Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1341402
S
Descr iption
Chemiluminescent acridine derivatives and their use in
luminescence immunoassays
The invention relates to chemiluminescent acridine deri-
vatives, processes for their preparation and their use in
luminescence immunoassays.
Luminescent compounds already have widely varying uses.
They are employed as indicators in bioassays, enzyme im-
munoassays and luminescence immunoassays (cf. W.P. Coffins
"Alternative Immunoassays", publishers John tiiley & Sons
Ltd., Chichester, 1985>, and are also used in nucleic acid
hybridization assays (cf. J.A. Matthews et al., "Analyti-
cal Biochemistry", 151, 205-209, 1985>. Chemiluminescent
compounds are moreover employed in "flow injection analysis",
in post column detectors in liquid chromatography, in flow
research and in artificial light sources.
In chemiluminescence immunoassays, two structural types
of chemiluminescent marking substances in particular have
achieved relatively great importance. These are on the
one hand the luminol and isoluminol derivatives described
by H.R. Schroeder et al., "Methods in Enzymology", Aca-
demic Press Inc., New York, Vol. LVII, 1978, 424 et seq.
and in British Patents 2,008,247 and 2,042,920, German
Patents 2,618,419 and 2,b18,511 and European Patent
Application 135,071. A review of the practical use of
isoluminol compounds as luminescence indicators is to be
found in W.G. Wood, J. Clin. Chem. Clin. Biochem. 22, 1984
905-918.
On the other hand, acridinium ester compounds have also
3S found use as chemiluminescent marking substances.' Such
acridinium esters are known from U.S. Patent 3,352,791,
British Patents 1,31b,363 and 1,461,877 and European Patent
Application 82,b3b. The use of acridinium esters as
134102
marking substances in immunoassays is described by Weeks
et al., Clin. Chem. 29l8 (1983), 1474-1479. The use of
phenanthridinium esters as a marking substance in lumin-
escence immunoassays is already known from European Patent
Application 170,415.
The chemiluminescence of acridinium esters can be started
by addition of alkaline N202 solution. A convincing
explanation of the mechanism of chemiluminescence has been
given by F. McCapra, Acc. Chem. Res. 9, 201, 1976. thus,
the nature of the leaving group is evidently decisive both
for the light quantum yield and for the hydrolytic stabi-
lity.
Compared with the luminol and isoluminol compounds, the
acridinium esters already known to date have the advantage
of a higher light quantum yield, which is also not im-
paired by proteins bonded to the indicator (cf. Weeks
et al., Clin. Chem. 29/8 (1983), 1474-1479).
zo
Although the acridinium phenyl esters known from European
Patent Application 82,b35 are distinguished by a high
detection sensitivity when the chemiluminescence is sti-
mulated by mild oxidizing agents, they have disadvantages
which interfere with their use in practice. In particu-
lar, the phenyl ester bond is very unstable in aqueous
systems, even already at room teeperature. Moreover,
under the oxidation conditions stated therein, the acri-
dinium phenyl esters exhibit photoemission which largely
subsides, that is to say to the extent of mare than 95%,
only after about 10 seconds. In comparison with this,
other non-isotopic assay methods have far shorter mea-
surement times and thereby allow a higher sample through-
put.
The object of the present invention was therefore to pro-
vide new acridinium derivatives which exhibit faster reac-
tion kinetics with a high light quantum yield and thereby
allow short measurement times for luminescence immunoassay.
1 3~1 40 2
_ 3 _
It has now been found that these requirements are met by
chemiluminescent acridinium derivatives of the formula I
R1
.~ (I)
a
/ /
O = C - R4
in which R1 is hydrogen, an alkyl, alkenyl or alkinyl
radical with 1 to 10 carbon atoms or a benzyl or aryl
group, R2 and R3 are hydrogen, an alkyl. group with 1
to 4 carbon atoms, a substituted or unsubstituted amino
group, a carboxyl, alkoxy, cyano or vitro group or halo-
gen, R4 represents a radical in which a sulfonamide group
is bonded directly via the nitrogen to the carbonyl group,
or is a thioalkyl or thioaryl radical of the formula II
X -- RS (II)
in which X is a branched or unbranched aliphatic group
or an aromatic group, which can also contain heteroatoms,
and R5 is a reactive group which can undergo selective
bonding under gentle conditions with amino, carboxyl or
thiol groups or other functional groups in substances of
biological interest, and A~ is an anion which does not
impair _.th~ chemiluminescence.
Further details of the invention are described below with
the help of the examples illustrated in the accompanying
drawings in which:
Figure 1 is a graph showing the kinetics of the
photoemission of an antibody conjugate of the acridinium
thioester prepared according to the process of the
invention;
~ 3~~ 40 2
;3 A
Figure 2 is a graph showing the kinetics of the
photoemission of the antibody conjugate of
4-(2-succinimidyl-axycarbonyl-ethyl)-phenyl-10-methyl-
acridinium 9-carboxylate methosulfate; and
Figure 3 is a graph showing the typical course of a standard
curve of an immuna-chemiluminometric assay (ICMA) for the
a-Fetoprotein (AFP).
