A NOVEL SPECTROFLUOROMETRIC METHOD AND COMPOUNDS THAT ARE OF VALUE FOR THE METHOD

Abstract: A spectrofluorometric method for the detection of target structures by the use of two or more fluorescent lanthanide chelate probes for specifically marking each of said structures. The probes used differ with respect to their excitation or emission wavelength maxima. Also disclosed are certain lanthanide chelates that can be used in the method, either for synthesizing the probes or to be used directly as probes. The chelating ligand of these novel probes comprises two heterocyclic rings (preferably pyridyl) linked together so that jointly they can coordinate (chelate) one metal ion. The bridge linking the two rings together permits electron delocalization between the rings. Attached to their 6-positions the two rings carry chelating groups having heteroatoms (N, O or S) that participate in the chelation of the metal ion. Preferably the chelating groups at the 6-positions are -CH2N(CH2COOH)2, -CH2N(CH2CH2COOH)2, (a) or (b).

Description: A novel spectrofluorometric method and compounds that are of value for the method Field of the Invention This invention presents a new spectrofluorometric method utilizing two or more fluorescent probes for the detection of structures (target structures) that can be specifically marked by the probes. The method falls under the well- known concept of multiple labeling. The invention also provides new compounds that can be used either directly as probes in spectrofluorometric methods or as intermediates for the synthesis of the probes. The compounds may also have other uses. The novel compounds comprise chelating structures or ester, salt and chelate forms thereof in which one or more of the chelating heteroatoms have been transformed to said forms. Time-resolved fluorometry is a fluorescence technique in which the lifetime of the emission occurring after a pulsed light excitation is monitored at a cer¬ tain wavelength. In order to be able to utilize the benefits of time-resolved fluorescence, the labels should be fluorophores with a fluorescence lifetime considerably longer than that of the components giving rise to the background signal. The fluorescent half life of certain lanthanide chelates, such as europium, is 5-6 orders of magnitude longer than that of conventional fluo¬ rescent labels. The decay time and intensity of lanthanide fluorescence are very much dependent on the structure of the ligands which chelate the metal and on the physical environment around it. Lanthanide chelates have already found wide application in a variety of biological assays. In the only commer¬ cial methods so far, however, a twc-stage procedure for developing the fluo¬ rescent signal is used. These methods comprise a biospecific affinity reaction of a reactant labeled with lanthanide chelates, followed by the release of the metal ion and formation of a new strongly fluorescent chelate (Dakubu et al. , Clin. Biochem.Anal. 14, 71-101 (1984) and Lbvgren et al. , In: Alternative Immunoassays, Ed: Collins, W.P., John Wiley & Sons Ltd. (1985) 203-17). This can be explained by the difficulties to combine good absorption and energy transfer properties, strong chelating capacity and ability to form strong covalent bonds with labeled molecules to the same ligand. Most of the now existing immunological or hybridization assays utilize reac¬ tions on solid supports but for technical purposes it is preferable to detect the fluorescence in homogeneous solutions. Therefore since fluorescent markers thus have to be released anyway from the solid matrix the aforesaid two-step system could find extensive application. Fluorescent lanthanide chelates have been recognized as being very promising labels. Used as markers in i muno- assays they could be detected with a sensitivity at least at the same level as is commonly shown for 12 I. This could, if properly realized, open up the possibility of utilizing them as markers in areas which until now have used traditional fluorescent labels. Possible candidates for such fields of appli¬ cation are: 1) Homogeneous fluorometric immunoassays 2) Nucleic acid sequencing 3) Fluorescence microscopy 4) Homogeneous hybridization assays for nucleic acid detection 5) Cytometry 6) Nucleic acid and protein finger printing Fluorescent chelates described heretofore, being composed of chemically diffe¬ rent ligands, differ inter se in many aspects, but in most cases their fluo¬ rescence decay time is long enough to permit their use in time-resolved fluo¬ rescence assays. While it is commonly recognized that a high stability cons¬ tant and intensive fluorescence i.e. high quantum yield, are desirable for a fluorescent chelate, some other often-discussed properties are still a natter of controversy. One of them is the dependence of the emitted light on the en¬ vironment that surrounds the label. In our opinion this should be seen not as a negative, nonpredictable effect but as a new property making it possible to use fluorescent chelates in new areas, e.g. in the field of various kinds of homogeneous assays. It is apparent from the literature that most of the efforts have been focused on the development of more 'highly fluorescent and more stable chelates. When aspects of excitation versus emission of certain chelates were investigated, the only object of interest was to develop a che¬ late with a relatively long wavelength excitation maximum while at the same time preserving the large Stokes shift typical of rare earth metals. To our knowledge there are no reports on systematic studies aiming at the development of a group of chelates differing substantially in their fluorescent properties by different excitation or emission spectra, yet being derivatives of one and the same metal. This would give the possibility of using such chelates simul¬ taneously as multiple labels in the same experiment, provided that the shapes of the excitation spectra would not substantially coincide with each other. The multiple labeling technique using traditional organic fluorescent markers is a well established method. A commonly accepted disadvantage is the compara¬ tively fast rate of fluorescence decay of the labels employed, thus creating substantial background problems. It has been shown earlier that the use of long decay time fluorescent chelates of certain rare earth metals may drasti¬ cally diminish this problem simply by permitting effective measurement of the marker fluorescence after the background signal has decayed. For some experi¬ ments, when separation of labeled components is necessary or preferred prior to their determination, and multiple labeling of all the components is the method of choice, it is advantageous to use labels with a minimal degree of difference in their physicochemical properties. For example, this requirement - not always easy to fulfill - was the main difficulty in the nonisotopic approach to nucleic acid sequencing (Hood et al. 1986, Nature pp. 674-679). Obviously there is a need for a family of fluorescent markers that do not substantially differ with regard to their chemical structure, yet exhibiting a wide spectrum of excitation or/and emission wavelengths. Another aspect of the problem, often not emphasized enough, is the dependence of the emitted fluo¬ rescent light on the type of the functional, reactive group connected to the fluorophore in order to allow its coupling to biological materials. It has been shown earlier (EP-A-171 ,978) that the introduction of one nitro group to the side phenyl ring system in the" europium chelate of 4,7-diphenyl-l ,10- phenanthroline-2,9-dicarboxylic acid diminishes the fluorescence by 60 %. The reduction of this nitro group, which results in the introduction of an amino group sharing its electrons through π-type bonds with the rest of the chelate, is combined with an even greater decrease of fluorescence - in fact only 0.27 % of the fluorescence of the unsubstituted compound is preserved. Recently another type of fluorescent chelates has been published (EP-A-195,413) where the substituted 4-phenyl pyridine was used as a chromophore. Since superiority of these compounds was claimed over all other fluorescent chelates and no fluorescent data was presented, we decided to check their value in practice. The compounds of the following structures have been synthesized T ( 1 ( II ) (III ) and their fluorescent properties have been measured. We found again the same tendency toward decreasing intensity of fluorescent light as the ground struc¬ ture (I) is converted to (II) and (III). In particular, compound (II) in the form of the Eu 3 chelate exhibits 8 % of (I) fluorescence and compound (III) only 1.8 %. Even greater differences were noted for appropriate Tb 3_r chelates. In this case the chelated compound (II) shows only 0.006 % fluorescence as compared to the fluorescence of the terbium chelate of (I) . It is therefore clear that a successfully employed reactive group should, if possible, always be attached in a position separating it from the chromophore in such a way that its electronic Interaction with the rest of the molecule can be neglected - preferably through an aliphatic carbon chain. If this is done correctly it will also ensure that the fluorescent properties observed in simple nonreac- tive chelates (without any additional reactive group) will remain essentially the same if such a reactive group is connected to the chelate or if a thus- functionalized chelate combines with biological material. Several aromatic structures have been proposed to function as light absorbing units - chromo- phores. Phenanthrolines have been suggested as already mentioned (EP-A-171,978) . Phenanthrolines, being a chromophoric part of cryptates, are also presented in FR-A-2,570,703. Other chromophoric units have also been suggested, such as: functionalized pyridines (EP-A-195,413, EP- 203,047), substituted phenols (EP-A-68,875) , unsubstituted terpyridines (J. Am. Chem. Soc. 8_6_ (23), 5117-5125, 1964), indole- and benzyl-EDTA (Photochemistry and Photobiology 39_ (6), 763-769, 1984) and p-amino salicylic acid (Analyst 109, 1449- 1450, 1984). Bipyridine is known to be an excellent chromophore but also a very poor chelator in itself. Attempts have been made to introduce one or more bipyridines into a cryptate chelating system (FR-A-2,570,703) as well as into other chelating systems (WO-A-87/04523). Closely related to the present invention is the description of acidic comp- lexone which is a derivative of bipyridine, see Chem. Berichte 118, 22-27, 1985. This paper describes however only the simplest ligand in bipyridine series, and no fluorescent rare earth metal chelate. In addition, the paper does not suggest any possibility of attaching such ligands to any other mole¬ cule. Description of the invention One major aspect of the invention is an improved multiple labeling method for spectrofluorometric assays for the presence of specific chemical structures in a sample. The characteristic feature of this aspect of the invention is to simultaneously use at least two fluorescent lanthanide chelates as probes. The probes employed differ with respect to their excitation and/or emission wave length maxima so that they can be measured independently. Chelates having different ligands will differ in excitation wave lengths, and chelates having different