LUMINESCENT LANTHANIDE CHELATES

Field of the invention

The invention pertains to new luminescent lanthani chelates comprising three unsaturated heterocyclic rin covalently coupled to each other and additionally compri ing two chelating groups . This structure of three heter cyclic rings is formed of one or two 2, 6-pyridyle moieties and five-membered unsaturated heterocyclic ring

The new chelates of our invention find applications those areas that are classical for lanthanide chelates a known in the art. Moreover, these compounds are useful probes in time-resolved fluorescence microscopy, cytometr multilabelling techniques and process controls in industr

Description of the Prior Art

In immunoassays and DNA hybridization assays time-resolv luminescence spectroscopy using lanthanide chelates is we known (e.g. I.A.Hemmila, "Applications of Fluorescence Immunoassays" in J.D. inefordner and I.M.Kolthoff, Eds. Chemical Analysis, Vol 117, John Wiley & Sons, Inc., US 1991 and the references therein) . Stable luminesce lanthanide chelates also have other applications, e. fluorescence microscopy and cytometry. Because of the paramagnetic properties, these lanthanide chelates a useful as sensitive probes in magnetic resonance imagi

(MRI) . The radioactive isotopes of the metals such indium and stable chelating liσands on the macromoiecul

offer possibilities to use ligands of this invention in th treatment of diseases such as cancer.

Luminescent lanthanide chelates have previously bee suggested [macropolycycles: French Patent No . 2,570,703

(1986) and Eur. Patent Appl . 321,353 (1988); phenols: U. S.

Patent 4,670,572 (1987); coumarines: U. S. Patent 4,801,722

(1989) and U. S. Patent 4,794,191 (1988) ; - polypyridines:

U. S. Patent 4,837,169 (1989), U. S. Patent 4,859,777 (1989), Int . Pat . Appl . PCT/SE89/00073 (1989) and Int . Pat . Appl . PCT/SE89/00379 (1989); aryl pyridines: U. S. Pat . 4,761,481 (1988) and Int . Pat . Appl . PCT/WO89/04826; ethynyl pyridi¬ nes: U. S. Patent 4,920,195 (1990); phenanthrolines: U. S. Pat . 4 , 712, 563 (1988); salicylates: M.P.Bailey, B.F.Rocks and C.Riley, Analyst, 109, 1449 (1984)].

Stable luminescent lanthanide chelates, whose energy absorbing group is either one 2, 6-pyridylene and two five- membered unsaturated heterocyclic ring moieties, or two 2, 6-pyridylene moieties and one five-membered unsaturated heterocyclic ring moiety coupled to each other with a covalent bond between carbon atoms, are not known. Some basic structures comprising three heterocyclic rings covalently coupled to each other (including one - or two pyridine rings) have been synthesized (see e.g. V. Nair and K.H. Kim, J. Heterocyclic Chem . 13 (1976), 873; S. Kubota and Ξ. Ohtsuka, Tokushima Daiσaku Yakugaku Kenkyu Nempo 9 (1963) 15, CAS8:2449a; J.F. Geldhard and F. Lions, J. Orσ. Chem . 30 (1965) 318; R. Menasse, G. Klein and H. Ξrien- meyer, Helv. Chim . Acta, 38 (1955) 1289; H.A. Goodwin, Aust . J. Chem 27 (1964) 1366; R.J. Clark and J. Walker, J. Chem . Soc . C 6 (1966) 1354; S. Gronowitz and D. Peters, Heterccycies 30 (1) (1990) 645 and A.T. Parker, P. Singh and V. "irnevic , Aust. J. Chem. 44 (1991) 1041) . As such these

compounds are not stable enough for use with lanthanid ions in aqueous solution. A lanthanide chelate, containin one 2, 6-pyridylene and two 1, 3-pyrazolylene groups havin a covalent bond between the carbon and nitrogen atoms, ha been synthesized (M. Alanso, J . de Mendoza, M. Remuiήan, H. Roman and J.C. Rodriguez-Ubis, 2nd Conference on Method and Applications of Fl uorescence Spectroscopy, Graz, Austria, 14-17.10.1991, Pl/25) . The luminescence propertie of this chelate have not been published. The chelate ha no group for coupling to biologically active material an it cannot be used as such for applications mentioned i this invention.

The invention

The compounds of this invention are lanthanide chelate comprising three heterocyclic rings covalently coupled t each other (either one 2, 6-pyridylene and two five-membere unsaturated heterocyclic ring moieties or two 2, 6-pyridy lene and one five-membered unsaturated heterocyclic rin moiety) and two chelating groups so seated that the together chelate the same lanthanide ion even in aqueou solutions. Optionally these lanthanide chelates als contain a reactive group for coupling to biologicall active molecules.

The compounds of the invention are lanthanide chelate consisting of a lanthanide ion and a cheiatcr having common structure shown in Formula .

G, G _z G ₃ 1 I /

Ch-i-Cj- [ , 1 - [3 ] - [A ₂ ] -Ξ-Ch^ Formula I

The acid, ordinary salts and ester forms of Formula I a also novel and notable.

In Formula I, - represents a covalent bond between t carbon atoms and - represents a covalent bond.

E represents ethylene (CHJ or carbonyl (C=0) .