Substances of biological interest are to be understood as,
above all, antigens. This term also includes hormones,
steroids, medicaments, medicament metabolites, toxins,
alkaloids and also antibodies.
The anion which does not impair the chemiluminescence can
be a tetrafluoborate, perchlorate, halide, alkyl~~lfate,
halosulfonate, alkylsulfonate or arylsulfonate anion. Any
other anion can also be used, as long as it does not ex-
tinguish or weaken the chemiluminescence.
1341402
_~_
By aryl there are to be understood aromatic hydrocarbons,
in particular phenyl and naphthyl. By aromatic groups
containing heteroatoms there are to be understood aromatic
hydrocarbons which contain a nitrogen or oxygen atom, in
particular quinoline, indole, pyrrole and pyridine.
Halogens are to be understood as fluorine, chlorine, bro-
mine and iodine.
The substituted amino groups are preferably C1-C4-alkyl-
or C1-C4-dialkylamines, it being possible for the
alkyl radicals in turn to be monosubstituted, for example
by hydroxyl. The substituted amines can also be a morpho-
line radical. The alkoxy radicals mentioned for R2 and R3
are preferably those with 1-~ carbon atoms in the alkyl
part.
The branched or unbranched aliphatic groups mentioned for
X are preferably C1-C5-alkylene groups.
Acridinium derivatives in which X is a methylene, ethy-
lene, propylene or ortho-, meta- or para-phenylene group
are in general chosen. These groups can also carry hydro-
philic substituents containing heteroatoms in order to
improve the water-solubility of the acridinium derivative.
The substituent RS is of particular importance for the
possible uses of the acridinium derivatives according to
the invention. By a suitable choice of this group, the
acridine derivative is given such a high reactivity that
it can already undergo selective bonding under mild condi-
tions with a functional group of the biological substance
to be detected. Suitable reactive groups can be seen
from the following compilation:
13~14~2
- 5 -
0 O
g) -C - 0 - 2d
__
0 O~ SO (_) Y(-+)
3
_c _ o _ r~ _
Y(+)= H(+) (+>
O ~ alkali metal
0 O
a r ,,-
c) -C - p - N
O
d) -S02 - CH = CH2
(+)
e) S02 CH2-CH2-N V
halide
f) -N = C = S
~ -N3
(+)
NH2 halide
h ) -C ~
O ~ 0-R
i ) -N
C1 C1
0
k ) -C - 0 - ~ ~ - C~
0 C C1.
1 ) -C - p /~\ _N02
0 /~= N
m ) _C- N
0 N2
n) -C - C - CFA
1341402
- 6 -
In many cases, acridinium derivatives according to the
invention in which R5 is a group of the formula III
0
~ (III)
r
_ C _ Q _ N
0
t0 have proved to be suitable.
In addition, acridinium compounds in which R~ is a methyl
group are also preferred. Acridinium compounds according
to the invention which correspond to the formula IV CIV)
N~
B
A
0
zo T -
c o
Cm)
o~ s-x-~-o-N
0
in which A and ~( have the abovementioned meanings, are
therefore particularly frequently employed.
If the radical R4 in the acridinium derivatives according
to the invention is a sulfonamide radical, it can prefer
ably correspond to formula V
X _ R~
-N ~ (V)
~ ~p2 _ R6
or formula VI ,
1 341 44 2
R6
(VI)
~ S02 - X - R~
in which X and R5 have the abovementioned meanings and
R6 in formula V is an alkyl ar akenyl radical with ~1 to
carbon atoms, an amino group which is preferably mono-
or disubstituted by C1-C~-alkyl, or a morpholino, benzyl
'10 or aryl group, which can also be substituted by hydroxyl,
amino, alkoxy with 1 to 4 carbon atoms or aryloxy, and R6
in formula VI can additionally also denote hydrogen.
Typical representatives of this class of acridinium deri
vatives according to the invention correspond to the for
mula VII
CH3 cull)
~ '''
p E~
0
C 0
0~ \N - R - G - 0 - N
S02 0
in which A and X have the abovementioned meanings.
The present invention thus provides two new classes of
acridinium compounds, one class being distinguished by a
thioester grouping and the other by a sulfonamide struc-
ture. The acridinium acylsulfonamide derivatives have a
higher stability and the thiol esters faster kine~ics than
the previously known acridinium phenyl ester compounds.
9oth classes of compound are moreover distinguished by a
higher light yield.
1 X41 40 2
_8-
A significant advantage of the acridinium compounds accord-
ing to the invention with leaving groups containing thiol
esters in comparison with the acridinium phenyl esters
known from European Patent Application $2,b36 ties in the
S considerably faster reaction kinetics of the photoemis-
sion. Thus, the acridinium thioester according to_the
invention prepared according to Example 1 (compound b)
has a 10 times higher light yield at a measurement time of
1 second under the same oxidation conditions. This high
tight yield with measurement times which are at the same
time short allows a considerably higher sample throughput
in the luminometer.