rare earth metals will differ in emission wave lengths. The chelated lanthanide is Eu 3 , Tb 3 , Sm 3 or Dy 3 . This aspect of the invention may be applied to the same general type of spectrofluorometric techniques as set forth on page 2 that utilize a single probe. Heterogeneous hybridization assays and heterogeneous immunoassays are also included. Some of the methods require removal of probe excess, but others do not (e.g. the homogeneous methods). Generally the applicable assays comprise two steps, namely (i) marking the target structure by fluorescent probes, binding specifically to said structure and (ii) measuring the tluorescence from probes. The fluorescent pattern detected will be associated with the presence of the target structure. The sequence of the two steps may be interrupted by other steps such as separa¬ tion steps and/or addition of further reactants as in the case of imrαunoassays of various types. The compounds of the invention have the common structure given in formula (IV) and comprise also ordinary analogs thereof, such as acid, ester, salt and chela¬ te forms involving one or :aore of the chelating heteroatoms. Parent compound Formula IV Among the compounds of the invention it is possible to select several pairs o appropriate rare earth metal chelates which will differ enough with regard t their excitation spectra to enable one of them to be selectively excited in th presence of the other. Most of the compounds described have shown a relativel high fluorescence, comparable to those fluorescent chelates which have alread been described and used in different applications. Moreover, all the describe chelates, being derivatives of bipyridine, bipyrimidine or closely relate systems, differ very little as regards their biophysical properties such a molecular weight, hydrophobicity or electrophoretical mobility, which is of crucial importance when they are employed in experiments which involve multipl labeling. The invention provides methods for attaching a reactive function to such chela tes, a feature necessary for their coupling to a variety of biologically activ molecules. Moreover, this reactive function is connected to the chelate in suc a way that the fluorescence of the chelate is not changed either in the activ form before coupling or after its coupling to a biological molecule. We accom plished this by introducing the reactive group into the chelating chromophori part of the molecule via a aliphatic chain separating the two functions fro each other, or via a linker system composed of both aliphatic and aromati parts. This solution was the only acceptable one if the electronic status of th pyridine or bipyrimidine moiety was to be kept intact, in view of the very con siderable changes that may be caused by substituents connected to the light absorbing aromatic system. Also, the feature that the chelating part fluore¬ scence is independent of the presence of the attached reactive moiety has an additional advantage, namely it makes the usefulness of a given chelate as marker potentially predictable purely on the basis of the physicochemical pro¬ perties of its basic structure (chelate without a functional reactive group) : In formula (IV), n is an integer 0, 1, or 2, preferably 1, and speci¬ fies that the group X-Y is a substituent replacing a hydrogen anywhere in the parent compound (including also e.g. cases where X-Y is a substituent on or replacing any of R, 2 , R 3 , R. , R 5 , R & » When n is 0 the compound of our inven¬ tion is the parent compound in formula (IV). B is a bridge allowing i) electron delocalization between the heterocyclic rings, and ii) simultaneous coordination of the two shown ring nitrogens with a chelated metal ion so as to form a five- or six- membered ring. Examples of bridging groups fulfilling these criteria are a direct link, -0-, -NH- or -CO-. With respect to our experimental data a direct link and -CO- are to be prefer¬ red. A^, A 2 , A 3 , ^, 5 and 6 may all be carbon atoms which for our preferred com¬ pounds result in 2,2'-bipyridine or 2,2'-ketobipyridine structures. A,, A., A,, A , Ai j and g may also denote single nitrogen atoms. In the preferred embodiment only one in the group A, , A 2 , A 3 and/or only one in the group A. , A & , A.- is a nitrogen atom e.g. A, or A-. and/or A. or A,. For the case of B being a direct link this corresponds to the structure of 2-pyridino-2'-pyrimidines or 2,2'-bi¬ pyrimidines or the corresponding bipyridazine compounds. The 2,2'-bipyrimidines are usually symmetric but the synthetic methods employed by us do not exclude unsymmetrical bipyrimidines either. The mentioned nitrogen atoms may exist free or may be oxidized to form N-oxides. Each of Rι~Rg is nothing when the A to which it is attached is a nitrogen, and for each A that is a carbon ι~ g are selected among: a) hydrogen b) hydrocarbon group which may be straight, branched or cyclic, such as alkyl, alkenyl, alkynyl and an aromatic group (aryl) , and optionally con¬ taining additional structures not participating in the chelation like aromatic ring systems, ether, thioether, ester, amido, a ino (primary, secondary or tertiary), carboxy, halo, cyano etc., and other structures exhibiting heteroatoms, c) cyano, halo and nitro, and d) carboxylic acid (COOH), amido (C0NH 2 ), a ino (NH 2 ) , hydroxy (OH) and subs¬ tituted forms of these four groups in which a hydrogen has been replaced with a hydrocarbon group according to point b) above, and in case of the amino and hydroxy groups the hydrogens also have the possibility of being replaced with an acyl group, RCO, in which R is an hydrocarbon group according to point b) above. "Examples of alkyl are lower alkyls having less than 12 carbon atoms. Examples of aromatic groups are phenyl, quinolyl, naphthyl, pyridyl, pyrimi- dyl etc. all of which may contain additional substituents. By selecting alkenyl, alkynyl or an aromatic system directly bound or bound via a carbonyl or a nitro¬ gen atom to either one of the two heteroaromatic rings in formula (IV), the conjugated electron system can be extended to three, four or more aromatic rings or carbon atoms. The nitrogen at any position in the central aromatic system may also exist i oxidized form, e.g. N-oxide. Z and Z' represent identical or different chelating structures, each of whi comprises at least one, preferably more than two or three, heteroatoms havi a free pair of electrons and so positioned that the heteroatoms together wit the nitrogen atoms of the heteroaromatic rings shown are capable of chelatin a metal ion. Examples of efficient chelating heteroatoms are amino nitroge atoms (primary, secondary and tertiary amines), negatively charged oxyge atoms, e.g. in carboxylate anions (COO *" ), enolate anions (C=C-0~) phosphate or phosphonates. Another good chelating structure is the hydroxamate grou (CONOH). Efficient chelating also puts certain steric requirements on th bridge linking two chelating heteroatoms together. These heteroatoms should b placed at a distance of two atoms from each other, but three atoms distanc may be acceptable. In most cases the bridge contains 1, 2 or 3 aliphatic car bon atoms. Among particularly important Z and Z' structures may be mentione N-biscarboxymethyl and N-biscarboxyethyl amino groups and the analogous phos phate (-N(-CH 2 -0-P0 3 ) 2 ) and phosphonate (-N(-CH 2 -P0 3 ) 2 ). The chelatin heteroatoms (N and 0) may exist as the corresponding protonated forms and i the case of 0 also as ester forms such as lower alkyl (C,-C 6 ) or benzyl es ters. In view of spectrofluorometric considerations Z and Z' are preferabl chelated to Eu 3+ , Tb 3+ , Dy 3" * " or Sm 3+ . X-Y represents an inert organic group in which X is an inert and stable bridg and Y is (a) a functional group or (b) a residue of an organic compound (Y' that has properties retained in the compound of formula (IV) (n 1 or 2 after is has been coupled covalently to the parent compound. The term "inert above means that the group or bridge characterized by this adjective does no have any efficient chelating heteroatom closer at on a distance of four atom from the heteroatoms participating in the chelation of a metal ion. In actua practice this means that the four atoms are normally represented by a fou carbon chain. The term "stable" means that the bridge X does not deteriorat when the compounds of the invention are used, for instance the bridge does no easily undergo hydrolysis. In formula (IV), X-Y exists preferably as a substi tuent replacing a hydrogen in the Z and/or Z' groups, for instance in an N biscarboxymethyl group. X-Y may also exist as a substituent on or replacin any of R j _, R 2 , 3 , R 4 , R 5 and R 6 , and then preferably one of the Rs i replaced with X-Y. In addition to the requirement that the bridge X must have an aliphatic car¬ bon, preferably a methylene group (-CH 2 ~) , in the bridging chain when at¬ tached directly to a heterocyclic ring of formula (IV) , X may also contain at least one structural element selected from among the following: -NR- (secon¬ dary and tertiary amine), -C0NR- and -NRCO- (substituted amide), -S-S- (alipha¬ tic disulfide), -S- (aliphatic thioether), -0- (ether), -COO- and -00C- (es¬ ter), -N=N- (diaza) and pure hydrocarbon chain which may be straight, branched or cyclic and contain from 1 to 12 carbon atoms. The carbon chain may be purely aliphatic or purely aromatic (including phenyl, naphthyl, quinolyl, pyridyl and bipyridyl) , but it may also exhibit both types of structures, such as in alkyl- aryl, and other inert functional groups not participating in the chelation mentioned above. The symbol R in the substituted amide above represents prefer¬ ably hydrogen but may be alkyl, for instance an alkyl having less than 5 carbon atoms. The same applies to R in -NR-. Y may be selected from two main categories (A and B below) : A) Y may be the residue of an organic compound (Y') having certain properties which are substantially retained in the compound of formula (IV) (n = 0) . Th compound (Y') may be a biologically active molecule having the ability to participate in biospecific affinity reactions, such as between antigens (hap- tens) and the homologous antibody active components, complementary nuclei acids (RNA, DNA), lectins and carbohydrate structures, protein A and IgG etc. These types of biologically active molecules are often called targeting sub stances (targeting molecules). Usually they have been or can easily be deri vatized to contain functional groups permitting conjugation to diversifie types of compounds. The compound (Y') may also be a multifunctional organi compound which is bound by one of its functional groups to the bridge X so a to leave at least one of its remaining functional groups free for furthe derivatization. B) Y may be a functional group so selected that it can be made to react chemically with a functional group A of an organic compound (Y') so as t form a covalent linkage between Y' and a compound of formula (IV). The selec tion of Y depends on A and vice versa, but it is believed that any artisa can make the proper selection of mutually reactive groups. Y and A may b selected from among electrophilic and nucleophilic groups. If they are a pai of electrophilic groups or a pair of nucleophilic groups, it is possible fo instance to (a) employ oxidative coupling for forming the bond (e.g. -SH