One or two of [AJ , [B] and [AJ in a [AJ-[B]~[AJ struc ture represent a bivalent five-membered unsaturated hetero cyclic ring. Each of the remaining of [AJ , [B] and [AJ i 2, 6-pyridylene. Preferably either [AJ and [AJ or [B] ar 2, 6-ρyridylenes. The heteroatoms in five-membered unsatu rated heterocyclic rings are selected from the grou consisting of nitrogen, sulphur or oxygen. One heteroato in each ring is coordinating to the same lanthanide ion s that two five-membered rings are formed in which one membe is the lanthanide ion and two members are coordinatin heteroatoms of different rings [AJ , [B] and [AJ . Example of preferable five-membered unsaturated heterocycli ^■ bivalent σroucs include:

2, 5-furylene : J

^\' OA

2, 4-thiazolvlene :

2,5-thiazoiyiene

-thiazoiin-2, -viene:

3-thiazoiin-2, -ylene : J ----.

N

2, 4-oxazolylene:

2-oxazolin-2, 4-ylen

3-o:«:azolin-2,4-ylene

2, 4-imidazolylene:

2-imidazolin-2, 4-yle

3-imidazolin-2, 4-yle

N-NH l,2,4-triazol-3,5-ylene: ~-+l -^

N

1,3, 4-oxadiazol-2,5-ylene:

N-O

1, 2, 4-o:adiazol-3, 5- Iene:

~Nλ

..j-D -v -razciy -iene: or

In 2,6-pyridylenes and in five-membered unsaturated heter cyclic bivalent rings one hydrogen can be replaced with t appropriate group G,, G, and G ₃ .

The subs ituents G,, G _: and G ₃ can be selected from t group consisting of hydroxy, nitro, amino or lower alky substituted amino, lower aryl substituted amino or lowe acyl substituted amino, alkyl, aryl, alkyla yl, arylalkyl arylethynyl, such as phenylethynyl, alkoxy or arylox groups with the proviso alkyls contain 1-12 carbon atom and aryls are selected from phenyl, naphthyl and pyridyl G _r , G _z and G, can also be a group containing aryl (selecte from phenyl, naphthyl and pyridyl) and alkylene parts whic contains from 1 to 8 carbon atoms and additionally from to 4 other atoms such as oxygen, sulphur, nitrogen o phosphorus. Each of the above mentioned groups optionall contains amino, aminooxy, carboxyl, hydroxy, aldehyde o mercapto groups or an activated form made from them, suc as iso hiocyanato, isocyanato, diazoniu , bromoacetamido iodoacetamido, reactive esters (such as N-hydroxysuccin i ide, 4-nitrophenyl and 2,4-dinitrophenyl esters) pyridyl-2-dithio, 4-chloro-6-ethoxytriazon-2-ylamino o 4, 6-dichlorotriazon-2-ylamino. Other examples of suitabl groups to be used in the labelling of compounds exhibitin biological affinity are presented e.g. in R.F.Steiner an I.Weinryb (eds.), "Excited States of Proteins and Nuclei Acids" , Basingstibe Corp., London, 1971. One or two of th substituents G _lf G- and G- can also be attached to compound exhibiting biospecific affinity. Such molecule include e.g. proteins (such as enzymes), antibodies, antigens (haptens) , oiigo- and polynucieotides, lectins, receptors,, carbohydrate structures (such as dextrans) , protein A, IgG, drugs etc. This type of biologically activ comcounds are often called tarσet substances (tarσet

molecules) . The binding is performed in such a way tha these molecules still retain their biospecific affinity.

Ch, and Ch, represent identical or different chelatin groups, possibly linked together. Each of these chelatin groups comprises at least two heteroatoms that are coordi nated to the lanthanide ion and are selected from the grou consisting of oxygen and nitrogen. At least one of the sai coordinating heteroatoms in each of Chi and Ch, is formin a five-or six-membered ring together with the lanthanid ion and a coordinating heteroatom of one of [AJ , [B] an [AJ . The distance between each pair of heteroatom participating in the chelation and forming the same five or six-membered ring is two or three atoms, respectively

Examples of efficient chelating heteroatoms include amin nitrogens (primary, secondary and tertiary amine) an negatively charged oxygens (carboxylate anions, enolat anions, phosphates and phosphonates) . In most cases th bridge between the chelating heteroatoms contains 1, 2 o 3 saturated carbon atoms. Among particularly important C and Ch, structures are N,N-bis (carboxymethyl) amino [-N(CH,COO ^" ) J , N,N-bis (carboxyethyl) amino [-N(CH,CH.COO ^~ )J analogous phosphates [e.g. -N(CH,-0-P0 ₃ \' ^~ ) J and phosphonate [e.g. -N(CH _c -Pθ ^" )J and 2, 6-dicarboxypiperidin-l-yl. Alter natively, Ch and Ch, may form one or two bridges that cova lentiy connect the two outer heterocyclic rings ( [AJ an [A,] ) giving a macrocyciic chelating compound. The bridge bridges consist of saturated carbon, o:-:ygen cr nitroge atoms. The nitrogens and the oxygens are selected fro secondary or tertiary amino nitrogens and ether oxygens Preferably such a bridge together with the Ξ- [AJ - [B] - [A -Ξ system forms a carboxymethylated azacrow Fthe bridσe is e.σ. -N(CH,CCO ^~ ) CH,CH,N(CH,COO ^~ ) - cr

-N (CH,COO ^~ ) -] , a crypt ate [the bridge is e . g .

-N (CH,CH,-0-CH ₂ CH,-0-CH,CHJ ₂ N-] or a crown ether (the bridg is e . g . -0-CH,CH,-0-CH,CH,-0- or -0-CH,CH,-0-) .

In some compounds of the invention the chelating hetero atoms (nitrogen and oxygen) may exist as the correspondin protonated forms and for oxygen also as ester forms, suc as lower alkyl (C^-C^) , benzyl or tert-butyl- esters.