Figure 1 thus shows the kinetics of the photoemission of
an antibody conjugate of the acridinium thioester prepared
according to Example 1 (compound 6) and Figure 2 shows the
antibody conjugate of 4-(2-succinimidyl-oxycarbonylethyl>-
phenyl-10-methylacridinium 9-carboxylate methosulfate
(European Patent Application 82,636, page 10). 100 pl of
the particular tracer solutions are stimulated to chemi-
luminescence by additian of 350 ul of 0.05 M KCl/NaOH buffer,
pH 13, + 0.1% of H242 and recorded over a 10 second period.
In Figure 1, the maximum photoemission is already reached
after 0.6b second, and has already fallen again to half
after 0.88 second. In contrast, in Figure 2 the maximum
photoemission is reached only after 1.77 seconds and has
fallen again to half only after 2.88 seconds.
/ It is completely unexpected that acridinium 9-carboxylic
acid amides substituted by sulfonyl on the amide nitrogen
,Jhave an excellent chemiluminescence, since it is known
that acridinium 9-carboxylic acid amide, in contrast to
the acridinium 9-carboxylic acid esters, shows no chemi-
luminescence at all (cf. F. McCapra in W. Carruthers and
J.K. Sutherland: Progress in s~rganic Chem., Vol. 8, 231-
277, 1973, Butterworth, London>.
The acridinium 9-carboxylic acid thioesters according to
the invention can be prepared in the following way:
~ 341 40 2
- 9 -
Acridine or its derivatives with R' and/or R3 in the fused-
on phenyl rings are canverted into the 9-cyanoacridine in
ethanol/glacial acetic acid and potassium eyanide by the
process described by Lehmstedt and Hundertmark in Ber. 63,
1229 (1930). After recrystallization,, the acridine-9-
carboxylic acid or R2/R3-substituted acridine-9-carb-
oxylic acid is obtained from this product by reaction with
sulfuric acid and sodium nitrite in accordance with the
process described by Lehmstedt and Wirth in Ber. 61, 2044
(1928). Reaction of the acridine-9-carboxylic acid or
R2/R3-substituted acridine-9-carboxylic acid with
thionyl chloride gives the compound of the formula VIII
.N
'~ 5 R 2 r ~~, '~~~ ~ 3 ( V I I I )
i
v
i ~~.
0 Y
in which Y denotes chlorine. Instead of a halogen, an
oxycarbonyl-C1-CS-alkyl, oxycarbonylaryt or imidazolide
group can also be introduced for Y into compound VIII.
The R2/R3-substituted acridine derivatives can be syn-
thesized in a simple manner by processes which are known
from the literature. Such syntheses are described, for
example, in: Comprehensive Heterocyclic Chemistry; Editors
A. Katritzky, C.W. Rees, Vol. 2, 395 et seq, Pergamon
Press, 1984 or Hetercyclic Compounds, Vol. 9, Acridines
and Cond. 2nd Edition, R.M. Acheson, John Wiley ~ Sons,
1973.
The acid chloride (VIII) is then reacted with a thiol carb-
oxylic acid of the formula IX
,
Hs - x - caoH (Ix)
for example with 2-mercaptobenzoic acid, under alkaline
conditions to give the thiol ester carboxylic acid, which
1 X41 44 2
- 10 -
is then esterified with a compound suitable for prepara-
tion of the radical R5, for example with N-hydroxysuc-
cinimide. The acridine compound is then alkylated in the
10-position by processes which are knpwn from the litera-
ture. Trimethyloxonium tetrafluoborate is above all suit-
able for methylation, but good yields of the chemilumin-
escent acridinium compound are also obtained with dimethyl
sulfate, methyl fluorosulfonate, methyl toluenesulfonate
or methyl trifluoromethanesulfonate.
'I 0
To prepare the acridinium sulfonamide derivatives accord-
ing to the invention, the acridine-9-carboxylic acid
chloride (VIII) is likewise used as the starting sub-
stance. This compound is then reacted with a primary or
secondary sulfonamide, preferably with a protected sul-
fonamide carboxylic acid of the formula X
0
n
R - SOL - N - X - C - OZ
(x)
or of the formula XI
H
R6 - N - sot - x - coat c x I )
in which X and Rb have the abovementioned meanings and
Z is a radical which protects the carboxyl group and is
subsequently split off. N-Benzenesulfonylglycine benzyl
ester, for example, can be used as the protective group
for this reaction. The acid formed after the protective
group has been split aff is then converted into the radi-
cal R5 with a suitable coa~pownci, far example with N-hyd-
roxysuccinimide. The chemiluminescent acridinium compound
is obtained from this product by alkylation on the nitro-
gen in the 10-position by processes which are known from
the literature.
