4): m/z 488 (M+1). 
3-(N,N-Diethylamino)ethoxy-benz[b]acridine-12-carboxylic acid hydrochloride 
A solution of N,N-diethylaminoethyl 
3-(N,N-diethylamino)ethoxy-benz[b]acridine-12-carboxylate (60 mg, 0.123 
mmol) in 4 N sodium hydroxide (4 ml) and methanol (12 ml) was stirred at 
65.degree. C. under nitrogen for 16 hours, and then evaporated under 
reduced pressure to dryness. The residue was dissolved in water (10 ml); 
the solution was carefully acidified in an ice-water bath with 
concentrated HCl to pH 4. The resulting precipitate was collected and 
washed with diethyl ether (5 ml) to give 
3-(N,N-diethylamino)ethoxy-benz[b]acridine-12-carboxylic acid 
hydrochloride (32 mg, 61%). Rf 0.3 (silica gel, chloroform/methanol/water 
65:25:4). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-(N,N-Diethylamino)ethoxy-benz[b]acridine-12-carboxylate 
A mixture of 3-(N,N-diethylamino)ethoxybenz[b]acridine-12-carboxylic acid 
hydrochloride (29 mg, 0.069 mmol) and p-toluenesulfonyl chloride (29 mg, 
0.152 mmol) in pyridine (12 ml) was stirred at 80.degree. C. for 5 
minutes. The resulting homogeneous solution was cooled to 25.degree. C. 
and further treated with benzyl 4-hydroxy-3,5-dimethylbenzoate (20 mg, 
0.078 mmol). After stirring at 25.degree. C. under nitrogen for 16 hours, 
the solvent was removed under reduced pressure. The residue was purified 
on a preparative-TLC plate (2mm silica gel) developed with 15% 
methanol/chloroform. The orange band was collected and extracted with 10% 
methanol/chloroform. Evaporation of the solvents under reduced pressure 
gave (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-(N,N-diethylamino)ethoxy-benz[b]acridine-12-carboxylate (17 mg, 39%). Rf 
0.6 (silica gel, 10% methanol/chloroform). MS (FAB, Glycerol Matrix): m/z 
627 (M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 3-(N,N,Diethyl-N-methyl-ammonium) 
ethoxy-5-methyl-benz[b]acridinium-12-carboxylate Difluorosulfonate 
(3-QAE-LEAE-Bz) 
A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-(N,N-diethylamino)ethoxy-benz[b]acridine-12-carboxylate (13 mg, 0.0208 
mmol) in methylene chloride (1.9 ml) was treated with methyl 
fluorosulfonate (25 ul, 0.308 mmol). After 15 hours of stirring under 
nitrogen at 25.degree. C. the reaction mixture was slowly added to diethyl 
ether (5 ml). The precipitate was collected and washed with ether (10 ml) 
to give (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-(N,N-diethyl-N-methyl-ammonium)ethoxy-5-methylbenz[b]acridinium-12-carbo 
xylate difluorosulfonate (3-QAE-LEAE-Bz, 9 mg, 53%). MS (FAB, Glycerol 
Matrix): m/z 659 (M+3). 
EXAMPLE 7 
Preparation of (4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-Methoxy-5-methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate 
(2-MeO-LEAE-Bz) 
3-(4-Methoxy)anilino-2-naphthoic Acid 
A mixture of p-anisidine (84.7 g, 687.7 mmol) and 3-hydroxy-2-naphthoic 
acid (64.7 g, 343.9 mmol) was mechanically stirred at 160.degree. C. under 
nitrogen for 22 hours. After cooling to 130.degree. C., the mixture was 
treated with hot 1 N hydrochloric acid (1000 ml), stirred at 130.degree. 
C. for 10 minutes, and filtrated, when hot. The resulting cake was stirred 
with hot 0.5 N sodium carbonate (2200 ml) for 15 minutes and filtrated 
when hot. The filtrate was cooled and acidified to pH 6.5 with 
concentrated HCl in an ice-water bath. The precipitate was collected and 
washed with methanol (150 ml), yielding 3-(4-methoxy) anilino-2-naphthoic 
acid (14.8 g, 15%). Rf 0.6 (silica gel, 10% methanol/chloroform). MS (CI, 
CH.sub.4): m/z 294 (M+1). 
12-Chloro-2-methoxy-benz[b]acridine 
A mixture of 3-(4-methoxy)anilino-2-naphthoic acid (15.4 g, 49.44 mmol) and 
phosphorousoxy chloride (46 ml, 494.4 mmol) was refluxed at 120.degree. C. 
under nitrogen for 3.5 hours, and then evaporated under reduced pressure 
to dryness. The residue was taken into a mixed solvent containing 
chloroform (500 ml)/ice (450 g)/ammonium hydroxide (450 ml). The resulting 
two layers were separated. The aqueous layer was extracted with chloroform 
(3.times.250 ml). The combined chloroform layer was dried over calcium 
chloride and evaporated to dryness under reduced pressure, yielding 
12-chloro-2-methoxy-benz[b]acridine (12.5 g, 86%). Rf 0.8 (silica gel, 60% 
diethyl ether/hexane). MS (CI, CH4): m/z 294 (M+1). 
12-Cyano-2-methoxy-benz[b]acridine 
A mixture of 12-chloro-2-methoxy-benz[b]acridine (562 mg, 1.905 mmol), 
potassium cyanide (136 mg, 2.096 mmol) and copper(I) cyanide (86 mg, 0.953 
mmol) in methanol (3.7 ml) was stirred at 170.degree. C. in a sealed-tube 
for 4.5 hours. The resulting mixture was filtrated, and the solid was 
washed with chloroform/methanol (2:1, 10 ml). The combined filtrate was 
evaporated under reduced pressure to give a residue, which was 
flash-chromatographed on a silica column packed with chloroform and eluted 
with 1% methanol/chloroform to yield 12-cyano-2-methoxybenz[b]acridine 
(477 mg, 88%). Rf 0.6 (silica gel, 1% methanol/chloroform). MS (CI, 
CH.sub.4): m/z 285 (M+1). 
2-Hydroxy-benz[b]acridine-12-carboxylic acid hydrosulfate 
A mixture of 12-cyano-2-methoxy-benz[b]acridine (8.3 g, 29.1 mmol) and 50% 
sulfuric acid (v/v, 280 ml) was mechanically stirred under nitrogen at 
160.degree. C. for 48 hours. The resulting mixture was cooled and poured 
into ice-water (1800 ml). The precipitate was collected, washed with water 
(200 ml), and flash-chromatographed on a silica column packed with 
chloroform and eluted with 20% methanol/chloroform followed by 
chloroform/methanol/water (65:25:4) to give 
2-hydroxy-benz[b]acridine-12-carboxylic acid hydrosulfate (4.8 g, 43%). Rf 
0.4 (silica gel, chloroform/methanol/water 65:25:4). 
Methyl 2-Methoxy-benz[b]acridine-12-carboxylate 
To a solution of 2-hydroxy-benz[b]acridine-12-carboxylic acid hydrochloride 
(186 mg, 0.572 mmol) in methyl sulfoxide (4 ml) were added cesium 
carbonate (746 mg, 2.29 mmol) and iodomethane (143 ul, 2.29 mmol). The 
resulting mixture was stirred at 25.degree. C. under nitrogen for 4 hours 
and then treated with water (50 ml). The mixture was acidified in an 
ice-water bath with concentrated HCl to pH 6. The resulting precipitate 
was collected, washed with water (5 ml) and air-dried. The crude mixture 
was purified on 4 preparative-TLC plates (2 mm silica gel) developed with 
diethyl ether/hexane (5:1); the major orange band was collected and 
extracted with 10% methanol/chloroform. Removal of the solvents under 
reduced pressure gave methyl 2-methoxy-benz[b]acridine-12-carboxylate (35 
mg, 17%). Rf 0.8 (silica gel, diethyl ether/hexane 5:1). 
2-Methoxy-benz[b]acridine-12-carboxylic Acid 
A solution of methyl 2-methoxy-benz[b]acridine-12-carboxylate (35 mg, 0.10 
mmol) in 4N sodium hydroxide (3 ml) and methanol (9 ml) was stirred at 
65.degree. C. for 15 hours. The resulting mixture was evaporated under 
reduced pressure to dryness. The residue was dissolved in water (40 ml); 
the aqueous solution was acidified to pH 5 with concentrated HCl in an 
ice-water bath. The precipitate was collected and washed with water (5 
ml), yielding 2-methoxybenz[b]acridine-12-carboxylic acid (20 mg, 59%). Rf 
0.5 (silica gel, chloroform/methanol/water 65:25:4). MS (CI, CH4: m/z 304 
(M+1). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-Methoxy-benz[b]acridine-12-carboxylate 
A solution of 2-methoxy-benz[b]acridine-12-carboxylic acid (18 mg, 0.0529 
mmol) in pyridine (5 ml) was treated with p-toluenesulfonyl chloride (20 
mg, 0.106 mmol) at 25.degree. C. for 15 minutes and then benzyl 
3,5-dimethyl-4-hydroxybenzoate (27 mg, 0,106 mmol) was added. After 15 
hours of stirring at 25.degree. C. under nitrogen, the reaction mixture 
was evaporated under reduced pressure to remove the pyridine. The residue 
was purified on a preparative-TLC (2 mm silica gel) developed with diethyl 
ether/hexane (3:2). The orange band was collected and extracted with 5% 
methanol/chloroform. Removal of the solvents under reduced pressure gave 
(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-methoxy-benz[b]acridine-12-carboxylate (2.7 mg, 9%). Rf 0.5 (silica gel, 
60% diethyl ether/hexane). 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-Methoxy-5-methyl-benz[b]acridinium-12-carboxylate Fluorosulfonate 
(2-MeO-LEAE-Bz) 
A solution of 4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-methoxy-benz[b]acridine-12-carboxylate (2.5 mg, 0.0046 mmol) in 
methylene chloride (1 ml) was treated with methyl fluorosulfonate (3.7 ul, 
0.56 mmol) at 25.degree. C. with stirring under nitrogen for 15 hours. The 
reaction mixture was added to anhydrous diethyl ether (4 ml). The 
resulting precipitate was collected and washed with diethyl ether (5 ml) 
to afford (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
2-methoxy-5-methyl-benz[b]acridinium-12-carboxylate fluorosulfonate 
(2-MeO-LEAE-Bz) (1.5 mg, 49%). MS (FAB, Thioglycerol Matrix): m/z 556(M). 
