Phenothiazine derivatives, their production and use

There is disclosed a novel phenothiazine derivative of the general formula (I): ##STR1## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or different and are a C.sub.1-6 alkyl group and at least one of them has as a substituent a group which can react with amino group, thiol group or carboxyl group, and X.sup.- is a counter ion of the phenazathionium. Their production and the intermediates used in the production are also disclosed. The compound (I) is a derivative of methylene blue and is applicable to photodynamic therapy of cancer or immunoassays utilizing chemiluminescence.

FIELD OF THE INVENTION 
The present invention relates to novel phenothiazine derivatives, their 
production and use. The compounds of the present invention are derivatives 
of methylene blue, which is one of photosensitive dyes, and are capable of 
binding to antibody proteins and the like. Whereby the compounds of the 
present invention are expected to be applicable to photodynamic therapy of 
cancer or immunoassays utilizing chemiluminescence. 
BACKGROUND OF THE INVENTION 
As practical examples utilizing an oxidative characteristic reaction of 
photosensitive dyes, disinfection and sterilization of service water with 
methylene blue are well known. 
Various studies have been hitherto done on light excitation activities of 
photosensitive dyes such as xanthines (e.g., rose bengal, fluorescein, 
eosin, erythrosine, etc.), phenothiazines the representative example of 
which is methylene blue, porphyrins and the like. And, there is a tendency 
to increase in the use of photosensitive dyes in the fields of clinical 
drugs or diagnostics of cancer. 
Among these photosensitive dyes, methylene blue has extremely strong 
photo-oxidation activity and is advantageous in that it absorbs light of 
long wavelength (670 nm) which is readily passed through a living body 
tissue. As reported by Foote in "Mechanisms of Photosensitized Oxidation", 
Jan. 29, 1968, Vol. 162, pp. 963-970, it is considered that the activities 
due to excitation of methylene blue by light is mainly resulted from two 
reactions. That is, it is considered that, in the case of Type I light 
excitation reaction, an excited dye directly reacts with a substrate and 
that, in the case of Type II light excitation reaction, firstly, an 
excited triplet dye reacts with molecular oxygen to produce singlet 
oxygen, and then a substrate is oxidized with the singlet oxygen. Which 
type of the reaction occurs depends on concentrations of particular dye, 
dissolved oxygen and substrate to be used. 
Recently, among these photosensitive dyes, in particular, the use of 
porphyrins in treatment of cancer has been reported (Analytical Chemistry, 
Vol. 61, Dec. 15, 1989, pp. 1367-1375). According to this report, a 
porphyrin is injected into a cancer tissue or in the vicinity thereof and 
light energy is provided from outside of the body to excite the 
porphyrins. Whereby singlet oxygen is produced which causes a lethal 
effect on cancer cells. One of the important characteristics of these dyes 
is that the dyes can absorb light of long wavelength (&gt;650 nm) which can 
be passed through a living body tissue. 
Recently, as an attempt at such a cancer treatment, an improved technique 
has been reported in which specificity in an antigen-antibody reaction is 
utilized. That is, in this technique, a photosensitive porphyrin dye is 
bound to an antibody against cancer, whereby the cancer cells per se which 
are the antigen are attacked specifically (JO 2059-585-A, EP 252683). 
However, it is not easy to bind most of these photosensitive dyes to an 
antibody protein. 
Thus, if a highly reactive photosensitive dye is readily available which is 
easy to bind to proteins and is capable of absorbing light of long 
wavelength (&gt;650 nm), significant improvements are expected in this art. 
Another important use of photosensitive dyes is that in the field of 
diagnostics. For example, the photosensitive dye as described herein can 
be used for labeling an antibody, hapten or nucleic acid (DNA or RNA) 
probe. The labeled antibody, hapten or nucleic acid probe thus obtained is 
used to produce a signal indicating the original amount of an analyte in 
the course of a clinical chemical analysis. The signal is derived from the 
labeled photosensitive dye and a representative example thereof is color 
development, fluorescence or chemiluminescence. However, this technique 
has scarcely been employed because it is difficult to obtain a highly 
reactive derivative of photosensitive dye which is capable of covalently 
binding to a protein, hapten or nucleic acid. 
The desired derivative of a photosensitive dye is that having an active 
functional group which can readily react with a protein, hapten or nucleic 
acid under normal reaction conditions. One of these active functional 
groups is a succinimido ester group which can react with an amino group of 
proteins or nucleic acids. Another group is a maleimide group which can 
react with a thiol group of proteins. However, it is difficult to 
introduce these functional groups directly into methylene blue dye. This 
is clear from the fact that any methylene blue derivative capable of 
modifying proteins or nucleic acids is not yet found even though methylene 
blue has been studied for more than 50 years. 
When an aromatic compound having low molecular weight such as a dye is 
bound to a protein, hapten (e.g., thyroid hormone) or nucleic acid, 
problems often arise such as sedimentation of a protein-dye conjugate and 
non-specific binding of the protein, hapten or nucleic acid to the surface 
of a solid phase. These problems are caused by hydrophobic nature of the 
dye in an aqueous solution, which results in a low solubility of the 
protein-dye conjugate and non-specific absorption of the protein, hapten 
or nucleic acid to the surface of the solid phase. Thus, if any technique 
to improve such an instability of a protein-dye conjugate or to prevent 
such non-specific adsorption, it is possible to improve conventional 
techniques to a great extent. 
OBJECTS OF THE INVENTION 
Under these circumstances, the present inventors have synthesized and 
studied various reactive derivatives of methylene blue. As a result, it 
has been found that introduction of a hydrophilic group such as a carboxyl 
group or the like into the methylene blue skeleton is effective for 
improving instability of a protein-dye conjugate. Thus, the present 
invention has been completed. 
An object of the present invention is to provide a novel photosensitive dye 
derivative which is capable of modifying antibody proteins and the like. 
Another object of the present invention is to provide an immunoassay method 
using the novel photosensitive dye derivative. 
These objects as well as other objects and advantages of the present 
invention will become apparent to those skilled in the art from the 
following description with reference to the accompanying drawings.

SUMMARY OF THE INVENTION 
According to the present invention, there are provided: 
(1) A phenothiazine derivative of the general formula (I): 
##STR2## 
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same or different 
and are respectively a C.sub.1-6 alkyl group, at least one of which has as 
a substituent a group which can react with amino group, thiol group or 
carboxyl group, and X.sup.- is a counter ion of the phenazathionium; 
(2) A phenothiazine derivative of the general formula (II): 
##STR3## 
wherein at least one of R.sub.1 ', R.sub.2 ', R.sub.3 ' and R.sub.4 ' is a 
group of the formula: --(CH.sub.2).sub.m --CO--Y, the others are C.sub.1-6 
alkyl group, Y is a leaving group, m is an integer of 1 to 6 and X.sup.- 
is a counter ion of the phenazathionium; 
(3) An indaminethiosulfonic acid derivative of the general formula (III): 
##STR4## 
wherein R.sub.1 ', R.sub.2 ', R.sub.3 ' and R.sub.4 ' are as defined 
above; 
(4) A 2-amino-5-disubstituted aminophenyl thiosulfonic acid derivative of 
the general formula (IV): 
##STR5## 
wherein Y and m are as defined above, and R.sub.1 " is a C.sub.1-6 alkyl 
group; 
(5) An indaminethiosulfonic acid derivative of the general formula (V): 
##STR6## 
wherein R.sub.1 ', R.sub.2 ' and R.sub.3 ' are as defined above; (6) A 
process for producing the phenothiazine derivative of the general formula 
(I), which comprises the steps of: 
removing the group Y of the phenothiazine derivative of the general formula 
(II), and 
introducing a group which can react with amino group, thiol group or 
carboxyl group; 
(7) A process for producing the phenothiazine derivative of the general 
formula (I), which comprises the steps of: 
subjecting the indaminethiosulfonic acid derivative of the general formula 
(III) to oxidative ring closure with an inorganic oxidizing agent in 
water, an organic solvent or a mixed solvent thereof to obtain the 
phenothiazine derivative of the general formula (II), 
removing the group Y, and 
introducing a group which can react with amino group, thiol group or 
carboxyl group; 
(8) A process for producing a compound of the general formula (VII): 
##STR7## 
wherein B is succinimidoxycarbonyl group or maleimido group, m is an 
integer of 1 to 6 and n is 1 or 2, which comprises the steps of: 
reacting a succinimido ester derivative of the general formula (VI): 
##STR8## 
wherein B and m are as defined above, with a triethylamine salt of an 
acidic amino acid; 
(9) A method for the determination of an analyte in a sample comprising 
measuring a conjugate formed by coupling the phenothiazine derivative of 
the general formula (I) to an antigen or antibody; and 
(10) A labeled conjugate of an antigen or antibody comprising the 
phenothiazine derivative of the general formula (I) coupled to the antigen 
or antibody through amino group, thiol group or carboxyl group of the 
latter. 
DETAILED EXPLANATION OF THE INVENTION 
In R.sub.1 to R.sub.4 of the compound of the above general formula (I), the 
group which can react with amino group, thiol group or carboxyl group is 
not specifically limited. The group may be any one which can react with 
analytes in immunoassays and the like, such as proteins, haptens, 
peptides, nucleic acids, amino acids and the like. Examples of the group 
which can react with amino group include carboxyl group, halogenocarbonyl 
groups (e.g., chlorocarbonyl group, etc.), azidocarbonyl group, 
cyanocarbonyl group, alkoxycarbonyloxycarbonyl groups (e.g., 
ethoxycarbonyloxycarbonyl group, isobutoxycarbonyloxycarbonyl group, 
etc.), substituted phenoxycarbonyl groups (e.g., p-nitrophenoxycarbonyl 
group, 2,4-dinitrophenoxycarbonyl group, pentachlorophenoxycarbonyl group, 
2,4,5-trichlorophenoxycarbonyl group, pentafluorophenoxycarbonyl group, 
etc.), 1-imidazolylcarbonyl group, 1-benzotriazolyloxycarbonyl group, 
5-norbornene-2,3-dicarboximidoxycarbonyl group, succinimidoxycarbonyl 
group, maleimidoxycarbonyl group, aspartic acid residue, glutamic acid 
residue and the like. Examples of the group which can react with thiol 
group include maleimido group and the like. Examples of the group which 
can react with carboxyl group include an aminoalkylcarbamoyl groups (e.g., 
aminoethylcarbamoyl group, aminodecylcarbamoyl group, etc.) and the like. 
In view of the object of the present invention, at least one of R.sub.1 to 
R.sub.4 groups should be such a reactive group. Further, in view of the 
object of the present invention, the compound of the general formula (I) 
is preferably hydrophilic. The number of such a reactive group is suitably 
selected with taking into account the combination with the other groups of 
R.sub.1 to R.sub.4. The C.sub.1-6 alkyl group represented by R.sub.1, 
R.sub.2, R.sub.3 and R.sub.4 respectively may be the same or different and 
are a straight or branched chain C.sub.1-6 alkyl group such as methyl, 
ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, 
neopentyl, hexyl, isohexyl or the like. In view of the structural 
similarity to methylene blue and hydrophilic nature, methyl and ethyl are 
especially preferred. The C.sub.1-6 alkyl group of R.sub.1 ' to R.sub.4 ' 
and the other groups included in the present specification is as defined 
above. 
As the group represented by X in the above general formulas (I) and (II) 
which can form a counter ion of the phenazathionium, there are mentioned 
halogens (e.g., chlorine, bromine, iodine, fluorine), hydroxyl, perchloric 
acid, sulfuric acid, nitric acid, trifluoroacetic acid, hydrofluoroboric 
acid and the like. 
The leaving group represented by Y of the group --(CH.sub.2).sub.m --CO--Y 
in the above general formulas (II), (III), (IV) and (V) may be a 
conventional leaving group used in the field of peptide synthesis, 
.beta.-lactam antibiotic synthesis or the like. Examples thereof include 
lower alkoxy groups having 1 to 4 carbon atoms such as methoxy, ethoxy, 
propoxy, sec-butoxy, tert-butoxy and the like; aralkyloxy groups such as 
benzyloxy, p-nitrobenzyloxy, p-methylthiobenzyloxy, diphenylmethyloxy, 
4-pyridylmethoxy and the like; and 1-phenoxy-ethoxy group and the like. 
The compound of the general formula (I) can be obtained, for example, by 
removing Y of the phenothiazine derivative of the general formula (II) and 
then introducing the group which can react with amino group, thiol group 
or carboxyl group. For example, it can be produced according to the 
process illustrated in Chart 1. 
##STR9## 
That is, 
(1) Among the phenothiazine derivatives of the general formula (I), the 
compound wherein at least one of R.sub.1 to R.sub.4 is a group which can 
react with amino group can be obtained according to a known method by 
subjecting the compound of the general formula (II) to hydrolysis, 
catalytic reduction or acidolysis and then converting the resulting 
carboxylic acid derivative into its halide, azide, mixed acid anhydride or 
activated ester. 
Examples of the group represented by --COA in the Chart 1 which can react 
with amino group include the groups include those described with respect 
to the above R.sub.1 to R.sub.4 other than aspartic acid residue and 
glutamic acid residue. 