HS- — ■ -S-S-) or (b) convert one of the groups of the pair chemically to group of the opposite type. An example of the latter case is the activatio with bifunctional coupling reagents (also called activation reagents). If is nucleophilic and A electrophilic or vice versa these two groups can usual ly be reacted with each other without any preceding activation. Most nucleo philic groups comprise a heteroato having an electron pair available fo reaction with an electron deficient atom (electrophilic group). Examples of suitable functional groups include isothiocyanato, bromoacetamido, iodoacetamido, succinamido, pyridyldithio, mercapto, carboxyl and its activ esters (e.g. N-hydroxysuccinimido or _£_-nitrophenyl) j hydroxyl, aldehyde, amino, diazonium, tosyl, mesytylyl, trexyl, phosphodiester or phosphotriester. Other functional groups are known to those skilled in the art. In a compound according to the invention it is imperative that all the groups mentioned can coexist. However, the reactive group Y does not necessarily hav to coexist with a chelate form of the invention. For some purposes the chelatin part of the molecule may be temporarily protected e.g. in the form of an este so that the protected ligand will be coupled to the target molecule, and after deblocking may finally form the desired labeled product. The protective group is chosen in accordance with known principles (see for instance Protective Groups in Organic Synthesis; Greene TN; John Wiley & Sons Inc; USA (1981)). Factors to be taken into account when the group is chosen are inter alia the stability of the compound with which Y is to be reacted, the reactivity of Y and the type of structure formed upon reaction of Y and the compound intended. One aspect of the invention comprises the use of a compound of formula (IV) (n^O) for binding the fluorescent structure of the parent compound covalently to an organic compound (Y f ) having at least one functional group A which is reac¬ tive with Y. After the covalent binding reaction, and if an acid, ester or salt form as defined above has been used, the product is converted in a manner known per se to the appropriate chelate. The reaction conditions for the binding de¬ pend on the pair of functional groups (Y) and (A), Y' and the compound of formu¬ la (IV) used etc. and are known per se. In this aspect of the invention A.-Ag, R,-R 6 , Z, Z', X, Y, n and have the aforesaid meanings except that Y can only be a functional group and n^O. Protective groups known per se are sometimes required in order to perform the binding. See the above-mentioned text-book. The invention is defined in the appended claims which are part of the descrip¬ tion. The invention will now be illustrated by way of the synthesis and use of a larg number of compounds. Their structure as well as the synthetic routes for thei preparation are shown on separate formula pages (53-64). In case of discrepan cies between a given name and the corresponding structural formula, the latte has precedence. Example 1 (Scheme 1), x=y = H 6,6'-Dimethyl-2,2'-bipyridine (1) Ref. G.R.Newkome et al., J.Inorg.Nucl.Chem. , 43, 1529-1531(1981) 6-Bromo-2-methylpyridine (49.0 g, 0.285 moles), benzyltriethylammonium chloride (11.6 g, 0.0509 moles), sodium formate (25.7 g, 0.408 moles), 10 % palladium on carbon (1.7 g) , 32 % sodium hydroxide (29 ml) and water (86 ml) were refluxed for 24 hours. The mixture was filtered and the aqueous phase was extracted with methylene chloride. After evaporation of solvent the product was distilled. Yield: 59 % m.p. 82-83°C (Lit. 88-89°C) UV (in ethanol): 290 n , 237 nm iHNMR (400 MHz, CDC1..): 2.61 (s, 6 H); 7.11 (d, 2 H, J = 7.6 Hz); 7.64 (t, 2 H, 7.6 Hz); 8.18 (d, 2 H, J = 7.6 Hz) Example 2 (Scheme 2) 2-Bromo-4-methylpyrimidine (2) Ref. D.D.Bly et al., J.Org.Che ., 27, 2945- (1962) To the mixture containing 2-amino-4-methylρyrimidine (40.0 g, 0.367 moles), sodium bromide (180 g, 1.75 moles), sodium nitrite (60.0 g, 0.870 moles) and water (160 ml), 100 ml of concentrated hydrobromic acid was added slowly. The temperature was kept below 0°C during the addition. After stirring for four hours at -5°C - +5°C the temperature was raised to room temperature and the mixture was neutralized with concentrated sodium hydroxide solution. The product was extracted from the aqueous phase with chloroform. After evaporation of the solvent the product was distilled in vacuum. Yield: 23 % b.p. 69-70°C/0.6 mm UV (in ethanol): 255 nm IHNMR (60 MHz, CDC1 3 ): 2.54 (s, 3 H); 7.17 (d, 1 H, J = 5 Hz); 8.42 (d, 1 H, 5 Hz) Example 3 (Scheme 2) 4,4'-Dimethyl-2,2'-bipyrimidine (3) According to: M. Tiecco et al., Synthesis 736 (1984) Nickel chloride hexahydrate (8.48 g, 35.7 mmoles) and triphenylphosphine (37.4 g, 143 mmoles) were dissolved in 180 ml dimethylformamide at 50°C. After bubbling nitrogen through the solution for 20 min 2.30 grams (0.0352 gr.-atoms) zinc powder was added and the mixture was stirred for one hour. 2-Bromo-4- methylpyri idine (2) (6.25 g, 36.1 mmoles) was added and the mixture was kept at 50°C for 3.5 hours. After pouring the mixture into 500 ml of diluted ammonia solution the organic material was extracted with chloroform. The product was extracted from chloroform with 1-N hydrochloric acid. The hydrochloric acid solution was made alkaline and extracted with chloroform. The solvent was evapo¬ rated and the product was purified by means of flash chromatography. Yield: 12 % UV (in ethanol) : 248 nm IHNMR (60 MHz, CDC1-.): 2.72 (s, 6 H); 7.28 (d, 2 H, J = 5 Hz); 8.86 (d, 2 H, J = 5 Hz) Example 4 -j-Bromo-6,6'-dimethyl-2,2'-bipyridine (4) According to: G.R. Newkome et al., J. Inorg. Nucl. Chem. 43, 1529 (1981) 2,5-Dibromo-6-methylpyridine (1.25 g, 5.0 mmoles), sodium formate (0.51 g, 7.5 mmoles), 10 % palladium on carbon (30 mg), benzyltriethylammonium chloride (0.20 g, 0.88 mmoles) and 8 % sodium hydroxide solution (2.0 ml) were refluxed for two days. The mixture was filtered and the aqueous phase was extracted with ethy- lene chloride. The organic phase was dried, the solvent was evaporated and the product was purified by means of flash chromatography (silica, ethylene chlo¬ ride) . The main product was a monobromo substituted compound. Yield: 35 % UV (in ethanol): 296 nm, 251 nm, 245 nm 1-HNMR (400 MHz, CDC1 3 ) : 2.61 (s, 3 H); 2.73 (s, 3 H) ; 7.16 (d, 1 H, J = 7.6 Hz); 7.67 (t, 1 H, 7.6 Hz); 7.88 (d, 1 H, J = 8.5 Hz); 8.12 (d, 1 H, J = 8.5 Hz); 8.19 (d, 1 H, J = 7.6 Hz) Example 5 (Scheme 3) 3,3'-Dihydroxy-6,6'-dimethyl-2,2'-bipyridine (5) Ref. J.Rebek et al. , J.Heterocycl.Chem. , 17, 749-751 (1980) 6-Methyl-3-hydroxypyridine (23.9 g, 0.219 moles), lead dioxide (98.3 g, 0.411 moles) and toluene (1 liter) were combined and refluxed for five hours. The hot solution was filtered and evaporated to dryness. The residue was refluxed with hexane and filtered while hot. The solution was evaporated to dryness giving the product as a yellow powder. Yield: 9.3 % UV (in ethanol): 350 nm, 246 nm iHNMR (400 MHz, CDC1-.): 2.52 (s, 6 H) ; 7.10 (d, 2 H, J = 8.2 Hz); 7.31 (d, 2 H, J = 8.2 Hz) Example 6 (Scheme 3) 3,3'-Dibenzoyloxy-6,6'-dimethyl-2,2'-bipyridine (6) 3,3'-Dihydroxy-6,6'-dimethy1-2,2'-bipyridine (5) (0.22 g, 1.02 mmoles) was dis¬ solved in 12 ml pyridine and benzoylchloride (0.30 g, 2.13 mmoles) was added. After stirring for 1.5 hours at room temperature, 100 ml chloroform was added and the solution was extracted with 100 ml saturated sodium hydrogen carbonate solution. The chloroform phase was dried and evaporated to dryness. The product was crystallized as a grey powder from cyclohexane. Yield: 51 % UV (in ethanol): 276 nm, 232 nm iHN R (400 MHz, CDC1-.) : 2.33 (s, 6 H); 7.17 (d, 2 H, J = 9 Hz); 7.43 (t, 4 H, J = 8 Hz); 7.58 (t, 2 H, J = 8 Hz); 7.60 (d, 2 H, J = 9 Hz); 8.01 (d, 4 H, J = 8 Hz) Example 7 (Scheme 4) 3,3'-Dicarboxy-6,6'-dimethyl-2,2'-bipyridine (7) According to: F.L. Wimmer et al., Org. Prep. Proced. 15, 368 (1983). Neocuproin hydrochloride (2.5 g, 12.1 mmoles), sodium hydroxide (0.76 g) , potassium permanganate (4.51 g) and water (60 ml) were refluxed for 24 hours. After filtration the solvent was evaporated to half volume. The pH was adjus¬ ted to 2 with concentrated hydrochloric acid and some activated charcoal was added. After 10 minutes charcoal was filtered off and the product was precipita¬ ted with ethanol. The precipitated powder was filtered and crystallized from ethanol. Yield: 22 % UV (in ethanol): 270 n , 226 nm --HNMR (400 MHz, DMSO): 2.60 (s, 6 H); 7.58 (d, 2 H, J - 8.1 Hz); 8.38 (d, 2 H, J = 8.1 Hz) Example 8 (Scheme 4) 3,3'-Bis(carbethoxy)-6,6'-dimethyl-2,2'-bipyridine (8) Thionyl chloride (0.5 ml) was added to 10 ml of dry ethanol. After 15 minutes, 3,3'-dicarboxy-6,6'-dimethyl-2,2'-bipyridine (740 mg, 2.7 mmoles) was added and the mixture was refluxed overnight. The mixture was poured onto ice and the solution was neutralized with sodium hydrogen carbonate. The product was extrac¬ ted with ethyl acetate, dried and evaporated. Yield: 16 % UV (in ethanol): 270 nm, 227 n --HNMR (400 MHz, CDC1 3 ) : 1.01 (t, 6 H, J - 7.0 Hz); 2.63 (s, 6 H); 4.08 (q, 4 H, J = 7.0 Hz); 7.28 (d, 2 H, J = 8.1 Hz); 8.28 (d, 2 H, J = 8.1 Hz) Example 9 (Scheme 5, R=C 6 H 5 -CH 5 -CH=-CH=styryl) 5,6-Dioxo-l,10-diphenyl-deca-l,3,7,9-tetraene (9) Ref. P.Karrer et al. , Helv.Chi .Acta, , 28, 1181-1184 (1945) A mixture of 2,3-butanedione (30.0 g, 0.348 moles), trans-cinnamaldehyde (92.0 g, 0.696 moles), ethanol (100 ml) and piperidine (2 ml) was refluxed overnight. After cooling to 0°C the crystallized product was filtered, washed with cold ethanol and recrystallized from ethanol. Yield: 24 % UV (in ethanol): 367 nm X HNMR (400 MHz , CDC1 - ) : 6.93-7.08 (m, 6 H) ; 7 .34-7 .40 (m, 6 H) ; 7 .49-7 .51 ( dd , 4 H, J = 1 .5 Hz & 8 .0 Hz) ; 7 .58-7 .65 (m, 2 H) Example 10 (Scheme 5) ή,6'-Dimethyl-4,4'-distyryl-2,2'-bipyridine (10) Ref. F. Krδhnke, Synthesis, 1-24 (1976) 5,6-Dioxo-l,10-diphenyldeca-l,3,7 ,9-tetraene (9) (15.7 g, 50 mmoles), acetonyl- pyridinium chloride (17.2 g, 100 mmoles), ammonium acetate (100 g) and methanol (500 ml) were refluxed for two hours. After cooling the product was filtered, washed with ethanol and crystallized from dimethyl formamide. Yield: 14 % UV (in ethanol): 311 nm X HNMR (60 MHz, CDC1 3 ): 2.68 (s, 6 H); 7.20-7.61 (m, 16 H); 8.34 (s, 2 H) Example 11 (Scheme 6, starting material) 6-Bromo-2-dimethoxymethyl pyridine (11) 6-Bromo-2-pyridinecarboxaldehyde (j.Am.Chem.Soc. 91, (11), 3500 (1970)) (11.38 g, 62.2 mmoles) was dissolved in a mixture of dry methanol (200 ml) and tri- methyl orthoformate (26.5 g, 250 mmoles). After addition of p-toluenesulfonic acid raonohydrate (250 mg) the mixture was refluxed for 1 h, cooled and neutrali¬ zed by addition of pyridine (5 ml). Evaporation of solvent and distillation of the product under reduced pressure yielded pure dimethylacetal as a colorless liquid. Yield: 96% X HNMR (60 MHz, CDCI3): 3.36 (s, 6 H); 5.25 (s, 1 H); 7.46 (m, 3 H) Example 12 (Scheme 6) Bis(6-dimethoxyme hyl-2-pyridyl)ketone (12) 6-Bromo-2-dimethoxymethyl pyridine (11) (14.2 g, 61 mmoles) was dissolved in dry diethyl ether (200 ml) and cooled down to -70°C in a round bottom three-necked flask equipped with reflux condenser and dropping funnel while a gentle flow of dry argon was passing through the magnetically stirred mixture. Butyllithium (25.1 ml (2.6M), 65.3 mmoles) was added dropwise