EXAMPLE 8 
Preparation of (2,6-Dimethyl-4succinimidyloxycarbonyl)phenyl 
5-Methyl-2-(trimethylammonium)ethoxy-benz[b]acridinium-12-carboxylate 
Difluorosulfonate (2-QAE-LEAE-NHS) 
N,N-Dimethylaminoethyl 
2-(N,N-Dimethylamino)ethoxybenz[b]acridine-12-carboxylate 
To a solution of 2-hydroxy-benz[b]acridine-12-carboxylic acid hydrochloride 
(360 mg, 1,108 mmol) in methyl sulfoxide (11 ml) were added cesium 
carbonate (3.61 g, 11.08 mmol) and N,N-dimethylaminoethyl bromide 
hydrobromide (1.03 g, 4.432 mmol). After 15 hours of stirring at 
60.degree. C. under nitrogen, the reaction mixture was diluted with methyl 
sulfoxide (20 ml) and filtered to remove the insoluble impurities. The 
filtrate was concentrated under reduced pressure to a small volume, which 
was separated on a preparative-TLC plate (2 mm silica gel) by developing 
with chloroform/methanol/water (47:48:5). The desired orange band was 
collected and extracted with 25% methanol/chloroform. Removal of the 
solvents under reduced pressure gave N,N-dimethylaminoethyl 
2-(N,N-dimethylamino)ethoxy-benz[b]acridine-12-carboxylate (86 mg, 18%). 
Rf 0.6 (silica gel, chloroform/methanol/water 65:25:4). MS (FAB, Glycerol 
Matrix): m/z 432 (M+1). 
2-(N,N-Dimethylamino)ethoxy-benz[b]acridine-12-carboxylic acid 
A solution of N,N-dimethylaminoethyl 
2-(N,N-dimethylamino)ethoxy-benz[b]acridine-12-carboxylate (86 mg, 0.20 
mmol ) in 4 N sodium hydroxide (7.3 ml) and methanol (22 ml) was stirred 
at 65.degree. C. for 1 hour and at 35.degree. C. for 15 hours. The 
reaction mixture was evaporated under reduced pressure to dryness. The 
residue was washed with water (5 ml) and air-dried, yielding 
2-(N,N-dimethylamino)ethoxybenz[b]acridine-12-carboxylic acid (23 mg, 
32%). Rf 0.3 (silica gel, chloroform/methanol/water 65:25:4). MS (FAB, 
Glycerol Matrix): m/z 361 (M+1). 
Succinimidyl 3,5-Dimethyl-4-hydroxy-benzoate 
A solution of 3,5-dimethyl-4-hydroxybenzoic acid (5.0 g, 30.0 mmol) in 
N,N-dimethylformamide (150 ml) was cooled to 0.degree. C. and treated with 
N-hydroxysuccinimide (3.45 g, 30.0 mmol) and 1,3-dicyclohexylcarbodiimide 
(6.81 g, 33.0 mmol). The solution was stirred under nitrogen at 0.degree. 
C. for 2 hours and then at 25.degree. C. for 16 hours. The resulting 
mixture was stirred with 0.5 ml of acetic acid for 15 minutes, and then 
filtered to remove the insoluble urea. The filtrate was evaporated under 
reduced pressure to dryness. The dried material was washed with diethyl 
ether (100 ml) and suspended in boiling ethyl acetate (200 ml). The 
suspension, when hot, was filtered to remove insoluble impurities. The 
filtrate was concentrated and suspended in hot ethyl acetate/methylene 
chloride (1:1, 200 ml) and cooled to give an off-white powder in 2.91 g 
(37%). Rf 0.6 (silica gel, diethyl ether). 
(2,6-Dimethyl-4-succinimidyloxycarbonyl)phenyl 
2-N,N-Dimethylamino)ethoxy-benz[b]acridine-12-carboxylate 
A solution of 2-(N,N-dimethylamino)ethoxy-benz[b]acridine-12-carboxylic 
acid (30 mg, 0.0833 mmol) in pyridine (4 ml) was treated with 
p-toluenesulfonyl chloride (31.8 mg, 0,166 mmol) at 25.degree. C. for 10 
minutes, followed by addition of succinimidyl 
3,5-dimethyl-4-hydroxybenzoate (32 mg, 0.0833 mmol). After 15 hours of 
stirring at 25.degree. C. under nitrogen, the solution was diluted with 
chloroform (10 ml), quickly washed with water (3.times.4 ml) and 
evaporated under reduced pressure to dryness. The residue was purified on 
two preparative-TLC plates (1 mm silica gel) developed with 10% 
methanol/chloroform. The desired orange band was collected and extracted 
with 10% methanol/chloroform. Removal of the solvents under reduced 
pressure gave (2,6-dimethyl-4-succinimidyloxycarbonyl)phenyl 
2-(N,N-dimethylamino)ethoxy-benz[b]acridine-12-carboxylate (7 mg, 14%). Rf 
0.8 (silica gel, 10% methanol/chloroform). MS (FAB, Thioglycerol Matrix): 
m/z 606 (M+1). 
(2,6-Dimethyl-4-succinimidyloxycarbonyl)phenyl 
5-Methyl-2-(trimethylammonium)ethoxy-benz[b]acridinium-12-carboxylate 
Difluorosulfonate (2-QAE-LEAE-NHS) 
A solution of 2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
2-(N,N-dimethyl amino)ethoxybenz[b]acridine carboxylate (1.9 mg, 0.0031 
mmol) in anhydrous methylene chloride (4 ml) was treated with methyl 
fluorosulfonate (3.8 ul, 0.0465 mmol). The solution was allowed to stir at 
25.degree. C. under nitrogen for 16 hours. The resulting precipitate was 
collected and washed with diethyl ether (2 ml) to give 
(2,6-dimethyl-4-succinimidyloxycarbonyl)phenyl 
5-methyl-2-(trimethylammonium)ethoxy-benz[b]acridinium-12-carboxylate 
difluorosulfonate (2-QAE-LEAE-NHS) (1.0 mg, 39%). 
EXAMPLE 9 
Preparation of (4-Benzyloxycarbony1-2,6-dimethyl)phenyl 
5-(3-Sulfopropyl)-benz[b]acridinium-12-carboxylate (NSP-LEAE-Bz) 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-(3-Sulfopropyl)-benz[b]acridinium-12-carboxylate (NSP-LEAE-Bz) 
A mixture of (4-benzylcarboxyl-2,6-dimethyl)phenyl 
benz[b]acridine-12-carboxylate from Example 1 (30 mg, 0.0587 mmol) and 
1,3-propane sulton (600 mg, 4.9 mmol) was flushed with nitrogen and kept 
in a sealed tube. The tube was heated with stirring at 180.degree. C. for 
5 hours and then cooled. The resulting mixture was purified by 
reverse-phase preparative-HPLC on a C-18 column (YMC SH-344-15, S-15, 
128), eluted under gradient condition with 25% to 40% acetonitrile in 0.05 
M aqueous trifluoroacetic acid from 0 to 20 minutes, 40% to 90% over 5 
minutes, and maintaining 90% acetonitrile for another 10 minutes. The 
fraction with retention time of 17 minutes was collected and evaporated 
under reduced pressure to give 3.2 mg of the title compound (NSP-LEAE-Bz). 
MS (FAB, Glycerol Matrix): m/z 634 (M+1). 
EXAMPLE 10 
Preparation of (2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
2-Methoxy-5-(2-sulfoethyl)-benz[b]acridinium-12-carboxylate 
(2-MeO-NSE-LEAE-NHS) 
(2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
2-Methoxy-benz[b]acridinium-12-carboxylate 
A mixture of 2-methoxy-benz[b]acridine-12-carboxylic acid from Example 7 
(208 mg, 0.6118 mmol) in pyridine (20 ml) was treated with 
p-toluenesulfonyl chloride (233 mg, 1.2235 mmol) at 25.degree. C. for 10 
minutes, followed by addition of succinimidyl 
3,5-dimethyl-4-hydroxy-benzoate (161 mg, 0.6118 mmol). After 24 hours of 
stirring under nitrogen at 25.degree. C., the pyridine was removed by 
evaporation under reduced pressure. The resulting mixture was 
flash-chromatographed on a silica column packed and eluted with diethyl 
ether. The crude product collected was further purified on a Chromatotron 
plate (1 mm silica gel) by elution with diethyl ether to yield 80 mg (24%) 
of the pure product. Rf 0.8 (silica gel, ethyl acetate/hexane 2:1). MS 
(FAB, Thioglycerol Matrix): 549 (M+1). 
2,6-Dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
2-Methoxy-5-(2-sulfoethyl)-benz[b]acridinium-12-carboxylate 
(2-MeO-NSE-LEAE-NHS ) 
A mixture of (2,6-dimethyl-4-N-succinimidyloxycarbonyl)phenyl 
2-methoxy-benz[b]acridine-12-carboxylate (23 mg, 0.04197 mmol) and 
ethylenesulfonyl chloride (prepared according to the procedure of C. S. 
Rondestredt Jr., J. Amer. Chem. Soc., 76, 1926 (1954)) (378 ul, 4,197 
mmol) was stirred at 25.degree. C. under nitrogen for 63 hours. The 
resulting mixture was purified by reverse-phase preparative-HPLC on a C-18 
column, eluted under gradient condition with 35% acetonitrile in 0.05M 
aqueous trifluoroacetic acid from 0 to 10 minutes, 35% to 80% from 10 to 
30 minutes, 80% to 90% from 30 to 35 minutes, and remaining 90% for 
another 5 minutes. The fraction with retention time of 25 minutes was 
collected and evaporated under reduced pressure to give 2.2 mg (8%) of the 
title compound (2-MeO-NSE-LEAE-NHS). MS (FAB, Thioglycerol Matrix): m/z 
657 (M+1). 