Normally, hydrolysis of the ester of the formula (II) can be carried out in 
a 1N solution of a mineral acid or caustic alkali at a reaction 
temperature of 20.degree. to 30.degree. C. The reaction time required is 
normally 20 minutes to 48 hours. In general, however, the yield is low 
under these conditions because degradation of the phenothiazine skeleton 
proceeds simultaneously. This tendency is especially pronounced under 
alkaline conditions. When the leaving group in the compound (II) is an 
aralkyloxy group such as benzyloxy, p-nitrobenzyloxy, 4-pyridylmethoxy, 
diphenylmethyloxy or the like, it can be removed by catalytic reduction. 
Normally, the reaction is carried out by treatment with a palladium 
catalyst in an inert solvent at ordinary temperature and pressure in a 
stream of hydrogen. Although, when the ester group is removed, the 
phenothiazine skeleton is also reduced to form a phenothiazine derivative 
in the form of leuco, it is oxidized with air rapidly to the original 
phenothiazine. On the other hand, an alkoxy group such as tert-butoxy, an 
aralkyloxy group such as p-methylthiobenzyloxy, diphenylmethyloxy or the 
like, or 1-phenoxyethoxy group can be removed readily by treatment in 
formic acid or trifluoroacetic acid. Normally, the reaction is completed 
in 30 to 60 minutes at a reaction temperature of 0.degree. to 25.degree. 
C. The organic acid can be used alone or as a mixture with an inert 
solvent. Among the leaving groups in the general formula (II), in view of 
the ease of the removal and purification after the removal, tert-butoxy 
group is the most preferred. That is, when the leaving group is 
tert-butoxy group, the product can be used in the next step merely by 
concentrating it to dryness after completion of the reaction. If further 
purification is required, the product can be subjected to silica gel 
column chromatography (eluent: MeCN/H.sub.2 O/1N HCl=40:5:1). 
The carboxylic acid derivative thus obtained can be converted into a 
reactive derivative such as an acid chloride, an acid azide, a mixed acid 
anhydride or an activated ester or the like in order to coupling the 
carboxylic acid derivative with a compound having an amino group. 
This step is further illustrated in detail below. 
Acid chloride method 
The carboxylic acid derivative is reacted with a chloride such as thionyl 
chloride, oxalyl chloride, trichloromethylchloroformate (diphosgene), 
bis-trichloromethylcarbonate (triphosgene) or the like in an inert solvent 
such as dichloromethane, chloroform, carbon tetrachloride, benzene or the 
like at 0.degree. to 25.degree. C. to obtain the corresponding acid 
chloride. Then the acid chloride is subjected to coupling with an amino 
compound in the presence of an organic tertiary amine such as 
triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine or 
the like. 
Acid azide method 
A hydrazide derivative derived from the carboxylic acid derivative is 
reacted with sodium nitrite in an acidic solution according to the method 
of Curtius [Chem. Ber., 35, 3226 (1902)]. The reaction mixture is 
neutralized. Then the resulting acid azide is extracted with a solvent and 
subjected to the coupling, or the neutralized reaction mixture can be 
subjected to the coupling in situ. Alternatively, this azidation can be in 
a non-aqueous solvent by using an alkyl nitrite instead of sodium nitrite 
[J. Rudinger, et al., Collect. Czech. Chem. Commun., 26, 2333 (1961)]. 
Further, this derivative can also be synthesized by using 
diphenylphosphoryl azide (DPPA) [T. Shioiri et al., J. Am. Chem. Soc., 94, 
6203 (1972)]. 
Mixed acid anhydride method 
As an acid component which forms a mixed acid anhydride with a carboxylic 
acid derivative, a monoalkyl carbonate is preferred. The mixed acid 
anhydride can be obtained by reacting a carboxylic acid derivative with 
ethoxycarbonyl chloride [T. Wieland et al., Ann. Chem., 572, 190 (1951); 
R. A. Boissonnas, Helv. Chim. Acta., 34, 874 (1951)] or isobutoxycarbonyl 
chloride [J. R. Vaughan et al., J. Am. Chem. Soc., 74, 676 (1952)] in the 
presence of an organic tertiary amine at -15.degree. to -5.degree. C. The 
mixed acid anhydride can react directly with an amino compound. 
Activated ester method 
A phenyl ester having an electron-withdrawing substituent is known as one 
of the activated esters used in peptide synthesis and is also applicable 
to the carboxylic acid derivative in the present invention. That is, 
esters formed with p-nitrophenol, 2,4-dinitrophenol, pentachlorophenol, 
2,4,5-trichlorophenol, pentafluorophenol and the like are preferred. On 
the other hand, O-acylhydroxylamine esters are known as the other 
important activated ester. Examples thereof include ester derivatives of 
1-hydroxybenzotriazole [W. Koenig et al., Chem. Ber., 103, 788 (1970)], 
N-hydroxysuccinimide [G. W. Anderson et al., J. Am. Chem. Soc., 85, 3039 
(1963); ibid. 86, 1839 (1964)], N-hydroxymaleimide (which can be produced 
according to the same manner as that of N-hydroxysuccinimide) or 
N-hydroxy-5-norbornene-2,3-dicarboximide [M. Fujino et al., Chem. Pharm. 
Bull., 22, 1857 (1974)] and the like. Further, esters formed with 
N-hydroxyphthalimide [G. H. L. Nefkeus et al., J. Am. Chem. Soc., 83, 1263 
(1961)] can be used according to the same manner. These activated ester 
derivatives can be obtained by reacting a mixture of both reactants with a 
condensing agent such as N,N-dicyclohexylcarbodiimide (DCC) or 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC: water 
soluble carbodiimide) or the like. 
In addition to the above methods, the reactive derivatives can be obtained 
as follows. 1-Acylimidazole can be obtained by the reaction with 
1,1'-carbonyldiimidazole (CDI) [S. Staab, Angew. Chem. Internat. Edit., 1, 
351 (1962)]. Acylcyanide can be obtained by the reaction with 
diethylphosphoric acid cyanide (DEPC) [T. Shioiri et al., Tetrahedron 
Lett., 1973, 1595]. Any reactive derivative can be used directly for the 
reaction with an amino derivative. 
Among the phenothiazine derivatives of the general formula (I) thus 
obtained, the compounds wherein at least one of R.sub.1 to R.sub.4 is a 
group which can react with amino group, can form peptide bond with amino 
compounds such as proteins, haptens, nucleic acids, amino acids and the 
like by selecting the above appropriate coupling method. The condensation 
method is not specifically limited and can be selected appropriately 
depending upon nature of a particular amino compound. 
In particular, as the coupling method which can also be used in a 
water-containing solvent, the azide method, the mixed acid anhydride 
method or the activated ester method is preferred. In view of stability, 
high reactivity of the reactive derivative and facility in purification 
after completion of the reaction, esters of O-acylhydroxylamines, 
especially, N-hydroxysuccinimide are most preferred. 
The succinimido ester can be produced, for example, as follows. The 
monoester can be obtained by reacting the equivalent amount of 
dicyclohexylcarbodiimide (DCC) or 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) at 
20.degree. to 40.degree. C. for 1 to 48 hours in the presence of 
N-hydroxysuccinimide (2 to 3 equivalents) in an anhydrous aprotic solvent 
such as acetonitrile, dimethylacetamide or dimethylformamide. In the case 
of a dicarboxylic acid, a mixture containing the monoester and diester the 
major portion of which is the monoester is obtained. In order to obtain 
the diester, it is preferred to use the reagent in an amount of 3 to 6 
equivalents. 
In these esterification, especially succinimide esterification, it is 
preferred to use an activated ester of N-hydroxysuccinimide such as 
N,N'-disuccinimidocarbonate (DSC) (H. Ogura et al., Tetrahedron Lett., 
1979, 4745) or N,N'-disuccinimidooxalate (DSO) [H. Ogura, Tetrahedron 
Lett., 24, 4451 (1983)]. Normally, it is preferred to use the above 
activated ester in an amount of 1.5 to 3 equivalents in the case of the 
monoesterification or 3 to 6 equivalents in the case of the 
diesterification in an inert solvent such as acetonitrile, 
tetrahydrofuran, dioxane, acetone or the like in the presence of an 
organic base such as pyridine, triethylamine or the like in an amount of 2 
equivalents based on the activated ester. 
Normally, the reaction is completed in 1 to 3 hours at 20.degree. to 
30.degree. C. When the starting material is a dicarboxylic acid 
derivative, it is often difficult to produce the monoester only. These two 
esters can be separated by column chromatography using Sephadex.RTM. LH-20 
(eluent: acetonitrile). The succinimido ester thus obtained is reacted 
with triethylamine salt of aspartic acid or glutamic acid in MeCN/H.sub.2 
O (3:1) at 25.degree. C. for 10 minutes to obtain the compound of the 
general formula (VII) or the general formula (VII') in the Chart 1. 
(2) Among the phenothiazine derivatives of the general formula (I), the 
compound wherein at least one of R.sub.1 to R.sub.4 is a group which can 
react with thiol group, namely a maleimido group, for example, the 
compound having a maleimidoalkyl group as R.sub.4 (the compound of the 
general formula (VI) and (VII) wherein B is a maleimido group) can be 
obtained by treating the compound of the general formula (II) wherein 
R.sub.4 ' is a maleimidoalkyl group obtained from the general formula (V) 
(i.e., the compound of the general formula (III) wherein R.sub.4 ' is a 
maleimidoalkyl group) according to the same manner as that of the compound 
having a group which can react with amino group. 
(3) Among the compounds of the general formula (I), the phenothiazine 
derivative wherein at least one of R.sub.1 to R.sub.4 is a group which can 
react with carboxyl group can be obtained by reacting the reactive 
derivative of phenothiazine carboxylic acid described (I) above with a 
diamino compound of the formula H.sub.2 N--(CH.sub.2).sub.p --NH.sub.2 
(wherein p is an integer of 2 to 10). The monoacylaminoalkylamine thus 
obtained is further reacted with another carboxyl group or its reactive 
derivative. 
The compound of the general formula (II) which is the starting material for 
the production of the compound of the general formula (I) can also be 
obtained by the process as shown in the Chart 2 according to a per se 
known synthesis method of methylene blue [A. Bernthsen, Annalen der 
Chemie, 251, 1 (1888); I. Tomioka, J. Chem. Ind. Tokyo Japan, 20, I-2 
(1917)]. 
##STR10## 
wherein at least one of R.sub.1 ', R.sub.2 ', R.sub.3 and R.sub.4 ' is 
--(CH.sub.2).sub.m --CO--Y, the other groups are a C.sub.1-6 alkyl group, 
Y is a leaving group and X.sup.- is a counter ion of the phenazathionium. 
However, the synthetic intermediate of the compound of the present 
invention is much more lipophilic than methylene blue, and hence any 
satisfactory result is not obtainable by mere application of the synthesis 
method of methylene blue wherein all the reaction solvents are water. 
Thus, as the reaction solvent, water or an organic solvent, especially 
acetonitrile or acetone is preferred. If necessary, the solvent is used in 
combination with them. As an oxidizing agent used in each oxidation step, 
a conventional inorganic oxidizing agent such as a dichromate, ferric 
chloride, manganese dioxide or the like can be used. As an oxidizing agent 
used in oxidation in a water-containing organic solvent which is 
characteristic of the present invention, a dichromate or manganese dioxide 
is preferred. 
More detailed illustration of each step is set forth below. 
A monoalkylaniline (wherein the alkyl is methyl or ethyl) is subjected to 
N-alkylation and nitrosation followed by reduction according to a 
conventional manner to obtain N,N-dialkyl-p-phenylenediamine. The 
N,N-dialkyl-p-phenylenediamine is reacted with a dichromate in an amount 
of 0.3 to 4.0 equivalents, preferably 0.3 to 2.0 equivalents in water or a 
water-containing organic solvent such as acetone, acetonitrile or the like 
in the presence of sodium thiosulfate in an amount of 1.2 to 3.5 
equivalents, aluminium sulfate in an amount of 1.0 to 3.0 equivalents and 
an acid (e.g., hydrochloric acid, sulfuric acid or acid potassium hydrogen 
sulfate) in an amount of 8.0 to 12.0 equivalents at a reaction temperature 
of 0.degree. to 40.degree. C., preferably 5.degree. to 25.degree. C. to 
obtain the compound of the general formula (IV). This compound can be 
produced by using manganese dioxide in an amount of 3.0 to 10.0 
equivalents instead of a dichromate. 
The compound of the general formula (IV) is reacted with a dichromate in an 
amount of 0.6 to 3.0 equivalents, preferably 0.6 to 1.5 equivalents at 
0.degree. to 40.degree. C., preferably 5.degree. to 25.degree. C. in 
water, water-containing acetone or water-containing acetonitrile in the 
presence of the compound of the general formula (VIII) in an equivalent 
amount and an acid (e.g., hydrochloric acid, sulfuric acid or potassium 
hydrogen sulfate) in an amount of 1.0 to 3.0 equivalents to obtain an 
indaminthiosulfonic acid derivative of the general formula (III). Further, 
as an oxidizing agent to be used in this reaction, manganese dioxide in an 
amount of 3.0 to 10.0 equivalents can also be used. 