The acridinium compounds obtained can then be reacted with
a substance of biological interest, for example an antigen,
~ ~4~ 4a 2
- ,~ _
an antibody, a hormone, a medicament, a medicament meta-
bolite, a toxin or an alkaloid to give a luminescent com-
pound. The acridinium derivative is thereby bonded either
directly or via a bridge molecule, such as polylysine,
polyglutamic acid or polyvinylamine, to the substance of
biological interest to form a stable immunologically act-
ive conjugate. This conjugate is also called a tracer
and is used in the luminescence immunoassays described
below. At least one immunologicalLy active component which
is immobilized on a solid phase and also the luminescent
tracer are required for the luminescence immunoassay
according to the invention far the determination of an
antigenic substance in a liquid sample by a competitive
process or a sandwich process. The luminescence immuno-
assay can now be carried out in various ways.
One possibility is to incubate the immobilized antibodies
which react specifically with the antigen with a sample
of the liquid under investigation and a conjugate of the
antigen and a chemiluminescent acridinium derivative (anti-
gen tracer), to separate off the sample and the non-bonded
tracer, to bring the bonded tracer together with an
oxidizing agent in order to cause photoemission and then
to determine the amount of antigen present from the photo-
emission intensity measured.
Another possibility far carrying out the luminescence
immunoassay comprises incubating an immobilized antibody
which reacts specifically with the antigen with a sample
of the liquid under investigation, a conjugate of a sec-
ond antibody which reacts specifically and a chemilumin-
escent acridinium derivative, separating off the sample
and the non-bonded marked conjugate, bringing the bonded
marked conjugate together with an oxidizing agent in order
3~ to cause photoemission and determining the amount of
antigen present from the photaemission intensity measured.
The luminescence immunoassays mentioned above can also be
carried out by separating off the liquid under investigation
1341402
- 12 -
from the immobilized antibody before addition of the
marked conjugate.
In other luminescence immunoassays which can be carried
out according to the invention, the antigen and not the
antibody is immobilized. Thus, an immobilized antigen
which reacts specifically with the antibody can be incu-
bated with a sample of the liquid under investigation and
a solution of a conjugate of the antibody and a chemi-
luminescent acridinium derivative, the sample and the
non-bonded marked conjugate can then be separated off and
the bonded marked conjugate can subsequently be brought
together with an oxidizing agent. Photoemission then
occurs, and the amount of antigen present can be deter-
mined from its intensity.
Another variant comprises incubating an immobilized anti-
gen which reacts specifically with the antibody with a
solution of a conjugate of the antibody and a chemilumi-
nescent acridinium derivative, separating off the unreacted
marked conjugate, adding a sample of the liquid under in-
vestigation, subsequently separating off the sample again,
bringing the bonded marked conjugate together with an oxi-
dizing agent in order to cause photoemission and then
determining the amount of antigen present from this photo-
emission.
Finally, the luminescence immunoassay can also be carried
out by a procedure in which an immobilized antigen which
reacts specifically with the antibody is incubated with
a solution of a conjugate of the antibody and a chemi-
luminescent acridinium derivative, a sample of the liquid
under investigation is added, the sample and the non-
bonded conjugate are separated off, the bonded marked con-
jugate is brought together with an oxidizing agent and
the amount of antigen present is then determined from the
photoemission measured.
1 341 40 2
- 13 -
The preparation of the acridinium compounds according to
the invention is shown by Examples 1 to 3.
Example 1
9-Cyanoacridine (1)
3.3 ml of glacial acetic acid are added to acridine (10 g)
in 45 ml of ethanol and a solution of 5.25 g of potassium
cyanide in 8 ml of water are added dropwise, the reaction
mixture is heated under reflux for 2 hours and cooled and
the volatile constituents are stripped off in vacuo. The
residue is stirred with 30 ml. of 2 N NaOH, filtered off
with suction, washed twice with 2 N NaOH and water and
left to stand in the moist state in air for some time.
'15 The crude product is stirred into methylene chloride, un-
dissolved substance is filtered off with suction and washed
with methylene chloride, the combined organic phases are
concentrated and the crude 9-cyanoacridine is recrystal-
lized from n-butyl acetate.
Yield: 50% melting point: 183-5°C
IR: 2230 cm 1
Acridine-9-carboxylic acid (2)
9-Cyanoacridine (5 g) is slowly added in portions to
40 ml of concentrated H2S04 and the mixture is heated
at 90-95°C for 2 hours and, after addition of 8.5 g of
NaN02, is stirred at this temperature for a further 2 hours.
The hot solution is poured onto 620 ml of ice water, with
rapid stirring, and the precipitate is filtered off with
suction and dissolved in the smallest possible amount of
2 N NaOH. The solution is filtered, the filtrate is aci-
dified with 50% strength H2S04 and the acridine-9-carb-
oxylic acid which has precipitated out is filtered off
with suction and dried in vacuo.