EXAMPLE 11 
Preparation of [2,6-Dimethyl-4-(2-methoxyiminoethyl)]phenyl 
2-Methoxy-5-methylbenz[b]acridinium-12-carboxylate Dichloride 
(2-MeO-LEAE-Imidate) 
(4-Cyanoethyl-2,6-dimethyl)phenyl 2-Methoxy-benz[b]acridine-12-carboxylate 
A suspension of 2-methoxy-benz[b]acridine-12-carboxylic acid from Example 7 
(100 mg, 0.2941 mmol) in pyridine (15 ml) was treated with 
p-toluenesulfonyl chloride (112 mg, 0.5882 mmol) at 0.degree. C. for 40 
minutes, followed by addition of triethylamine (164 ul. 1.1789 mmol) and 
4-cyanoethyl-2,6-dimethyl-phenol (prepared according to the procedure of 
E. Jexova et al, CS 158810, Jul. 15, 1975; CA 84(13):898299)(51 mg, 0.2914 
mmol). After 18 hours of stirring at 25.degree. C. under nitrogen, the 
reaction solution was evaporated under reduced pressure to remove the 
pyridine. The residue was purified on 4 preparative-TLC plates (2 mm 
silica gel) developed twice with ethyl acetate/hexane (3:4). The major 
orange band was collected and extracted with methanol/chloroform (1:30). 
Removal of the solvents under reduced pressure gave the title compound (75 
mg, 55%). Rf 0.5 (silica gel, ethyl acetate/hexane 2:3). MS (FAB, 
Thioglycerol Matrix): m/z 461 (M+ 1). 
(4-Cyanoethyl2,6-dimethyl)phenyl 2-Methoxy-5-methyl 
benz[b]acridinium-12-carboxylate Fluorosulfonate 
A solution of (4-cyanoethyl-2,6-dimethyl)phenyl 
2-methoxy-benz[b]acridine-12-carboxylate (20 mg, 0.04338 mmol) in 
methylene chloride (1 ml) was treated with methyl flurosulfonate (17.5 ul, 
0.2169 mmol) at 25.degree. C. with stirring under nitrogen for 18 hours. 
The reaction mixture was added to anhydrous diethyl ether (5 ml). The 
resulting precipitate was collected and purified by reverse-phase 
preparative-HPLC on a C-18 column, eluted under gradient condition with 
40% to 80% acetonitrile in 0.05M aqueous trifluoroacetic acid from 0 to 30 
minutes, and remaining 80% acetonitrile for another 40 minutes. The 
fraction with retention time of 24 minutes was collected and evaporated 
under reduced pressure to give the product (15.7 mg, 65%). MS (FAB, 
Thioglycerol Matrix): m/z 475 (M). 
[2,6-Dimethyl-4-(2-methoxyiminoethyl)]phenyl 2-methoxy- 5-methyl 
benz[b]acridinium-12-carboxylate Dichloride (2-MeO-LEAE-Imidate) 
A solution of (4-cyanoethyl-2,6-dimethyl)phenyl 2-Methoxy-5-methyl 
benz[b]acridinium-12-carboxylate fluorosulfonate (4 mg, 0.00697 mmol) in 
anhydrous methanol (0.5 ml) was treated with anhydrous hydrogen chloride 
(gas) at 0.degree. C. for 10 minutes. The reaction solution was then 
reduced by blowing with nitrogen to a small volume; and the concentrate 
was added to anhydrous diethyl ether (3 ml). The resulting precipitate was 
collected and washed with diethyl ether (5 ml), yielding the title 
compound (2-MeO-LEAE-Imidate) (1.5 mg, 37%). 
PREATION OF THE ANALOGS OF ACRIDINIUM ESTER 
EXAMPLE 12 
Preparation of (4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 
10-Methyl-acridinium-9-carboxylate Fluorosulfonate (DIPAE-Bz) 
(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl Acridine-9-carboxylate 
A mixture of acridine-9-carboxylic acid hydrochloride (74 mg, 0.33 mmol) in 
thionyl chloride (3 ml, 41.1 mmol) was refluxed at 110.degree. C. under 
nitrogen for 2 hours. After cooling, the solution was evaporated under 
reduced pressure to dryness. The solid was washed with anhydrous diethyl 
ether (5 ml) to give acridine-9-carbonyl chloride hydrochloride. This acid 
chloride was dissolved in anhydrous pyridine (4 ml), followed by addition 
of benzyl 3,5-diisopropyl-4-hydroxy-benzoate (102 mg, 0.33 mmol) and 
4-N,N-dimethylamino-pyridine (16 mg, 0.13 mmol). After 16 hours of 
stirring at 25.degree. C. under nitrogen, the solution was evaporated 
under reduced pressure to dryness. The residue was purified on a 
Chromatotron plate (1 mm silica gel) by elution with 20% diethyl 
ether/hexane to yield (4 -benzyloxycarbonyl-2,6-diisopropyl)phenyl 
acridine-9-carboxylate (64 mg, 38%). Rf 0.6 (silica gel, 20% ethyl 
acetate/toluene). MS(EI): m/z 517(M). 
(4-Benzyloxycarbonyl-2,6-diisopropyl)phenyl 
10-Methyl-acridinium-9-carboxylate Fluorosulfonate (DIPAE-Bz) 
A solution of (4-benzyloxycarbonyl-2,6-diisopropyl)phenyl 
acridine-9-carboxylate (62 mg, 0.120 mmol) in anhydrous methylene chloride 
(3 ml) was treated with methyl fluorosulfonate (97 ul, 1.198 mmol). After 
21 hours of stirring at 25.degree. C. under nitrogen, the solution was 
treated with anhydrous diethyl ether (10 ml). The resulting precipitate 
was collected, washed with diethyl ether (20 ml) and crystallized from 
acetonitrile/diethyl ether to give 
(4-benzyloxycarbonyl-2,6-diisopropyl)phenyl 
10-methylacridinium-9-carboxylate fluorosulfonate (DIPAE-Bz) (20 mg, 26%) 
EXAMPLE 13 
Preparation of (4-Benzyloxycarbony1-2,6-dimethyl)phenyl 
3-Methoxy-10-methyl-acridinium-9-carboxylate Fluorosulfonate 
(3-MeO-DMAE-Bz) 
Methyl 3-Methoxy-acridine-9-carboxylate 
To a solution of 3-hydroxy-acridine-9-carboxylic acid (2 g, 8,368 mmol) in 
methyl sulfoxide (50 ml) was added at 25.degree. C. cesium carbonate (10.9 
g, 33.47 mmol), followed by slow addition of iodomethane (2.08 ml, 33.47 
mmol). After 2 hours of stirring at 25.degree. C. under nitrogen, the 
mixture was poured into water (500 ml). The precipitate was collected, 
washed with water (200 ml) and air-dried. The resulting mixture was 
flash-chromatographed on silica column packed with chloroform and eluted 
with 1% methanol/chloroform, followed by 2% methanol/chloroform, to give 
the crude product. This crude product was further purified on six 
preparative-TLC plates (2 mm silica gel) by elution with 5% 
methanol/chloroform. The major band was collected and extracted with 5% 
methanol/chloroform. Removal of the solvents under reduced pressure gave 
methyl 3-methoxyacridine-9-carboxylate (1.05 g, 47%). Rf 0.7 (silica gel, 
5% methanol/chloroform). 
3-Methoxy-acridine-9-carboxylic Acid Hydrochloride 
A solution of methyl 3-methoxy-acridine-9-carboxylate (900 mg, 3.37 mmol) 
in 4N sodium hydroxide (10 ml) and methanol (30 ml) was stirred at 
65.degree. C. under nitrogen for 14 hours, cooled and evaporated under 
reduced pressure to dryness. The solid was dissolved in water (100 ml); 
the aqueous solution was washed with diethyl ether (4.times.50 ml) and 
acidified in an ice-water bath with concentrated HCl to pH 3. The 
resulting precipitate was collected, washed with water (200 ml) and 
air-dried, to give 3-methoxy-acridine-9-carboxylic acid hydrochloride (710 
mg, 73%). Rf 0.6 (silica gel, chloroform/methanol/water 65:25:4). 
(4-Benzyloxycarbonyl-2,6-dimethylphenyl 3-Methoxyacridine-9-carboxylate 
To a suspension of 3-methoxy-acridine-9-carboxylic acid hydrochloride (150 
mg, 0,519 mmol) in pyridine (25 ml) was added at 0.degree. C. 
p-toluenesulfonyl chloride (198 mg, 1.038 mmol). After stirred for 10 
minutes, the suspension turned homogeneous; and then benzyl 
3,5-dimethyl-4-hydroxybenzoate (132 mg, 0,519 mmol) was added. The 
solution was stirred at 65.degree. C. under nitrogen for 2 hours and at 
25.degree. C. for additional 20 hours, and evaporated under reduced 
pressure to dryness. The residue was suspended in chloroform (100 ml), 
washed with 5% ammonium hydroxide (4.times.50 ml), water (2.times.50 ml), 
brine (1.times.50 ml) and dried over anhydrous magnesium sulfate. Removal 
of the chloroform under reduced pressure gave a crude mixture, which was 
purified on 2 preparative-TLC plates (2 mm silica gel) developed with 
toluene/ethyl acetate (4:1). The major band was collected and extracted 
with 10% methanol/chloroform. Evaporation of the solvents under reduced 
pressure yielded (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-methoxy-acridine-9-carboxylate. Rf 0.7 (silica gel, 20% ethyl 
acetate/toluene). MS: (CI CH.sub.4) m/z 492 (M+1). 