The compound of the general formula (II) can be obtained by subjecting the 
indaminthiosulfonic acid derivative of the general formula (III) to 
oxidative ring closure with an inorganic oxidant in water, an organic 
solvent or a mixed solvent thereof. More specifically, the compound of the 
general formula (III) is treated in water, acetone, acetonitrile or a 
water-containing solvent thereof with manganese dioxide in an amount of 
5.0 to 20.0 equivalents, preferably 10.0 to 15.0 equivalents, more 
preferably in the presence of a catalytic amount of copper sulfate at 
20.degree. to 100.degree. C. to obtain the novel methylene blue derivative 
of the general formula (II). Furthermore, in the steps illustrated in the 
Chart 2, the compound (II) can be produced from the compound (IV) without 
isolating the intermediate (IV) and (III) by adding required reagents to 
the slurry of each step. 
The compound of the present invention can be coupled to various proteins 
such as an antibody fragment, avidin (basic sugar protein) and the like. 
When the photosensitive dye derivative is used for treatment of cancer, it 
is necessary to select a protein or biomolecule which is required for the 
growth of cancer cells and readily uptaken by cancer cells such as 
transferrin protein (iron transport protein). 
For this coupling, it takes 1 minute to 24 hours, preferably 4 to 12 hours. 
The coupling temperature is preferably 1.degree. to 80.degree. C. Although 
the coupling reaction is promoted at a higher temperature, heat 
denaturation of the protein is also caused. Therefore, normally, the 
temperature is suitably 1.degree. to 24.degree. C. The pH during the 
coupling is 4 to 10, preferably 6 to 8. Although, normally, the coupling 
reaction is carried out in an aqueous solution, there can also be used a 
solvent which does not have strong nucleophilicity and does not react with 
the reactive derivative such as alcohols, acetone and dimethylformamide. 
Likewise, any buffer salts for controlling the pH which does not react 
with the reactive derivatives can be used. For example, phosphates and the 
like are suitable, whereas tris(hydroxymethyl)aminoethane and the like are 
not suitable. 
Virtually all proteins have a primary amino group which can react with the 
compound of the present invention. When the compound of the present 
invention having a group which can react with an amino group is reacted 
with a compound having an amino group such as proteins, haptens or the 
like, the reaction is preferably carried out in the presence of a 
condensing agent in the case that the group which can react with an amino 
group above is a carboxyl group. Examples of the condensing agent include 
DCC, WSC and the like. It is necessary to ensure that no impurities having 
a mercapto group except reduced state of cysteine of a protein is present 
in a solution of the protein during its coupling to the maleimido 
derivative. An antibody protein is particularly recommended in the 
application to immunoassays or photodynamic therapy of cancer. The molar 
ratio of the reactive photosensitive dye derivative based on a protein 
during the coupling is more than 1, preferably 10 to 300. 
After the reactive dye derivative is coupled to a protein, the protein-dye 
conjugate thus formed can be separated from the unreacted dye by, for 
example, gel filtration chromatography. The protein-dye conjugate thus 
prepared can be stored in an aqueous solution for a long period. In a 
higher pH region (above 10), however, the dye is unstable and cannot be 
stored. 
The reactive dye derivative can be coupled to low molecular weight haptens 
such as medicaments or derivatives thereof, hormones or nucleic acids 
under the same conditions as those described above. The conjugate thus 
formed can be purified by means of chromatography and the like which can 
distinguish a product from unreacted reagents. 
In the application of the protein-dye conjugate to immunoassays, the 
conjugate is irradiated with light of appropriate wavelength which can 
excite the dye, and oxidative chemiluminescence is detected which is 
produced by the product from the excited dye in combination with a 
sensitive reagent such as luminol according to a conventional manner. 
When the protein-dye conjugate is used for photodynamic therapy of cancer, 
semiconductor laser light (670 nm) is especially preferred because light 
spectrum of the light source agrees well with absorption spectrum of the 
dye and it is readily available. 
According to the photosensitive dye derivatives of the present invention, 
covalently coupling of antibody proteins and the like which has been 
difficult can be readily attained. Further, the protein-dye conjugate 
precipitation and nonspecific binding to the surface of a solid phase 
which is a problem in covalently coupling of antibody proteins and the 
like can be avoided. 
The following Reference Examples and Examples further illustrate the 
present invention in detail but are not to be construed to limit the scope 
thereof. 
Reference Example 1 
N-(3-Ethoxycarbonylpropyl)-N-methyl-p-phenylenediamine 
Ethyl 4-(N-methylanilino)butyrate (J.C.S. Perkin I, 1972, 1803) (22 g) was 
dissolved by addition of conc. hydrochloric acid (60 ml) under 
ice-cooling. To the solution was added a solution of sodium nitrite (8 g) 
in water (10 ml) dropwise at 0.degree. to 4.degree. C. with vigorous 
stirring. The mixture was stirred at the same temperature for 30 minutes. 
Then zinc powder (30 g) was added at below 10.degree. C. The resulting 
mixture was further stirred for 30 minutes at 15.degree. C. The zinc was 
filtered off. The filtrate was made alkaline with sodium bicarbonate and 
then was extracted with ether. The ether layer was dried over anhydrous 
sodium sulfate and concentrated. The residue was subjected to column 
chromatography on silica gel and developed with ethyl acetate/hexane (1:1) 
to obtain N-ethoxycarbonylpropyl-N-methyl-p-phenylenediamine (9 g). 
NMR (CDCl.sub.3) .delta.: 1.21 (3H, t, J=7 Hz), 1.84 (2H, quintet, J=7 Hz), 
2.30 (2H, t, J=7 Hz), 2.80 (3H, s), 3.1-3.5 (2H, br.), 3.20 (2H, t, J=7 
Hz), 4.10 (2H, q, J=7 Hz), 6.63 (4H, s). 
IR (neat) .nu.: 3450, 3350, 1730, 1520, 1370, 1265, 1175, 815 cm.sup.-1. 
Reference Example 2 
Ethyl 5-(N-methylanilino)valerate 
A mixture of N-methylaniline (16 g) and ethyl 5-bromovalerate (30.2 g) was 
stirred at 110.degree. C. for 22 hours. Then the mixture was made alkaline 
with an aqueous sodium bicarbonate. The resulting mixture was extracted 
with ethyl acetate, dried over anhydrous magnesium sulfate and 
concentrated. The residue was distilled under reduced pressure to obtain 
ethyl 5-(N-methylanilino)valerate (28.6 g). 
b.p: 124.degree.-128.degree. C./0.4 mmHg 
NMR (CDCl.sub.3) .delta.: 1.25 (3H, t, J=7.2 Hz), 1.66 (4H, m), 2.33 (2H, 
t, J=8 Hz), 2.91 (3H, s), 3.32 (2H, t, J=7 Hz), 4.12 (2H, q, J=7.2 Hz), 
6.67 (3H, m), 7.22 (2H, m). 
Reference Example 3 
Ethyl 6-(N-methylanilino)hexanoate 
A mixture of N-methylaniline (25 g) and ethyl 6-bromohexanoate (50 g) was 
stirred at 120.degree. C. for 24 hours. After cooling, an aqueous sodium 
bicarbonate was added to make the mixture alkaline. Then the resulting 
mixture was extracted with ethyl acetate, washed with brine and dried over 
anhydrous sodium sulfate. The mixture was concentrated under reduced 
pressure and distilled to obtain ethyl 6-(N-methylanilino)hexanoate (40.5 
g). 
b.p.: 141.degree.-143.degree. C./0.9 mmHg 
NMR (CDCl.sub.3) .delta.: 1.25 (3H, t, J=7.2 Hz), 1.37 (2H, m), 1.65 (4H, 
m), 2.30 (2H, t, J=7.4 Hz), 2.92 (3H, s), 3.30 (2H, t, J=7.4 Hz), 4.13 
(2H, q, J=7.2 Hz), 6.67 (3H, m), 7.22 (2H, m). 
Reference Example 4 
tert-Butyl 4-chlorobutyrate 
4-Chlorobutyryl chloride (14.1 g) was added dropwise with stirring under 
ice-cooling to a mixture of tert-butanol (18.5 g) and dimethylaniline 
(18.2 g). The resulting mixture was stirred for 2 hours in an oil bath 
(100.degree. C.). Ether (200 ml), water (100 ml) and 1N hydrochloric acid 
(50 ml) were added to the reaction mixture and mixed with shaking. The 
organic layer was washed successively with 1N hydrochloric acid, an 
aqueous sodium bicarbonate and brine and dried over anhydrous magnesium 
sulfate. The solvent was distilled off and then the residue was subjected 
to distillation under reduced pressure to obtain tert-butyl 
4-chlorobutyrate (13.2 g). 
b.p.: 94.degree.-95.degree. C./22 mmHg 
NMR (CDCl.sub.3) .delta.: 1.45 (9H, s), 2.05 (2H, quintet, J=7 Hz), 2.41 
(2H, t, J=7 Hz), 3.59 (2H, t, J=7 Hz). 
Reference Example 5 
tert-Butyl 4-(N-methylanilino)butyrate 
A mixture of tert-butyl 4-chlorobutyrate (10.72 g) obtained in Reference 
Example 4 and N-methylaniline (7.72 g) was stirred for 21 hours in an oil 
bath (100.degree. C.). A saturated solution (50 ml) of sodium bicarbonate 
was added to the reaction mixture. The mixture was extracted with ether 
(300 ml), dried over anhydrous magnesium sulfate and concentrated. Then 
the residue was subjected to distillation under reduced pressure to obtain 
tert-butyl 4-(N-methylanilino)butyrate (4.40 g). 
b.p.: 140.degree.-147.degree. C./1.5 mmHg 
NMR (CDCl.sub.3) .delta.: 1.45 (9H,s), 1.86 (2H, quintet, J=7 Hz), 2.26 
(2H, t, J=7 Hz), 2.92 (3H, s), 3.35 (2H, t, J=7 Hz), 6.6-6.8 (3H, m), 
7.1-7.3 (2H, m). 
Reference Example 6 
N-(3-tert-Butoxycarbonylpropyl)-N-methyl-p-phenylenediamine 
tert-Butyl 4-(N-methylanilino)butyrate (8.00 g) obtained in Reference 
Example 5 was dissolved in 80% acetic acid (30 ml). To the solution was 
added dropwise a solution of sodium nitrite (2.44 g) in water (3 ml) with 
stirring at below 10.degree. C. The resulting mixture was stirred for 30 
minutes at the same temperature. Then acetic acid (30 ml) was added and 
zinc powder (19 g) was added at below 10.degree. C. The resulting mixture 
was stirred for 30 minutes and then filtered. The filtrate was 
concentrated under reduced pressure and mixed with an aqueous sodium 
bicarbonate and ether with shaking. The ether layer was dried over 
anhydrous magnesium sulfate and concentrated under reduced pressure. The 
residue was subjected to column chromatography on silica gel and eluted 
with hexane/ethyl acetate (2:1) to obtain 
N-tert-butoxycarbonylpropyl-N-methyl-p-phenylenediamine (3.21 g) as brown 
oil. 
NMR (CDCl.sub.3) .delta.: 1.44 (9H, s), 1.81 (2H, quintet, J=7 Hz), 2.55 
(2H, t, J=7 Hz), 2.50-3.50 (2H, br.), 2.80 (3H, s), 3.19 (2H, t, J=7 Hz), 
6.65 (4H, s). 
Reference Example 7 
3-[N-(4-Ethoxycarbonylbutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
Ethyl 5-(N-methylanilino)valerate (0.25 g) obtained in Reference Example 2 
was dissolved by addition of 1N hydrochloric acid (1 ml) and water (14 ml) 
under ice-cooling. Then 2-amino-5-dimethylaminophenylthiosulfonic acid 
[Ann. Chem., 251, 1 (1988)] (0.25 g) was added. A solution of sodium 
dichromate 2 hydrate (0.2 g) in water (2 ml) was added dropwise at 
5.degree. to 10.degree. C. and the mixture was stirred at the same 
temperature for 30 minutes. Acetic acid (0.03 ml) was added to the 
reaction mixture. The resulting mixture was stirred at room temperature 
for additional 1 hour. The resulting precipitate was filtered and washed 
with water. Then the precipitate was suspended in acetonitrile (20 ml). 
Manganese dioxide (0.25 g) and catalytic amount of copper sulfate were 
added and resulting mixture was heated under reflux for 30 minutes. The 
reaction mixture was filtered and the filtrate was concentrated to dryness 
under reduced pressure. The residue was subjected to column chromatography 
on silica gel and eluted with acetonitrile followed by a mixed solvent of 
CH.sub.3 CN/H.sub.2 O/1N HCl (40:5:1) to obtain 
3-[N-(4-ethoxycarbonylbutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.29 g). 
NMR (D.sub.2 O) .delta.: 1.28 (3H, t), 1.67 (4H, m), 2.48 (2H, m), 3.17 
(9H, br. s), 3.55 (2H, m), 4.20 (2H, q), 6.80-7.50 (6H, m). 