From a spectrofluorometric point of view the interestin chelate forms are such molecules where Cl- and Ch, togethe with E-[AJ-[B]~[A,]-E structure are chelated to th lanthanide ion, preferably to Eu ³⁺ , Tb ³ Dy ^3* or Sπr ^4" . Thes ligands can also be suitable for chelating such metal ions as Ru ^:+ , Os ²⁺ , Ir ³⁺ and Rh ³⁺ . As strong chelate formin compounds they are potential chelatόrs also for radioactive isotopes of different metals to be used in applications classical for radiotracers.

The new chelates of our invention find applications in the areas that are classical for lanthanide chelates. More¬ over, these compounds are useful as probes in time-resolved fluorescence microscopy, cytometry, nucleic acid sequenc¬ ing, nucleic acid and protein finger printing, homogeneous hybridization assays for nucleic acid detection, homogeneo¬ us fluorometric immunoassays, multilabelling techniques and process controls in industry.

Contrary to the many previously mentioned patents, full coordination number n ne can easily be achieved with the new lanthanide chelates of this invention. This implies that there are no water molecules coordinated to the lanthanide ion. The quenching effect of water is thus minimized and the decav time cf the luminescence is in ts

maximum. Thus the long-lived and very intensive lumine scence of the lanthanide chelates can be effectively deter mined after the short-lived background has decayed. In e.g microscopic applications and process controls in industr very stable (photo, thermodynamic and kinetic stability chelates are needed. As compared to the other kno lanthanide chelate structures, these stability requirement are best fulfilled with the chelates of this invention.

The smaller size of unsaturated five-membered heterocycle compared to e.g. pyridine and phenol makes the ligands o this invention more flexible. This means that lanthanid ion is better crowded with the chelator and the pi-elec trons of [AJ , [B] and [AJ and the free electron pairs o their heteroatoms are better delocalized over [AJ-[B]-[AJ .

The basicity of some five-membered unsaturated heterocycle (e.g. pK _a for pyridine is 5.25 and for imidazole 6.953 makes them better chelators than pyridine. Moreover, i some of the chelates the aromatic structure is negativel charged (e.g. compounds containing 1,2, 4-triazol-3, 5 ylene) . Besides that this stabilizes chelates, it additio nally changes their adsorption properties diminishin unspesific binding to column materials and plastics.

It is known that C-H stretchings of the ligand decrease th luminescence intensity of the lanthanide chelates. Th number of C-H bonds in the chelates cf this invention ha been reduced by five-membered heterocyclic ring moietie compared to pyridines and phenols.

As compared to the other lanthanide chelates these ne structures of this invention exhibit a greater extinctio

coefficient, longer excitation wavelengths and bett energy transfer from the ligand to the lanthanide io which lead to improvements in the detection sensitivity the luminescent labels.

The structures and the synthetic routes employed in t experimental part are shown in reaction schemes 1-14. T compounds 42 (Scheme 7), 57a and 57b (Scheme.9), 68 (Sche 10) and 80 are examples of compounds capable of binding biologically active molecules. The other schemes represe the synthesis of potential structures which can easily modified to such compounds with known methods. E. compound 32, the corresponding 2, 6-dicyanopyridine deriv tive and analogous compounds having a longer chain betwe the pyridine and the benzene rings can be used as versatile starting material for chelates that can coupled to compounds exhibiting biological affinity. I Example 84 compound 68 has been used for labelling o antibody. In Example 86 terbiu (III) labelled antibody ha been used for time-resolved fluorescence immunoassay an microscopy.

Modified iminodiacetic acid ester can be used instead o unsubstituted iminodiacetic acid ester as is taught in Int Pat. Appl . PCT/SE89/00379 (1989) .

General methods for the preparation of unsaturated five membered heterocycles are skill of art (see prior art an e.g. A.R.Katritzky, "Handbook of Heterocyciiz Chemiszry" Pergamon Press, Great Britain, 1986) .

The synthesis of phosphonic acids can be made using method described in the iitterature, see e.g. E.K. Fields, J. Am Chem. Soc. 74 (1952) 1525; . Ε.. Newkome, *3.£. Kiefer, 11

Matsumura and W.E. Puckett, J. Org. Chem . 50 (1985) 380

The new chelates of our invention can be used in tim resolved fluorescence immunoassays, DNA hybridizati assays and microscopy analogously to the methods describ in the litterature, see e.g. I.A. Hemmila, "Applications Fluorescence in Immunoassays" in J.D. Winefordner and I. Kolthoff, Eds., Chemi cal Analysis, Vol 117, John Wiley Sons, Inc., USA, 1991 and the references therein.

The method for the determination of an analyte in a samp comprises three steps: (a) contacting the sample with reactant exhibiting biospecific affinity towards t analyte to the formation of a complex comprising sa analyte and said reactant, the condition and amounts reactants being selected so that the amount of compl formed is a function of the amount of analyte in t sample, (b) quantitatively or qualitatively measuring t amount of complex formed by the use of a reactant th exhibits biospecific affinity for said complex and bei labelled with an analytically detectable group and ( relating the measured amount of the complex to the amou of analyte in the sample. The reactant labelled with t analytically detectable group complies with a lanthani chelate according to this invention.

Example 1. The synthesis of 2, 6-bis{5\'-[N,N-bis (methoxyca bonylmethyl)a inomethyl]-2\'-furyl}-4-(2", 4", 6"-trimethox phenyl)pyridine (1).