(4-Benzyloxycarbonyl-2,6-dimethylphenyl 
3-Methoxy-10-methyl-acridinium-9-carboxylate Fluorosulfonate 
(3-MeO-DMAE-Bz) 
A solution of (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-methoxy-acridine-9-carboxylate (45 mg, 0.0916 mmol) in anhydrous 
methylene chloride (2 ml) was treated with fluoromethyl sulfonate (74 ul, 
0,916 mmol). After 19 hours of stirring at 25.degree. C. under nitrogen, 
the solution was treated with anhydrous diethyl ether (6 ml). The 
resulting precipitate was collected and washed with diethyl ether (20 ml), 
yielding (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
3-methoxy-10-methyl-acridinium-9-carboxylate fluorosulfonate 
(3-MeO-DMAE-Bz) (41 mg, 74%). MS (FAB, Thioglycerol Matrix): m/z 506 (M) . 
EXAMPLE 14 
SYNTHESIS OF AN ABAC 
The preparations of an angular benz[a]acridinium ester, 
(4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-methylbenz[a]acridinium-12-carboxylate methosulfate and the 
intermediates are given below: 
Benz[a]acridine-12-carboxylic acid 
The procedure is essentially that reported by Martinet, J. and Dansette, A. 
in Bull. Soc. Chim., Fr., 45, 101 (1929) . 
N-Phenyl-.beta.-naphthylamine (22.9 g, 0.1 mol) (Aldrich, Cat#17,805-5) was 
mixed with diethylketomalonate (18 g, 0.1 mol) (Aldrich, Cat#D9,740-1) in 
5 ml of acetic acid and heated in an oil bath at 150.degree. C. for 45 
mins. The reaction mixture solidified upon cooling. The solid was 
transferred to a fritted funnel, washed thoroughly with ethyl alcohol, and 
dried in a desiccator under vacuum to give ethyl 
phenyl-1-benzo-4,5-dioxindol-3-carboxylate: mp 169.degree.-170.degree. C. 
A portion of this first intermediate (7 g, 0.02 mol) was further treated 
with 100 ml of 10% KOH, heated at reflux for 90 minutes and left at room 
temperature overnight. To this second reaction mixture was added 200 ml of 
1 N HCl. The resulting yellow precipitate was filtered, washed with 
boiling ethanol, dried to give 2.1 g (33.8%) of the title compound. MS 
(CI, CH4): m/z 274 (M+1). 
Benz[a]acridine-12-carbonyl chloride 
To benz[a]acridine-12-carboxylic acid (1 g, 3.66 mmol) obtained above was 
added 10 ml of thionyl chloride. The mixture was heated at 95.degree. C. 
for 3 hours, cooled, treated with 50 ml of benzene, and stored at 
4.degree. C. overnight. The precipitates were collected by filtration, 
washed with benzene, then ethyl ether, and dried in a desiccator under 
vacuum to give 300 mg (28%) of the acid chloride. 
(4-Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5-methyl-benz[a]acridinium-12-carboxylate methosulfate 
A solution of 4-benzyloxycarbonyl-2,6-dimethylphenol (0.27 g, 1 mmol) in 10 
ml of dry pyridine was treated with 32 mg, 0.26 mmol of 
4-dimethylaminopyridine (Aldrich, Cat.#10,770-0). To this solution was 
added benz[a]acridine-12-carbonyl chloride prepared above. The solution 
was heated at 100.degree. C. for 3 hours and evaporated to give a residue 
which was purified on 3 preparative TLC plates (EM Cat#5717) developed 
with 5% methanol in toluene/ethyl acetate (4:1) mixture. A fluorescent 
band with Rf slightly below that of the starting phenol was stripped, 
eluted with 5% methanol in chloroform, and the eluent evaporated to give 
433 mg of yellow intermediate, (4-benzyloxycarbonyl-2,6-dimethyl)phenyl 
benz[a]acridine-12-carboxylate. 
This intermediate was dissolved in 20 ml of trichloromethane, treated with 
4 ml of dimethyl sulfate and heated at 85.degree. C. for 48 hours and 
cooled. The yellow precipitate was filtered and washed with ether to give 
222 mg (37%) of the desired product. MS (FAB Thioglycerol Matrix): m/z 526 
(M)). 
PREATION OF CONJUGATES 
In the chemiluminescent compounds of the present invention, preferably at 
the R.sub.6 position, depending on which coupling moiety is selected, the 
AFAC label can be reacted directly with the specific binding partner, 
ligand, or hapten either in an aqueous or an organic medium. 
It is understood that alternate positions of the chemiluminescent compound 
may have a coupling moiety to be reacted with a binding partner to form a 
conjugate. 
The chemiluminescent labels can include an appropriate leaving group or an 
electrophilic functional group attached with a leaving group or functional 
groups which can be readily converted into such reactive groups, directly 
attached or connected via a spacer for attaching a substance to form a 
conjugate to be utilized in a test assay. An example of preparing the 
LEAE-anti-TSH conjugate is provided below. 
Preparation of LEAE-Anti-TSH conjugate 
A solution of a monoclonal anti-TSH antibody (2 mg, 0.013 umol) in 1.36 ml 
of 0.1M phosphate buffer, pH 8.0 was treated with a solution of LEAE-NHS 
(43 ug, 0.067 umole) in 240 ul of acetonitrile at room temperature for one 
hour. The conjugation reaction was stopped by adding a solution of lysine 
(10 mg) in 0.5 ml of 0.1 M phosphate buffer, pH 8. 
The LEAE-conjugated anti-TSH was purified by passing the reaction mixture 
through a Sephadex G-25 column (1.times.20 cm) packed and eluted with 10 
mM Phosphate, pH 8. The elution was monitored at 280 nm with a ISCO UV 
detector. The desired conjugate was collected when the first void volume 
peak was eluted out. 
Preparation of Oligonucleotide conjugate 
A method for conjugating binding parties, haptens, or ligands of 
luminescent labels to polynucleotides is described in EP-A-O 537 994 
(priority U.S. Ser. No. 775,399, filed Oct. 16, 1991), which is commonly 
assigned and incorporated herein by reference. 
LIGHT EMISSION SPECTRA 
The light emission spectra of LBAC's and the reference acridinium esters 
were determined by a Fast Spectral Scanning System (FSSS) of Photo 
Research (a division of Kollmorgen Corp) of Burbank, Calif., U.S.A. The 
experiment was carried out in a dark room. Each sample was dissolved in 
HPLC grade acetonitrile at the concentration of 1 mg/ml or higher and 
diluted with the same solvent to obtain the sample solution in the 
concentration specified. A typical determination utilized 10 to 100 ug of 
each compound, with the exception of the angular benz[a]acridinium ester 
(2 mg), separately or mixed together in 0.5 ml acetonitrile contained in 
13.times.100 mm borosilicate test tube. The tube was placed on a tube rack 
raised to a proper height. The FSSS optical head was placed in front of 
the tube at close distance and with its lense focused on the liquid in the 
tube. The sample solution was first treated with 0.35 ml of the Flashing 
Reagent #1 (Ciba Corning Diagnostics) containing 0.1 N HNO.sub.3 and 0.1% 
H.sub.2 O.sub. 2. The room was then darkened, and 0.35 ml of the Flashing 
Reagent #2 (Ciba Corning Diagnostics) containing 0.25 N NaOH and 0.2% 
ARQUAD was added to the reaction mixture immediately, see U.S. Pat. No. 
4,927,769 which is commonly assigned and incorporated herein by reference. 
The light which was generated instantaneously following the addition of 
the Reagent #2 was recorded by FSSS for 4 seconds except for 
2-MeO-LEAE-Imidate which was recorded for 30 seconds starting from split 
second before the Reagent #2 was added. The results of the various 
determinations are summarized in Table I. 
TABLE I 
______________________________________ 
Emission Max 
Range* 
Compound Quantity .sup..about. (nm) 
(nm) 
______________________________________ 
1. DMAE-Bz 20 ug 426-428 410-510 
2. 3-MeO-DMAE-Bz 50 ug 422 395-520 
3. DIPAE-Bz 20 ug 426 405-520 
4. ABAC 2 mg 436-440 410-530 
5. LEAE-Bz 50 ug 520-524 490-670 
6. DIP-LEAE-Bz 50 ug 520 485-670 
7. 2-MeO-LEAE-Bz 30 ug 550 510-700 
8. 3-EtO-LEAE-Bz 50 ug 508 470-660 
9. 3-QAE-LEAE-Bz 100 ug 544 470-680 
10. 2-QAE-LEAE-NHS 70 ug 550 510-700 
11. LEAC-Bz 50 ug 520 485-670 
12. NSP-LEAE-Bz 15 ug 516 482-655 
13. 2-MeO-NSE- 50 ug 546 500-700 
LEAE-NHS 
14. 2-MeO-LEAE- 100 ug 550 500-710 
Imidate 
______________________________________ 
.sup..about. The emission maximum for each compound could vary by 0-4 nm 
between different determinations. 
*Range is set for spectral region with signal intensity of above 5% of 
peak height. 
The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5methyl-benz[a]acridinium12-carboxylate methosulfate. 
Recorded emission spectra are shown in FIGS. 2A-2E, 3A-3J, and 4A-4D. FIGS. 
2A-2E and 3A-3J show individual emission spectra of chemiluminiscent 
compounds including an acridinium ring system and compounds including a 
benzacridinium ring system. The difference of the emission maxima between 
acridinium esters and LBAC's were found to range between 80-128 nm, while 
that between acridinium esters and the ABAC was about 8-14 nm. As shown in 
FIGS. 4A-4D, when the acridinium esters and LBAC's were mixed in a tube 
and flashed simultaneously, the resulting combined emission spectra showed 
the ideal summed up spectral profile, indicative of the non-interfering 
nature of these two groups of chemiluminescent emission signals. It is 
understood that these data may vary depending on the instrumentation 
utilized and the components of the instrumentation, particularly the 
filters. The major portions of the original constituting spectra which 
remained unchanged were indeed non-overlapping. These important physical 
characteristics fulfill the prerequisite for two or more subclasses of 
chemiluminescent compounds to be utilized in test assays for detecting 
and/or quantitating at least two substances in a test sample, and 
particularly to multianalyte clinical diagnostic assays. In the preferred 
method a benzacridinium compound is utilized as one component of the assay 
method and more specifically an N-alkylated benzacridinium compound. 