IR (KBr) .nu.: 3420 (br.), 1725, 1600, 1490, 1440, 1390, 1340, 1245, 1180, 
1140, 885 cm.sup.-1. 
Reference Example 8 
3-[N-(4-Ethoxycarbonylbutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
Ethyl 5-(N-methylanilino)valerate (0.2 g) obtained in Reference Example 2 
was dissolved in 50% acetonitril-water (10 ml). Acetic acid (0.1 ml), 
2-amino-5-dimethylaminophenylthiosulfonic acid (0.2 g) and manganese 
dioxide (0.5 g) were added. The mixture was stirred at room temperature 
for 2 hours and further at 80.degree. C. for 5 hours. Then the reaction 
mixture was filtered and washed with acetonitrile. The filtrate was 
concentrated to dryness under reduced pressure. The residue was subjected 
to column chromatography on silica gel and eluted with CH.sub.3 CN 
followed by CH.sub.3 CN/H.sub.2 O/1N HCl (40:5:1) to obtain 
3-[N-(4-ethoxycarbonylbutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.11 g). 
Reference Example 9 
3-[N-(4-Carboxybutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
3-[N-(4-Ethoxycarbonylbutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.7 g) was dissolved in 1N hydrochloric acid (10 ml). The 
mixture was allowed to react at room temperature for 48 hours. The 
reaction mixture was subjected to column chromatography on silica gel and 
eluted successively with water, CH.sub.3 CN/H.sub.2 O (10:1) and CH.sub.3 
CN/H.sub.2 O/1N HCl (40:5:1). The desired fraction was concentrated and 
then lyophilized to obtain 
3-[N-(4-carboxybutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.7 g). 
NMR (D.sub.2 O) .delta.: 1.67 (4H, br. s), 2.49 (2H, br. s), 3.08 (9H, br. 
s), 3.45 (2H, br. s), 6.60-7.40 (6H, m). 
IR (KBr) .nu.: 3650-2800, 1720, 1600, 1485, 1390, 1335, 885 cm.sup.-1. 
EXAMPLE 1 
3-[N-Methyl-N-(4-succinimidoxycarbonylbutyl)amino]-7-dimethylaminophenazath 
ionium chloride 
3-[N-(4-Carboxybutyl)-N-methylamino]-7-dimethylamino phenazathionium 
chloride (0.34 g) obtained in Reference Example 9 was dissolved in 
dimethylformamide (100 ml). The mixture was concentrated to about 30 ml 
under reduced pressure. Then N-hydroxysuccinimide (0.255 g) and WSC (0.425 
g) were added and the mixture was allowed to react at room temperature for 
3 days. The solvent was distilled off under reduced pressure. The residue 
was then dissolved in dichloromethane, washed with saturated brine and 
dried over anhydrous sodium sulfate. The mixture was concentrated under 
reduced pressure and the residue was dissolved by addition of 
dichloromethane (6 ml). Then ethyl acetate (100 ml) was added. The 
resulting precipitate was filtered off and dried to obtain 
3-[N-methyl-N-(4-succinimidoxy-carbonylbutyl)amino]-7-dimethylaminophenaza 
thionium chloride (0.2 g). 
NMR (DMSO-d.sub.6) .delta.: 1.74. (4H, br. s), 2.77 (2H, br. s), 2.82 (4H, 
s), 3.33 (3H, s), 3.38 (6H, s), 3.77 (2H, br. s), 7.45-7.60 (4H, m), 
7.80-7.98 (2H, m). 
IR (KBr) .nu.: 3425 (br.), 1810, 1780, 1735, 1600, 1395, 1355 (shoulder), 
1340, 855 cm.sup.-1. 
Reference Example 10 
3-[N-(5-Ethoxycarbonylpentyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
Ethyl 6-(N-methylanilino)hexanoate (1 g) obtained in Reference Example 3 
was dissolved by addition of 1N hydrochloric acid (4 ml) and water (50 ml) 
under ice-cooling. Then 2-amino-5-dimethylaminophenylthiosulfonic acid (1 
g) was added. To the mixture was added dropwise a solution of sodium 
dichromate 2 hydrate (0.8 g) in water (6 ml) at 5.degree. to 10.degree. C. 
The mixture was stirred at the same temperature for 30 minutes and further 
at room temperature for 1 hour. The resulting precipitate was filtered, 
washed with water and suspended in acetonitrile (80 ml). Manganese dioxide 
(1 g) and catalytic amount of copper sulfate were added and the resulting 
mixture was heated under reflux for 1 hour. The reaction mixture was 
filtered. The residue was washed with methanol and then the filtrate was 
concentrated to dryness under reduced pressure. The residue was subjected 
to column chromatography on silica gel and eluted with CH.sub.3 CN 
followed by CH.sub.3 CN/H.sub.2 O/1N HCl (40:5:1). The desired fraction 
was lyophilized to obtain 
3-[N-(5-ethoxycarbonylpentyl)-N-methylamino]-7-dimethylaminophenazathioniu 
m chloride (0.875 g). 
NMR (CDCl.sub.3) .delta.: 1.26 (3H, t, J=7 Hz), 1.49 (2H, m), 1.74 (4H, m), 
2.35 (2H, t, J=7.2 Hz), 3.46 (6H, s), 3.71 (2H, br. t), 4.13 (2H, q, J=7 
Hz), 7.28 (2H, m), 7.60-8.00 (4H, m). 
IR (KBr) .nu.: 3425 (br.), 1725, 1600, 1490, 1390, 1335, 1245, 1175, 1140, 
885 cm.sup.-1. 
EXAMPLE 2 
3-[N-Methyl-N-(5-succinimidoxycarbonylpentyl)amino]-7-dimethylaminophenazat 
hionium chloride 
3-[N-(5-Ethoxycarbonylpentyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.785 g) obtained in Reference Example 10 was dissolved in 1N 
hydrochloric acid (10 ml) and the mixture was allowed to react at room 
temperature for 16 hours. The reaction mixture was subjected to column 
chromatography on silica gel and eluted successively with water, CH.sub.3 
CN/H.sub.2 O/1N HCl (40:5:1). The desired fraction was lyophilized. The 
residue was dissolved in dimethylformamide (200 ml). Insoluble materials 
were filtered off and then the filtrate was concentrated to about 50 ml. 
To this solution was added N-hydroxysuccinimide (0.54 g) and WSC (0.9 g). 
The mixture was allowed to react at room temperature for 48 hours. The 
reaction mixture was concentrated under reduced pressure and then the 
residue was dissolved in dichloromethane. The solution was washed with 
saturated brine, dried over anhydrous sodium sulfate and concentrated 
under reduced pressure. The resulting residue was dissolved in 
dichloromethane (15 ml) and ethyl acetate (250 ml) was added thereto. The 
resulting precipitate was filtered and dried to obtain 
3-[N-methyl-N-(5-succinimidoxycarbonylpentyl)-amino]-7-dimethylaminophenaz 
athionium chloride (0.718 g). 
NMR (CDCl.sub.3) .delta.: 1.56 (2H, m), 1.83 (4H, m), 2.66 (2H, t, J=6.8 
Hz), 2.91 (4H, s), 3.40 (3H, s), 3.46 (6H, s), 3.73 (2H, m), 7.20-8.00 
(6H, m). 
IR (KBr) .nu.: 3425, 1810, 1780, 1735, 1600, 1390, 1335, 1210, 1140, 1070, 
885 cm.sup.-1. 
Reference Example 11 
3-[N-(3-tert-Butoxycarbonylpropyl)-N-methylamino]-7-dimethylaminophenazathi 
onium chloride 
tert-Butyl 4-(N-methylanilino)butyrate (0.50 g) obtained in Reference 
Example 5 was dissolved by addition of a solution of potassium 
hydrogensulfate (0.41 g) in water (25 ml) under ice-cooling. To this 
solution was added 2-amino-5-dimethylaminophenylthiosulfonic acid (0.50 
g). A suspension of sodium dichromate 2 hydrate (0.40 g) in water (5 ml) 
was added dropwise at 5.degree. to 10.degree. C. Then acetic acid (0.06 
ml) was added and the resulting mixture was stirred at the same 
temperature for 30 minutes. Further, it was allowed to react at room 
temperature for 1.5 hours. The reaction mixture was filtered and washed 
with water. Then the residue was suspended in acetone. Manganese dioxide 
(4.0 g) and copper sulfate (0.03 g) were added thereto and the mixture was 
heated under reflux for 1 hour. The reaction mixture was filtered and 
concentrated to dryness. The crude product thus obtained was subjected to 
column chromatography on silica gel and eluted with CH.sub.3 CN, 90% 
CH.sub.3 CN--H.sub.2 O and CH.sub.3 CN/H.sub.2 O/1N HCl (30:5:3) to obtain 
3-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-7-dimethylaminophenazath 
ionium chloride (0.33 g). 
m.p.: 94.degree.-98.degree. C. (dec.) 
NMR (D.sub.2 O) .delta.: 1.52 (9H, s), 1.87 (2H, m), 2.43 (2H, t, J=7 Hz), 
3.13 (3H, s), 3.17 (6H,s), 3.47 (2H, m), 6.81 (2H, br. s), 7.0-7.1 (2H, 
m), 7.25-7.4 (2H, m). 
IR (KBr) .nu.: 3380, 1720, 1600, 1390, 1335, 1140, 885, 615 cm.sup.-1. 
.lambda. max: 651 nm (in MeOH) 
Elemental Analysis for C.sub.23 H.sub.30 N.sub.3 O.sub.2 SCl.cndot.2H.sub.2 
O, Calcd.: C, 57.07; H, 7.08; N, 8.68 Found: C, 57.08; H, 6.70; N, 8.70 
Reference Example 12 
3-[N-(3-Carboxypropyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
3-[N-(3-tert-Butoxycarbonylpropyl)-N-methylamino]-7-dimethylaminophenazathi 
onium chloride (0.05 g) obtained in Reference Example 11 was dissolved in 
trifluoroacetic acid (3 ml). The mixture was allowed to react at room 
temperature for 1 hour. Trifluoroacetic acid was distilled off under 
reduced pressure. Then the residue was subjected to column chromatography 
on silica gel and eluted with CH.sub.3 CN followed by CH.sub.3 CN/H.sub.2 
O/1N HCl (30:5:3) to obtain 
3-[N-(3-carboxypropyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride (0.014 g). 
NMR (D.sub.2 O) .delta.: 1.79 (2H, m), 2.51 (2H, m), 3.00 (9H, br. s), 3.31 
(2H, m), 6.55-7.10 (6H, m). 
IR (KBr) .nu.: 3420, 1605, 1395, 1340, 1140, 885, 850, 800 cm.sup.-1. 
EXAMPLE 3 
3-[N-Methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-dimethylaminophenazat 
hionium chloride 
Trifluoroacetic acid (15 ml) was added to 
3-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-7-dimethylaminophenazath 
ionium chloride (0.17 g) obtained in Reference Example 11. The mixture was 
allowed to react at room temperature for 15 minutes. Trifluoroacetic acid 
was distilled off under reduced pressure and the residue was dissolved in 
acetonitrile (10 ml). Pyridine (0.19 ml) and DSO (0.32 g) were added 
thereto and the mixture was stirred at room temperature for 1.5 hours. The 
reaction mixture was concentrated to dryness. The residue was then 
dissolved in dichloromethane (50 ml). The resulting solution was mixed 
with saturated brine containing 1N HCl (5 ml) with shaking. Further, the 
mixture was washed with saturated brine, dried over anhydrous sodium 
sulfate and concentrated under reduced pressure. The residue was dissolved 
in dichloromethane and an excess amount of ethyl acetate was added thereto 
to form precipitate. The precipitate thus obtained was filtered off and 
dried to obtain 
3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-dimethylaminophenaza 
thionium chloride (0.12 g). 
m.p.: 125.degree.-130.degree. C. (dec.) 
NMR (CDCl.sub.3) .delta.: 2.11 (2H, m), 2.81 (2H, t, J=6 Hz), 2.91 (4H, s), 
3.37 (9H, br. s), 3.79 (2H, m), 7.23 (2H, d, J=9 Hz), 7.75 (2H, d, J=9 
Hz), 7.76 (2H, m). 
IR (KBr) .nu.: 3420, 1810, 1790, 1735, 1600, 1390, 1340, 1140, 885 
cm.sup.-1. 
Reference Example 13 
2-Amino-5-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenylthiosulfonic acid 
N-(3-Ethoxycarbonylpropyl)-N-methyl-p-phenylenediamine (8.97 g) obtained in 
Reference Example 1 was dissolved by addition of 1N sulfuric acid (76 ml) 
under ice-cooling. A solution of aluminium potassium sulfate 12 hydrate 
(17.1 g) in water (80 ml) and a solution of sodium thiosulfate 5 hydrate 
(9.88 g) in water (19 ml) were added at below 10.degree. C. Then a 
solution of potassium dichromate (3.65 g) in water (57 ml) was added 
dropwise at 10.degree. C. The cooling bath was removed and the mixture was 
stirred at room temperature for 1 hour. Then sodium bicarbonate was added 
to adjust the resulting mixture to pH 5. The reaction mixture was filtered 
and the filtrate was concentrated to dryness under reduced pressure. The 
residue was subjected to ODS (wherein octadecyl group is chemically bound 
to silanol of silica gel) column chromatography and eluted with 50% 
MeOH-H.sub.2 O. The desired fraction was lyophilized to obtain 
2-amino-5-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenylthiosulfonic 
acid (4 g). 