A mixture of 2, 6-bis (2\'-furyl)-4- (2", 4", 6"-trimethoxyph nyDpyridine (0.050 g, 0.13 mmol) , dimethyl iminodiaceta

(0.050 g, 0.32 mmol), 37 % formaline (24 μl, 0.32 mmol) a

_a

Scheme 1 The synt esis cf compound 2

acetic acid (1 mi) was stirred for five hours at 110°C After evaporation the residue was dissolved in dichloro methane, extracted with water and dried with sodiu sulfate. The product was purified with flash chromatograph (silica, chloroform) . The yield was 11 mg (12 %) .

^: H NMR (60 MHz, CDC1 ₃ ) : 3.60 (8 H, s) ; 3.67 (12 H, s) ; 3.7 (6 H, s) ; 3.88 (3 H, s) ; 4.03 (4 H, s) ; 6.24 (2 H, s) ; 6.3 (2 H, d, J = 3 Hz) ; 7.06 (2 H, d, J = 3 Hz) ; 7.50 (2 H, s UV (λ-^ in ethanol) : 331 & 284 nm

Example 2. The synthesis of 2, 6-bis{5\'-[N,N-bis (carboxy methyl) aminomethyl]-2\'-furyl}-4-(2", 4", 6"-trimethoxyphenyl)py ridine (2) .

A mixture of compound 1 (11 g, 15 umol) and 0.5 M potassiu hydroxide in ethanol (1 ml) was stirred for two hours. Th solution was neutralized with 1 M hydrochloric acid an evaporated to dryness. The residue was dissolved in water (0. ml) . UV (λ^ in water as free ligand) : 331 & 283 nm

UV λm _ax in water as europium chelate) : 338, 300 & 290 nm

Example 3. The synthesis of 2, 6-bis (4\' -carboxy-2\'-thiazolin

2\'-yl)pyridine (3) .

L-Cysteine (0.66 g, 5.4 mmol) was dissolved in water (5 ml and the solution was neutralized with sodium bicarbonate. mixture of 2, 6-dicyanopyridine (0.24 g, 1.9 mmol) in methano

(5 ml) was added to the solution of L-cysteine. After evapora tion of methanol the crystallized product was filtered an washed with acetone. The yield was 0.35 g (55 %) . - ^\' E NMR (60 MHz, D ₂ 0) : 3.23-3.85 (4 H, m) ; 5.20 (2 H, ~ , J =

Hz) ; 8.05-8.35 (3 H, m)

UV (λ_. _a: , in water as free iiσand) : 288 nm UV (.„, _ax i ⁿ water as europium (III) chelate) : 295 ά 24j nna

L-Cysteine

1. SOC1, 2. HN (CH,COO-tert-Bu) ₂ CF,COOH

_S c _h eme _! The synthesis cr compound 5

Example . The synthesis of 2, 6-bis { \' -[N,N-bis (terc-butoxy carbonylmethyl) aminocarbonyl] -2 \' -thiazolyl}pyridine (4) .

A mixture of compound 3 (0.35 g, 1.0 mmol) and thiony chloride (5 ml) was refluxed for one hour. After evaporatio to dryness the residue was dissolved in dry pyridine (6 ml) di-tert-butyl iminodiacetate (0.64 g, 2.6 mmol) was added an the solution was refluxed for two hours. The solution wa evaporated, dissolved in chloroform and filtered. The produc was purified with flash chromatography (silica, chloroform) . ^** H NMR (60 MHz, CDC1 ₃ ) : 1.45 (18 H, s); 1.51 (18 H, s); 4.2 (4 H, s); 4.63 (4 H, s); 7.86-8.27 (3 H, m) ; 8.33 (2 H, s) UV (A*. _x in ethanol) : 326 & 289 nm; mol wt (MS) : 787 (W)

Example 5. The synthesis of 2, 6-bis{4\' -[N,N-bis (carboxy methyl) aminocarbonyl] -2\'-thiazolyl}pyridine (5) .

A solution of compound 4 in trifluoroacetic acid was kept a room temperature overnight. After evaporation the residue wa triturated with diethyl ether and filtered.

^** H NMR (60 MHz, DMSO-d ₆ ) : 4.21 (4 H, s); 4.57 (4 H, s); 8.10

8.30 (3 H, ) ; 8.50 (2 H, s)

UV (λ- _ax in water as free ligand) : 325 & 278 nm

UV (λ^ in water as europium(III) chelate) : 325 & 278 nm

Example 6. The synthesis of 2, 6-bis (5\'-methyl-4\'-phenyl thiazol-2\'-yl)pyridine (6) .

A mixture of 2, 6-pyridinedithiodicarboxamide (0.76 g, 3. mmol), 2-bromo-i-phenyl-l-propanone (1.8 g, 8.4 mmol) , ethano (14 ml) and N,N-dimethylformamide (5 ml) was refluxed for 8. hours. -Solid material was filtered and washed with ethanol The suspension of the hydrobromic salt of the product in ho water (40 ml) was alkalized with 20 sodium bicarbonat

₃

30

HN (CH,COO-tert-Bu) .

→- 8

Scheme 3. The synthesis of compound 9

solution. The product was filtered and washed with water. T yield was 1.35 g (81 %) . M.p. 273°C. ^* Η NMR (400 MHz, CDC1 ₃ ) : 2.67 (6 H,s); 7.36-7.40 (2 H, m) 7.47-7.50 (4 H, m) ; 7.74-7.76 (4 H, m) ; 7.86 (1 H, t, J = 7. Hz); 8.21 (2 H, d, J = 7.7 Hz) UV (λ^ in acetonitrile) : 342 & 238 nm

Example 7. The synthesis of 2, 6-bis (5\' -bromomethyl-4\'-phenyl thiazol-2\'-yl)pyridine (7) .