As noted above, a luminometer for detecting and/or quantitating at least 
two chemiluminescent emission spectra is described in U.S. Ser. No. 
08/035,341. 
LIGHT EMITTING EFFICIENCY 
The light emitting efficiency of LBAC's, ABAC, and DMAE-Bz was determined 
on a Berthold luminometer (MLA-I) (Ciba Corning Diagnostics Corp.) fitted 
with a BG-38 filter with wavelength transmission range of about 320 to 650 
nm at transmission efficiency of 20 to 97%. (FIG. 5, Panel A). Alternate 
filters may be incorporated in luminometers to expand the range of 
transmission efficiency. 
Each sample was prepared in acetonitrile solution at 1 mg/ml, serially 
diluted to 10 ug/ml in acetonitrile and further on to 1 ng/ml, 0.1 ng/ml 
and 0.01 ng/ml in 10 mM phosphate buffer with 0.15M NaCl, 0.1% BSA. 0.05% 
NaN.sub.3, pH8. 
To determine the light emitting efficiency, 25 ul of blank (the buffer 
matrix) or each sample were flashed by injecting 0.35 ml each of the 
Flashing Reagent #1 and #2 sequentially. Light emission was integrated for 
2 seconds and results as means of duplicate determination are given in 
Table II. 
TABLE II 
__________________________________________________________________________ 
Compound Total Counts (RLU's)/2 sec 
(counter Molecular 
amount flashed (pg) 
RLU's/mol* 
ion) Weight 
0.25 pg 
2.5 pg 
25.0 pg 
(1 .times. E20) 
__________________________________________________________________________ 
DMAE-Bz 587 76,477 
769,477 
6,786,57 
1.8 
(CH.sub.3 SO.sub.4.sup.-) 
DIPAE-Bz 631 82,115 
845,660 
6,041,380 
2.1 
(FSO.sub.3.sup.-) 
3-MeO-DMAE-Bz 
605 -- 54,760 
523,380 
0.13 
(FSO.sub.3.sup.-) 
ABAC 613 -- 23,600 
143,400 
0.058 
(CH.sub.3 SO.sub.4.sup.-) 
LEAE-Bz 625 105,857 
1,037,943 
9,037,063 
2.6 
(FSO.sub.3.sup.-) 
DIP-LEAE-Bz 
681 29,930 
240,610 
2,413,320 
0.66 
(FSO.sub.3.sup.-) 
LEAC-Bz 766 79,873 
767,553 
6,312,163 
2.4 
(FSO.sub.3.sup.-) 
3-EtO-LEAE-Bz 
669 93,635 
883,785 
6,364,935 
2.4 
(FSO.sub.3.sup.-) 
3-QAE-LEAE-Bz 
854 16,765 
107,540 
1,005,855 
0.37 
(FSO.sub.3.sup.-) 
2-MeO-LEAE-Bz 
655 27,810 
223,995 
2,140,890 
0.59 
(FSO.sub.3 .sup.-) 
2-QAE-LEAE-NHS 
854 ND.sup..about. 
ND.sup..about. 
ND.sup..about. 
ND.sup..about. 
(FSO.sub.3.sup.-) 
NSP-LEAE-Bz 
633 22,715 
212,485 
2,250,520 
0.54 
2-MeO-NSE- 
646 14,853 
145,403 
1,503,100 
0.38 
LEAE-NHS 
2-MeO-LEAE- 
578 ND.sup..about. 
ND.sup..about. 
ND.sup..about. 
ND.sup..about. 
Imidate (2 Cl.sup..about.) 
__________________________________________________________________________ 
*counts/mol calculated from quantity of 2.5 pg. 
The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5methyl-benz[a]acridinium12-carboxylate methosulfate. 
.sup..about. ND = not determined prior to the establishment of final 
purity of the compound. 
From the data shown in Table II, the light emitting efficiency of the 
LBAC's was comparable to that of DMAE-Bz within the range of 0.21 to 1.39 
fold, depending on the substitutents on the benzacridinium nucleus and the 
phenoxy group. It should be noted these determinations were based on 
2-second signal collection and have not taken into account the flashing 
kinetics of the individual compounds, e.g. some compounds may take greater 
that 2 seconds to release most of their signals, the sensitivity of the 
photomultiplying tube, and the transmission efficiency of the optical 
filter(s) at different points of the spectral range. These findings, 
however, were totally unexpected in view of the much lower light emitting 
efficiency of the isomeric ABAC. This level of light emitting efficiency 
renders LEAC's useful in sensitive binding assays, including multi-analyte 
assays. 
KINETIC STUDY ON LIGHT EMISSION 
Due to the electronic and/or steric effects of different substituents on 
the phenoxy moiety, the acridinium and benzacridinium nucleus, it was 
anticipated that not all the DMAE analogs, ABAC and LEAC's would have the 
same flashing rates under identical conditions. In other words, within 2 
seconds of signal collection time different compounds were expected to 
release different percentages of total releasable signals. A time course 
study over a period of up to 10 seconds was conducted to determine these 
percentages, by flashing the compounds and normalizing all the signals 
collected for different lengths of time to that of 10 seconds. The results 
are summarized in Table III. 
TABLE III 
______________________________________ 
Percent signal released over 
different lengths of time 
Compounds 10.0 s 6.0 s 4.0 s 
2.0 s 
1.0 s 
0.5 s 
______________________________________ 
DMAE-Bz 100% 99% 96% 80% 48% 10% 
DIPAE-Bz 100% 98% 97% 89% 64% 14% 
3-MeO-DMAE-Bz 
100% 80% 70% 57% 49% 26% 
ABAC 100% 89% 73% 52% 20% 3% 
LEAE-Bz 100% 98% 97% 88% 71% 27% 
DIP-LEAE-Bz 100% 80% 67% 47% 26% 4% 
LEAC-Bz 100% 102% 98% 93% 82% 73% 
3-EtO-LEAE-Bz 
100% 95% 93% 85% 78% 45% 
3-QAE-LEAE-Bz 
100% 91% 88% 84% 75% 34% 
2-MeO-LEAE-Bz 
100% 92% 83% 65% 42% 16% 
2-QAE-LEAE-NHS 
100% 88% 76% 59% 37% 9% 
NSP-LEAE-Bz 100% 95% 96% 91% 72% 14% 
2-MeO-NSE- 100% 96% 90% 79% 58% 22% 
LEAE-NHS 
2-MeO-LEAE- 100% 76% 59% 40% 23% 6% 
Imidate 
______________________________________ 
The ABAC is (4Benzyloxycarbonyl-2,6-dimethyl)phenyl 
5methyl-benz[a]acridinium12-carboxylate methosulfate. 
As shown by the data of TABLE III, particularly at the 0.5 and 1 second 
intervals, the flashing kinetics varied widely for different DMAE analogs, 
ABAC and LEAC's. These data on release percentages should be utilized in 
comparing the light emission efficiency of the compounds for developing 
various assay utilizing the chemiluminescent compounds. 
MUTUALLY NON-INTERFERING LIGHT EMISSION 
Beside exhibiting discernable mutually non-interfering nature of their 
light emission spectra as mentioned above, DMAE and LEAE in the form of 
protein conjugates also demonstrated no mutual interactions in their light 
emissions during flashing as shown by no decrease or increase of the 
combined Relative Light Units (RLU) registered. 
The testing was carried out as follows: 
DMAE-anti-TSH and LEAE-anti-TSH were diluted in 10 mM phosphate buffer with 
0.15M NaCl, 0.1% BSA, 0.05% NaN.sub.3, pH 8 at two concentrations, such 
that 25 ul of the solutions would give about 200,000 and 1,000,000 RLU's, 
respectively, when they were flashed in the same manner on the Berthold 
luminometer equipped as described above. The light emission of each sample 
(25 ul) was measured separately and then the same volume of each were 
combined and measured again. In single sample determinations, an 
additional equal volume of the buffer was added to maintain the same 
sample volume as in the combined sample determinations. Results of the 
testing are summarized in Table IV. 
TABLE IV 
______________________________________ 
Single sample determination* 
Combined sample 
(RLU) determination* (RLU) 
DMAE-anti-TSH 
LEAE-anti-TSH 
Theoretical 
Found (%) 
______________________________________ 
860,617 1,017,200 1,877,817 1,835,350 
(98%) 
173,200 191,820 365,029 362,293 
(99%) 
______________________________________ 
*Each value was the mean of triplicate determinations. (RLU Relative 
light units) 
The results in Table IV show that the two tracers of different emission 
spectra were absolutely non-interfering between each other in their light 
emission. This characteristic further ensures their utility in 
multi-analyte binding assays. The LEAE of the preferred method is a 
N-alkylated benzacridinium compound. 
STABILITY of CONJUGATED LBAC'S 
LBAC-Anti-TSH conjugates were prepared and tested for their stability in 
aqueous media. DMAE-anti-TSH conjugate was also tested side by side. The 
retention of chemiluminescent activity as a function of temperature at 
various pH's (using citrate-phosphate buffer containing 0.1% BSA) was 
monitored over 7 day period. Proper concentrations of the above conjugates 
(0.8-1.4.times.10.sup.6 RLU's/25 ul) were placed in two sets of different 
buffers (pH 7.4, 8.0, 8.5, and 9.0). One set was kept at 
4.degree.-8.degree. C. as a control, while the other was subjected to 
37.degree. C. The buffered samples (25 ul) were flashed periodically as 
described above. The results are summarized in Table V. 