NMR (CH.sub.3 OD) .delta.: 1.22 (3H, t, J=7 Hz), 1.80 (2H, quintet, J=7 
Hz), 2.35 (2H, t, J=7 Hz), 2.96 (3H, s), 3.30 (2H, t, J=7 Hz), 4.10 (2H, 
q, J=7 Hz), 6.96 (2H, br. s), 7.23 (1H, br. s). 
IR (KBr) .nu.: 1730, 1625, 1505, 1220, 1030, 835 cm.sup.-1. 
Reference Example 14 
[N-(3-tert-Butoxycarbonylpropyl)-N-methylamino]-[N'-(3-ethoxycarbonylpropyl 
)-N'-methylamino]-indaminthiosulfonic acid 
tert-Butyl 4-(N-methylanilino)butyrate (0.997 g) obtained in Reference 
Example 5 was dissolved by addition of a solution of potassium 
hydrogensulfate (0.817 g) in water (10 ml) under ice-cooling. To this 
solution was added 
2-amino-5-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenylthiosulfonic 
acid (1.394 g) obtained in Reference Example 13. Then potassium dichromate 
(1.18 g) was added portionwise and the mixture was stirred at 5.degree. to 
10.degree. C. for 1 hour. The resulting precipitate was filtered, washed 
with water and dissolved in methanol. Insoluble materials were filtered 
off and the filtrate was concentrated under reduced pressure. The 
concentrate was subjected to ODS column chromatography and eluted with 50% 
CH.sub.3 CN--H.sub.2 O to obtain 
[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-[N'-(3-ethoxycarbonylpropy 
l)-N'-methylamino]indaminthiosulfonic acid. 
IR (KBr) .nu.: 3450, 3350, 1730, 1610, 1515, 1365, 1240, 1170, 1015, 820, 
615 cm.sup.-1. 
Reference Example 15 
3-[N-(3-tert-Butoxycarbonylpropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylpro 
pyl)-N-methylamino]-phenazathionium chloride 
[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-[N'-(3-ethoxycarbonylpropyl 
)-N'-methylamino]indaminthiosulfonic acid (0.60 g) obtained in Reference 
Example 14 was dissolved in acetone (25 ml). Manganese dioxide (3 g) was 
added thereto and the mixture was stirred at room temperature for 20 
hours. Manganese dioxide was filtered off and washed with methanol. The 
filtrate was concentrated under reduced pressure. The residue was 
subjected to column chromatography on silica gel and eluted with CH.sub.3 
CN/H.sub.2 O (20:1) followed by CH.sub.3 CN/H.sub.2 O/1N HCl (120:20:3). 
The desired fractions were collected and concentrated to obtain 
3-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylpr 
opyl)-N-methylamino]phenazathionium chloride (0.26 g). 
NMR (D.sub.2 O) .delta.: 1.30 (3H, t, J=7 Hz), 1.50 (9H, s), 1.83-1.90 (2H, 
hr.), 2.38-2.55 (2H, m), 3.15 (6H, br. s), 3.47 (2H, br. s), 4.20 (2H, q, 
J=7 Hz), 6.85 (2H, br. s), 7.11 (2H, m), 7.65 (2H, m). 
IR (KBr) .nu.: 1725, 1600, 1400, 1365, 1340, 1185, 1150, 890 cm.sup.-1. 
EXAMPLE 4 
3-[N-(3-Succinimidoxycarbonylpropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylp 
ropyl)-N-methylamino]phenazathionium chloride 
Trifluoroacetic acid (18 ml) was added to 
3-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylpr 
opyl)-N-methylamino]phenazathionium chloride (0.24 g) obtained in Reference 
Example 15 and the mixture was stirred at room temperature for 1 hour. The 
reaction mixture was concentrated to dryness under reduced pressure. 
Acetonitrile (15 ml), pyridine (0.33 ml) and DSC (0.52 g) were added to 
the residue and the resulting mixture was stirred at room temperature for 
2 hours. The reaction mixture was concentrated under reduced pressure. The 
residue was dissolved in dichloromethane (150 ml), washed with saturated 
brine containing 1N hydrochloric acid (6 ml), further washed with 
saturated brine, dried over anhydrous sodium sulfate and concentrated to 
dryness. The resulting residue was re-precipitated from 
dichloromethane-ethyl acetate to obtain 
3-[N-(3-succinimidoxycarbonylpropyl)-N-methylamino]-7-[N-(3-ethoxycarbonyl 
propyl)-N-methylamino]phenazathionium chloride (0.18 g). 
m.p. : 70.degree.-85.degree. C. (dec.) 
NMR (CDCl.sub.3) .delta.: 1.27 (3H, t, J=7 Hz), 2.04 (2H, m), 2.18 (2H, m), 
2,53 (2H, t, J=6.5 Hz), 2.89 (2H,m), 2.97 (4H, s), 3.40 (3H,s), 3.42 (3H, 
s), 3.83 (4H, m), 4.16 (2H, q, J=7 Hz), 7.36 (2H, t, J=10 Hz), 7.59 (2H, 
d, J=15 Hz), 7.85 (2H, dd, J=2,10 Hz). 
IR (KBr) .nu.: 3420, 1810, 1780, 1735, 1595, 1390, 1335, 1190, 1140, 885 
cm.sup.-1. 
Reference Example 16 
2-Amino-5-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]phenylthiosulfonic 
acid 
N-(3-tert-butoxycarbonylpropyl)-N-methyl-p-phenylenediamine (3.30 g) 
obtained in Reference Example 6 was dissolved in 1N sulfuric acid (50 ml) 
under ice-cooling. A solution of aluminium sulfate 14-18 hydrate (8.33 g) 
in water (15 ml), a solution of sodium thiosulfate 5 hydrate (9.31 g) in 
water (15 ml) and a solution of potassium dichromate 2 hydrate (1.27 g) in 
water (17 ml) were added successively at the reaction temperature of 
5.degree. to 10.degree. C. The resulting mixture was stirred at the same 
temperature for 30 minutes and then at room temperature for additional 1 
hour. Sodium bicarbonate was added to the reaction mixture to adjust to pH 
4 and the reaction mixture was concentrated to dryness under reduced 
pressure. Methanol (500 ml) was added to the concentrate and insoluble 
materials were filtered off. The filtrate was concentrated under reduced 
pressure. The residue was subjected to column chromatography on silica gel 
and eluted with ethyl acetate/methanol (15:1) followed by the same solvent 
system (5:1) to obtain 
2-amino-5-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]phenylthiosulfoni 
c acid (1.54 g) as a dark green viscous material. 
NMR (CDCl.sub.3) .delta.: 1.42 (9H, s), 1.73 (2H, m), 2.19 (2H, br. s), 
2.76 (3H, br. s), 3.15 (2H, br. m), 4.50 (3H, br. s), 6.50-7.40 (3H, m). 
Reference Example 17 
Bis[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]indaminthiosulfonic acid 
tert-Butyl 4-(N-methylanilino)butyrate (1.16 g) obtained Reference Example 
5 was dissolved in a solution of potassium hydrogensulfate (0.95 g) in 
water (90 ml). Then 
2-amino-5-[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]phenylthiosulfoni 
c acid (1.62 g) obtained in Reference Example 16 was added thereto under 
ice-cooling. A solution of potassium dichromate 2 hydrate (1.38 g) in 
water (10 ml) was added dropwise at 5.degree. to 10.degree. C. Acetic acid 
(0.14 ml) was added and the mixture was stirred at room temperature for 1 
hour. The resulting precipitate was filtered off and washed with water. 
The filtration residue was extracted with methanol (200 ml) and 
concentrated to dryness. The residue was subjected to column 
chromatography on silica gel and eluted with chloroform/methanol (20:1) to 
obtain 
bis[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]indaminthiosulfonic acid 
(0.48 g). 
NMR (CDCl.sub.3) .delta.: 1.45 (18H, s), 1.88 (4H, br.), 2.86 (4H, br. s), 
2.90-3.20 (4H, m), 3.20-3.70 (6H, br.), 6.50-7.70 (7H, m). 
Reference Example 18 
3,7-Bis[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]phenazathionium 
chloride 
Bis[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]indaminthiosulfonic acid 
(0.47 g) obtained in Reference Example 17 was dissolved in acetone (40 
ml). Manganese dioxide (2.0 g) and copper sulfate (0.03 g) were added and 
the mixture was heated under reflux for 30 minutes. The reaction mixture 
was filtered and the filtrate was concentrated to dryness under reduced 
pressure. The residue was subjected to column chromatography on silica gel 
and eluted successively with CH.sub.3 CN, 90% CH.sub.3 CN--H.sub.2 O and 
CH.sub.3 CN/H.sub.2 O/1N HCl (120:20:3). The desired fraction was 
concentrated to dryness. The residue was re-precipitated from 
dichloromethane-ethyl acetate to obtain 
3,7-bis[N-(3-tert-butoxycarbonylpropyl)-N-methyl-amino]phenazathionium 
chloride (0.11 g). 
m.p. : 142.degree.-145.degree. C. 
NMR (CDCl.sub.3) .delta.: 1.47 (18H, s), 2.00 (4H, quintet, J=7 Hz), 2.43 
(4H, t, J=7 Hz), 3.43 (6H, s), 3.79 (4H, t, J=7 Hz), 7.39 (2H, d, J=10 
Hz), 7.62 (2H, br. s), 7.91 (2H, s, J=10 Hz). 
IR (KBr) .nu.: 3430, 2970, 2920, 1720, 1595, 1395, 1335, 1240, 1240, 1140, 
980 cm.sup.-1. 
Elemental Analysis for C.sub.30 H.sub.42 N.sub.3 O.sub.4 SCl.cndot.3H.sub.2 
O, Calcd.: C, 57.17; H, 7.68; N, 6.67 Found: C, 57.29; H, 7.45; N, 6.67 
Reference Example 19 
3,7-Bis[N-(3-carboxypropyl)-N-methylamino]-phenazathionium chloride 
3,7-Bis[N-(3-tert-butoxycarbonylpropyl)-N-methylamino]phenazathionium 
chloride (0.11 g) obtained in Reference Example 18 was dissolved in 
trifluoroacetic acid (10 ml). The mixture was allowed to react at room 
temperature for 30 minutes. Then the reaction mixture was concentrated to 
dryness under reduced pressure to obtain 
3,7-bis[N-(3-carboxypropyl)-N-methylamino]phenazathionium chloride 
quantitatively as its trifluoroacetate. 
NMR (D.sub.2 O) .delta.: 1.86 (4H, br. s), 2.45 (4H, br. s), 2.80-3.70 (10 
H, br. m), 6.70-7.40 (6H, br. m). 
EXAMPLE 5 
3,7-Bis[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]phenazathionium 
chloride and 
3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-(3-carboxypropyl) 
-N-methylamino]phenazathionium chloride 
3,7-Bis[N-(3-carboxypropyl)-N-methylamino]phenazathionium chloride 
trifluoroacetate (0.11 g) obtained in Reference Example 19 was dissolved 
in acetonitrile (20 ml). Pyridine (0.14 ml) and DSC (0.21 g) were added 
and the resulting mixture was stirred at room temperature for 3 hours. The 
reaction mixture was concentrated under reduced pressure and then the 
residue was dissolved by addition of dichloromethane. The solution was 
washed with saturated brine, dried over anhydrous sodium sulfate and 
concentrated to dryness to obtain dark blue powder. The powder thus 
obtained was subjected to Sephadex.RTM. LH-20 column chromatography and 
eluted with acetonitrile. The fractions were concentrated to dryness 
respectively to obtain a bissuccinimido ester derivative, 
3,7-bis[N-(3-succinimidoxycarbonylpropyl)amino]phenazathionium chloride 
(0.1 g) from the firstly eluted fraction, and a monosuccinimido ester 
derivative, 
3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-(3-carboxypropyl) 
-N-methylamino]phenazathionium chloride (0.03 g) from the subsequently 
eluted fraction. 
Bissuccinimido ester 
m.p. : 115.degree.-120.degree. C. (dec.) 
NMR (DMSO-d.sub.6) .delta.: 1.88 (2H, m), 2.02 (2H, m), 2.38 (2H, t, J=7 
Hz), 2.87 (4H, s), 3.13 (2H, t, J=7 Hz), ca. 3.30 (3H, s), 3.36 (3H, s), 
3.76 (4H, m), 7.55 (4H, br. s), 7.95 (2H, d, J=10 Hz). 
IR (KBr) .nu.: 3410, 2930, 1810, 1775, 1735, 1595, 1390, 1335, 1190, 1140, 
880 cm.sup.-1. 