A mixture of compound 6 (0,50 g, 1.2 mmol), N-bromosuccinimi (0.42 g, 2.4 mmol), α,α\'-azoisobutyronitrile (21 mg, 0.1 mmol) and benzene (140 ml) was refluxed for six hours. Th reaction mixture was evaporated and the product was purifie with flash chromatography (silica, dichloromethane) . The yiel was 0.10 g (15 %) . M.p. 223°C. ^: H NMR (400 MHz, CDC1 ₃ ) : 4.88 (4 H, s); 7.45-7.48 (2 H, m) ^" .52-7.56 (4 H, ) ; 7.82- ^~ .85 (4 E, m; ; ^" -.91 (1 H, t, J = 7.

Hz ) ; 8 . 29 ( 2 K, d, J = 7 . 8 Hz)

UV (λ _Bax in ethanol) : 340, 316 & 243 nm

Example 8. The synthesis of 2, 6-bis{5\'-[N, -bis (tert-butoxy carbonyI ethy1)aminomethy1]-4 ^r -phenylthiazol-2\'-y1}- pyridine (8) .

A mixture of compound 7 (90 mg, 0.15 mmol), di-tert-buty iminodiacetate (76 mg, 0.31 mmol), dry potassium carbonat (0.21 g, 1.5 mmol) and dry acetonitrile (30 ml) was refluxe for six hours. The reaction mixture was iltered and th filtrate was evaporated to dryness. The residue was dissolve in chloroform (5ml) , washed with water (2 x 2 ml) and drie with sodium sulfate. Evaporation left a pure product. Th yield was 0.14 g (100 %) . M.p. 121-123°C. ^r H NMR (400 MHz, CDC1 ₃ ) : 1.43 (36 H, s); 3.54 (8 H, s) ; 4.3 (4 H, s); 7.35-7.39 (2 H, m) ; 7.44-7.47 (4 H, m) ; 7.72-7.74 (4 H, ); 7.86 (1 H, t, J = 7.8 Hz); 8.26 (2 H, d, J = 7.8 Hz) UV (λ^ in ethanol) : 340, 312 & 239 nm

Example 9. The synthesis of 2,6-bis{5\'-[N,N-bis (carboxy- methyl)aminomethyl]-4\'-phenylthiazol-2\'\'-yl>-pyridine (9) .

A solution of compound 8 (100 mg, 0.11 mmol) in trifluoro- acetic acid (2 ml) was kept 1.5 hours at room temperature.

After evaporation the residue was triturated with diethyl ether and filtered. The yield was 30 mg (40 %) . M.p. 185°C

(dec.) .

^X H NMR (400 MHz, DMSO-d ₆ ) : 3.56 (8 H, s) ; 4.29 (4 K, s) ; 7.42- 7.45 (2 H, m) ; 7.49-7.53 (4 H, m) ; 7.72-7.74 (4 H, m) ; 8.13 (1 H, t, J = 7.9 Hz); 8.26 (2 H, d, J = 7.9 Hz) UV (λ,_ _ax in water as free ligand) : 340, 305 & 237 nm UV (λ^ in water as europium(III) chelate) : 350, 310 & 240 nm

15

11 X 0 16 X NH

12

HN (CH,COO-ϋert-Bu)

13

CF OOH

14

Scheme . The synthesis or compounds 14 and 16

Example 10. The synthesis of 2, 6-bis [N- (I\' -phenyl-1\' -propanon- 2\'-yl) aminocarbonyl]pyridine (10) .

2-Amino-l-phenyl-l-propanone hydrochloride (1.93 g, 10.3 mmol) 5 was added in small portions to a mixture of 2 , 6-pyridine- dicarbonyl dichloride (1.05 g, 5.20 mmol) and pyridine (25 ml) . After refluxing for 15 min the reaction mixture was evaporated to dryness. The residue was dissolved in chloroform

(50 ml) , washed with saturated sodium bicarbonate (20 ml) and 0 dried with sodium sulfate. The product was purified with flash chromatography (silica, petroleum ether/ethyl acetate, 1/1) .

The yield was 1.60 g (72 %) . M.p. 71°C.

^: H NMR (400 MHz, CDC1 ₃ ) : 1.65 (3 H, d, J = 7.3 Hz); 1.66 (3 H, d, J = 7.3 Hz); 5.79 (1 H, quintet, J = 7.3 Hz); 5.80 (1 H, 5 quintet, J = 7.3 Hz); 7.55 (4 H, t, J = 7.4 Hz); 7.65 (2 H, t, J = 7.4 Hz); 8.06 (1 H, t, J = 7.8 Hz); 8.11-8.14 (4 H, m) ;

8.39 (1 H, d, J = 7.8 Hz); 8.39 (1 H, d, J = 7.8 Hz); 8.94 (1

H, d, J = 7.3 Hz); 9.02 (1 H, d, J = 7.3 Hz)

UV (λ _n ^ in ethanol) : 244 nm 0

Example 11. The synthesis of 2, 6-bis (4\'-methyl-5\' -phenyl- oxazol-2- ^" -yl)pyridine (11) .

A mixture of compound 10 (1.56 g, 3.65 mmol) and phosphorus 5 oxychloride (55 ml) was refluxed for 23 hours. After evapo¬ ration to dryness the residue was treated with water and the mixture was neutralized with 1 M sodium hydroxide. The crude product was filtered, washed with water and finally purified with flash chromatography (silica, 5% methanol in chloroform) . C The yield was 1.26 g (88 %) . M.p. 161-163°C.