Yield: 95% melting point: 288-9oC
IR: 3440(br>, 3200(br>, 2600-2500Cbr), 1980; 1650; 1605;
1420 cm 1
1 X41402
- 14 -
Acridine-9-carboxylic acid chloride hydrochloride (3)
Acridine-9-carboxylic acid (5 g) is added in portions to
50 ml of freshly distilled SOCl2 and the mixture is heated
under reflux for 5 hours; the solution, which is then
'.i clear, is concentrated by distillation until a precipitate
starts to form, and precipitation is brought to completion
by addition of cyclohexane and cooling. The precipitate
is filtered off with suction and dried in vacuo to give
acridine-9-carboxylic acid chloride hydrochloride.
Yield: 90% melting paint: 223°C
Elemental analysis (calculated as Cl4HgClNO x HCt)
calculated C 60.5 H 2.8 N 5.0 Cl 25.5
found C 59.4 H 3.3 N 5.0 Cl 25.2
(Phenyl-2'-carboxylic acid)acridine-9-thiocarboxylate (4)
Acridine-9-carboyxlic acid chloride hydrochloride (30 g>
is suspended in 720 ml of methylene chloride, thiosalicylic
acid (17.7 g) and SO ml of triethylamine are added and
the solution, which becomes clear, is subsequently stir-
red at room temperature for 10 minutes. After the solvent
has been stripped off, 35 g of sodium carbonate and 1400 ml
of water are added to the residue, the resulting solu-
lion is concentrated until a precipitate appears and this
is filtered off with suction. The filtrate is saturated
with NaCI and the precipitate which thereby separates out
is likewise filtered off with suction. The aqueous solu-
tion of the combined precipitates is acidified with gla-
cial acetic acid at 80°C and the product which has preci
pitated out is filtered off with suction and dried in
vacuo.
Yield: 80% melting point: 261-5oC
NMR (DMSO, 100 MHz): 8= 7.6-8.4 ppm, complex multiplet
IR: 1b80 cm ~ (s), 1720 (m) 1260 (s>
2'-(Succinimidoyloxycarbonyl)phenylacridine-9-thiocarboxy-
late (5)
3.2 g of N-hydroxysuccinimide are added to a suspension
1 X41 40 2
_ 15 _
of 10 g of the thiol ester carboxylic acid in 190 ml of
dry tetrahydrofuran at 0°C, b.9 g of dicyclohexylcarbo-
diimide (DCC) are then added at -20°C and the mixture is
subsequently stirred at -20°C for 2 hours and then at room
temperature overnight. After addition of 0.28 ml of
glacial acetic acid, the mixture is s~:irred far 1 hour,
ethyl acetate (25 ml) is then added and the precipitate
is filtered off. The filtrate is concentrated and recrys-
tallization from chlorobenzene gives pale yellow 2'-(suc-
cinimidoyloxycarbonyl)phenylacridine-9-thiocarboxylate.
Yield: 80% melting point: 198-20l?°C
IR: 1810 cm 1, 1786, 1745, 1225, 1205
NMR (DMSO, 100 MHz): ~= 2.95 ppm (s, 4H), 7.7-8.4 ppm
(m, 12H)
2'-(Succinimidoyloxycarbonyl)phenyl-10-methylacridinium
9-thiocarboxylate tetrafluoborate (b)
3 g of N-hydroxysuccinimide ester (5> are heated at 80°C
with 7.8 g of trimethyloxonium tetrafluoborate in 40 ml
of 1,2-dichloroethane for 8 hours and the mixture is sub-
sequently stirred overnight at room temperature. The pre-
cipitate is filtered off and extracted by boiling with
1,2-dichloroethane. The combined organic phases are
concentrated and the residue is recrystallized from
acetone-diisopropyl ether.
Yield: 40% melting point: 245°C
IR: 3440 cm 1 (br), 1800, 1780, 1740(s), 1b70, 1065(s)
NMR (DMSO, 100 MHz); cS= 3.0 ppm (s, 4H), 4.95 ppm (s,
slightly spread, 3H), 7.9-8.6 (m, 10H>, 9.9 ppm (d, 2N)
Example 2
The preparation of succinimidoyloxycarbonylmethyl-10-
methylacridinium 9-thiocarboxylate tetrafluoborate, start-
ing from the acid chloride (3> and thioglycolic acid is
carried out analogously to the synthesis of (6). The
yields of the individual synthesis steps and characteriz-
ation of the products (7) to (9) by spectroscopy are
given below:
X41 40 2
- 1b -
Carboxymethylacridine-9-thiocarboxylate (7)
Yield: b0% melting point: 218oC (with decomposition)
IR: 3440 cm 1 (6r), 2400(6r), 1950(6r>, 1710(m), 1660(s),
1070(m)
NMR (DMSO, 100 MHz): 6= 4.25 ppm Cs, 2H); 7.6-$.4 (m, 8H)
Succinimidoyloxycarbonylmethylacridine-9-thiocarboxylate ($)
Yield: 80%
IR: 3440 cm 1 (6r), 2930, 1820, 1785, 1740(s), 1205,
1165
NMR (DMSO, 100 MHz): b= 2.95 ppm (s, 4H), 4.77 ppm (s,
2H), 7.6-8.3 ppm (m, 8H)
Succinimidoyloxycarbonylmethyl-10-methylacridinium 9-
thiocarboxylate tetrafluoborate (9)
Yield: 40% melting point: 250oC
IR: 3440 cm 1 (6r), 1810, 1780, 1735(s), 1538, 1350, 1060
NMR (DMSO, 100 MHz): ~= 2.9 ppm Cs, 4H), 4.8 (s, 2H),
4.9 pmm, (s, 3H), 7.7-9.0 (m, 8H)
Example 3
N-Benzenesulfonyl-N-(benzyloxycarbonylmethyl)acridine-9-
carboxylic acid amide C10)
130 g of 4-(dimethylamino)-pyridine and 6 ml of triethyl-
amine are added to 3.3 g of N-benzenesulfonylglycine benzyl
ester in 110 ml of tetrahydrofuran, 3 g of acridine-9-
carboxylic acid chloride hydrochloride are added after 10
minutes and the suspension formed is heated under reflux
for b hours. The precipitate is filtered off with suction,
the solvent is stripped off and the residue is taken up
in methylene chloride and stirred briefly with 2 N NaOH.