TABLE V 
______________________________________ 
Relative Stability* of Conjugates 
pH Compds 1 day 3 days 
5 days 7 days 
______________________________________ 
7.4 I 93% 98% 91% 95% 
II 66% 56% 51% 44% 
III 67% 78% 77% 80% 
IV 89% 94% 101% 96% 
V 66% 46% 
8.0 I 99% 104% 96% 98% 
II 106% 106% 80% 83% 
III 79% 69% 68% 71% 
IV 102% 114% 140% 138% 
V 45% 13% 
8.5 I 92% 102% 89% 81% 
II 111% 97% 110% 82% 
III 81% 76% 78% 86% 
IV 134% 142% 150% 156% 
9.0 I 90% 95% 70% 71% 
III 94% 68% 51% 71% 
IV 133% 140% 149% 130% 
______________________________________ 
Compounds I-V are DMAEanti-TSH, LEAEanti-TSH, 2MeO-LEAE antiTSH, 
3Eto-LEAE-anti-TSH, and Nonortho-substituted AEanti-TSH, respectively. Th 
stability data for Nonortho-substituted AEanti-TSH were equivalent to tha 
reported earlier in U.S. Pat. No. 4,745,181. 
*Relative Stability is defined by expressing the percentage 
chemiluminescent activity of 37.degree. C. samples relative to that of th 
corresponding 4-8.degree. C. samples. For example, at pH 8, after 7 days 
of storage, the DMAEanti-TSH and LEAEanti-TSH 37.degree. C. samples 
retained 87% and 83% activity, respectively, in comparison with the 
corresponding 4.degree. C. samples, while the nonortho-substituted 
acridinium ester retained only 13% activity in comparison with the 
corresponding 4 .degree. C. sample. 
The stability study summarized in Table V demonstrates that the stabilizing 
effect of ortho-substitution on the phenoxy ring not only applies to the 
class of acridinium esters, it also benefits the LBAC's series to about 
the same extent with regard to maintaining their chemiluminescent activity 
in aqueous media at or near pH 8 under prolonged heat-stress conditions as 
required for commercial binding assay products. Listed in great contrast 
is the stability data of the non-ortho-substituted acridinium ester 
conjugate at pH 8. A non-ortho-substituted LEAC would likely also have 
poor stability in aqueous media. 
Signal-to-Noise in Binding Assays 
LEAE-anti-TSH was employed as tracer in a TSH assay. Performance was 
assessed by determining signal-to-noise (S/N) ratio. The performance of 
DMAE-antiTSH was also compared side by side. The assay was configured as 
follows: 
100 microliters of either of the above conjugates was incubated for two 
hours at room temperature with 100 ul of a TSH standard (Ciba Corning 
Diagnostics Corp., Medfield, Mass.). Incubations were done separately with 
five standards containing either 0, 0.5, 1.0, 16 or 100 uIu/ml of TSH. A 
second incubation was then performed by adding 500 ul of MAGIC.RTM. 
magnetic particle immobilized with sheep anti-TSH (Ciba Corning 
Diagnostics Corp.) to the above mixture, then waiting for 30 minutes at 
room temperature. 
A wash was done first by magnetically separating the particles from the 
solution, decanting the solution, then adding 500 ul of water, followed by 
another magnetic separation. The washed particles were resuspended in 100 
ul of water. Flashing and counting were done according to the 
above-described procedures. The results are provided in Table VI using 
ratios of the counts with a TSH standard containing TSH versus the zero 
TSH standard. 
TABLE VI 
______________________________________ 
S/N at various Standards 
Conjugate 
0.4 uIu/ml 
1.0 uIu/ml 
16 uIu/ml 
100 uIu/ml 
______________________________________ 
DMAE-anti- 
10.0 20.9 202.3 669.4 
TSH 
LEAE-anti- 
5.4 8.4 87.4 282.6 
TSH 
______________________________________ 
The results given in Table VI indicate that LEAE conjugate can be utilized 
in an immunoassay format to provide a dose-response curve and, therefore, 
allows the development of useful assays. 
DUAL-ANALYTE SIMULTANEOUS IMMUNOASSAY 
Instrumentation 
One embodiment of a Dual-PMT Luminometer (DPL) utilized to demonstrate the 
hardware of DPL includes at least two photo multiplyer tube (PMT) 
assemblies, an injection pump for Flashing Reagent #2, and a cube-shape 
light tight chamber designed for holding a disposable cuvette. At two 
opposite sides of the chamber, two cylindrical PMT tube assemblies are 
separately attached such that light of two different spectral ranges 
generated inside the cuvette can be individually registered by the PMT 
assemblies. The top of the cuvette-holding chamber is hinged to allow the 
cuvette to be manually inserted and removed. In addition, the top also has 
a fixed probe attached for the purpose of injecting the Flashing Reagent 
#2 into the cuvette. Within each PMT assembly an optical filter selected 
for particular spectral range, and is inserted between the cuvette and the 
PMT tube. 
Alternate embodiments and configurations of DPLs may be designed for 
semi-automated and automated detection of two or more chemiluminescent 
compounds or conjugates in a test sample. A luminometer as a component on 
an automated analyzer is described in EP-A-0 502 638 noted above. 
Essential to the discrimination or discernability of two or more emitted 
light spectra are the choices of a plurality of optical filters with 
proper wavelength cutoffs. 
Filters of this type are widely available from commercial vendors and may 
be modified, i.e. by lamination or specifically manufactured to be 
incorporated in a PMT assembly for detection and/or quantitation of 
spectral signals of the conjugates. Careful selection of filters will 
enhance the ability to discern emission signals and with appropriate 
correction may allow multiple signals with the emission overlap to be 
discerned. 
For the purpose of running a simultaneous LH/FSH dual-immunoassay as 
disclosed below, a long pass filter (P/N LL-500 of Corion, Holliston, 
Mass.) and a short pass filter (P/N P70-450 also of Corion) were chosen to 
match with the two different spectral ranges of light generated from a 
pair of tracers, LEAE-anti-LH and DMAE-anti-FSH, which were prepared in 
the same manner as described above for LEAE-anti-TSH and DMAE-antiTSH, 
respectively. The transmittance curves for the two filters are shown in 
FIG. 5, panels B and C. The choice of the optical filters should take into 
consideration the requirements on maximal signal transmittance and minimal 
signal cross-talk. Optical filters with more desirable transmittance 
profile and cut-off may be selected to maximize the transmission of light 
emitted from the tracers and/or to fit better with the emission spectral 
ranges of particular chemiluminescent compounds so as to improve the 
Percent Cross Talk (PCT) as described below. For example Corion's 
laminated CS550/CS600 filter (FIG. 5, panel D) was found to be a better 
replacement for filter P70-450 as the short pass filter matching with the 
long pass filter LL-500 for the determination of the pair of DMAE and LEAE 
tracers. Not only was the registered RLU's for DMAE tracer found to 
increase by more than 2 fold as a result of this filter's use, the 
Percentage Cross Talks, as shown in Table VII, were also greatly improved. 
Furthermore, as more LEAE derivatives with even longer emission maxima 
were developed, e.g. 2-MeO-LEAE, long pass filters such as LL-520 (FIG. 5 
E) would be a better choice than filter LL-500 for enabling further 
reduction of the PCT. 
For system controlling, which generally includes the basic functions of 
parameter setting, execution and registration of flashing, signal 
correction as described below as a function of filters used and the 
chemiluminescent compounds utilized, and data display, a personal computer 
unit containing proper software is utilized and connected to the DPL. 
Percentage Cross-Talks (POT's) Determination 
As mentioned above the two optical filters installed in two separate PMT 
assemblies on the DPL were intended to gate the emitted lights of two 
different spectral ranges: the long pass filter is to match with the 
longer emission from LEAE tracer and the short pass filter with the 
shorter emission from DMAE tracer. However, as illustrated by FIGS. 6 and 
7, because of the minor overlap between the transmittance curves and the 
emission spectra of the cross-matching pairs, light signals generated by 
one tracer can be picked up by the primary PMT intended for it but also in 
small percentage by the secondary PMT intended for the other tracer, and 
vice versa. That portion of signal of one tracer, that can be registered 
by the secondary PMT, must be quantitated separately in term of percentage 
for each tracer prior to their use in a dual-analyte immunoassay, in order 
that the apparent RLU's can be corrected and the pure signal of each 
tracer detected by each PMT assembly be obtained when the two tracers were 
flashed simultaneously in the same tube. 
Table VII shows the determined PCT's of several pairs of tracers. 
Anti-FSH-DMAE and anti-LH-LEAE were used in the simultaneous LH/FSH 
dual-analyte assay described below. Other pairs of tracers were included 
to demonstrate that through the selection of acridinium and benzacridinium 
compounds of wider separation in their emission maxima and proper choice 
of optical filters, minimal PCT's ideal for multi-analyte assay can be 
realized. The PCT's were obtained by dividing the minor signal from the 
secondary PMT by the major signal from the primary PMT in each case, and 
multiplying the results by 100%. 
The concentrations of the samples were randomly selected such that the 
primary signals fell in the range of 100,000 to 1,500,000 RLU's per 25 ul 
sample. Each determination was made by sequentially pipeting 25 ul of one 
tracer solution, 300 ul of Flashing Reagent #1 into the cuvette, vortexing 
the resulting solution briefly, inserting the cuvette into the PMT 
housing, and performing the flashing by injecting 300 ul of Flashing 
Reagent #2 through the key-board control. 
TABLE VII 
______________________________________ 
Determination of Percent Cross-Talk (PCT) 
Long Short 
Pass Signals 
Pass Signals 
Sample (RLU's) (RLU's) PCT (%) 
______________________________________ 
SET (I)*: 
LH tracer 538 688 
diluent 444 396 
Anti-LH-LEAE 
6.25 E5 4.18 E4 6.7 
5.78 E5 3.91 E4 6.8 
6.19 E5 4.18 E4 6.8 
Aver. 6.8 
FSH tracer 242 288 
diluent 268 340 
Anti-FSH-DMAE 
2.32 E4 1.59 E5 14.5 
2.42 E4 1.65 E5 14.7 
2.60 E4 1.75 E5 14.8 
Aver. 14.7 
SET (II) : 
Anti-LH-LEAE 
1.27 E6 5.52 E4 4.4 
1.25 E6 5.40 E4 4.3 
Ave. 4.4 
Anti-FSH-DMAE 
4.62 E4 4.28 E5 10.8 
4.60 E4 4.23 E5 10.9 
Ave. 10.9 
SET (III)+: 
Buffer.sup..about. 