^: H NMR (400 MHz, CDC1 _? ) : 2.56 (6 H, s); 7.37 (2 H, t, J = 7.6 Hz); 7.49 (4 H, t, J = 7.6 Hz); 7.80 (4 H, d, J = 7.6 Hz); 7.95 (1 H, t, J = 8.0 Hz); 8.20 (2 H, d, J = 8.0 Hz) UV (λ- _nax ⁱⁿ ethanol) : 350, 295, 260 & 220 (sh) nm

Example 12. T-tie synthesis of 2, 6— is (4 ^* "—bxromome ^* c yl—5\'—phenyi oxazol-2 ^r — l)pyridine (12) .

A mixture of compound 11 (0.63 g, 1.6 mmol), N-bromo succinimide (0.57 g, 3.2 mmol), α,α\'-azoisobutyronitrile (2 mg, 0.18 mmol) and carbon tetrachloride (10 ml) was refluxe for three hours. After evaporation the solid material wa washed several times with a mixture of petroleum ether an ethyl acetate (5/3). The yield was 0.44 g (50 %) . M.p. 234 237°C.

^X H NMR (400 MHz, CDC1 ₃ ) : 4.71 (4 H, s) ; 7.46 (2 H, t, J = 7. Hz); 7.55 (4 H, t, J = 7.3 Hz); 7.88 (4 H, d, J = 7.3 Hz); 8.00 (1 H, t, J = 7.9 Hz); 8.28 (2 H, d, J = 7.9 Hz) UV (λ^ in ethanol) : 353, 293 & 262 nm

Example 13. The synthesis of 2,6-bis{4\'-[N,N-bis ( ert-butoxy carbonylmethyl)a inomethyl]-5 ^r -phenyloxazol-2\'-y1}pyridin (13) .

A mixture of compound 12 (0.28 g, 0.50 mmol), di-tert-butyl iminodiacetate (0.25 g, 1.0 mmol), dry potassium carbonat

(0.69 g, 5.0 mmol) and dry acetonitrile (50 ml) was refluxe overnight. The reaction mixture was filtered and the filtrate was evaporated to dryness. The residue was dissolved in chloroform (15 ml) , washed with water (2 5 ml) and dried with sodium sulfate. The product was purified with flash chromatography (silica, petroleum ether/ethyl acetate, first 5/1 then 5/2) . The yield of an oily product was 0.21 g (61 %) . ^** H NMR. (400 MHz, CDC1 ₃ ) : 1.43 (36 H, s) ; 3.58 (8 H, s); 4.21 (4 K, s); 7.38 (2 H, t, J = 7.6 Hz); 7.49 (4 H, t, J = 7.6 Hz); 7.94 (1 H, t, J = 7.8 Hz); 8.01 (4 H, d, J = 7.6 Hz); 8.28 (2 H, d, J = 7.8 Hz) UV ^ _ax in ethanol): 335(sh) , 301 £ 262 nm

Example 14. The synthesis of 2, 6-bis{ \' -[N,N-bis (carboxy- methyl) aminomethyl] -5\'-phenyloxazol-2\' -yl}pyridine (14) .

A solution of compound 13 (0.21 g, 0.24 mmol) in trifluoro- acetic acid (6.5 ml) was kept 1.5 hours at room temperature.

After evaporation the residue was triturated with diethyl ether and filtered. The yield _. was 0.13g (81 %) . M.p. 173°C

(dec. ) .

^X H NMR (400 MHz, DMSO-d ₆ ) : 3.65 (8 H, s) ; 4.18 (4 H, s); 7.47 (2 H, t, J = 7.8 Hz); 7.56 (4 H, t, J = 7.8 Hz); 8.01 (4 H, d, J = 7.8 Hz); 8.19-8.23 (1 H, m) ; 8.28-8.30 (2 H, m) UV (λ _max in water as free ligand) : 332, 310 & 259 nm UV (λm _ΛX in water as europium(III) chelate) : 350 & 243 nm

Example 15. The synthesis of 2, 6-pyridinedicarboxyamidine dihydrochloride (15) .

A mixture of 2 , 6-dicyanopyridine (7.0 g, 54 mmol), sodium methoxide (0.59 g, 11 mmol) and methanol (50 ml) was stirred for 2.5 hours at room temperature. Ammonium chloride (5.9 g, 110 mmol) was added and the reaction mixture was stirred for three days. The product was filtered and washed with diethyl ether. The yield was 12.0 g (94%) . M.p. >310°C (dec) . ^l E NMR (400 MHz, D,0) : 8.32-8.49 (3 H, m) UV (λ^ in water) : 273 & 224 nm

Example 16. The synthesis of 2, 6-bis (4\' -methyl-5\' -phenyl- imidazol-2\'-yl)pyridine (16) .

0.5 M Potassium hydroxide (17 ml) was added to a mixture of compound 15 (0.50 g, 2.1 mmol) , 2-bromo-l-phenyl-l-propanone

(0.89 g, 4.2 mmol), N,N-diisopropylethylamine (1.1 g, 8.4 mmol) and chloroform (10 ml) . After refluxing for one day the solid material was filtered, washed with chloroform and the filtrate was evaporated to dryness. The product was purified

with, flash ciiromatograpiiy (silica, first chloroform, then % methanol in chloroform) . The yield of an oily product wa 0.17 g (21 %) .