and the tempera¬ ture of the reaction mixture was kept below -60°C. After completion of this addition the mixture was stirred for an additional 1 h, and ethyl chloroformate (4.52 g, 41.7 mmoles) dissolved in dry diethyl ether was then introduced at a rate such that the temperature did not exceed -60°C The yellow suspension was stirred for 45 min at -60°C and then additionally for 15 min at -40°C. The reaction was quenched with methanol (20 ml) and the reaction mixture was poured into saturated aqueous sodium hydrogen carbonate (300 ml). The ether phase was separated and the aqueous phase was extracted twice with 100 ml of dichloro- methane. The combined organic phase was evaporated and coevaporated with toluene which yielded crude title compound (12). --HNMR (60 MHz, CDC1 3 ): 3.38 (s, 12 H); 5.30 (s, 2 H); 8.18-7.64 (m, 6 H) Example 13 (Scheme 6) Bis(6-formyl-2-pyridyl)ketone (13) Crude bis(6-dimethoxymethyl-2-pyridyl)ketone (12) was dissolved in dioxane (40 ml) and water (25 ml). Concentrated hydrochloric acid (3 ml) was added and the magnetically stirred mixture was boiled for 15 min. The dark solution was then poured into saturated sodium hydrogen carbonate solution (100 ml) and extracted with chloroform (3x100 ml) . The residue after evaporation of the combined ex¬ tracts as filtered through a short silica gel column using 4% Et0H/CHCl 3 as a solvent and the fractions containing the product were evap.orated. The product was crystallized from hot toluene (100 ml). Yield: 65% (based on compound 11) L HNMR (60 MHz, CDC1 3 ): 8.48-8.11 ( , 6 H); 10.07 (s, 2 H) Example 14 (Scheme 6) Bis(6-hydroxymethyl-2-pyridyl)ketone (14) To the compound (13) (2.5 g, 52.5 mmoles) in dry ethanol (50 ml) sodium -boro- hydride (400 mg, 52.5 mmoles) dissolved in dry ethanol (30 ml) was added drop- wise at 0°C with gentle stirring. A satisfactory ratio of diol (14)/triol was obtained when about 2/3 of the borohydride had been added. Acetone (20 ml) was added for destroying unreacted reducing agent and the mixture was evaporated, dissolved in chloroform/ethanol 1:1 and extracted with saturated sodium hydrogen carbonate. The organic phase was evaporated, coevaporated with toluene and puri¬ fied by silica gel column chromatography using Et0H/CHCl 3 1:9 as an eluting solvent. Yield: 48% X HNMR (60 MHz, CDC1 3 + CD 3 0D): 4.77 (s, 4 H); 7.98-7.27 ( , 6 H) Example 15 (Scheme 8) 6,6'-Dimethyl-2,2'-bipyridine-N-oxide (15) 6,6'-Dimethy1-2,2'-bipyridine (1.97 g, 0.0107 moles, Example 1) was dissolved in chloroform (10 ml). m-Chloroperbenzoic acid (1.85 g, 0.0107 moles) was dissol¬ ved in chloroform (40 ml), and added slowly to the bipyridine solution at 0-5°C. After stirring for two hours at room temperature the solution was extracted twice with saturated sodium hydrogen carbonate solution and three times with water. The chloroform phase was dried and evaporated. The product was purified by flash chromatography. Yield: 70 % UV (in ethanol): 270 nm, 250 nm IHNMR (400 MHz, CDC1 3 ): 2.61 (s, 3 H) ; 2.62 (s, 3 H); 7.19 (d, 1 H, J = 8 Hz); 7.28 (d, 1 H, J = 5 Hz); 7.69 (t, 1 H, J = 8 Hz); 7.97 (t, 1 H, J = 5 Hz); 8.53 (d, 1 H, J = 8 Hz) Example 16 (Scheme 8) 6,6'-Dimethyl-2,2'-bipyridine-N,N'-dioxide (16) 6,6'-Dimethy1-2,2'-bipyridine (10.0 g, 0.054 moles) was dissolved in chloroform (50 ml). m-Chloroperbenzoic acid (21.0 g, 0.122 moles) was dissolved in chloro¬ form (200 ml) and the solutions were combined. After stirring at room temperatu¬ re overnight the solvent was evaporated and the product was purified by means of flash chromatography (silica, 0 - 30 % methanol/chloroform). Yield: 68 % UV (in ethanol): 263 nm, 225 nm iHNMR (400 MHz, CDC1-.): 2.58 (s, 6 H) ; 7.23 (t, 2 H, J = 8 Hz); 7.34 (dd, 2 H, J = 8. Hz & 2 Hz); 7.36 (dd, 2 H, J = 8 Hz & 2 Hz) Example 17 (Scheme 8) 6,6 τ -Dimethyl-4-nitro-2,2'-bipyridine-N-oxide (17) 6,6'-Dimethyl-2,2 r -bipyridine-N-oxide (1.50 g, 0.00749 moles) was dissolved in concentrated sulfuric acid (8.0 ml) and fuming nitric acid (6.0 ml) and the mixture was heated at 100°C for four hours. The solution was poured slowly into ice-water and the pH was adjusted to 5.5 with 10 % sodium hydroxide. The product was filtered and dried. Yield: 53 % m.p. 160-163°C UV (in ethanol): 337 nm, 294 nm, 233 nm IHNMR (400 MHz, CDC1_,) : 2.62 (s, 3 H) ; 2.65 (s, 3 H); 7.27 (d, 1 H, J = 8 Hz); 7.75 (t, 1 H, J = 8 Hz); 8.10 (d, 1 H, J = 3 Hz); 8.56 (d, 1 H, J = 8 Hz); 8-93 (d, 1 H, J = 3 Hz) Example 18 (Scheme 8) ( ,6-Dimethyl-4, '-dinitro-2,2'-bipyridine-N,N'-dioxide (18) 6,6'-Dimethyl-2,2'-bipyridine-N,N'-dioxide (6.84 g, 0.0316 moles) was stirred with concentrated sulfuric acid (33.5 ml) and fuming nitric acid (25.0 ml) at 100° C for five hours. The pH was adjusted to 5.5 with 10 % sodium hydroxide. The precipitated product was filtered and dried in vacuum. Yield: 48 % UV (in ethanol): 336 nm X HNMR (400 MHz, DMS0): 2.48 (s, 6 H); 8.53 (d, 2 H, J = 4 Hz ); 8.61 (d, 2 H, J = 4 Hz) Example 19 (Scheme 8) 4-Bromo-6,6'-dimethyl-2,2'-bipyridine (19) 6,6'-Dimethyl-4-nitro-2,2'-bipyridine-N-oxide (0.50 g, 2.0 mmoles) was dissolved in acetyl bromide (10.0 ml) and phosphorus tribromide (2.5 ml). After refluxing for 1.5 hours the mixture was poured into 100 g ice and the solution was neutra¬ lized with 30 % sodium hydroxide solution. The mixture was extracted with chlo¬ roform, the chloroform phase was dried and evaporated. The product was purified by means of flash chromatography. UV (in ethanol): 291 nm, 242 nm, 217 nm iHNMR (400 MHz, CDC1-,): 2.59 (s, 3 H) ; 2.62 (s, 3 H) ; 7.17 (d, 1 H, J = 7.6 Hz); 7.33 (d, 1 H, 1.5 Hz); 7.68 (t, 1 H, J = 7.6 Hz); 8.17 (d, 1 H, J = 7.6 Hz); 8.41 (d, 1 H, J - 1.5 Hz) Example 20 (Scheme 8) 4~Amino-6,6'-dimethyl-2,2'-bipyridine (20) 6,6'-Dimethyl-4-nitro-2,2'-bipyridine-N-oxide (17) (2.0 g, 8.2 mmoles) was dis¬ solved in methanol (20 ml). 10 % palladium on carbon (0.36 g) was added followed by slow addition of sodium borohydride (3.6 g, 95 mmoles). After one hour the reaction mixture was filtered and the solution was evaporated. The residue was dissolved in 0.1 N sodium hydroxide solution and the product was extracted with chloroform from the water phase. The chloroform phase was dried and evaporated. Yield: 80 % m.p. 101-103°C UV (in ethanol): 284 nm, 236 nm IHNMR (400 MHz, CDC1-.): 2.48 (s, 3 H); 2.61 (s, 3 H); 6.40 (d, 1 H, J = 2.1 Hz); 7.12 (d, 1 H, J = 7.6 Hz); 7.47 (d, 1 H, J = 2.1 Hz); 7.65 (t, 1 H, 7.6 Hz); 8.13 (d, 1 H, J = 7.6 Hz) Example 21 6,6'-Dimethyl-4-ethoxy-2,2'-bipyridine-N-oxide (21) " 6,6'-Dimethyl-4-nitro-2,2'-bipyridine-N-oxide (17) (1.0 g, 4.1 mmoles) was added to sodium ethoxide solution made from (0.19 g, 0.0083 g-atoms) sodium and 25 ml ethanol. The mixture was stirred at 70 C for 30 minutes. After neutralization with concentrated hydrochloric acid the reaction mixture was filtered and evaporated to dryness. The product was purified by means of flash chromatography (silica, 0 %-50 % methanol/chloroform). Yield: 90% UV (in ethanol): 272 nm, 239 nm X HNMR (400 MHz, CDC1 3 ): 1.45. (t, 3 H, J = 7.0 Hz); 2.58 (s, 3 H); 2.61 ( s , 3 H) ; 4.14 (q, 2 H, J = 7.0 Hz) ; 6.84 (d , 1 H, J » 3.4 Hz) ; 7.20 (d , 1 H , J = 7.7 Hz) ; 7.54 (d , 1 H, J = 3.4 Hz) ; 7.70 (t , 1 H, 7 .7 Hz) ; 8.65 (d , 1 H , J = 7 .7 Hz) Example 22 (Scheme 8) 6,6'-Dimethyl-4-ethoxy-2,2'-bipyridine (22) 6,6'-Dimethyl-4-ethoxy-2,2'-bipyridine-N-oxide (21) (0.96 g, 3.93 mmoles) was dissolved in chloroform (34 ml). After addition of phosphorus tribromide (3.0 ml) the mixture was refluxed for 1.5 hours. The solution was poured on ice, some chloroform was added and the phases were separated. The chloroform phase was extracted with water. The aqueous phases were combined, made alkaline with sodium hydroxide solution, and extracted with chloroform. Evaporation of the organic phase followed by silica gel chromatography yielded pure title product. Yield: 69 % UV (in ethanol): 286 nm, 243 nm iHNMR (400 MHz, CDC1-.) : 1.45 (t, 3 H, J = 7.0 Hz); 2.56 (s, 3 H); 2.62 (s, 3 H); 4.18 (q, 2 H, J = 7.0 Hz); 6.67 (d, 1 H, J = 2.1 Hz); 7.14 (d, 1 H, J = 7.6 Hz); 7.67 (t, 1 H, J = 7.6 Hz); 7.75 (d, 1 H, J ■ 2.1 Hz); 8.16 (d, 1 H, J = 7.6 Hz). Example 23 (Scheme 9) 4~(2-hydroxyethoxy)-6,6'-dimethyl-2,2'-bipyridine-N-oxide (23) Sodium hydride (2.07 g, 51.8 mmoles) was added to 1,2-dihydroxyethane (100 ml). After 15 minutes 6,6'-dimethyl-4-nitro-2,2'-bipyridine-N-oxide (17) (6.35 g, 25.9 mmoles) was added and the mixture was stirred at 60°C for 4 hours. Excess of 1,2-dihydroxyethane was removed by evaporation in high vacuum and the product was purified by flash chromatography. Yield: 79 % --HNMR (60 MHz, CDC1 3 ): 2.52 (s, 3 H); 2.57 (s, 3 H); 3.83-4.06 (m, 4 H); 6.80 (d, 1 H, J = 4 Hz); 7.16 (d, 1 H, J = 8 Hz); 7.45 (d, 1 H, J = 4 Hz); 7.66 (t, 1 H, J = 8 Hz); 8.27 (d, 1 H, J = 8 Hz) Example 24 (Scheme 9) 4~(2-hydroxyethoxy)-6,6'-dimethyl-2,2'-bipyridine (24) 4-(2-hydroxyethoxy)-6,6'-dimethy1-2,2'-bipyridine-N-oxide (23) (2.40 g, 9.22 mmoles) was dissolved in methanol (75 ml). 10 % palladium on carbon (0.54 g) was added and then slowly sodium borohydride (3.24 g, 85.6 mmoles). Palladium on carbon was filtered out and the solvent was evaporated. The product was purified by means of flash chromatography (silica, 5-50 % methanol/chloroform). Yield: 91 % IHNMR (60 MHz, CDC1 3 ): 2.57 (s, 3 H); 2.61 (s, 3 H); 3.98-4.25 (m, 4 H); 6.69 (d, 1 H, J = 2 Hz); 7.14 (d, 1 H, J - 7 Hz); 7.67 (t, 1 H, J = 7 Hz); 7.81 (d, 1 H, J = 2 Hz); 8.19 (d, 1 H, J = 7 Hz) Example 25 (Scheme 9) 4-(2-benzoyloxyethoxy)-6,6'-dimethyl-2,2'-bipyridine (25) 4-(2-Hydroxyethoxy)-6,6'-dime hy1-2,2'-bipyridine (24) (1.0 g, 4.1 mmoles) was dissolved in dry pyridine (20 ml), and benzoyl chloride (0.63 g, 4.5 mmoles) was added. After 30 minutes chloroform (50 ml) was added and the solution was ex¬ tracted with saturated sodium bicarbonate. The chloroform phase was dried and the solvent was evaporated and coevaporated with dry toluene. The product was purified by means of flash chromatography (silica, 0 - 50 % methanol/dichloro- methane) . Yield: 56 % m.p. 81-83°C iHNMR (60 MHz, CDC1.,) : 2.55 (s, 3 H) ; 2.60 (s, 3 H) ; 3.96-4.22 (m, 4 H); 6.71 (d, 1 H, J = 2 Hz); 7.13 (d, 1 H, J = 8 Hz); 7.36-7.56 (m, 3 H) ; 7.65 (t, 1 H, J = 8 Hz); 7.86 (d, 1 H, J = 2 Hz); 7.98-8.14 (m, 2 H); 8.20 (d, 1 H, J = 8 Hz) Example 26 (Scheme 10, starting material) 4,4' ,6,6'-Tetramethyl-2,2'-bipyridine (26) This compound has been synthesized in a method analogous to the one of Example 1 using 2-bromo-4,6-dimethyl pyridine as a starting material. UV (in ethanol): 289 nm, 241 nm L HNMR (400 MHz, CDC1 3 ): 2.35 (s, 6 H) ; 2.56 (s, 6 H) ; 6.94 (s, 2 H) ; 8.00 (s, 2 H) Example 27 (Scheme 10) 4,4'-Bis(2-phenylethyl)-6,6'-dimethyl-2,2'-bipyridine (27) To liquid ammonia (20 ml) at -50°C enough sodium was added to maintain a blue colored solution. Iron (III) nitrate (20 mg) was added and then sodium (0.19 g, 0.008 gr.ato ). After 45 minutes 4,4' ,6,6'-tetramethy1-2,2'-bipyridine (26) (1.06 g, 5.00 mmoles) was added in small portions and the mixture was stirred for 1.5 hours at -50°C. Benzyl bromide (1.88 g, 11.0 mmoles) was added at -50°C during 2 hours. The mixture was allowed to warm and water (50 ml), ethanol (20 ml) and chloroform (30 ml) was added. The organic phase was extrac¬ ted with saturated ammonium sulfate solution, dried with magnesium sulfate and evaporated. By addition of ethyl acetate some pure compound was precipitated. The rest was purified by means of flash chromatography (silica, 0 - 6 % metha¬ nol/dichloromethane). Yield: 53 % UV (in ethanol): 290 nm, 241 nm- HlNMR (400 MHz, CDC1 3 ) : 2.60 (s, 6 H); 2.98 (bs, 8 H); 6.97 (s, 2 H) ; 7.18 - 7.35 (m, 10 H); 8.07 (s, 2 H) Example 28 (Scheme 10) 4,4'-Bis(2-(p-nitrophenyl)-ethyl)-6,6'-dimethyl-2,2'-bipyridine (28) 4,4'-Bis(2-phenylethyl)-6,6'-dimethyl-2,2'-bipyridine (27) (3.0 g, 7.6 mmoles) was dissolved in tetrahydrofurane (30 ml). 