After drying over MgS04, the organic phase is concen-
trated and the resulting residue is recrystallized from
toluene/heptane.
1 ~4~ 40 2
- 17 -
Yield: 70% melting point: 58oC
IR: 3440 cm-1 (br), 1735, 1680, 1357, 1165
NMR (DMSO, 100 MHz), b= 5.2 ppm (s, 2H), 5.3 ppm (s, 2H),
7.0-8.4 ppm (m, 18H)
N-Benzenesulfonyl-N-(carboxymethyl)acridine-9-carboxylic
acid amide (11)
1 g of N-benzenesulfonyl-N(benzyloxycarbonylmethyl)acri-
dine-9-carboxylic acid amide in 60 ml of glacial acetic
'10 acid is hydrogenated at room temperature and normal
pressure with the addition of 2 ml of concentrated HCl
and Pd/C (10%); when the reaction has ended, the catalyst
is filtered off with suction and concentration of the fil-
trate gives the carboxylic acid as a yellow solid.
NMR (DMSO, 100 MHz): 8~. 5.0 ppm (s, 2H), 7.1-8.5 ppm
(m, 13H)
The compound (11) is reacted with N-hydroxysuccinimide
analogously to the preparation of (5) to give N-benzene-
sulfonyl-N-(succinimidoyloxycarbonylmethyl)acridine-9-carb-
oxylic acid amide (12). t12) is quaternized with tri-
methyloxonium tetrafluoborate, as described for (6>, to
give N-benzenesulfonyl-N-(succinimidoyloxycarbonylmethyl)-
10-methylacridinium 9-carboxylic acid amide tetrafluobor-
ate (13>.
Example 4
N-Phenyl-N(4-benzyloxycarbonylbenzenesulfonyl)acridine-9-
carboxylic acid amide (14)
360 mg of 4-(dimethylamino)pyridine and 16.6 ml of tri-
ethylamine are added to 11 g of benzyl 4-(N-phenylsulf-
amido)benzoate in 300 ml of methylene chloride, 8.34 g of
acridine-9-carboxylic acid chloride hydrochloride (3) are
added after 10 minutes and the mixture is heated under
reflux for 16 hours. The cooled solution is stirred
briefly with 2 N NaOH and the organic phase is separated
off, washed with water, dried over Na2S04 and concentrated.
The residue is recrystallized from toluene/heptane.
:~41 40 2
- 18 -
Yield: 70% melting point: 1b1 - 1fi3°C
NMR (DMSO, 100 MHz): b= 5.5 ppm (s, 2H), ~S= 6.8-8.6 ppm
(m, 22H)
N-Phenyl-N-(4-carboxybenzenesulfonyl)acridine-9-carboxylic
acid amide hydrobromide (1S>
8.58 g of N-phenyl-N-(4-benzyloxycarbanylbenzenesulfonyl)-
acridine-9-carboxylic acid amide (14) are heated at 60°C
in 30 ml of 33% strength HBr in glacial acetic acid for
2 hours and, after cooling, 60 ml of diisopropyl ether
are added and the precipitate is filtered off with suction
and dried in vacuo.
Yield: 95% melting point: 255°C
NMR (DMSO, 100 MHz): b = 6.8-9 ppm (m)
N-Phenyl-N-(4-succinimidoyloxycarbonylbenzenesulfonyl)-
acridine-9-carboxylic acid amide (1b)
2.8 ml of triethylamine are added to 5.63 g of N-phenyl-
N-(4-carboxybenzenesulfonyl)acridine-9-carboxylic acid
amide hydrobromide (1S) in 250 ml of tetrahydrofuran, the
mixture is cooled to -15°C and 0.9b ml of ethyl chloro-
formate is added. The mixture is subsequently stirred
for 20 minutes, 1.15 g of Nwhydroxysuccinimide are added
and the mixture is stirred at -15°C for 3 hours, allowed
to thaw to room temperature and is subsequently stirred
overnight. The precipitate is filtered off with suction,
the filtrate is concentrated, the residue is taken up in
methylene chloride and the resulting solution is washed
with water, NaHC03 solution and water and dried over
Na2S04. The organic phase is concentrated and the resi-
due is recrystallized from toluene.