^X H NMR (400 MHz, CDC1 ₃ ) : 2.28 (6 H, s) ; 7.21 (2 H, t, J = 7. Hz); 7.30 (4 H, t, J = 7.3 Hz); 7.49-7.52 (4 H, m) ; 7.67 ( H, t, J = 7.8 Hz); 8.05 (2 H, d, J = 7.8 Hz) UV (-^ in ethanol) : 353, 306 & .266 nm mol wt (MS) : 391 (M ^* )

18

Scheme 5. The svnthesis of compound 18

Example 17. The synthesis of 2, 6—bis{l ^r -[N,N-bis (methoxycar- bonyImethyl)aminometh I]—3 ^r —p razolyl}pyridine (17) .

A mixture of 37% formaline (0.81 g, 10 mmol), methanol (40 ml) and dimethyl iminodiacetate (1.6 g, 10 mmol) was evaporated

to dryness. The residue was dissolved in methanol (40 ml) an evaporated once again. 2, 6-Bis (3\' -pyrazolyl) pyridine (1.1 g, 5.0 mmol) was added and the reaction mixture was stirred fo 20 hours at 110°C. The product was purified with flash chroma tography (silica, triethylamine/petroleum ether/ethyl acetate 1/5/3) . The yield was 1.54 g (55 %) .

^** H NMR (60 MHz, CDC1 ₃ ) : 3.67 (12 H, s); 3.67 (8 H, s); 5.20 ( H, s); 7.06 (2 H, d, J = 2 Hz); 7.61 (2 H, d, J = 2 Hz); 7.74 8.02 (3 H, m) mol wt (MS) : 557 (M ⁺ )

UV (λ^ _x in ethanol) : 301 & 251 nm

Example 18. The synthesis of 2, 6-bis{l\'-[N,N-bis (carboxy methyl) aminomethyl] -3 \'-pyrazolyl}pyridine (18) .

A mixture of compound 17 (1.0 g, 1.8 mmol), 0.5 M potassi hydroxide in ethanol (50 ml) was stirred for three hours water (0.5 ml) was added and the reaction mixture was stirre for one hour. The reaction mixture was evaporated to drynes and the residue was dissolved in water (2 ml) . The soluti was acidified with 2 M hydrochloric acid and triturated wit ethanol. The solid material was filtered, washed with ethano and the filtrate was evaporated to dryness. The product wa purified with flash chromatography (silica, acetonitrile water, 4/1) . After evaporation the product was crystallize from water. The yield was 0.19 g (21%) .

^X H NMR (60 MHz, DMSO-d : 3.64 (8 H, s); 5.46 (4 H, s); 6.97 7.05 (2 H, m) ; 7.75-7.92 (5 H, m) UV (λ _^ in water as free ligand) : 301 & 234 nm

UV (λ^ _as in water as europium(III) chelate) : 313, 263, 254 234 nm

(CH ₃ ) ₃ SiCN

PhCOCl

32

25, 34

m-ClPBA

19 (R = H) , 26, 35

R

20, 27, 36

21, 28, 37

BrCH,COO- tert-Bu

22, 29, 38

23, 30, 39,

Scheme 6. The synthesis cf compounαs 23, 30 and 39

Example 19. The synthesis of 2,4-di(2\'-pyridyl)thiazole N\',N\'- dioxide (19) .

Λi-Chloroperbenzoic acid (50-55 %, 25.9 g, about 75 mmol) was added in small portions during 24 hours to a mixture of 2,4- di(2\'-pyridyl)thiazole (2.39 g, 10.0 mmol) and dichloromethane

(400 ml) . After stirring for 24 hours, the reaction mixture was washed with 10 % sodium carbonate (3 x 150 ml) and water

(150 ml) . The combined water fractions were extracted with chloroform (150 ml) . The combined organic fractions were dried with sodium sulfate and evaporated to dryness. The yield was

2.71 g (100 %) .

^** H NMR (60 MHz, CDC1 ₃ ) : 7.18-7.66 (4 H, m) ; 7.87-8.04 (1 H, m) ; 8.35-8.76 (3 H, m) ; 9.53 (1 H, s) mol wt (MS) : 271 (M ^* )

UV (λ^ in ethanol) : 324 & 249 nm

Example 20. The synthesis of 2,4-bis (6 ^, -cyano-2 ^* \'-pyridyl)- thiazole (20) .

Trimethylsilylcyanide (20 ml, 150 mmol) was added during five minutes to a mixture of compound 19 (2.71 g, 10.0 mmol) and dichloromethane (110 ml) . After stirring for five minutes benzoyl chloride (7.2 ml, 60 mmol) was added and the reaction mixture was stirred for 9 days. After concentration to half of its original volume, 10 % potassium carbonate (300 ml) was added and the reaction mixture was stirred for half an hour. The product was filtered, washed with water and cold dichloro¬ methane. The yield was 1.84 g (64 %) . mol wt (MS) : 289 (M ⁺ )

UV (λ_. _aκ in ethanol ) : 301 , 271 & 252 nm

Example 21. The synthesis of 2, 4-bis ( 6 \' -aminomethy1-2\' -pyri dyl) thiazole pentahydrochloride (21) .

1 M Borane in tetrahydrofuran (52 ml, 52 mmol) was adde during 10 minutes to a mixture of compound 20 (1.16 g, 4.0 mmol) and dry tetrahydrofuran (50 ml) . After stirring for 2 hours, the extra borane was distroyed by adding methanol. Th mixture was evaporated to dryness and methanol saturated wit hydrogen chloride (70 ml) was added. After stirring for on hour, the product was filtered and washed with cold methanol. The yield was 0.80 g (42 %) .