65 % nitric acid (0.81 g) was added and the solution was evaporated. Concentrated sulfuric acid (26 ml) was added and the solution was stirred at 60°C for 30 minutes. The reaction mixture was poured slowly onto 200 ml ice. Water solution was neutralized with solid sodium carbonate and then extracted with chloroform/ethanol solution. The organic phase was dried and evaporated. The product was purified by means of flash chromato¬ graphy. Yield: 34 % UV (in ethanol): 285 nm, 251 nm IHNMR (60 MHz ,CDC1 3 ) : 2.59 (s, 6 H); 3.05 (bs, 6 H); 6.95 (s, 2 H); 7.34 (d, 4 H, J = 9 Hz); 8.08 (s, 2 H); 8.15 (d, 4 H, J = 9 Hz) Example 29 Procedures for introducing reactive halomethyl groups into bipyridines or bipyrimidines A) GENERAL PROCEDURE FOR DIRECT HALOGENATION OF DIMETHYL COMPOUNDS WITH N-HALOSUCCINIMIDES Ref. G.R.Newkome et al., J.Org.Chem. , 48, 5112-5114 (1983) Dimethyl compound (4.0 mmoles), was dissolved in carbon tetrachloride (100 ml). N-Bromosuccinimide (9.0 mmoles) and a catalytic amount of dibenzoyl peroxide were added and the mixture was refluxed overnight. The mixture was filtered and the solution was evaporated to dryness. The product was purified by flash chro¬ matography. E) GENERAL PROCEDURE FOR SYNTHESIS OF BIS HALOMETHYL BIPYRIDINES AND BIPYRIMIDINES VIA BIS ACETATE (Scheme II) i) General procedure for N,N'-dioxide synthesis Ref. W. Sont et al. Org. Prep. Proced. Int. 243-244 (1980) A 6,6'-Dimethy1-2,2'-bipyridine (or an analogous dimethyl-bipyrimidine) (5 mmoles) was dissolved in chloroform (100 ml). m-Chloroperbenzoic acid (15 mmoles) was added and the solution was stirred for 3 hours. After extraction with saturated sodium hydrogen carbonate solution, the chloroform phase was dried and evaporated to dryness. The product was purified by means of flash chromatography. ii) General procedure for diacetate synthesis from N,N-dioxide Ref. G.R.Newkome, J.Org.Chem., 47, 4116-4120 .(1982) A 6,6'-Dimethy1-2,2'-bipyridine-N,N'-dioxide (3 mmoles) was dissolved in acetic anhydride (20 ml) and the solution was refluxed for 0.5 hours. Acetic anhydride was evaporated and the residue was partitioned between saturated sodium hydrogen carbonate and chloroform. The organic phase was dried, evaporated and the product was purified by flash chromatography. ill) General procedure for dialcohol synthesis Diacetate (1 mmol) was dissolved In acetone (10 ml) and 1 N sodium hydroxide (10 ml) was added. After stirring for 1 hour at room temperature the solution was neutralized with hydrochloric acid. Acetone was evaporated and the water phase was extracted with chloroform. Chloroform was evaporated and the product was purified by flash chromatography. iv) General procedure for dibromide or dichloride synthesis from dialcohol Dialcohol (1 mmol) was dissolved in chloroform (15 ml) and phosphorus tribromide or phosphorus trichloride (1.5 mmol) was added. After refluxing for two hours the mixture was cooled and extracted with saturated sodium hydrogen carbonate. The chloroform phase was dried and evaporated to dryness. Purification by flash chromatography gave pure dibromide. C) GENERAL PROCEDURE FOR INTRODUCTION OF BIS-CHLOROMETHYL GROUPS DIRECT FROM DI N,N'-OXIDE (Scheme II) Ref. T.Sakamoto et al. , Heterocycl., 20, 991-994 (1983) A 6,6'-Dimeth 1-2,2'-bipyridine-N,N'-dioxide (1 mmole) (or an analogous bipy- rimidine-N j N 1 - dioxide) was dissolved in 1,4-dioxane (20 ml) and phosphorus oxychloride (7 mmoles) was added. The mixture was refluxed for two hours and then evaporated to dryness. The product was purified by flash chromatography. .Example 30 6,6'-Bis(chloromethyl)-2,2'-bipyridine (30) Ref. G.R.Newkome et al. , J.Org.Chem., 48, 5112-5114 (1983) According to general direct halogenation procedure of Example 29A 6,6'-dimethy1-2,2'-bipyridine (1) (0.26 g, 2.3 mmoles), N-chlorosuccinimide (0.47 g, 5.5 mmoles), dibenzoylperoxide (3.6 mg) and tetrachloromethane (7.2 ml) were refluxed for 24 hours. The mixture was filtered and the solvent was evapo¬ rated. The product was purified by flash chromatography (silica, cyclohexane/ ethylene chloride 9:1). Yield: 12 % UV (in ethanol): 287 nm, 238 nm iHNMR (400 MHz, CDC1..): 4.73 (s, 4 H); 7.47 (d, 2 H, J = 7.5 Hz); 7.80 (t, 2 H, J - 7.5 Hz); 8.38 (d, 2 H, J- = 7.5 Hz) Example 31 (Scheme 14) 4-Bromo-6,6'-bis(bromomethyl)-2,2'-bipyridine (31) Qsing the general procedure (Example 29 A) for halogenation of dimethyl com¬ pounds with N-halosuccinimides and using (19) as a starting material. UV (in ethanol): 290 nm iHNMR (400 MHz, CDC1-.): 4.56 (s, 3 H); 4.62 (s, 3H); 7.49 (dd, 1 H, J = 7.8 & 1.0 Hz); 7.63 (d, 1 H, J - 1.7 Hz); 7.82 (t, 1 H, J = 7,8 Hz); 8.35 (dd, 1 H, J = 7.8 & 1.0 Hz); 8.57 (d, 1 H, J = 1.7 Hz) Example 32 (Scheme 9) 4-(2-benzoyloxyethoxy)-6,6'-bis(bromomethyl)-2,2'-bipyridine (32) Using the general procedure for halogenation of dimethyl compounds with N-halo¬ succinimides (Example 29 A) and compound (25) as a starting material Yield: 20 % iHNMR (60 MHz, CDC1 3 ): 4.54 (s, 2 H) ; 4.59 (s, 2 H) ; 4.43-4.73 (m, 4 H) ; 7.02 (d, 1 H, J = 2 Hz); 7.43 (dd, 1 H, J = 1 & 8 Hz); 7.36-7.56 ( , 3 H) ; 7.78 (t, 1 H, J = 8 Hz); 7.99 (d, 1 H, J - 2 Hz); 7.99-8.15 (m, 2 H) ; 8.38 (dd, 1 H, " J = 1 & 8 Hz) Example 33 (Scheme 10) i) 4,4'-Bis-(2-(p-nitrophenyl)-ethyl)-6,6'-dimethyl-2,2'-bipyridine-N,N-dioxide __ Using the general procedure of Example 29 B i), applying it to compound (28) Yield: 46 % UV (in ethanol): 269 nm iHNMR (60 MHz, CDC1 3 ) : 2.53 (s, 6 H); 3.00 (bs, 8 H); 7.12 (bs, 4 H); 7.30 (d, 4 H, J = 9 Hz); 8.17 (d, 4 H, J = 9 Hz) ii) 4 ,4'-Bis ( 2- ( p-nitrophenyl ) -ethyl)-6 ,6 '-bis(acetoxymethyl)-2 , 2' -bipyridine (34) Using the general procedure for diacetate synthesis according to Example 29 B il) starting from N,N'-dioxide (33) Yield: 54 % UV (in ethanol) : 286 nm, 251 nm iHNMR ( 60 MHz , CDCi . 2.17 ( s , 6 H) ; 3.09 (bs , 8 H) ; 5.27 ( s , 4 H) ; 7.12 (s , 2 H) , 7 .34 (d, 4 H, J = 9 Hz) ; 8.16 (d , 4 H, J = 9 Hz) ; 8.23 (s , 2 H) iii) 4,4'-Bis(2-(p-nitrophenyl)-ethyl)-6,6'-bis(hydroxymethyl)-2,2'- bipyridine Using the general procedure for diol synthesis (Example 29 B iii) and compound (34) as a starting material Yield: 90 % UV (in ethanol): 284 nm i-HNMR (60 MHz, CDC1 3 ): 3.10 (bs, 8 H); 4.81 (s, 4 H); 7.21 (s, 2H); 7.34 (d, 4 H, J = 9 Hz); 8.17 (d, 4 H, J = 9 Hz); 8.17 (s, 2 H) iv) 4,4'-Bis(2-(p-nitrophenyl)-ethyl)-6,6'-bis(chloromethyl)-2,2 1 -bipyridine __ Using the general procedure of Example 29 B iv) applying it to the compound (35) as a starting material .Yield: 36 % UV (in ethanol): 278 nm, 253 nm iHNMR (60 MHz, CDC1 3 ): 3.10 (bs, 8 H); 4.70 (s, 4 H) ; 7.26 (s, 2 H); 7.34 (d, 4 H, J = 9 Hz); 8.17 (d, 4 H, J = 9 Hz); 8.24 (s, 2 H) Example 34 i) 2 » 2'-Dimethy1-4,4'-bipyrimidine-N,N'-dioxide (37) The synthesis was carried out as according to the general description in Example 29 B i). 2,2'-Dimethyl-4,4'-bipyrimidine (150 mg, 0.81 mmoles) (obtained according to F. Effenberger, Chem. Ber. 98, 2260-2265 (1986) see scheme 7) was dissolved in chloroform (2 ml). m-Chloroperbenzoic acid (420 mg, 2.4 mmoles) dissolved in chloroform (5 ml) was added slowly and the mixture was stirred overnight. Sol¬ vent was evaporated and the product was purified by flash chromatography. Yield: 72 % UV (in ethanol): 368 nm, 295 nm iHNMR (400 MHz, CDC1 3 + CH 3 0H): 2.81 (s, 6 H); 8.29 (d, 2 H, J = 7 Hz); 8.51 (d, 2 H, J = 7 Hz) ii) 2,2'-Bis(chloromethylene)-4,4'-bipyrimidine (38) The synthesis was carried out as according to the general description in Example 29 C. 2,2'-dimethyl-4,4'-bipyrimidine-N,N'-dioxide (70 mg, 0.32 mmoles), phosphorus oxychloride (300 mg, 2.0 mmoles) and 1,4-dioxane (7 ml) were refluxed for four hours. The solvent was evaporated and the product was purified by short column chromatoghaphy (5 % methanol/chloroform) Yield: 41 % UV (in ethanol): 285 nm, 276 nm iHNMR (400 MHz, CDC1 3 ): 4.85 (s, 4 H); 8.41 (d, 2 H, J = 5 Hz); 9.00 (d, 2 H, J = 5 Hz) Example 35 -4,4'-Dimethyl-2,2'-biρyrimidine-N,N'-dioxides (39) According to the procedure of Example 29 B i). 4,4'-Dimethyl-2,2'-bipyrimidine (3) (210 mg, 1.13 mmoles) and m-chloroperbenzoic acid (1.4 g, 8.4 mmoles) were dissolved in chloroform (15 ml) and refluxed for five hours. The undissolved m-chloroperbenzoic acid was filtered out and the products (- three different N,N-dioxides) were purified by means of flash chro¬ matography. Isomer 1: Yield: 29 mg UV (in ethanol): 312 nm, 274 nm iHNMR (60 MHz, CDC1 3 ) : 2.61 (s, 6 H); 7.42 (d, 2 H, J =- 5 Hz); 8.26 (d, 2 H, J = 5 Hz) Isomer 2: Yield: 50 mg UV (in ethanol): 316 nm, 272 nm iHNMR (60 MHz, CDClg): 2.59 (s, 6 H); 7.44 (d, 1 H, J - 5 Hz); 7.44 (d, 1 H, J = 7 Hz); 8.28 (d, 1 H, J » 5 Hz); 8.49 (d, 1 H, J = 7 Hz) Isomer 3: Yield: 32 mg UV (in ethanol): 320 nm, 273 nm iHNMR (60 MHz, CDCI3) : 2.59 (s, 6 H); 7.29 (d, 2 H, J = 7 Hz); 8.47 (d, 2 H, J = 7 Hz) ii) 4,4'-Bis(chloromethylene)-2,2'-bipyrimidine (40) According to the procedure of Example 29 C 4, '-di ethyl-2,2 , -bipyrimIdine- ,N , -dioxide (39) (150 mg, 0.23 mmoles) (isomer 2) was dissolved in 1,4-dioxane (4 ml) and phosphorus oxychloride (400 μl, 1.6 mmoles) was added. The mixture was refluxed for two hours and then evaporated to dryness. Some ethanol was added and the mixture was evaporated again. The pro¬ duct was purified by flash chromatography (silica, 0 - 5 % methanol/chloroform) Yield: 31 % UV (in ethanol): 248 nm i-HNMR (400 MHz, CDC1 3 ) : 4.85 (s, 4 H); 7.76 (d, 2 H, J = 5 Hz); 9.09 ( , 2 H) Both of the other isomeric N,N'-dioxides gave the same dichloride under iden¬ tical conditions. Example 36 (Scheme 6) BJs(6-bromomethyl-2-pyridyl)ketone (41) Bis(6-hydroxymethyl-2-pyridyl)ketone (14) (410 mg, 1.68 mmoles) suspended in dichloromethane (15 ml) was stirred at room temperature in a 50 ml round bottom flask. Phosphorus tribromide (1.82 g, 6.72 mmoles) was added at once and the mixture was refluxed for 5 min. The cooled mixture was partitioned between satu¬ rated sodium hydrogen carbonate and chloroform. The organic phase was collected, evaporated, coevaporated with toluene, and purified by flash chromatography using chloroform as solvent. Yield: 87% UV (in dichloromethane): 282 nm X HNMR (60 MHz, CDCl 3 ); 4.55 (s, 4 H); 8.16-7.22 (m, 6 H) Example 37 (Scheme 12) L-Lysine ethyl ester (42) Thionyl chloride (5.0 ml, 8.06 g, 68 mmoles) was dropped into 500 ml of ice- cooled dry ethanol. The stirred mixture was kept for 20 min at this temperature and L-lysine hydrochloride (20 g, 109 mmoles) was added. The mixture was then refluxed for 3 h and concentrated to a volume of about 200 ml. 200 ml of diethylether was added and the crystallized product filtered off. Yield: 97 % dihydrochloride ' Example 38 (Scheme 12) " ω-N-(4-Nitrobenzoyl)-L-lysine ethyl este r (43) L-lysine HC1 (5 g, 27.4 mmoles) dissolved in 50 ml of water was titrated with 5 M NaOH to pH 10.5. 