Monosuccinimido ester 
m.p. 129.degree.-133.degree. C. (dec.) 
NMR (DMSO-d.sub.6) .delta.: 2.01 (4H, m), 2.84 (4H, s), 2.88 (4H, t, J=7 
Hz), ca. 3.30 (6H, s), 3.80 (4H, m), 7.56.(4H, br. s), 7.96 (2H, d, J=10 
Hz). 
IR (KBr) .nu.: 3400, 2930, 1810, 1775, 1730, 1595, 1390, 1335, 1190, 1140, 
1065, 880 cm.sup.-1. 
EXAMPLE 6 
3-[N-Methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-[3-(1,3-dicarboxyp 
ropylcarbamoyl)propyl]-N-methylamino]phenazathionium chloride 
3,7-Bis[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]phenazathionium 
chloride (20 mg) obtained in Example 5 was dissolved in acetonitrile (2 
ml). Glutamic acid (8 mg), triethylamine (15 .mu.l) and a solution of 50% 
acetonitrile-water (2 ml) were added. The resulting mixture was stirred 
for 10 minutes at room temperature. 1N hydrochloric acid (0.1 ml) was 
added to the reaction mixture. Then the mixture was subjected to 
Sephadex.RTM. LH-20 column chromatography and eluted with acetonitrile. 
The desired fraction was concentrated to dryness to obtain 
3-[N-methyl-N-(3-succinimidoxycarbonylpropyl)amino]-7-[N-[3-(1,3-dicarboxy 
propylcabamoyl)propyl]-N-methylamino]phenazathionium chloride (10.8 mg) as 
blue powder. 
NMR (DMSO-d.sub.6) .delta.: 1.75-2.05 (6H, m), 2.20-2.35 (4H, m), 2.84 (4H, 
s), 2.85-2.95 (2H, m), 3.60-3.85 (6H, m), 4.15-4.30 (1H, m), 7.50-7.60 
(3H, m), 7.90-8.10 (3H, m). 
IR (KBr) .nu.: 3420, 2970, 2940, 2670, 2490, 1810, 1735, 1660, 1595, 1390, 
1340, 1145, 1035, 880 cm.sup.-1. 
Reference Example 20 
N-(5-Ethoxycarbonylpentyl)-N-methyl-p-phenylenediamine 
N-(5-Ethoxycarbonylpentyl)-N-methylaniline (10 g) obtained in Reference 
Example 3 was dissolved in 80% acetic acid (41.3 ml). A solution of sodium 
nitrite (3.05 g) in water (3.8 ml) was added thereto with stirring at 
below 10.degree. C. Then acetic acid (37.5 ml) was added to the reaction 
mixture and zinc powder (23.8 g) was added over about 40 minutes, while 
keeping the mixture at below 0.degree. C. The reaction mixture was poured 
into ice water (200 ml). The resulting mixture was filtered and the zinc 
powder was washed with ethyl acetate. The filtrate was extracted with 
ethyl acetate and the extract was neutralized with an aqueous sodium 
bicarbonate, washed with saturated brine, dried over anhydrous magnesium 
sulfate and concentrated under reduced pressure. The residue was subjected 
to column chromatography on silica gel and eluted with hexane/ethyl 
acetate (2:1) to obtain the desired compound (2.76 g) as purplish red 
viscous oil. 
NMR (CDCl.sub.3) .delta. (ppm): 1.25 (3H, t, J=7.0 Hz), 1.33 (2H, quintet, 
J=7.5 Hz), 1.54 (2H, quintet, J=7.5 Hz), 1.65 (2H, quintet, J=7.5 Hz), 
2.29 (2H, t, J=7.5 Hz), 2.80 (3H,s), 2.90-3.50 (2H, br.), 3.16 (2H, t, 
J=7.5 Hz), 4.12 (2H, q, J=7.0 Hz), 6.65 (5H, s). 
IR (neat) .nu.: 3430, 3350, 2930, 1730, 1515, 1260, 1175, 810 cm.sup.-1. 
Reference Example 21 
3,7-Bis[N-(5-ethoxycarbonylpentyl)-N-methylamino]phenazathionium chloride 
N-(5-Ethoxycarbonylpentyl)-N-methyl-p-phenylenediamine (1.32 g) was 
dissolved in water (65 ml) containing sulfuric acid (0.75 g). A solution 
of sodium thiosulfate 5 hydrate (2.18 g) in water (5 ml) and manganese 
dioxide (0.86 g) were added with stirring under ice-cooling. After 20 
minutes, additional sodium thiosulfate 1/2 hydrate (4.36 g) and manganese 
dioxide (1.92 g) were added and the resulting mixture was stirred for 1 
hour. The reaction mixture was adjusted to pH 4 with 1N sulfuric acid. A 
solution of N-(5-ethoxycarbonylpentyl)-N-methylaniline (1.70 g) in 1N 
sulfuric acid (10 ml) was added at the same temperature. Further, 
additional manganese dioxide (2.08 g) and 25% sulfuric acid (6 ml) was 
added and the mixture was stirred for 1.5 hours. Then the reaction mixture 
was adjusted to pH 5 with 1N sodium hydroxide. Manganese dioxide (0.77 g) 
and copper sulfate (0.29 g) were added and the resulting mixture was 
stirred at 90.degree. C. for 40 minutes. The reaction mixture was 
filtered. The residue was washed with water and then extracted with 
acetone (300 ml). The solution extracted with acetone was concentrated. 
The residue was extracted with chloroform and dried over anhydrous sodium 
sulfate. After concentration, the residue was subjected to column 
chromatography on silica gel and eluted successively with CH.sub.3 CN, 90% 
CH.sub.3 CN--H.sub.2 O and CH.sub.3 CN/H.sub.2 O/1N HCl (120:20:3) to 
obtain the desired compound (30 mg). 
NMR (CDCl.sub.3) .delta. (ppm): 1.26 (6H, t, J=7.0 Hz), 1.50 (4H, m), 1,72 
(4H, m), 2.35 (4H, t, J=7.0 Hz), 3.41 (6H, s), 3.71 (4H, m), 4.13 (4H, q, 
J=7.0 Hz), 7.23 (2H, br. d, J=9.0 Hz), 7.80 (2H, br. s), 7.91 (2H, br. d, 
J=9.0 Hz). 
IR (KBr) .nu.: 1720, 1600, 1395, 1340, 1140, 885 cm.sup.-1. 
Reference Example 22 
3,7-Bis[N-(5-carboxypentyl)-N-methylamino]phenazathionium chloride 
3,7-Bis[N-(5-ethoxycarbonylpentyl)-N-methylamino]phenazathionium chloride 
(0.32 g) was dissolved in 90% acetonitrile (20 ml). 1N Hydrochloric acid 
(40 ml) was added to this solution and the resulting mixture was stirred 
at room temperature for 20 hours. The reaction mixture was concentrated 
under reduced pressure. The residue was subjected to column chromatography 
on silica gel and eluted successively with 90% CH.sub.3 CN--H.sub.2 O and 
CH.sub.3 CN/H.sub.2 O/1N HCl (30:5:1) to obtain the desired compound (0.32 
g) as deep blue powder. 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.25-1.50 (4H, m), 1.50-1.75 (8H, m), 
2.24 (4H, t, J=7.0 Hz), 3.50 (6H, s), 3.65-3.80 (4H, m), 7.35-7.50 (2H, 
m), 7.54 (2H, br. s), 7.93 (2H, br. d, J=9.0 Hz ). 
IR (KBr) .nu.: 1635, 1600, 1485, 1390, 1340, 1240, 1145, 885 cm.sup.-1. 
EXAMPLE 7 
3,7-Bis[N-(5-succinimidoxycarbonylpentyl)-N-methylamino]phenazathionium 
chloride and 
3-[N-(5-succinimidoxycarbonylpentyl)-N-methylamino]-7-[N-(5-carboxypentyl) 
-N-methylamino]phenazathionium chloride 
Method A 
3,7-Bis[N-(5-carboxypentyl)-N-methylamino]phenazathionium chloride (0.15 g) 
was dissolved in trifluoroacetic acid (10 ml) and the solution was 
concentrated to dryness. The trifluoroacetate thus obtained was dissolved 
in acetonitrile (30 ml) and DSC (0.92 g) and pyridine (0.85 ml) were 
added. The resulting mixture was stirred at room temperature for 15 hours. 
The reaction mixture was concentrated to dryness and the residue was 
dissolved by addition of dichloromethane. The solution was washed with 
saturated brine twice, dried over anhydrous sodium sulfate and 
concentrated. The residue was subjected to Sephadex.RTM. LH-20 column 
chromatography and eluted with acetonitrile to obtain a bissuccinimido 
ester derivative (38 mg) as a first fraction and a monosuccinimido ester 
derivative (36 mg) as a second fraction. 
Method B 
3,7-Bis[N-(5-carboxypentyl)-N-methylamino]phenazathionium chloride (0.32 g) 
was dissolved in dimethylformamide (5 ml). N-Hydroxysuccinimide (0.42 g) 
and WSC (0.70 g) were added and the mixture was stirred for 3 hours. The 
reaction mixture was concentrated to dryness under reduced pressure and 
then extracted with dichloromethane. The dichloromethane layer was washed 
with saturated brine twice, dried over anhydrous sodium sulfate and 
concentrated. The residue was subjected to Sephadex.RTM. LH-20 column 
chromatography and eluted with acetonitrile. The desired fractions were 
concentrated. The concentrate was dissolved in a small amount of 
dichloromethane and ethyl acetate was added to obtain a bissuccinimido 
ester derivative (83 mg) as purplish deep blue powder. 
Bissuccinimido ester 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.40-1.55 (4H, m), 1.65-1.75 (8H, m), 
2.71 (4H, t, J=7.0 Hz), 2.82 (8H, s), 3.31 (6H, s), 3.75 (4H, br. t, J=7.0 
Hz), 7.51 (2H, br. s), 7.54 (2H, br. d, J=9.5 Hz), 7.93 (2H, d, J=9.5 Hz). 
IR (KBr) .nu.: 1810, 1780, 1735, 1595, 1390, 1335, 1200, 1135, 1065, 880 
cm.sup.-1. 
Monosuccinimido ester 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.35-1.65 (4H, m), 1.65-1.80 (8H, m), 
2.24 (2H, t, J=7.0 Hz), 2.71 (2H, t, J=7.0 Hz), 2.82 (4H, s), 3.35 (6H, 
s), 3.74 (4H, br. t, J=7.0 Hz), 7.50 (2H, s), 7.52 (2H, br. d, J=9.5 Hz), 
7.92 (2H, d, J=9.5 Hz). 
IR (KBr) .nu.: 1810, 1780, 1735, 1595, 1390, 1335, 1200, 1065, 880 
cm.sup.-1. 
EXAMPLE 8 
3-[N-(5-Succinimidoxycarbonylpentyl)-N-methylamino]-7-[N-[5-(1,3-dicarboxyp 
ropylcarbamoyl)pentyl]-N-methylamino]phenazathionium chloride 
A solution prepared from glutamic acid (27 mg), water (3 ml) and 
triethylamine (51 .mu.l) was added to a solution of 
3,7-bis[N-(5-succinimidoxycarbonylpentyl)-N-methylamino]phenazathionium 
chloride (72 mg) in acetonitrile (6 ml). After 5 minutes, 1N hydrochloric 
acid (0.35 ml) was added. The resulting mixture was subjected to 
Sephadex.RTM. LH-20 column chromatography and eluted with acetonitrile to 
obtain the desired compound (16 mg). 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.35-1.60 (4H, m), 1.60-1.80 (8H, m), 
1.80-2.00 (2H, m), 2.15 (2H, t, J=6.0 Hz), 2.27 (2H, t, J=7.0 Hz), 2.71 
(2H, t, J=7.0 Hz), 2.82 (4H, s), 3.35 (6H, s), 3.72 (4H, br. s), 4.20 (1H, 
m), 7.40-7.55 (4H, m), 7.92 (1H, d, J=9.5 Hz), 8.04 (1H, d, J=9.5 Hz). 
IR (KBr) .nu.: 1810, 1780, 1735, 1650, 1595, 1390, 1335, 1235, 1200, 1135, 
1065, 880 cm.sup.-1. 
Reference Example 23 
N-(3-Maleimidopropyl)-N-methylaniline 
N-(3-Aminopropyl)-N-methylaniline [S. L. Shapiro et al., J. Am. Chem. Soc., 
81, 3081 (1958)] (8.3 g) was dissolved in dichloromethane (50 ml). Maleic 
anhydride (6 g) was added portionwise under ice-cooling and the mixture 
was stirred at room temperature for 1 hour. The solvent was distilled off 
and then acetic anhydride (20 ml) was added. The resulting mixture was 
allowed to stand at room temperature for 2 days. The reaction mixture was 
stirred at 60.degree. C. for 2 hours and further at 100.degree. C. for 2 
hours. The mixture was concentrated under reduced pressure and the residue 
was extracted with ethyl acetate. The organic layer was neutralized with 
an aqueous sodium bicarbonate, washed with saturated brine and dried over 
anhydrous sodium sulfate. After concentration, the residue was subjected 
to column chromatography on silica gel and eluted with hexane/ethyl 
acetate (4:1). The desired compound thus obtained was recrystallized from 
the same solvent to obtain the compound as yellow needles (4.1 g). 
m.p.: 87.degree.-88.degree. C. 