348 278 
350 284 
Anti-TSH-2-MeO- 
9.90 E5 1.29 E4 1.3 
LEAE 9.88 E5 1.29 E4 1.3 
Ave. 1.3 
Anti-FSH-DMAE 
1.17 E4 3.77 E5 3.1 
1.17 E4 3.81 E5 3.1 
Ave. 3.1 
SET (IV)#: 
Buffer.sup..about. 
187 608 
182 429 
Anti-TSH-2-MeO- 
2.12 E5 6.26 E3 2.9 
LEAE 2.0 E5 5.95 E3 2.9 
Aver. 2.9 
Anti-TSH-DMAE 
2.93 E4 5.39 E5 5.4 
3.12 E4 5.83 E5 5.4 
Aver. 5.4 
______________________________________ 
*Optical filters mounted on the DPL: LL500 & P70450. 
Optical filters mounted on the DPL: LL500 & Laminated CS550/CS-600. 
+Optical filters mounted on the DPL: LL520 & Laminated CS550/CS-600. 
#Optical filters mounted on the DPL: LL520 & P70450. 
.sup..about. The buffer was 10 mM PBS/0.1% BSA/0.05% NaN.sub.3, pH 8.0. 
The constancy of the PCT over a wide range of RLU's is critical in the 
multi-analyte assay signal correction. Table VIII shows that when the 
laminated CS550/CS600 filter and LL520 filter were used to gate the short 
pass and long pass signals, respectively, the PCT for anti-TSH-DMAE has 
the mean of 2.96% with standard deviation of 0.16% over RLU range of 
10,000 to 7,000,000 counts or broader, while the PCT for anti-CKMB-LEAE 
has the mean of 4.79% with standard deviation of 0.23% over RLU range of 
50,000 to 7,000,000 counts or broader. 
TABLE VIII 
______________________________________ 
Constancy of Percent Cross-Talk 
Short Long 
Pass Signal 
Pass Signal 
PCT Mean/SD 
Sample (RLU's) (RLU's) (%) (%) 
______________________________________ 
Anti-TSH-DMAE 
6,689,796 217,010 3.24 
6,545,872 212,956 3.25 
6,645,674 210,262 3.16 
1,469,500 43,710 2.97 
1,469,770 45,004 3.06 
1,450,042 43,492 3.00 
303,944 8,922 2.81* 
302,876 8,788 2.78* 
287,632 8,928 2.98* 
59,468 2,106 2.95* 
58,816 2,072 2.93* 
60,314 1,996 2.73* 
12,298 692 2.77* 
11,956 692 2.86* 
12,420 716 2.96* 
2.96/0.16 
Buffer Diluent 
738 496 
702 320 
Anti-CKMB-LEAE 
320,758 6,663,484 4.81 
315,344 6,497,756 4.85 
320,224 6,528,242 4.91 
61,374 1,350,584 4.54 
61,514 1,329,548 4.63 
60,036 1,330,152 4.51 
13,044 264,526 4.71* 
11,968 244,106 4.67* 
12,324 245,542 4.78* 
3,312 56,120 4.84* 
3,586 54,906 5.45* 
3,220 53,928 4.87* 
4.79/0.23 
Buffer Diluent 
652 372 
622 346 
______________________________________ 
*Correction was made in consideration of the additional signal contribute 
by the buffer and system noise. 
Equations for Correcting the Apparent RLU's due to Cross-Talks in 
Dual-Tracer Determination 
When DMAE and LEAE derivatives or tracers are mixed and flashed 
simultaneously, the observed long and short pass signals can be broken 
down as follows: 
EQU S(s)=S(DMAE)+S'(LEAE)+b1 (1) 
EQU S(1)=S(LEAE)+S'(DMAE)+b2 (2) 
Where, S(s) and S(1) are the observed short and long pass signals, 
respectively; S(DMAE) and S(LEAE) are the portions of signals due to DMAE 
and LEAE in the observed short and long pass signals, respectively. They 
will also be referred to as the corrected DMAE and LEAE signals; S'(DMAE) 
and S'(LEAE) are portions of the long and short pass signals due to DMAE 
and LEAE cross-talking, respectively; b1 and b2 are the combined signals 
due to assay components and system noise in the absence of DMAE and LEAE 
tracers, respectively. 
Since the PCT's (represented by k1 and k2 below) are constants for any 
particular DMAE and LEAE tracers, there exist the following relationships: 
EQU S'(DMAE)=k1.times.S(DMAE) (3) 
EQU S'(LEAE)=k2.times.S(LEAE) (4) 
Where k1, k2 are the PCT's for the DMAE and LEAE tracers, respectively. 
Substitute equation (4) into (1): 
EQU S(s)=S(DMAE)+k2.times.S(LEAE)+b1 or S(DMAE)=S(s)-k2.times.S(LEAE)-b1(5) 
Substitute equations (5) into (3) and (3) into (2): 
EQU S(1)=S(LEAE)+k1.times.[S(s)-k2.times.S(LEAE)-b1+b2= 
S(LEAE)+k1.times.S(s)-k1.times.k2.times.S(LEAE)-k1.times.b1+b2 
Rearranging: 
##EQU1## 
Equations (5) and (6) will yield the corrected short pass signal due to 
DMAE tracer and long pass signal due to LEAE tracer, respectively. For the 
purpose of demonstrating the feasibility of conducting a simultaneous 
LH/FSH dual-analyte assay, the determination of the combined matrix and 
system noises, b1 and b2 was found not to be significant. They were 
therefore both assigned a 0 value in the signal corrections for the 
following examples of the dual-analyte assays. 
Simultaneous Immunoassay for Luteinizing Hormone (LH) and Follicle 
Stimulating Hormone (FSH) 
One objective of the invention is to provide a method for simultaneously 
detecting and/or quantitating two or more substances or analytes in a 
single sample through the utilization of two different chemiluminescent 
labels or conjugates. 
In an example of one embodiment, the assay system utilizes a DMAE labelled 
FSH antibody and a LEAE labelled LH antibody. The following examples 
demonstrate that LH and FSH standard curves and sample recovery are 
identical within the limits of experimental error when each analyte is 
assayed as a single analyte by introduction of one chemiluminescent tracer 
into the assay system, or in a dual analyte system which employs two 
chemiluminescent tracers. 
The examples further show that tracers prepared from a pair of a DMAE and a 
LEAC can be utilized in a simultaneous assay of two substances for which a 
corresponding binding partner, e.g. antibody, is available. 
EXAMPLE 15 
Single FSH assay using Dual-Analyte Immunoassay System 
The Magic Lite FSH kit components and protocol (Ciba Corning Diagnostics) 
were modified such that the assay could be performed as a single or dual 
analyte assay depending on the tracer selection. A solid phase consisting 
of paramagnetic particles (PMP) coupled to anti-FSH antibodies and PMP 
coupled to anti-LH antibodies was prepared by removing the buffer diluent 
from the Magic Lite FSH kit solid phase and resuspending these particles 
in Magic Lite LH kit solid phase (Ciba Corning Diagnostics Corp.). The kit 
tracer, anti-FSH-DMAE, was diluted 1:2 in Magic Lite LH kit tracer buffer. 
Standards for calibration contained both FSH and LH. Standards were 
prepared by spiking known concentrations of purified human FSH and human 
LH into a horse serum basepool. Nominal standard values were 0, 0.9, 2.2, 
4.4, 8.8, 21.9, 43.8, 87.5, 140.0, 201.0 mIu/ml of FSH. Nominal LH 
concentrations were 0, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 160.0, 
230.0 mIu/ml LH. Samples for analysis were prepared by spiking a human 
serum pool with varying concentrations of both purified human FSH and 
human LH. Additionally, serum based multi-constituent calibrators 
containing human FSH and human LH were used as samples. 
To perform the assay, 50 ul of each standard or sample and 200 ul of 
diluted FSH tracer were vortex mixed and incubated for 30 minutes at room 
temperature. 500 ul of the combined anti-FSH/anti-LH solid phase was 
added, vortex mixed and incubated for 30 minutes at room temperature. The 
reacted solid phase was magnetically separated for 3 minutes in a Magic 
Lite rack (Ciba Corning Diagnostics Corp.), see European Patent 136126, 
and the supernatant decanted. The reacted solid phase was next washed with 
1.0 ml of distilled water, separated for 3 minutes. The supernatant was 
decanted, and 100 ul of distilled water added. Each sample was manually 
transferred to a cuvette, and counted for 5 seconds on the DPL described 
above. The results (in RLU's) obtained from the short pass (DMAE) channel 
were used to calculate FSH concentration in each sample. Concentrations 
were calculated by using 10-point calibration with a spline data reduction 
routine. Each standard and sample-was assayed in replicates of three. 
RLU's and % CVC for this assay are shown in Table IX under the heading FSH 
single-analyte assay. FSH sample recovery is shown in Table X under the 
heading FSH single-analyte assay. The FSH standard curve presented as % 
B/Bmax vs log FSH concentration is shown in FIG. 8 labelled as FSH 
single-analyte assay. 
EXAMPLE 16 
Single LH Assay using Dual-analyte Immunoassay System 
The solid phase reagent, standards, and samples described in Example 15 
were used to perform an LH assay. The anti-FSH-DMAE tracer was replaced 
with an anti-LH-LEAE tracer which was diluted 1:2 in Magic Lite FSH kit 
tracer diluent. The assay methodology described in Example 13 was applied 
to this assay, except that the RLU results obtained from the Long pass 
(LEAE) channel were used to calculate LH sample concentrations. 
The assay was calibrated using nine of the standards described in Example 
15, excluding the 1.0 mIu/ml LH standard. Results for this assay are shown 
in Table XI and Table XII under the heading LH single-analyte assay. The 
standard curve is shown in FIG. 9 labelled as LH single-analyte assay. 