4-Nitrobenzoyl chloride (6.6 g, 36 mmoles) in dioxane (50 ml) and 5 M NaOH were slowly added keeping the vigorously stirred reaction mixture at pH 10.5. After complete addition and disappearance of the pink color the reaction mixture was acidified with cone. HC1 to pH 2 and extracted four times with diethylether. The aqueous phase was concentrated to dryness, coevaporated twice with 200 ml of dry ethanol and suspended in 250 ml of dry ethanol pre¬ viously treated with 10 ml of thionyl chloride. The mixture was refluxed for 3 h, filtered and evaporated. The residual material was partitioned between saturated sodium hydrogen carbonate and chloroform/ethanol 1:1 and the organic phase was dried over magnesium sulphate yielding a crude product which was purified by flash chromatography using 5% EtOH/chloroform as eluent. Yield: 12% iHNMR (60 MHz, CDC1 3 ): 8.25 (d, 2 H, J=9 Hz); 7.93 (d, 2 H, J=9 Hz); 6.87 (s, broad, 1 H); 4.17 (q, 2 H, J = 7 Hz); 3.30-3.60 (m, 3 H); 1.40-1.75 ( , 8 H); 1.24 (t, 3 H, J = 7 Hz) Example 39 (Scheme 12) α-N-(Methoxycarbonylmethyl)-o-N-(4-nitrobenzoyl)-L-lysine etftyl ester (44) Compound (43) (0.54 g, 1.7 mmoles) was coevaporated with toluene, dissolved in dry acetonitrile (10 ml) and bromacetic acid methylester (0.265 g, 1.7 mmoles) was added followed by pulverized dry sodium carbonate (2.0 g). The mixture was refluxed for 3 h. Filtration of the inorganic salts and evaporation of the acetonitrile gave an oily crude product which was purified by flash chromatography. Yield: 68% iHNMR (60 MHz, CDC1 3 ) : 8.25 (d, 2 ' H, J=9 Hz); 7.93 (d, 2 H, J=9 Hz); 6.63 (s, broad, 1 H); 4.13 (q, 2 H, J -= 7 Hz); 3.68 (s, 3 H); 3.30-3.60 (m, 3 H); " 1.40-1.75 ( , 7 H); 1.24 (t, 3 H, J = 7 Hz). Example 40. (Scheme 12) ω-N-Monomethoxytrityl°L°lysine ethyl este r (45) Dry triethylamine (1.8 ml, 18 mmoles) was added to a suspension of (42) (1.5 g, 6 mmoles) in 20 ml of dry pyridine. To this mixture stirred at RT, solid monomethoxytrityl chloride (1.96 g, 6 mmoles) (MMTrCl) was added in small portions during a period of 1 h whereupon the mixture was stirred for addi¬ tional 2 h. A standard sodium bicarbonate work-up followed by extraction with chloroform yielded a crude product contaminated by α-MMTr isomer. The pure title product was easily isolated by flash column chromatography due to the large Rf difference between the isomers. Yield: 48% iHNMR (400 MHz, CDC1 3 ): 7.5-6.75 (m, 14 H); 4.18 (q, 2 H, J = 7 Hz); 3.78 (s, 3 H); 3.45-3.37 (m, 1 H); 2.14-2.10 (t, 2 H, J=7 Hz); 1.75-1.35 (m, 9 H); 1.26 (t, 3 H, J » 7 Hz) Example 41. (Scheme 12) g -N-(Methoxycarbonylmethyl)- ω -N-monomethoxytrityl-L-lysine ethyl ester (46) A partially protected L-lysine derivative (45) (1.0 g, 2.13 mmoles) was con¬ verted to product (46) using the method described in Example 39. Yield: 70% iHNMR (400 MHz, CDC1 3 ): 7.46-6.77 (m, 14 H); 4.19-4.14 (q, 2 H) ; 3.77 (s, 3 H); 3.70 (s, 3 H); 3.31-3.45 (q, 2 H, J = 7 Hz); 3.22-3.25 (t, 1 H) ; 2.09-2.12 (t, 2 H); 1.35-1.70 (m, 6 H); 1.23-1.27 (t, 3 H) Example 42. (Scheme 12) ω-N-Trifluoroacetyl-L-lysine ethyl este r (47) Compound (42) (2.0 g, 8.1 mmoles), dissolved in 10 ml of dry ethanol was trea¬ ted with dry triethylamine (4.09 g, 40.4 mmoles). Ethyl trifluoroacetate (1.5 g, 10.5 mmoles) was added to the stirred suspension formed, and the mixture was refluxed for 6 h. All volatile substnces were then evaporated and the residue partitioned bet¬ ween saturated sodium hydrogen carbonate and chloroform/ethanol 1:1. The combined organic phase (5x60 ml) was evaporated, coevaporated with toluene and flash chromatographed to get the title product in the form of a colorless oil. Yield: 87% iHNMR (400 MHz, CDC1,.) : 7.10 (t, 1 H, exchangeable); 4.21-4.16 (q, 2 H) ; 3.45-3.40 (m, 1 H); 3.38-3.31 (m, 2 H); 1.84 (s, 2 H, exchangeable); 1.82-1.40 (m, 6 H); 1.28 (t, 3 H) Example 43. (Scheme 12) g-N-(Methoxycarbonylmethyl)- u j-N-trifluoroacetyl-L-lysine ethyl ester (48) L-lysine derivative (47) (1.0 g, 3.7 mmoles) was converted to the product (48) in a method analogous to Example 39. Yield: 83% iHNMR (60 MHz, CDC1 3 +CD 3 0D): 4.4-4.0 (q, 2 H); 3.68 (s, 3 H); 3.5-3.1 (m, 5 H); 1.8-1.4 (m, 6 H); 1.23 (t, 3 H) Example 44. (Scheme 12) ω-N-(4-Hydroxybutyryl)-L-lysine ethyl este r (49) L-lysine ethyl ester, 2 HC1 (42) (2 g, 8.1 mmoles) in 30 ml of dry ethanol was treated with dry triethylamine (5.63 ml, 40.5 mmoles) and γ-butyrolactone (0.7 g, 8.1 mmoles) and the suspension obtained was refluxed for 3 h. Evaporation of volatile substances and coevaporation with toluene yielded a crude product which was purified by flash chromatography using 20% metha- πol/chloroform as solvent. Yield: 73% iHNMR (400 MHz, CDC1 3 +CD 3 0D) : 4.30-4.22 (q, 2 H); 3.72-3.77 (m, 1 H); 3.58- 3.65 (t, 2 H); 3.18-3.28 (m, 2 H); 2.30-2.36 (t, 2 H); 1.40-2.00 (m, 8 H); 1.28-1.34 (t, 3 H) Example 45. (Scheme 12) - g -N-(Methoxycarbonylmethyl)- M -N-(4-hydroxybutyryl)-L-lysine ethyl ester (50) Compound (49) (1.22 g, 4.68 mmoles) in 20 ml of dry acetonitrile was converted to product (50) in a reaction analogous to that in Example 39. Yield: 64% iHNMR (400 MHz, CDC1 ) : 6.25 (s, broad, 1 H); 4.16-4.21 (q, 2 H); 3.73 (s, 3 H); 3.67-3.69 (t, 2 H); 3.33-3.49 ( , 2 H); 3.20-3.30 (m, 3 H); 2.34-2.37 (t, 2 H); 1.40-1.90 (m, 8 H); 1.26-1.30 (t, 3 H) Example 46 (Scheme 13) GENERAL PROCEDURE FOR SYNTHESIS OF UNFUNCTIONALIZED TETRAESTER Bis(nalomethyl) compound (1.0 mmole), di-t-butyl, diethyl or dimethyl iminodi- acetate (2.5 mmoles), sodium carbonate (250 mg) and acetonitrile (10 ml) are refluxed overnight. The mixture is filtered, solvent is evaporated and the product is purified by flash chromatography. Example 47 (Scheme 9) 4-(2-benzoyloxyethoxy)-6,6'-bis(N,N-bis(ethoxycarbonylmethyl)aminomethyl)-2,2'- bipyridine_ (51) Using the general procedure of tetraester synthesis of Example 46 and compound (32) as a starting material. Yield: 63 % iHNMR (60 MHz, CDCI3): 1.23 (t, 12 H, J = 7 Hz); 3.65 (s, 8 H) ; 4.10 (s, 4 H); 4.16 (q, 8 H, J = 7 Hz); 4.50-4.77 ( , 4 H); 7.24 (d, 1 H, J = 2 Hz); 7.56 (d, 1 H, J = 8 Hz); 7.39-7.60 ( , 3 H); 7.79 (t, 1 H, J = 8 Hz); 7.97-8.16 (m, 2 H) ; 8.02 (d, 1 H, J = 2 Hz); 8.31 (d, 1 H, J = 8 Hz) Example 48 (Scheme 9) 4~(2-hydroxyethoxy)-6,6'-bis(N,N-bis(ethoxycarbonylmethyl)amino ethyl)-2,2'- bipyridine (52) 4-(2-benzoyloxyethoxy)-6,6'-bis(N,N-bis(ethoxycarbonylmethyl)aminomethy1- 2,2'- bipyridine (220 mg, 0.31 mmoles) was added to a solution containing ethanol (20 ml) and thionyl chloride (1.5 ml). The mixture was refluxed overnight and then evaporated. The residue was dissolved in chloroform and it was extracted with saturated sodium bicarbonate solution. The product was purified by means of flash chromatography (silica, 0 - 5 % methanol/chloroform) . Yield: 36 % iHNMR (60 MHz, CDCI3): 1.25 (t, 12 H, J = 7 Hz); 3.65 (s, 8 H) ; 4.11 (s, 4 H); 4.17 (q, 8 H, J = 7 Hz); 3.99-4.35 (m, 4 H); 7.21 (d, 1 H, J = 2 Hz); 7.54 (dd, 1 H, J = 2 & 7 Hz); 7.77 (t, 1 H, J = 7 Hz); 7.90 (d, 1 H, J = 2 Hz); 8.28 (dd, 1 H, J = 2 & 7 Hz) Example 49 (as in Scheme 13) 4,4'-Bis( , -bis(t-butoxycarbonylmethyl)aminomethyl)-2,2'-bipyrimidine (53) 4,4'-Bis(chloromethyl)-2,2'-bipyrimidine (18.0 mg, 0.0793 mmoles), di-t-butyl iminodiacetate (76.3 mg, 0.31 mmoles), sodium carbonate (210 mg) and aceto¬ nitrile (3 ml) were refluxed overnight. The mixture was filtered, the solvent was evaporated and the product was purified by flash chromatography (silica, 0 - 5 % methanol/chloroform). Yield: 47 % UV (in ethanol): 244 nm iHNMR (60 MHz, CDC1 3 ): 1.45 (s, 36 H); 3.50 (s, 8 H); 4.23 (s, 4 H); 7.97 (d, 2 H, J = 5 Hz); 8.97 (d, 2 H, J = 5 Hz) Example 50 (Scheme 14) 4-Bromo-6,6'-bis(N, -bis(t-butoxycarbonylmethyl)aminomethyl)-2,2'-bipyridine _ Using the general procedure of Example 46 and compound (31) as a starting mate¬ rial. UV (in ethanol): 291 nm, 242 nm IHNMR (400 MHz, CDClg): 1.47 (s, 36 H); 3.52 (s, 8 H); 4.10 (s, 2 H); 4.11 (s, 2 H); 7.66 (d, 1 H, J = 7.6 Hz); 7.78 (t, 1 H, J = 7,6 Hz); 7.83 (s, 1 H); 8.27 (d, 1 H, J = 7.6 Hz); 8.49 (s, 1 H) Example 51 ~2 , 2'-Bis(N,N-bis(t-butoxycarbonylmethyl)-aminomethyl)-4,4'-bipyrimidine (55) Using the general procedure of Example 46 and compound (38) as a starting material. UV (in ethanol): 276 nm iHNMR (400 MHz, CDC1 3 ) : 1.45 (s, 36 H) ; 3.68 (s, 8 H); 4.34 (s, 4 H); 8.34 (d, 2 H, J = 5.1 Hz); 8.91 (d, 2 H, J = 5.1 Hz) Example 52 6,6'-Bis(N,N-bis(ethoxycarbonylmethyl)-aminomethyl-2-pyridyl)ketone (56) This compound has been prepared using the ' general procedure of Example 46, applying it to compound (41) as a starting material. Yield: 82% UV (in CH 2 C1 2 ): 280 nm iHNMR (60 MHz, CDCl-.): 1.21 (t, " 12 H); 3.60 (s, 8 H); 4.08 (s, 4 H); 4.15 (q, 8 H); 7.95-7.80 (m, 6 H) Example 53 (Scheme 13) GENERAL PROCEDURE FOR THE SYNTHESIS OF REACTIVE LIGAND OR CHELATE EMPLOYING DERIVATIVES OF α, ω DIAMINO CIDS AS STARTING MATERIAL a) The appropriate dihalomethyl bipyridine or bipyrimidine (1 mmol) in dry ace¬ tonitrile (10 ml) is reacted with 1 mmol of one of compounds (44), (46), (48) or (50) in the presence of 2 g powderized dry sodium carbonate at room tempe¬ rature with vigorous stirring. After overnight stirring the resulting mixture composed of unreacted dihalomethyl derivative, monohalomethyl diester, and tetraester is evaporated, coevaporated with toluene and flash chromatographed to give pure monohalomethyl diester. b) The obtained monohalomethyl diester (0.2 mmoles) is coevaporated with dry acetonitrile, dissolved in 3 ml of acetonitrile, and 1 g of powderized sodium carbonate is added followed by iminodiacetic acid diethyl ester (0.25 mmoles). The mixture is refluxed overnight, filtered and evaporated. The desired functionalized tetraester is obtained after short column chromato¬ graphy. c) If compound (44) is used as a starting material in Example 53 a) the nitro group has to be reduced to amino prior to ester group hydrolysis and chelate formation. This is performed in the following way: Solid sodium borohydride (0.3 mmoles) is added to the mixture from Example 53 b) followed by 0.2 g palladium on carbon (10%) in 5 ml of dry ethanol. The resultant mixture is stirred at " room temperature for 10 min and partitioned between saturated sodium hydrogen carbonate and chloroform. The evaporated organic extracts are flash chromatographed to yield the respective tetraester containing a reactive amino group. d) If compound (50) is used as a starting material in Example 53 a) its side chain hydroxyl group has to be activated in order to allow subsequent coup¬ ling of such a ligand to the other hydroxyl containing materials. This can be performed in several ways known to those who a ' re skilled in the art. Here we present one of the possibilities which can be applied for instance to direct coupling of an activated ligand to oligonucleotides synthesized by solid phase method. 2-Cyanoethyl-N,N-diisopropylaminophosphochloridate (2 mmol) is added to a mixture of hydroxytetraester from Example 53 b) and diisopropylethylamine (4.6 mmoles) in anhydrous dichloromethane (10 ml). After 15 min of stirring at room temperature the mixture is washed with cold sodium hydrogen carbo¬ nate, extracted with dichloromethane, evaporated and purified by silica gel column chromatography using petroleum ether/triethylamine 9:1 as the eluting solvent. Example 54 (Scheme 13) GENERAL PROCEDURE FOR TETRAACID SYNTHESIS STARTING FROM DIETHYL OR TETRAETHYL ESTER Bis(N,N-bis(ethoxycarbonylmethyl)aminomethyl) derivative (0.25 mmoles) was dis¬ solved in acetone (10 ml). 