NMR (CDCl.sub.3) .delta. (ppm): 1.89 (2H, quintet, J=7.3 Hz), 2.91 (3H, s), 
3.33 (2H, t, J=7.3 Hz), 3.57 (2H, t, J=7.3 Hz), 6.68 (3H, m), 6.69 (2H, 
s), 7.21 (2H, m). 
IR (KBr) .nu.: 1700, 1600, 1510, 1410, 1410, 1370, 1220, 830, 750, 690 
cm.sup.-1. 
Elemental Analysis for C.sub.14 H.sub.16 N.sub.2 O.sub.2, Calcd.: C, 68.83; 
H, 6.60; N, 11.47 Found : C, 68.95; H, 6.77; N, 11.49 
Reference Example 24 
3-[N-(3-Maleimidopropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylpropyl)-N-met 
hylamino]phenazathionium chloride 
N-(3-Maleimidopropyl)-N-methylaniline (0.25 g) was dissolved in acetone (10 
ml) and 1N hydrochloric acid (1 ml) and water (9 ml) were added. Then 
2-amino-5-[N-(3-ethoxycarbonylpropyl)-N-methylamino]phenylthiosulfonic 
acid (0.35 g) was added and further a solution of sodium dichromate 2 
hydrate (0.2 g) in water (2 ml) was added dropwise with stirring. The 
resulting mixture was stirred at the same temperature for 30 minutes. 
Acetic acid (0.03 ml) was added and further stirred at room temperature 
for 30 minutes. Then catalytic amount of copper sulfate and manganese 
dioxide (0.25 g) were added. The mixture was stirred at room temperature 
for 30 minutes and further at 70.degree. C. for 1 hour. After cooling, the 
reaction mixture was filtered. The residue was washed with ethanol and the 
washings were combined with the filtrate and the mixture was concentrated 
under reduced pressure. The residue was extracted with ethanol and 
insoluble materials were filtered off. The filtrate was concentrated. Then 
residue was subjected to column chromatography on silica gel and eluted 
with CH.sub.3 CN followed by CH.sub.3 CN/H.sub.2 O/1N HCl (40:5:1) to 
obtain the desired compound (0.31 g). 
NMR (CDCl.sub.3) .delta. (ppm): 1.28 (3H, t, J=7.2 Hz), 1.78 (4H, m), 2.55 
(2H, m), 3.39 (3H, s), 3.46 (3H, s), 3.60-3.95 (6H, m), 4.17 (2H, q, J=7.2 
Hz), 6.80 (2H, s), 7.15-8.00 (6H, m). 
IR (KBr) .nu.: 1720, 1700, 1590, 1390, 1330, 1225, 1180, 1140, 880 
cm.sup.-1. 
EXAMPLE 9 
3-[N-(3-Maleimidopropyl)-N-methyamino]-7-[N-(3-carboxypropyl)-N-methylamino 
]phenazathionium chloride 
3-[N-(3-Maleimidopropyl)-N-methylamino]-7-[N-(3-ethoxycarbonylpropyl)-N-met 
hylamino]phenazathionium chloride (0.3 g) was dissolved in a mixture of 
acetonitrile (2 ml) and 1N hydrochloric acid (6 ml). The solution was left 
open for 2 days. The resulting precipitate having metallic luster was 
filtered off, washed with ether and dried to obtain the desired compound 
(0.25 g). 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.90 (4H, m), 2.93 (2H, t, J=6.8 Hz), 
3.23 (3H, s), 3.35 (3H, s), 3.54 (2H, m), 7.01 (2H, s), 7.40-7.65 (4H, m), 
7.85-8.00 (2H, m). 
IR (KBr) .nu.: 1735, 1700, 1595, 1390, 1330 cm.sup.-1. 
EXAMPLE 10 
3-[N-(3-Maleimidopropyl)-N-methylamino]-7-[N-[3-(1,3-dicarboxypropylcarbamo 
yl)propyl]-N-methylamino]phenazathionium chloride 
3-[N-(3-Maleimidopropyl)-N-methylamino]-7-[N-(3-carboxypropyl)-N-methylamin 
o]phenazathionium chloride (51 mg) was dissolved in dimethylformamide (5 
ml) and dried over 4 .ANG. molecular sieve. N-Hydroxysuccinimide (25 mg) 
and WSC (50 mg) were added thereto and the resulting mixture was allowed 
to react at room temperature for 23 hours. This reaction mixture was added 
to a solution prepared from L-glutamic acid (17.7 mg), dimethylformamide 
(5 ml), water (0.5 ml) and triethylamine (34 .mu.l) under ice-cooling. The 
resulting mixture was stirred for 1 hour. The mixture was concentrated 
under reduced pressure. Then the residue was dissolved in water (5 ml) and 
1N hydrochloric acid (0.24 ml) was added to the solution. The resulting 
solution was subjected to column chromatography on silica gel and eluted 
with CH.sub.3 CN/H.sub.2 O (9:1) followed by CH.sub.3 CN/H.sub.2 O/1N HCl 
(30:15:1). The desired fraction was concentrated. The residue was 
subjected to Sephadex.RTM. LH-20 column chromatography and eluted with 
CH.sub.3 CN/H.sub.2 O (10:1). The desired fractions was lyophilized to 
obtain the desired compound (26 mg). 
NMR (D.sub.2 O) .delta. (ppm): 1.60-2.30 (6H, m), 2.45 (4H, m), 3.14 (6H, 
s), 3.35-3.65 (6H, m), 4.35 (1H, t), 6.80-7.50 (6H, m), 6.92 (2H, s). 
IR (KBr) .nu.: 1730, 1700, 1640, 1595, 1390, 1330 cm.sup.-1. 
Reference Example 25 
N-(3-Cyanopropyl)-N-methylaniline 
To N-methylaniline (25 g) was added 4-chlorobutyronitrile (25 ml) and the 
mixture was stirred at 110.degree. C. for 24 hours. After cooling, the 
mixture was neutralized with an aqueous sodium bicarbonate and extracted 
with ethyl acetate. The organic layer was washed with saturated brine, 
dried over anhydrous sodium sulfate and concentrated. The residue was 
subjected to distillation under reduced pressure to obtain the desired 
compound (21.5 g) as pale yellow oil. 
b.p.: 118.degree.-121.degree. C./0.5 mmHg. 
NMR (CDCl.sub.3) .delta. (ppm): 1.96 (2H, quintet, J=7.0 Hz), 2.40 (2H, t, 
J=7.0 Hz), 2.96 (3H, s), 3.47 (2H, t, J=7.0 Hz), 6.74 (3H, m), 7.25 (2H, 
m). 
IR (neat) .nu.: 2950, 2875, 2825, 2250, 1600, 1505, 1370, 1190, 750, 690 
cm.sup.-1. 
Reference Example 26 
N-(4-Aminobutyl)-N-methylaniline 
Lithium aluminium hydride (2 g) was added to dry ether (50 ml) in a stream 
of nitrogen, and a solution of N-(3-cyanopropyl)-N-methylaniline (9 g) in 
dry ether (10 ml) was added dropwise. The mixture was stirred for 1 hour 
and then heated under reflux for 2 hours. After decomposition of the 
excess reducing agent with ethyl acetate, the organic layer was washed 
with water, dried over anhydrous sodium sulfate and concentrated to obtain 
the desired compound (9.8 g) as colorless oil. 
NMR (CDCl.sub.3) .delta. (ppm): 1.54 (4H, m), 2.73 (2H, t, J=7.0 Hz), 2.93 
(3H, s), 3.33 (2H, t, J=7.0 Hz), 6.70 (3H, m), 7.22 (2H, m). 
Reference Example 27 
N-(4-Maleimidobutyl)-N-methylaniline 
N-(4-Aminobutyl)-N-methylaniline (7.2 g) was dissolved in dichloromethane 
(70 ml) and maleic anhydride (4 g) was added thereto. The mixture was 
stirred at room temperature for 2 hours. The solvent was distilled off and 
then acetic anhydride (10 ml) was added. The resulting mixture was stirred 
at 100.degree. C. for 5 hours. After concentration, an aqueous sodium 
bicarbonate was added to the residue to neutralize it. The mixture was 
extracted with ethyl acetate. The organic layer was washed with saturated 
brine, dried over anhydrous sodium sulfate and concentrated under reduced 
pressure. The residue was subjected to column chromatography on silica gel 
and eluted with hexane/ethyl acetate (2:1) to obtain the desired compound 
(2.0 g) as yellow needles. 
m.p.: 66.degree.-67.degree. C. 
NMR (CDCl.sub.3) .delta. (ppm): 1.60 (4H, m), 2.91 (3H, s), 3.33 (2H, t, 
J=6.8 Hz), 3.54 (2H, t, J=6.8 Hz), 6.68 (2H, s), 6.68 (3H, m), 7.22 (2H, 
m). 
IR (KBr) .nu.: 1695, 1600, 1505, 1410, 1365, 750, 690 cm.sup.-1. 
Elemental Analysis for C.sub.15 H.sub.18 N.sub.2 O.sub.2, Calcd.: C, 69.74; 
H, 7.02; N, 10.84 Found: C, 69.98; H, 7.20; N, 10.92 
Reference Example 28 
N-(4-Ethoxycarbonylbutyl)-N-methyl-p-phenylenediamine 
N-(4-Ethoxycarbonylbutyl)-N-methylaniline (2.35 g) obtained in Reference 
Example 2 was dissolved in 1N hydrochloric acid (30 ml) and to the 
solution was added dropwise a solution of sodium nitrite (0.76 g) in water 
(5 ml) at 0.degree. to 4.degree. C. The mixture was stirred at the same 
temperature for 30 minutes. To the resulting mixture was added 3N 
hydrochloric acid (15 ml) followed by zinc powder (2 g) at 20.degree. to 
27.degree. C. The mixture was stirred at the same temperature for 
additional 1 hour. Sodium bicarbonate was added to the reaction mixture to 
neutralized it. The reaction mixture was extracted with ethyl acetate. The 
organic layer was washed with saturated brine, dried over anhydrous sodium 
sulfate and then concentrated. The concentrate was subjected to flush 
column chromatography on silica gel and eluted with hexane/ethyl acetate 
(1:1) to obtain the desired compound (1.7 g) as brown oil. 
NMR (CDCl.sub.3) .delta. (ppm): 1.25 (3H, t, J=7.2 Hz), 1.61 (4H, m), 2.31 
(2H, t, J=7.1 Hz), 2.81 (3H, s), 3.18 (2H, t, J=6.5 Hz), 4.12 (2H, q, 
J=7.2 Hz), 6.65 (4H, s). 
Reference Example 29 
2-Amino-5-[N-(4-ethoxycarbonylbutyl)-N-methylamino]phenylthiosulfonic acid 
N-(4-Ethoxycarbonylbutyl)-N-methyl-p-phenylenediamine (1.7 g) was dissolved 
in 1N hydrochloric acid (13.6 ml) and to the solution was added a solution 
of aluminium sulfate 14-18 hydrate in water (10 ml). The mixture was 
ice-cooled. A solution of sodium thiosulfate 5 hydrate (2 g) in water (8 
ml) was added thereto and further a solution of sodium dichromate 2 
hydrate (0.798 g) in water (5 ml) was added dropwise. The resulting 
mixture was stirred for 30 minutes under ice-cooling. Then acetic acid 
(0.6 ml) was added and the mixture was further stirred at room temperature 
for 2 hours. The reaction mixture was concentrated to dryness. The 
concentrate was made alkaline with an aqueous sodium bicarbonate and 
insoluble materials were filtered off. The filtrate was adjusted to pH 4 
with acetic acid, concentrated to about 50 ml and cooled. The resulting 
crystalline precipitate was filtered off, washed with cold water and dried 
to obtain the desired compound (2.1 g). 
NMR (DMSO-d.sub.6 +D.sub.2 O) .delta. (ppm): 1.64 (3H, t, J=7.0 Hz), 1.51 
(4H, m), 2.30 (2H, m), 3.01 (3H, s), 3.39 (2H, m), 4.04 (2H, q, J=7.0 Hz), 
6.80-7.40 (3H, m). 
IR (KBr) .nu.: 1720, 1620, 1250, 1180, 1110, 630 cm.sup.-1. 