EXAMPLE 17 
Simultaneous LH/FSH Assay using Dual-analyte Immunoassay System 
Solid phase reagent, standards, and samples described in Example 15 and 16 
were used to perform a dual label LH/FSH assay in a single tube. The 
tracer consisted of the Magic Lite FSH kit tracer, anti-FSH-DMAE diluted 
1:2 in the anti-LH-LEAE tracer. The assay methodology was the same as that 
described in Example 15. The raw RLU's from each channel was 
mathematically corrected for cross-talk prior to concentration 
calculations. Corrected RLU's and concentrations resulting from these 
corrected RLU's are shown in Tables IX-XII, and are labelled as 
dual-analyte assay. Mean sample recovery for single analyte vs. dual 
analyte assays are compared by t-test in Tables X and XII. The FSH and LH 
standard curves are shown in FIGS. 8 and 9 and labelled as FSH and LH 
dual-analyte assay, respectively. 
Assays and Assay Formats 
The present invention relates to chemiluminescent compounds and more 
particularly, the use of two or more chemiluminescent conjugates to 
simultaneously detect two or more substances in a test sample. The 
disclosure teaches the use of benzacridinium compounds and preferably 
N-alkylated benzacridinium compounds in such assays. 
A test substance includes any component(s) or analytes sought to be 
detected and/or quantitated in a test sample, including but not limited 
to, more than one component of a single structure, e.g. more than one 
portion of a nucleic acid sequence or different loci of a chromosome, 
genome or molecule, where the components or analytes may be of biological 
or industrial origin, such as nucleic acids, proteins, ligands, haptens or 
other materials or compounds to which an appropriate assay method can be 
formatted. It is understood that the test sample and/or substance may need 
to be pretreated to render it assayable by a test method. The test 
substances and quantities thereof sought to be detected may limit the 
types of assays which can be performed because of, for example, 
sensitivity concerns, but not the use of chemiluminescent labels for 
detection. Various internal standards or controls may be added to a test 
sample for detection and/or quantitation to assess the performance of the 
assay. Diagnostic assays exemplified by immunoassays, hybridization assays 
and amplification assays have increasingly incorporated chemiluminescent 
labels in their formats. Designs and formats of such assays are well known 
by those skilled in the art and extensively published in the technical and 
patent literature, for example, an assay format may require the separation 
of a reaction product or unreacted agent to a transfer tube for detection 
and/or quantitation. Such separation techniques may be useful for 
competitive assays, solid phase assays or to limit interferents. 
In one embodiment of the invention, two or more chemiluminescent conjugates 
are utilized as labels in an amplification assay. Representative 
amplification assays include but should not be limited to polymerase chain 
reaction (PCR), autocatalytic replication of recombinant RNA and 
amplification of midivariant DNA or RNA. See EP-A-O 481 704 (priority U.S. 
Ser. No. 598,269 (Oct. 16, 1990), abandoned) which is commonly assigned 
and incorporated herein by reference. Such methods, as taught in the 
technical and patent may be made adaptable to incorporate chemiluminescent 
labels, and particularly two or more chemiluminescent labels for detection 
of target sequences of interest. The advantage of using a multi-label 
method is to detect and/or quantitate a plurality of target sequences or 
one or more target sequences and an internal standard. An example of such 
a method includes providing a test sample suspected of containing one or 
more target sequences, amplifying the target sequences, providing at least 
two chemiluminescent conjugates, each chemiluminescent conjugate being 
associated with a target sequence(s) and simultaneously detecting and/or 
quantifying amplified target sequences by emissions of at least two 
chemiluminescent conjugates. In another step of this method an internal 
reference, control or control system may be added to the assay to insure 
assay performance and results. The internal reference may be amplified as 
well as the target sequences. 
The use of chemiluminescent labels for such assays serves to demonstrate 
the utility of this invention. 
The chemiluminescent compounds of this invention are adapted to be packaged 
in kit form for commercial sale. The chemiluminescent labels of these kits 
may be conjugated to appropriate substances or materials which are 
specific to the substances sought to be detected in the test samples. 
Appropriate functional groups may be added to the chemiluminescent 
compounds for use in various assays and other applications. Examples of 
assays for which the methods of the present invention may be utilized 
include but should not be limited to: assays including at least two 
antibodies of different specifities; assays including at least two 
antigens; assays including at least one antigen and at least one antibody; 
and assays for molecules indicative of cancer, infectious diseases, 
genetic abnormalities, genetic disposition, genetic assessment and to 
monitor medicinal therapy. 
It is to be understood that various other modifications will be apparent to 
and can readily be made by those skilled in the art, given the disclosure 
herein, without departing from the scope and materials of this invention. 
It is not, however, intended that the scope of the claims appended hereto 
be limited to the description as set forth herein, but rather that the 
claims be construed as encompassing all features of patentable novelty 
which reside in the present invention, including all features which would 
be treated as equivalents thereof by those skilled in the art to which the 
invention pertains. It is also noted that the examples given therein are 
intended to illustrate, and not to limit the invention. 
TABLE IX 
______________________________________ 
MEAN RLU's and % CVC FOR MODIFIED MAGIC LITE 
FSH ASSAYS 
FSH SINGLE FSH RESULTS DUAL 
SAMPLE ANALYTE ASSAY ANALYTE ASSAY* 
(VALUE) MEAN RLU % CVC MEAN RLU % CVC 
______________________________________ 
S1 (0) 1336 4.3 610 6.7 
S2 (0.9) 1891 5 1309 14.4 
S3 (2.2) 3202 10.9 2375 6.7 
S4 (4.4) 5155 9.4 4189 3.9 
S5 (8.8) 9005 2.6 7702 2.4 
S6 (21.9) 
19637 5.5 16942 5.2 
S7 (43.8) 
35491 0.7 30563 4 
S8 (87.5) 
56844 1.7 51969 5 
S9 (140.0) 
74850 0.9 66499 0.5 
S10 (201.0) 
87531 0.8 77003 0.5 
SAMPLE 1 9962 7.3 8417 2.8 
SAMPLE 2 16779 4.9 14777 0.8 
SAMPLE 3 29683 13 28721 4.5 
SAMPLE 4 62549 2.2 55878 2.4 
SAMPLE 5 93019 1.1 84182 3.3 
SAMPLE 6 103290 3 93297 1.2 
LOW 1756 9 1160 9.8 
MULTI-CAL 
HIGH 80308 2.4 71082 0.6 
MULTI-CAL 
TOTAL 564200 571392 
COUNTS 
______________________________________ 
*corrected RLU 
TABLE X 
__________________________________________________________________________ 
FSH SAMPLE RECOVERY: SINGLE ANALYTE ASSAY VS. DUAL ANALYTE ASSAY 
FSH SINGLE FSH DUAL CRITICAL T-VALUE 
ANALYTE ASSAY 
ANALYTE ASSAY 
95% C.I. 
SAMPLE MIU/ML 
% CVD 
MIU/ML 
% CVD 
T-VALUE 
__________________________________________________________________________ 
SAMPLE 1 9.923 8.6 9.737 3.2 0.36 +/-4.30 
SAMPLE 2 18.291 
5.7 18.668 
1 0.62 +/-4.30 
SAMPLE 3 35.235 
15.1 40.708 
5.3 -1.65 +/-4.30 
SAMPLE 4 101.951 
3.5 98.877 
4.2 0.98 +/-3.18 
SAMPLE 5 &gt;&gt; &gt;&gt; 
SAMPLE 6 &gt;&gt; &gt;&gt; 
MULTI-CAL LOW 
0.693 30.8 0.697 20.1 -0.2 +/-3.18 
MULTI-CAL HIGH 
163.132 
5.4 163.812 
1.4 -0.13 +/-4.30 
__________________________________________________________________________ 
TABLE XI 
______________________________________ 
MEAN RLU's AND % CVC FOR MODIFIED MAGIC LITE 
LH ASSAYS 
LH SINGLE LH RESULTS DUAL 
SAMPLE ANALYTE ASSAY ANALYTE ASSAY* 
(VALUE) MEAN RLU % CVC MEAN RLU % CVC 
______________________________________ 
S1 (0) 17,883 14.2 18,477 10.6 
S3 (2.5) 24,310 1.9 22,818 2.7 
S4 (5.0) 27,007 6.5 25,118 11.9 
S5 (10.0) 
34,713 11.6 28,934 6.2 
S6 (25.0) 
42,440 4.6 41,543 5.3 
S7 (50.0) 
65,787 2.9 71,140 6.2 
S8 (100.0) 
115,760 6.8 113,694 1.9 
S9 (160.0) 
161,767 4.4 164,225 4.4 
S10 (230.0) 
223,569 4.8 205,347 4.5 
SAMPLE 4 42,157 2.8 40,519 8.2 
SAMPLE 5 82,934 4.3 83,592 5.5 
SAMPLE 6 110,502 2.8 113,285 1.9 
MULTI-CAL 
86,189 3.5 90,451 2.9 
HIGH 
TOTAL 2,250,526 2,322,672 
COUNTS 
______________________________________ 
*CORRECTED RLU 
TABLE XII 
__________________________________________________________________________ 
LH SAMPLE RECOVERY: SINGLE ANALYTE ASSAY VS. DUAL ASSAY 
LH SINGLE LH RESULTS CRITICAL 
ANALYTE ASSAY 
DUAL ANALYTE ASSAY* 
T-VALUE 
SAMPLE MIU/ML 
% CVD 
MIU/ML 
% CVD 
T-VALUE 
95% C.I. 
__________________________________________________________________________ 
SAMPLE 4 21.347 
7 22.342 
15.3 -0.46 +/-4.30 
SAMPLE 5 67.392 
5.7 64.829 
7.5 0.72 +/-3.18 
SAMPLE 6 97.375 
3.6 97.984 
2.5 -0.25 +/-3.18 
MULTI-CAL HIGH 
70.906 
4.5 72.153 
4 -0.5 +/-3.18 
__________________________________________________________________________