1 N sodium hydroxide solution (10 ml) was added. After 1 hour the solution was neutralized with concentrated hydrochloric acid and most of the salts were precipitated by adding acetone. After filtration the solution was evaporated to dryness. Example 55 (Scheme 13) GENERAL PROCEDURE FOR TETRAACID SYNTHESIS STARTING FROM TETRA-t- BUTYL ESTER Bis(N,N-bis(t-butoxycarbonylmethyl)aminomethyl) derivative (0.25 mmoles) was dissolved in trifluoroacetic acid (10 ml). After five hours of stirring trifluoroacetic acid was evaporated and some diethyl ether was added. The product was filtered and dried. Example 56 GENERAL PROCEDURE FOR EUROPIUM OR TERBIUM CHELATE SYNTHESIS Tetra acid (0.20 mmoles) was dissolved in water and the pH was adjusted to 5.0- 5.5. Europium or terbium chloride (0.20 mmoles) dissolved in water was added. After stirring for 30 minutes the pH was adjusted to 8.0 and the precipitate was filtered out. Most of the water was evaporated under reduced pressure and the product was precipitated by adding acetone. Example 57 (Scheme 14) PROCEDURE FOR PHENYLETHYNYLATION OF BIPYRIDINE TETRAESTERS Ref. J.Kankare et al. , EP-A-203,047. 4-Bromo-6,6'-bis(N,N-bis(t-butoxycarbonylmethyl)aminomethyl)-2,2'-bipyridine (750 mg, 1.0 mmole) or the corresponding 4,4'-dibromo derivative (414 mg, 0.50 mmoles), bis(triphenylphosphine)palladium(II) chloride (14.0 mg, 0.020 mmoles) and copper(I) iodide (7.6 mg, 0.040 mmoles) were dissolved in dry triethylamine (15 ml). After bubbling nitrogen gas through the system for 10 min substituted alkyne (1.2 mmoles) was added. After stirring at 40 - 50°C usually for 3 hours the solution was filtered and the precipitate was washed with dry triethylamine. Solvents were evaporated and the residue was dissolved in chloroform. After extraction with water the chloroform phase was dried and evaporated. The product was purified by means of flash chromatography. 4-(p-Aminophenylethynyl)-6,6'-bis(N,N-bis(t-butoxycarbonylmethyl)aminomethyl)- 2,2'-bipyridine (57) ~~ Using p-aminophenyl acetylene as the substituted alkyne UV (in ethanol): 338 nm, 314 nm, 294 nm, 231 nm iHNMR (400 MHz, CDC1-.): 1.47 (s, 18 H); 1.48 (s, 18 H); 3.53 (s, 4 H) ; 3.55 (s, 4 H); 4.12 (s, 2 H) ; 4.13 (s, 2 H) ; 6.65 (d, 2 H, J = 8.7 Hz); 7.36 (d, 2 H, J = 8.7 Hz); 7.64 (d, 1 H, J = 7.8 Hz); 7.67 (d, 1 H, J. • 1.4 Hz); 7.78 (t, 1 H, J = 7.8 Hz) ; 8.30 (d, 1 H, J = 7.8 Hz) ; 8.37 (d, 1 H, J = 1.4 Hz) Example 58 (Scheme 14) 4-(p-Aminophenylethynyl)-6,6'-bis(N,N-bis(carboxymethyl)aminomethyl)-2,2'- bipyridine (58) Using the method of Example 55 with compound (57) as a starting material. UV (in water) : 297 nm L HNMR (400 MHz, DMS0) : 3.68 (s, 4 H); 3.70 (s, 4 H) ; 4.22 (s, 2 H); 4.30 (s, 2 H); 6.62 (d, 2 H, J = 8.0 Hz); 7.33 (d, 2 H, J = 8.0 Hz); 7.65 (s, 1 H); 7.66 (d, 1 H, J = 7.5 Hz); 8.01 (t, 1 H, J = 7.5 Hz); 8.29 (d, 1 H, J - 7.5 Hz); 8.33 (s, 1 H) Example 59 (Scheme 14) 4-(p-Aminophenylethynyl)-6,6 * -bis(N,N-bis(carboxymethyl)aminomethyl)-2,2'- bipyridine as europium chelate (59) The synthesis was carried out using the general procedure for europium chelate synthesis according to Example 56 applied to compound (58) UV (in water): 317 nm, 240 nm Example 60 GENERAL PROCEDURE FOR IS0THI0CYANATE CHELATE SYNTHESIS Ref.Mikola et al., EP-A-0139,675 .Amino chelate (0.05 mmoles) was dissolved in 3 ml water and sodium hydrogen carbonate (20 mg) was added. This chelate solution was added slowly to the thio- phosgene solution (20 μl in chloroform (3 ml)) and stirred for 30 minutes. The phases were separated and the aqueous phase was extracted with chloroform. The aqueous phase was evaporated to a small volume and the product was precipitated with acetone and dried. Example 61 (Scheme 14) 4-(p-Isothiocyanatophenylethynyl)-6,6'-bis(N,N-bis(carboxymethyl) minomethyl)- 2,2'-bipyridine as europium chelate (60) Using the isothiocyanate synthesis of Example 60 applied to chelate (59). UV (in water): 317 nm Example 62 (Scheme 9) synthesis of (61)-phosphitamidate derivative of compound (52) This synthesis was carried out essentially according to the description of Example 53d) using compound (52) as a starting material. Yield: 72% iHNMR (400 MHz, CDCI3): 1.20 (d, 6 H, J = 2 Hz); 1.21 (d, 6 H, J = 2 Hz); 1.25 (t, 6 H, J = 7 Hz); 1.27 (t, 6 H, J = 7 Hz); 2.65 (t, 2 H, J = 5 Hz); 3.67 (s, 8 H); 3.60 - 3.70 (m, 2 H) ; 3.82 - 3.92 ( , 2 H); 3.93 - 4.00 (m, 1 H) ; 4.04 - 4.11 (m, 1 H); 4.10 (s, 2 H); 4.15 (s, 2 H); 4.17 (q, 8 H, J = 7 Hz); 4.30 (t, 2 H, J = 5 Hz); 7.18 (d, 1 H, J = 2 Hz); 7.59 (d, 1 H, J = 8 Hz); 7.76 (t, 1 H, J = 8 Hz); 7.89 (d, 1 H, J - 2 Hz); 8.29 (d, 1 H, J = 8 Hz) 31 PNMR (CDCI3): 148.9 (s) Example 63 (Scheme 10) 4,4'-(Bis(2-(p-nitrophenyl)-ethyl)-6,6'-bis(N,N-bis(ethoxycarbonylmethyl)amino¬ methyl)^,2'-bipyridine (62) .The synthesis was carried out using the general method for tetraester synthesis as in Example 46 applied to compound (36) Yield: 45 % UV (in ethanol): 282 nm iHNMR (60 MHz, CDC1 3 ) : 1.25 (t, 12 H, J = 7 Hz); 3.09 (bs, 8 H); 3.62 (s, 8 H); 4.12 (s, 4 H); 4.18 (q,- 8 H, J = 7 Hz); 7.35 (d, 4 H, J = 9 Hz); 7.43 (s, 2 H) ; 8.15 (d, 4 H, J = 9 Hz); 8.16 (s, 2 H) Example 64 (Scheme 10) 4,4'-Bis(2-(p-aminophenyl)-ethyl)-6,6'-bis(N,N-bis(ethoxycarbonylmethyl)amino¬ methyl)-2,2'-bipyridine_ (63) The synthesis was carried out essentially according to the description of Example 53 c), using compound (62) as a starting material. Yield: 28% iHNMR (60 MHz, CDC1-.): 1.25 (t, 12 H, J=7 Hz); 2.91 (bs, 8 H); 3.70 (s, 8 H); 4.12 (s, 4 H); 4.18 (q, 8 H, J=7 Hz); 6.64 (d, 4 H, J=8 Hz); 6.99 (d, 4 H, J=8 Hz); 7.30 (s, 2 H); 8.10 (s, 2 H) Example 65 (Scheme 10) 4,4'-Bis(2-(p-aminophenyl)-ethyl)-6,6'-bis(N,N-bis(carboxymethyl)aminomethyl)- 2,2'-bipyridine (64) The synthesis was carried out using the general method according to the descrip¬ tion of Example 54 and using compound (63) as a starting material. UV (in water): 300 nm, 289 nm, 235 nm L HNMR (60 MH, DMSO) : 3.00 (bs, 8 H); 3.35 (s, 8 H); 3.64 (s, 4 H); 6.65 (d, 4 H, J=8 Hz); 7.02 (d, 4 H, J=8 Hz); 7.28 (s, 2 H); 8.10 (s, 2 H); Example 66 (Scheme 10) 4,4'-Bis(2-(p-aminophenyl)-ethyl)-6,6'-bis(N,N-bis(carboxymethyl)aminomethyl)- 2,2'-bipyridine as europium chelate (65) The synthesis was carried out using the general procedure for europium chelate synthesis (Example 56) with compound (64) as a starting material. UV (in water): 310 nm, 299 nm, 234 nm Example 67 (Scheme 10) 4,4'-Bis(2-(p-isothiocyanatophenyl)-ethyl)-6,6'-bis(N,N-bis(carboxymethyl)amino¬ methyl)-2,2'-bipyridine europium chelate (66) The synthesis was carried out using the general procedure for isothiocyanate chelate synthesis (Example 60) applied to compound (65). UV (in water): 318 nm, 280 nm Example 68 (Scheme 5) 6,6'-Dimethy1-4,4'-diphenyl-2,2'-bipyridine (67) Ref. F.Kroehnke, Synthesis, 1 - 24 (1976) 2.62 g (10.0 mmoles) 1,6-diphenyl-3,4-dioxo-butane-l,5-diene, 3.43 g (20 mmoles) acetonyl pyridinium chloride, 20 g ammonium acetate and 100 ml methanol were refluxed for four hours. After cooling to 0°C the precipitated product was filtered and recrystallized from methanol. Yield: 40 % UV (in ethanol): 302 nm, 246 nm - • HNMR (60 MHz, CDC13): 2.70 (s, 6 H); 7.41 - 7.79 (m, 12 H); 8.50 (s, 2 H) Example 69 (Scheme 15A) 6,6'-Bis(bromomethyl)- ,4'-diphenyl-2,2'-bipyridine (68) This compound was synthesized according to the general procedure of Example 29 for halogenation of dimethyl compounds with N-halosuccinimides. Yield: 32 % UV (in ethanol): 299 nm, 252 nm L HNMR (60 MHz, CDC1 3 ): 4.71 (s, 4 H); 7.45 - 7.74 (m, 12 H); 8.68 (d, 2 H, J = 2 Hz) Example 70 (Scheme 15A) 4,4'-Diphenyl-6-(N-(methoxycarbonylmethyl)-N-(l-(5-(p-nitrobenzamido))-l- (ethoxycarbonyl)-pentyl)-aminomethyl)-6'-bromomethy1-2,2'-bipyridine (compound (69)) and 4,4'-diphenyl-6,6'-Bis(N-(methoxycarbonylmethyl)-N- (l-(5-(p-nitrobenzamido))-l-(ethoxycarbonyl)-pentyl)aminomethyl)-2,2'- bipyridine (compound (70)) The general procedure of Example 53a gave both diester and tetraester compounds when 6,6'-bis(bromomethyl)-4,4'-diphenyl-2,2'-bipyridine (68) and modified iminodiacetic acid ester (compound 44) were used in the ratio 1:1.2. :, Compound (69) Yield: 41 % iHNMR (60 MHz, CDC13) : 1.26 (t, 3 H, J = 7 Hz); 1.56 - 1.90 (m, 6 H) ; 3.30 - 3.54 (m, 3 H); 3.54 (s, 3 H); 3.75 (s, 2 H); 4.14 (s, 2 H); 4.19 (q, 2 H, J = 7 Hz); 4.70 (s, 2 H) ; 7.36 - 7.85 ( , 12 H) ; 7.78 (d, 2 H,J = 9 Hz); 8.12 (d, 2 H,J = 9 Hz); 8.60 (d, 1 H, J = 2 Hz); 8.63 (d, 1 H, J = 2 Hz) Compound (70) Yield: 21 % iHNMR (60 MHz, CDC1 3 ) : 1.28 (t, 6 H, J = 7 Hz); 1.56 - 1.90 (m, 12 H) ; 3.23 - 3.50 (m, 6 H); 3.56 (s, 6 H) ; 3.74 (s, 4 H) ; 4.14 (s, 4 H); 4.20 (q, 4 H, J = 7 Hz); 7.36 - 7.80 (m, 12 H); 7.80 (d, 4 H, J - 9 Hz); 8.10 (d, 4 H, J = 9 Hz); 8.57 (d, 2 H, J « 2 Hz) Example 71 (Scheme 15A) 4,4'-Diphenyl-6-(N-(methoxycarbonylmethyl)-N-(l-(5-(ρ-nitrobenzamido))-l- (ethoxycarbonyl)-pentyl)-aminomethyl)-6'-(N,N-bis(ethoxycarbonylmethyl) aminomethyl)-2,2'-bipyridine (71) This compound was synthesized following the general procedure for tetraester synthesis according to Example 53b using compound (69) and diethyl iminodiace- tate as starting materials. Yield: 51 % UV (in ethanol): 300 nm, 252 nm iHNMR (400 MHz, CDC1 3 ): 1.21 (t, 6 H, J = 7 Hz); 1.27 (t, 3 H, J = 7 Hz); 1.50 - 1.90 (m, 6 H); 3.35 - 3.50 (m, 3 H); 3.57 (s, 3 H); 3.71 (s, 6 H); 4.14 (q, 6 H, J = 7 Hz); 4.18 (s, 2 H); 4.19 (s, 2 H); 7.45 - 7.83 (m, 12 H); 7.73 (d, 2 H, J = 8 Hz); 8.11 (d, 2 H, J = 8 Hz); 8.58 (d, 2 H, J = 2 Hz) Example 72 (Scheme 15B) 4,4'-Diphenyl-6,6 , -bis(N-(methoxycarbonylmethyl)-N-(l-(5-(p-aminobenzamido))- l-(ethoxycarbonyl)-pentyl)aminomethyl)-2,2'-bipyridine (72) Compound (70) was reduced with palladium on carbon and sodium borohydride accor¬ ding to the general description.of Example 53c. Yield: 76 % UV (in ethanol): 270 nm, 256 nm iHNMR (60 MHz, CDC1 3 ): 1.25 (t, 6 H, J = 7 Hz); 1.43 - 1.87 (m, 6 H) ; 3.22 - 3.52 (m, 6 H); 3.61 (s, 6 H); 3.71 (s, 4 H); 4.16 (q, 4 H, J = 7 Hz), 4.19 (s, 4 H) ; 6.55 (d , 4 H, J - 8 Hz ) ; 7.38 -7 .91 ( , 12 H) , 7.53 (d , 4 H , J Hz) ; 8.62 (d, 2 H, J = 2 Hz) Example 73 (Scheme 15) 4,4'-Diphenyl-6-(N-(methoxycarbonylmethyl)-N-(l-(5-(p-aminobenzamido))-l- (ethoxycarbonyl)-pentyl)aminomethyl)-6'-(N,N-bis(ethoxycarbonylmethyl)a ino- methyl)-2,2'-bipyridine (73~) This compound was produced in a synthesis analogous to that of Example 72 and using compound (71) as a starting material. Yield: 79 % UV (In ethanol): 252 nm iHNMR (60 MHz, CDC1 3 ): 1.25 (t, 9 H, J = 7 Hz); 1.47 - 1.87 (m, 6 H); 3.25 - 3.47 (in, 3 H); 3.61 (s, 3 H); 3.69 (s, 4 H); 3.71 (s, 2 H) ; 4.16 (q, 6 H, J = 7 Hz); 4.22 (s, 4 H) ; 6.57 (d, 2 H, J = 8 Hz); 7.39 - 7.91 (m, 12 H) ; 7.53 (d, 2 H, J = 8 Hz); 8.60 (d, 1 H, J = 2 Hz); 8.63 (d, 1 H, J = 2 Hz) Example 74 (Scheme 15) 4,4'-Dipheny1-6,6'-bis(N-(carboxymethyl)-N-(l-(5-(p-aminobenzamido))-1-carboxy- pentyl)aminomethyl)-2,2'-bipyridine and corresponding europium chelate (74) Using compound (72) as a starting material the title compound was obtained by means of the general procedure for hydrolysis of tetraesters (Example 55) and europium chelate formation (General Example 56). UV (as sodium salt): 261 nm UV (as Eu 3+ complex): 330 nm, 315 nm, 268 nm Example 75 (Scheme 15) 4, '-Diphenyl-6-(N-(carboxymethyl)-N-(l-((5-(p-aminobenzamido))-l-carboxy- pentyl)aminomethyl)-6'-(N,N-bis(carboxymethyl)aminomethyl)-2,2'-bipyridine and, corresponding europium chelate (75) Using with compound (71) as a starting material the title compounds was obtained by means of the general procedures for hydrolysis of tetraesters (Example 55) and europium chelate formation (as in General Example 56). UV (as sodium salt): 310 nm, 256 nm UV (as Eu 3 complex): 330 nm, 315 nm, 261 nm Example 76 (Scheme 16) 4-Ethoxy-6,6'-bis(bromomethyl)-2,2'-bipyridine (76~Ϊ This compound was synthesized according to the general procedure of Example 29A for halogenation of dimethyl compounds with N-halosuccinimides, and using com¬ pound (22) as a starting material. Yield: 19 % UV (in ethanol): 286 nm, 225 nm iHNMR (400 MHz, CDC1 3 ) : 1.45 (t, J=7.0 Hz); 4.19 (q, J=7.0 Hz); 4.55 (s); 4.60 (s); 6.95 (d, J=2.3 Hz); 7.43 (dd, J=0.9 Hz & 7.6 Hz); 7.77 (t, J=7.6 Hz); 7.90 (d, J=2.3 Hz); 8.35 (dd, J=0.9 Hz & 7.6 Hz) Example 77 (Scheme 16) 4-Ethoxy-6-(N-(me hoxycarbonylmethyl)-N-(l-(5-(p-nitrobenzamido))-!