Yield: SO% melting point: 226°C (decomposition)
NMR (DMSO, 100 MHz>: 8= 2.95 ppm (s, 4H), 8= 6.8-8.T
ppm (m, 17H)
N-Phenyl-N-(4-succinimidoyloxycarbonylbenzenesulfonyl)-10-
methylacridinium 9-carboxylic acid amide fluorosulfonate (17>
1.16 g of N-phenyl-N-(4-succinimidoyloxycarbonylbenzene-
sulfonyl)acridine-9-carboxylic acid amide (16) are stirred
~ 341 40 2
- 19 -
in 60 ml of 1,2-dichloroethane with 0.3 ml of methyl
fluorosulfonate at room temperature for 24 hours and the
precipitate which has separated out is filtered off with
suction and dried in vacuo.
Yield: 65%
IR: 3420 cm 1 (br), 3100(br), 1805(w), 1770(m), 1745(s>,
1700(m>, 1385(m), 1280(m), 1255(s), 1230(s), 1205(s)
NMR (DMSO, 100 MHz): b= 2.95 ppm (s, 4H), s= 4.75 ppm
(s, br, 3H), ~= 7.0-9.0 ppm (m, 17H)
Mass spectrum: m/z - 594 . M+ (ration)
Example 5
The preparation of N-(4-methoxyphenyl)-N-(4-succinimidoyl-
oxycarbonylbenzenesulfonyl)-10-methylacridinium 9-carb-
oxylic acid amide fluorosulfonate (21) starting from
benzyl 4-CN-(4'-methoxyphenyl)sulfamido7-benzoate and
acridine-9-carboxylic acid chloride hydrochloride (3> is
carried out analogously t° the synthesis of (17) (see
Example 4). The yields of the individual synthesis steps
and the characterization by spectr°scopy are given below.
N-(4-Methoxyphenyl)-N-(4-benzyloxycarbonyl-benzenesulfonyl)-
acridine-9-carboxylic acid amide (18)
Yield: ?0% melting point: 182-183'°C
NMR (DMSO, 100 MHz): ~= 3.S ppm (s, 3H), &= 5.5 ppm (s,
2H), b= 6.35-6.63 ppm (d, br, 2H), ~_ 1.05-7.2 ppm (d,
br, 2H), 8= 7.35-8.5 ppm (m, 17H)
N-(4-Methoxyphenyl)-N-(4-carboxybenzenesulfonyl)acri-
dine-9-carboxylic acid amide hydrobromide (19)
Yield: 95% melting point: 273°C (decomposition)
NMR (DMSO, 100 MHz): &= 3.5 ppm (s, 3H), ~= 6.4-b.b ppm
(d, br, 2H>, b= 7.05-7.2 ppm (d, br, 2H), s= 7.7-8.5 PPm
(m, 12H)
N-(4-Methoxyphenyl)-N-(4-succinimidoyloxycarbonylbenzene-
sulfonyl)acridine-9-carboxylic acid amide (20)
Yield: 50% melting point: 232-234°C
NMR (DMSO, 100 MHz): &= 2.95 ppm Cs, 4H), 6= 3.5 ppm
1 341 40 2
- 20 -
(s, 3H), b= b.4-b.b ppm (d, br, 2H), ~= 7.05-7.25 ppm
(d, br, 2H), S= 7.8-8.6 ppm (m, 12H)
IR: 3050 cm-1 1805 (w), 1780(m>, 1740(s>, 1700(m),
1505(m), 1370(m), 1250(m), 1200(s), 1185
N-(4-Methoxyphenyl)-N-(4-succinimidoyloxycarbonylbenzene-
sulfonyl)-10-methylacridinium-9-carboxylic acid amide
fluorosulfonate (21)
The substance does not precipitate out on reaction and
is obtained by concentrating the solution and stirring
the residue with diisopropyl ether.
Yield: 80%
NMR (DMSO, 100 MHz): S= 2.95 ppm (s, 4H), b= 3.5 ppm (s,
3H), b= 4.8 ppm (s, br, 3H>, &= 6.45-b.7 ppm (d, br,
2H), b= 7.2-7.4 ppm (d, br, 2H), ~= 7.7-9 ppm (m, 12H)
Mass spectrum: mJz - 624 M+ (can on)
IR: 3440 cm 1 (br), 3100, 2950, 1805Cw), 1775(m), 1740(s),
1b95(m), 1610(m), 1505(m), 1375(rn), 1280(m), 1250(s),
1205(s).