Reference Example 30 
3-[N-(4-Maleimidobutyl)-N-methylamino]-7-[N-(4-ethoxycarbonylbutyl)-N-methy 
lamino]phenazathionium chloride 
N-(4-Maleimidobutyl)-N-methylaniline (0.26 g) obtained Reference Example 27 
was dissolved in acetone (10 ml), and 1N hydrochloric acid (1 ml) and 
water (9 ml) were added thereto. The mixture was ice-cooled. To the 
mixture was added 
2-amino-5-[N-(4-ethoxycarbonylbutyl)-N-methylamino]phenylthiosulfonic acid 
(0.4 g) followed by a solution of sodium dichromate 2 hydrate (0.2 g) in 
water (1 ml). The resulting mixture was stirred for 30 minutes. Acetic 
acid (0.03 ml) was added and the mixture was further stirred at room 
temperature for 1 hour. Then catalytic amount of copper sulfate and 
manganese dioxide (0.25 g) were added, and the resulting mixture was 
stirred at room temperature for 30 minutes and further at 70.degree. C. 
for 1 hour. After cooling., the reaction mixture was filtered and the 
residue was washed with methanol and water. The filtrate was concentrated 
to dryness and mixed with saturated saline and chloroform with shaking. 
The organic layer was dried over anhydrous sodium sulfate and 
concentrated. The residue was subjected to column chromatography on silica 
gel and eluted with CH.sub.3 CN followed by CH.sub.3 CN/H.sub.2 O/1N HCl 
(40:5:1). The desired fraction was concentrated. Acetonitrile was added to 
the concentrate and insoluble materials were filtered off. The filtrate 
was concentrated to dryness to obtain the desired compound (0.289 g). 
NMR (CDCl.sub.3) .delta. (ppm): 1.26 (3H, t, J=7.0 Hz), 1.77 (8H, m), 2.41 
(2H, t, J=5.7 Hz), 3.40 (3H, s), 3.61 (2H, t, J=6.0 Hz), 3.77 (4H, m), 
4.13 (2H, q, J=7.0 Hz), 6.78 (2H, s), 7.65-7.96 (6H, m). 
IR (KBr) .nu.: 1720, 1700, 1595, 1390, 1330, 1140, 880 cm.sup.-1. 
EXAMPLE 11 
3-[N-(4-Maleimidobutyl)-N-methylamino]-7-[N-(4-carboxybutyl)-N-methylamino] 
phenazathionium chloride 
3-[N-(4-Maleimidobutyl)-N-methylamino]-7-[N-(4-ethoxycarbonyl)butyl-N-methy 
lamino]phenazathionium chloride (0.287 g) was dissolved by addition of 
acetonitrile (2 ml) and 1N hydrochloric acid (6 ml). The solution was left 
open for 44 hours. The resulting precipitate having metallic luster was 
filtered off, washed with ether and dried to obtain the desired compound 
(0.246 g). 
NMR (DMSO-d.sub.6) .delta. (ppm): 1.61 (8H, m), 2.30 (2H, t, J=6.0 Hz), 
3.76 (4H, m), 7.03 (2H, s), 7.52 (4H, br. s), 7.85-8.00 (2H, m). 
IR (KBr) .nu.: 1740, 1700, 1390, 1330 cm.sup.-1. 
EXAMPLE 12 
3-[N-(4-Maleimidobutyl)-N-methylamino]-7-dimethylaminophenazathionium 
chloride 
N-(4-Maleimidobutyl)-N-methylaniline (0.26 g) obtained in Reference Example 
27 was dissolved in acetone (10 ml), and 1N hydrochloric acid (1 ml) and 
water (10 ml) were added thereto. The mixture was ice-cooled. To the 
mixture was added 2-amino-5-dimethylaminophenylthiosulfonic acid (0.25 g). 
Then a solution of sodium dichromate 2 hydrate (0.2 g) in water (2 ml) was 
added dropwise. The resulting mixture was stirred at the same temperature 
for 30 minutes. Then acetic acid (0.03 ml) was added thereto and the 
mixture was further stirred at room temperature for 1 hour. Acetone was 
distilled off under reduced pressure. Precipitated insoluble materials 
having metallic luster were filtered and washed with water. The materials 
were suspended in 50% acetone-water (30 ml), and catalytic amount of 
copper sulfate and manganese dioxide (0.25 g) were added to the 
suspension. The mixture was stirred at room temperature for 1 hour and 
further at 70.degree. C. for 1 hour. After cooling, the reaction mixture 
was filtered. The residue was washed successively with acetone, water and 
ethanol. The filtrate was concentrated to dryness. Then the residue was 
subjected to column chromatography on silica gel and eluted with CH.sub.3 
CN followed by CH.sub.3 CN/H.sub.2 O/1N HCl (40:5:1). The desired fraction 
was concentrated. Chloroform (50 ml) and ethanol (4 ml) were added to the 
residue. Insoluble materials were filtered off and then the filtrate was 
concentrated to dryness to obtain the desired compound (0.42 g). 
NMR (DMSO-d.sub.6 +D.sub.2 O) .delta. (ppm): 1.60 (4H, m), 3.31 (3H, s), 
3.36 (6H, s), 3.47 (2H, m), 3.72 (2H, m), 7.00 (2H, s), 7.40-7.60 (4H, m), 
7.85-7.96 (2H, m). 
IR (KBr) .nu.: 1700, 1590, 1390, 1350, 1330 cm.sup.-1. 
EXAMPLE 13 
Coupling of succinimidomethylene blue derivatives to BSA and denatured BSA 
Bovine serum albumin (Miles Inc., Kankakee, Ill., USA) (50 mg) was 
dissolved in 25 mM phosphate buffer (pH 7, 25 ml) containing 0.5% sodium 
lauryl sulfate and 0.25% .beta.-mercaptoethanol. The solution was heated 
at 90.degree. C. for 10 minutes. The reaction mixture was cooled and then 
subjected to Sephadex.RTM. G-50 column gel filtration, and eluted with 25 
mM phosphate buffer to separate the denatured protein from the reducing 
agent. 
All buffers used hereinafter are 0.1M phosphate buffers (pH 7) unless 
otherwise stated. 
The succinimidomethylene blue derivatives 1, 2, 3, 4, 5, 6, 7 and 8 (each 2 
mg) were dissolved in the buffer (1 ml). The buffer (0.5 ml) containing 
BSA (1 mg) was added to each solution (0.5 ml) and the mixture was 
incubated at 6.degree. C. overnight. The buffer (0.75 ml) containing the 
denatured BSA (0.5 mg) was added to each solution (0.25 ml) and the 
mixture was incubated at 6.degree. C. overnight. Lysine (2 mg) was added 
to a solution of the succinimidomethylene blue derivative 1 which had been 
incubated overnight. Each solution was subjected to gel filtration using 
Sephadex.RTM. G-50 column and the unreacted methylene blue derivative was 
removed. 
The concentration of the protein in each conjugate the solution was 
determined by dye-binding assat (Biorad Laboratories, Richmond, Calif., 
U.S.A.). The amount of the coupled methylene blue derivative was estimated 
from absorbance at 662 nm. 
Table 1 shows the number of the methylene blue derivatives incorporated 
into BSA and into denatured BSA (MB/BSA), and their relative 
chemiluminescence activities. 
The chemical structures of the methylene blue derivatives are as follows: 
##STR11## 
TABLE I 
______________________________________ 
Deriv- 
BSA Denatured BSA 
ative MB/ Relative MB/ Relative 
No. BSA Chemiluminescence 
BSA Chemiluminescence 
______________________________________ 
1 36 0.16 13.7 1.07 
2 9.77 0.08 6.7 0.72 
3 27 0.12 8.0 0.71 
4 22 0.23 7.7 2.01 
5 32 0.25 9.2 1.05 
6 * 0.11 8.1 1.43 
7 * 0.20 9.0 0.75 
8 * 0.55 8.0 2.70 
______________________________________ 
*formation of precipitate 
EXAMPLE 14 
Coupling of succinimidomethylene blue derivatives to IgG 
The succinimidomethylene blue derivatives 2, 4, 6 and 8 (each 2 mg) were 
dissolved in the buffer (1 ml). Each solution was mixed with the buffer 
(0.5 ml) containing 0.5 mg of IgG (anti-alpha fetoprotein antibody A4-4, 
manufactured by Wako Pure Chemical Industries, Ltd., Osaka, Japan) so that 
the concentration of the derivative became 15, 50 or 200-fold molar ratio 
relative to IgG and the mixture was incubated at 6.degree. C. overnight. 
Each solution was subjected to Sephadex.RTM. G-50 gel filtration, and the 
unreacted methylene blue derivative was removed. The concentration of IgG 
and the methylene blue derivative (MB) was determined according to the 
method as described above. The antibody binding activity was determined by 
a competitive reaction between IgG-methylene blue derivative conjugate and 
IgG-HRP conjugate in ELISA of alpha fetoprotein. 
The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Deriv- 
ative 
No. Relative IgG 
(Molar MB/IgB Chemiluminescence 
Binding 
Ratio) binding ratio 
Activity Activity (%) 
______________________________________ 
2 (15X) 
0.64 1.7 100 
2 (50X) 
2.0 5.9 ND 
2 (200X) 
4.3 19.4 16 
4 (15X) 
1.6 4.9 100 
4 (50X) 
5.3 16.4 100 
4 (200X) 
part 19.4 20 
precipitated 
6 (15X) 
2.1 4.8 100 
6 (50X) 
precipitate 6.3 100 
6 (200X) 
precipitate 9.3 15 
8 (15X) 
3.1 3.0 100 
8 (50X) 
precipitate 4.8 15 
8 (200X) 
precipitate 1.6 13 
______________________________________ 
ND: not determined 
EXAMPLE 15 
ELISA using IgG-methylene blue derivative conjugate 
A 10% solution of latex of 3.2 .mu.m in average diameter (Sigma Chemical 
Co., St. Lous, Mo. U.S.A.) (0.5 ml) was washed with water (5 ml) twice to 
prepare the solid phase. The latex thus obtained was washed with 0.1M 
carbonate buffer (pH 9.5, 5 ml) and then suspended in the same buffer (5 
ml) again. The suspension was incubated at 56.degree. C. for 20 minutes 
and then anti-alpha fetoprotein antibody (A2-95, manufactured by Wako Pure 
Chemical Industries, Ltd., Osaka, Japan) (1 mg) was added thereto. The 
resulting mixture was incubated for 40 minutes at 56.degree. C. and then 
further for 2 hours at room temperature. The mixture was washed with 0.1M 
phosphate buffer (pH 6, 5 ml) containing 0.2% Tween-20 followed by 
phosphate buffered saline (5 ml, 50 mM sodium phosphate, 0.8% sodium 
chloride, pH 7) containing 25% Block Ace (Snow Brand Products, Sapporo, 
Japan). The mixture was stored in phosphate buffered saline containing 25% 
Block Ace, 0.5% bovine serum albumin and 0.02% merthiolate. 
In ELISA procedure, the 2% latex solution (50 .mu.l) prepared above was 
mixed with IgG-methylene blue derivative conjugate (5 .mu.l) obtained by 
incubating succinimidomethylene blue derivative 4 of 50-fold molar ratio 
with respect to IgG and phosphate buffered saline (250 .mu.l) containing 
10% Block Ace. Various concentrations of alpha fetoprotein was added 
thereto, and the resulting mixture was incubated at 37.degree. C. for 30 
minutes. Each of the mixtures was washed with phosphate buffered saline 
containing 0.1% Tween-20 four times followed by phosphate buffered saline. 
To each of the resulting mixtures was added 0.1M sodium hydroxide (0.4 ml) 
containing 1 mM luminol. Then the suspension was transferred to a plastic 
tube for the measurement by an Optically Pumped Chemiluminescence (OPC) 
method as described in (Japanese Patent Application No. 2-249879 
corresponding to U.S. patent application Ser. No. 07/757,958 and European 
Application No. 91115648.7). The results are shown in FIG. 1. In FIG. 1, 
the ordinate axis indicates photons counted per minute (CPM) and the 
abscissa axis indicates the amount of alpha fetoprotein. From the results, 
the chemiluminescent output linearly increases in accordance with the 
amount of alpha fetoprotein. 
EXAMPLE 16 
ELISA using Fab'-BSA-methylene blue derivative conjugate 
In this example, antibody fragment Fab'-methylene blue derivative conjugate 
was prepared by using bovine serum albumin as a carrier protein. 
F(ab').sub.2 (1 mg) prepared from anti-alpha fetoprotein antibody was 
reduced by incubating it in 1 ml of 0.1M phosphate buffer (pH 6.8) 
containing 20 mM dithiothreitol. The excess dithiothreitol was removed by 
gel filtration in the same buffer. The protein thus obtained was reacted 
with N,N'-bis(3-maleimidopropyl)-2-hydroxy-1,3-propanediamine (2 mg) at 
37.degree. C. for 30 minutes. The excess reagent was removed by gel 
filtration. According to the above-described method, bovine serum albumin 
(1 mg) was denatured, reacted with succinimidomethylene blue derivative 4 
and then reduced with dithiothreitol. The protein was recovered from the 
gel filtration and mixed with the Fab' prepared above. The resulting 
mixture was incubated at 6.degree. C. overnight. According to the same 
manner as that described in Example 15, the Fab'-BSA-methylene blue 
derivative conjugate thus prepared was used for the assay of 
alpha.alpha.fetoprotein. The results are shown in FIG. 2. In FIG. 2, the 
ordinate axis indicates CPM and the abscissa axis indicates the amount of 
alpha fetoprotein. As is apparent from the results, the chemiluminescent 
output linearly increases in accordance with the amount of alpha 
fetoprotein.