- (ethoxycarbonyl)-pentyl)-aminomethyl)-6'-bromomethy1-2,2'-bipyridine (77) This compound was synthesized using the general procedure of Example 53a. 4-Ethoxy-6,6'-bis(bromomethyl)-2,2'-bipyridine (76) and modified iminodiacetic acid ester (compound 44) were used as starting materials. Yield: 38 % UV (in ethanol): 286 nm, 220 nm iHNM (400 MHz, CDC1 3 ) : 1.30 (t, J=7 Hz); 1.45 (t, J=7 Hz); 1.55-1.68 (m) ; 1.75- ' 1.83 (m); 3.45 (m); 3.50 (m); 3.55 (d, J=15 Hz); 3.64 (s); 3.66 (d, J-15 Hz); 3.94 (d, J-15 Hz); 4.03 (d, J-15 Hz); 4.18 (q, J=7 Hz); 4.19 (q, J=7 Hz); 4.58 (s); 6.83 (s); 7.45 (s); 7.62 (d, J=7 Hz); 7.76 (t, J=7 Hz); 8.00 (d, J=9 Hz); 8.25 (d, J=9 Hz); 8.28 (d, J=7 Hz); 8.35 (s) Example 78 (Scheme 16) 4-Ethoxy-6-(N-(methoxycarbonylmethyl)-N-(l-(5-(p-nitrobenzamido))-!-(ethoxy- carbonyl)-pentyl)-aminometh l-6 * -(N,N-bis(ethoxycarbonylmethyl) mino ethy1- 2,2'-bipyridine (78) * This compound was synthesized following the tetraester synthesis general procedure of Example 53b using compound (77) and diethyl i inodiacetate as starting materials. Yield: 62 % UV (in ethanol) : 287 nm, 222 nm iHNMR (400 MHz, CDC1-.): 1.26 (t, J=7 Hz); 1.28 (t, J=7 Hz); 1.46 (t, J=7 Hz); 1.59 (m); 1.76 (m); 3.35 ( ); 3.49 (m); 3.58 (d, J=15 Hz); 3.65 (s); 3.66 (s); 3.67 (s); 3.71 (d, J-15 Hz); 3.96 (d, J-15 Hz); 4.05 (d, J-15 Hz); 4.09 (s); 4.14 (q, J-7 Hz); 4.17 (q, J=7 Hz); 6.88 (s); 7.15 (d, J=2 Hz); 7.50 (d, J-7 Hz); 7.75 (d, J=7 Hz); 7.83 (d, J=2 Hz); 8.01 (d, J-9 Hz); 8.24 (d, J-7 Hz); 8.26 (d, J=9 Hz) Example 79 (Scheme 16) 4-Ethoxy-6-(N-(methoxycarbonylmethyl)-N-(l-(5-(ρ-aminobenzamido))-l- (ethoxycarbonyl)-pentyl)-aminomethyl)-6'-(N,N-bis(ethoxycarbonylmethyl)amino¬ methyl)^,2'-bipyridine (79) This compound was synthesized using the nitrogroup reduction general procedure of Example 53c having compound (78) as a starting material. Yield: 71 % UV (in ethanol): 286 nm, 222 nm iHNMR (400 MHz, CDC1 3 ): 1.26 (t, J-7 Hz); 1.28 (t, J-7 Hz); 1.46 (t, J-7 Hz); 1.60 (m); 1.75 (m) ; 3.41 (m); 3.44 (m); 3.54 (d, J-15 Hz); 3.62 (d, J-15 Hz); 3.64 (s); 3.69 (s); 3.92 (d, J-15 Hz); 4.01 (s); 4.04 (d, J=15 Hz); 4.16 (q, J-7 Hz); 4.17 (q, J-7 Hz); 6.30 (s); 6.64 (d, J-9 Hz); 7.16 (d, J-2 Hz); 7.50 (d, J-7 Hz); 7.63 (d, J-9 Hz); 7.78 (t, J-7 Hz); 7.80 (d, J=2 Hz); 8.24 (d, J-7 Hz) Example 80 (Scheme 16) 4-Ethoxy-6-(N-(carboxymethyl)-N-(l-(5-(p-aminobenzamido))-l-carboxypentyl)amino¬ methyl)-6'-(N,N-bis(carboxymethyl)-aminomethyl)-2,2'-bipyridine (80) and corresponding europium chelate (81) Compound (79) was hydrolyzed using the general method in Example 54 and the europium chelate was formed according to Example 56 starting from compound (80). UV (as sodium salt in water): 295 nm, 286 nm, 225 nm UV (as europium chelate in water): 308 n , 290 nm, 230 nm Example 81 (Scheme 16) 4-Ethoxy-6-(N-(carboxymethyl)-N-(l-(5-(p-isothiocyanatobenzamido))-l-carboxy- ■ pentyl)aminomethyl)-6'-(N,N-bis(carboxymethyl)-aminomethyl)- 2,2'-bipyridine as europium chelate (82) The amino group in compound (81) was converted to isothiocyanato group using the general procedure in Example 60. UV (in water): 309 nm, 290 nm, 228 nm Example 82 (Scheme 6) 6~(N-(carboxymethyl)-N-(l-(5-(p-isothiocyanatobenzamido))-l-carboxypentyl)- aminomethyl)-6 f -N,N-bis(carboxymethyl)aminomethyl-2,2'dipyridyl keton e This compound was prepared using essentially the same procedure as for the synthesis of compound (82). Bis(6-bromomethyl-2-pyridyl)ketone (41) was subjec¬ ted to the reaction with modified iminodiacetic acid (44) according to Example 77. Following the procedure of Examples 78 - 79 - 80 it was converted to an aminoform of europium chelate. The amino group was reacted with thiophosgene analogically to the procedure of Example 81 to form the active isothiocyanate chelate (83). Example 83 Fluorometric method employing lanthandde chelates of the invention in multiple labeling Monoclonal antibodies to human laminine and keratine (10 mg) were separately labeled with a 100-fold molar excess of the isothiocyanate derivatives of Eu chelates (82) and (83). The labeling was done in carbonate buffer, 50 mM, pH 9.8 by incubating overnight. The labeled antibodies were then purified from the free chelates by gel filtration through Sephadex G50 column (1.5x30 cm) eluting with Tris-HCl (50 mM, pH 7.75) containing 9 g of NaCl and 0.5 g of NaN 3 per liter. The bound chelate was quantified by measuring the Eu 3+ after cation dissociation with acid (Hemmila et al., Anal.Biochem. 1984; 137: 335-43). Cryostat sections of tissues were fixed in periodate-lysine-paraformaldehyde fixative. Immunofluorescence stainings were done according to standard proce¬ dures. The labeled antibodies were used in dilutions from 1:10 to 1:1000 and incubated with slides overnight at +4°C. The sections were dehydrated with ethanol and viewed uncovered with a fluorescence microscope (Laitz Dialux) equipped with an epi illumination filter block and mercury lamp. For excita¬ tions filters with bandpass from 340 nm to 380 nm were used and for emission a filter with narrow bandpass at 613 nm. The fluorescence of the chelates was measured according to Kankare et al. (Eur. Pat. Appl. 86 85 0172.7). The relative fluorescence of the chelates was calculated using the formula: I • c • k ■ = : where I , and I_ are pre-exponential terms for chelate I„ • c , • k , che Eu Eu che che and Eu 3 (aq) (used as reference), c , and c„ are their concentrations, and che Eu k and their decay constants, respectively. For testing the solvent effect of the fluorescence of the chelates, the fluor¬ escence was measured in borate buffer, pH 8.5 and in ethanol. The fluorescence properties of some chelates are presented in table 1. Table 1. Fluorescence properties of some 6,6'-bis(N,N-bis(carboxymethyl)aminomethyl)- 2,2'-bipyridines 1 - Log (relative fluorescence) 2 = Excitation wavelength (nm) 3 = Decay time (μs) CI- JNL Br SCHEME ! SCHE-.I: SCHEMT, 3 SCHEME 4 SCHEME 5 (10) R = Sf/r/l (67) R = Phenyl BuLi SCHEME 6 SCHEME 7 CO lysine = CF 3 C0 2 tf SCHEME 1_j = OCH,CH,LH.C=0

Claims: Claims 1. In a spectrofluorometric method for the detection of different target struc¬ tures by the use of two or more fluorescent probes for specifically labeling said structures, the improvement that said probes comprise fluorescent lan¬ thanide chelate structures differing in excitation and/or emission wave length maxima. 2. The method of claim 1, wherein at least two of said probes differ with respect to their excitation wave length maxima. 3. The method of claim 1, wherein at least two of said probes differ with respect to their emission wave length maxima. 4. The method of any of claims 1-3, wherein at least one of said probes has the structure of the following formula (IV). 5. Chelating compound having the structure of the following formula (IV): Substituent Parent compound Formula IV where i) ^- \ ~--s are s: -- n S-'- e atoms selected from the group consisting of a carbon atom and a nitrogen atom, preferably only one of A, , A 2 , 3 and/or only one of A,., A , A 6 being nitrogen, such as A^ or A 3 and A^ or A fi ; ii) n is an integer 0, 1 or 2, preferably 1; iii) Each of R, to R g is nothing when the A to which it is attached is a nitro¬ gen; and for each A that is a carbon atom R^ to R 6 are selected from the group consisting of a) hydrogen and b) carbon-, oxygen- and/or nitrogen- containing groups; iv) Z and Z' represent identical or different structures, each of which compri¬ ses at least one heteroatom having a free pair of electrons so that said at least one heteroatom together with the nitrogen atoms of the heterocyclic rings shown in formula (IV) are capable of chelating a metal ion; v) B denotes a bridge allowing a) electron delocalization between the hetero¬ cyclic rings shown in formula (IV), and b) simultaneous coordination of the two nitrogen atoms shown in formula (IV) to the chelated metal ion so that a five- or six-membered ring is formed, vi) ■ specifies that the group X-Y is a substituent replacing a hydrogen anywhere in the parent compound; and -vii) X-Y represents an organic group which is inert to said chelation, and in which X is an inert and stable bridge and Y is (a) a functional group, or (b) a residue of an organic compound (Y') that has properties conferred on the compound of formula (IV) in which n =- 0; with the proviso that said bridge has an aliphatic carbon atom separating said group Y from one of the heterocyclic rings shown in formula (IV) when X-Y is a substituent; or an acid, ester, salt and chelate form of structure (IV) in which one or more of the said chelating heteroatoms have been transformed to one of said forms; and with the further proviso that for chelates having chelating metal ions other than lanthanides at least one of ^-Rg is not hydrogen for n = 0, Z = Z' = -N-(CH 2 -C00~) 2 and all A-_-A 6 » carbon. 6. The compound of claim 5, c h a r a c t e r i z e d in that Z and Z' are equal. 7. The compound of either of claims 5-6, c h a r a c t e r i z e d in that the said at least one heteroatorα in Z and Z' is selected from the group consis¬ ting of nitrogen, such as in primary, secondary and tertiary amines, nega¬ tively charged oxygen, such as in COO "" , phosphonates and phosphates. 8. The compound of any of claims 5-7, c h a r a c t e r i z e d in that one of the said heteroatoms in Z and Z' is a nitrogen atom linked to one of the heterocyclic rings shown in formula (IV) via an aliphatic carbon bridge having at most two carbon atoms. 9. The compound of any of claims 5-8, c h a r a c t e r i z e d in that at least one of Z and Z' is an N-biscarboxymethyl amino group. 10. Compound according to any of claims 5-9, c h a r a c t e r i z e d in that n=l, and X-Y replaces one of R,-R g , while the A to which X-Y is bound is carbon. 11. Compound according to any of claims 5-10, c h a r a c t e r i z e d in that X-Y replaces a hydrogen in at least one of Z and Z' . 12. The compound of any of claims 5-11, c h a r a c t e r i z e d in that Y is a residue of a molecule capable of participating in a biospecific affi¬ nity reaction.

Inventors: KWIATKOWSKI MAREK, MUKKALA VELI-MATTI, SUND CHRISTIAN, KWIATKOWSKI, MAREK, MUKKALA, VELI-MATTI, SUND, CHRISTIAN
Applicants: WALLAC OY
Application year: 1989
Main classification: G01N33
IPC codes: C07D213, C07D239, C07F5, C07F9, C09K11, G01N21, G01N33