2'. 0
Example b
The preparation of N-(4-methoxyphenyl3-N-(3-succinimidoyl-
oxycarbonylbenzenesulfonyl)-10-methylacridinium 9-carb-
oxylic acid amide fluorosulfonate (25) starting fram
benzyl 3-CN-(4'-methoxyphenylsulfamidoJ-benzoate and
acridine-9-carboxylic acid chloride hydrochloride (3) is
carried out analogously to the synthesis of (17) (see
Example 4). the yield of the individual. synthesis steps
and the characterization by spectroscopy are given below.
N-(4-Methoxyphenyl)-N-(3-benzyloxycarbonylbenzenesulfonyl)-
acridine-9-carboxylic acid amide (22>
Yield: 70% melting point: 168-170oC
NMR (DMSO, 100 MHz): b= 3.5 ppm (s, 3H), ~ 5.45 ppm (s,
2H), b= 6.5 ppm (s, br, 2H), &= 7.1 ppm Ibr, 2H),
8= 7.3-8.8 ppm (m, 17H)
1 341 40 2
- z1 -
N-(4-Methoxyphenyl>-N-(3-carboxybenzenesulfonyl>acridine-
9-carboxylic acid amide hydrobromi_de (23)
Yield: 90% melting point: 264°C
NMR (DMSO, 100 MHz): 8= 3.5 ppm (s, 3H), b= 6.4-b.b ppm
(d, br, 2H), 8= 7.0-7.2 ppm (d, br, 2H), b= 7.b-8.8 ppm
(m, 12H) .
N-(4-Methoxyphenyl)-N-(3-succinimidoyloxycarbonylbenzene-
sulfonyl)acridine-9-carboxylic acid amide (24)
Yield: 50% melting point: 223-225°C
NMR (DMSO, 100 MHz): S= 2.95 ppm (s, 4H), b= 3.5 ppm (s,
3H), S= 6.4-6.6 ppm (d, br, 2H), b= 7.0'7.2 ppm (d, br,
2H), 8=7.4-8.9 ppm (m, 12H)
IR: 3500 cm 1 (br), 3060, 2950, 2840, 1805 (w>, 1785(m),
1740(s), 1700(m>, 1510(m), 1380(m), 1250(m>, 1205(m>,
1165(m)
N-(4-Methoxyphenyl)-N-C3-succinimidoyloxycarbonylbenzene-
sulfonyl)-10-methylacridinium 9-carboxylic acid amide
fluorosulfonate (25)
Yield: 90%
NMR (DMSO, 100 MHz): 8= 2.95 ppm (s, 4H), 8= 3.55 ppm
(s, 3H), b= 4.8 ppm (s, br, 3H), S= 6.45-b.7 (d, br, 2H),
b= 7.05-7.3 ppm (d, br, 2H), S= 7.5-8.9 ppm (m, 12H)
IR: 3500 cm 1 (br), 3080, 2950, 1805 (w), 1780(m),
1740(s), 35 1700(m>, 1b10(m), 1510(m), 1380(m), 1250(s),
1205(s), 1170(s)
Mass spectrum: m/z - 624 M+ (cation)
Example 7
Tracer preparation for a-fetoprotein chemiluminescence
immunoassay
100 ul of antibodies (1 mg/ml), 11.5 Nl of the acridinium
thioester prepared according to Example 1 (compound 6)
(1 mg/ml in DMSO and 600 ~l of conjugation buffer (0.01 M
phosphate, pH 8.0 » are incubated far 15 minutes. 200 ul
of lysine (10 mglml) are then added and the mixture is
1 341 40 2
''' - 2 2
incubated for a further 15 minutes. This batch is trans-
ferred to a F~D 10 column (~ephadex'* G 25 medium, 0.1 M
phosphate, pH 6.3 as the mobile phase). 10 drops/fraction
are collected. The individual fractions are tested for
their chemiluminescence activity after appropriate dilu-
tion (350 ul of oxidizing agent; 0.1X of H202 in 0.1 N
NaOH). The tracer fractions (1st activity peak) are pooled
and stored at 4°C.
Example 8
a-Fetoprotein chemiluminescence immunoassay procedure
50 ul of standard/sample and 150 ul of buffer (phosphate;
0.1 M pH 6.3, 1~ of Tween* ,~0, 0.1~ of bovine serum albumin,
0.1 M NaCI, 0.01X of NaN3) are shaken for 15 minutes in
tubes coated with monoclonal anti-AFP antibodies. Wash-
ing with 2 x 1 ml partions of buffer is then carried out.
200 ul of tracer are added in an appropriate dilution and
the mixture is shaken for 15 minutes. washing with 2 por-
tions of 1 ml of buffer is again carried out. The chemi-
luminescence measurement is started with 350 ul of oxi-
dizing agent (0.1% of H202 in 0.1 N Na~H, measurement
time of 2 seconds).
Figure 3 shows the typical course of a standard curve of
an immuno-chemiluminometric assay (TCMA) for the a-fetopro-
tein (AFP).
* denotes trade mark