^** H NMR (60 MHz, D,0) : 4.56 (2 H, s); 4.57 (2 H, s); 7.55-7.7 (2 H, m) ; 8.00-8.47 (4 H, m) ; 8.56 (1 H, s) UV (λ _^x in water) : 320 (sh) , 292 & 244 nm

Example 22. The synthesis of 2, 4-bis{ 6\'-bis [N, -bis ( ert butoxycarbonylmethyl) aminomethy1] -2\'-pyridyl}thiazole (22) .

A mixture of compound 21 (0.48 g, 1.0 mmol), N,N-diisoprop ylethylamine (2.6 ml, 15 mmol), tert-butyl bromoacetate (0.7 g, 4.0 mmol) and acetonitrile (20 ml) was refluxed for 2 hours. After evaporation, the residue was dissolved in chloro form (50 ml), washed with water (3 x 20 ml) and dried wit sodium sulfate. The product was purified with flash chromato graphy (silica, petroleum ether/ethyl acetate, 5/3.) . Th yield was 0.47 g (63 %) .

^** H NMR (400 MHz, CDC1 ₃ ) : 1.48 (18 H, s); 1.48 (18 H, s); 3.5 (8 H, s); 4.11 (2 H, s); 4.12 (2 H, s); 7.59 (1 H, d, J = 7. Hz); 7.71 (1 H; d, J = 7.6 Hz); 7.79 (1 H, t, J = 7.6 Hz); 7.81 (1 H, t, J = 7.6 Hz); 8.13 (1 H, d, J = 7.6 Hz); 8.13 ( H, d, J = 7.6 Hz); 8.20 (1 H, s) mol wt (MS) : 753 (M ^τ )

Example 23. The synthesis of 2, -bis{6\'-bis [N,N-bis (carboxy methyl)aminomethyl]-2 \'-pyridyl}thiazole (23) .

This compound (23) was synthesized using a method analogous to the synthesis described in Example 9. The yield was 100 %.

-E NMR (400 MHz, DMSO-d ₆ ) : 3.63 (4 H, s) ; 3.72 (4 H, s) ; 4.12

(2 H, s); 4.21 (2 H, s); 7.56 (1 H, d, J = 7.6 Hz); 7.69 (1

H, d, J = 7.6 Hz); 7.99 (1 H, t, J = 7.6 Hz); 8.03 ( 1 H, t,

J = 7.6 Hz); 8.14 (1 H, d, J » 7.6 Hz); 8.18 (1 H, d, J = 7.6 Hz) ; 8.39 (1 H, s)

UV λ _najj in water as free ligand) : 325 (sh) , 294 & 247 nm

UV (λ _∞ in water as europium(III) chelate) : 331, 295 & 243 nm

Example 24. The synthesis of 4-phenylpyridine-2-tiocarboxamide (24) .

Absolute ethanol saturated with ammonia (10 ml) was added to a cold solution of 2-cyano-4-phenylpyridine (1.8 g, 10 mmol) and absolute ethanol (30 ml) . The mixture was saturated with hydrogen sulfide. After stirring overnight, the solution was concentrated to 10 ml. The cold mixture was filtered and washed with cold ethanol. The yield was 1.69 g (79 %) . -B. NMR (60 MHz, CDC1 ₃ ) : 7.69-7.94 (6 H, m) ; 8.83 (1 H, d, J = 5 Hz); 9.24 (1 H, d, J = 2 Hz) UV (λ^ _ax in ethanol) : 322 & 241 nm

Example 25. The synthesis of 2-(4 -phenyl-2\'-pyridyl)-4-(2\' \' - pyridyl)thiazole (25) .

A mixture of compound 24 (1.07 g, 5.00 mmol), 2-(bromoacetyl)- pyridine (1.00 g, 5.00 mmol) and ethanol (20 ml) was refluxed for three hours. A cold mixture was filtered and washed with cold ethanol. The suspension of the hydrobromic salt of the product in hot water (40 ml) was alkalized with solid sodium carbonate. The product was filtered and washed with cold

water. The yield was 1.25 g (79 %) . ^l E NMR (400 MHz, CDC1 ₃ ) : 7.25-7.29 (1 H, m) ; 7.47-7.51 (1 H, m) ; 7.52-7.58 (3 H, m) ; 7.76-7.79 (2 H, m) ; 7.80-7.85 (1 H, dt, J = 2 & 8 Hz); 8.23 (1 H, s); 8.30 (1 H, d, J = 8 Hz) 8.57 (1 H, d) ; 8.65-8.69 (2 H, m)

UV (λ^ _x in ethanol) : 320, 282 (sh) & 249 nm

Example 26. The synthesis of 2- (4\'-phenyl-2\'-pyridyl) -4- (2\' \' pyridyl)thiazole N\' ,N\' \' -dioxide (26) .

This compound (26) was synthesized using a method analogou to the synthesis described in Example 19. The yield was 93 % ^l E NMR (400 MHz, CDC1,) : 7.23-7.27 (1 H, m) ; 7.41-7.46 (1 H m); 7.48-7.52 (1 H, m) ; 7.55-7.60 (3 H, m) ; 7.74-7.77 (2 H m); 8.41 (1 H, d) ; 8.45 (1 H, d) ; 8.69 (1 H, dd) ; 8.90 (1 H d); 9.58 (1 H, s)

Example 27. The synthesis of 2-(6\'-cyano-4\'-pheny1-2\' -pyri dyl) -4-(6\' \'-cyano-2"-pyridyl)thiazole (27) .

This compound (27) was synthesized using a method analogou to the synthesis described in Example 20. The yield was 65 % ^l E NMR (400 MHz, DMSO-d _€ ) : 7.50 (1 H, t, J = 8 Hz); 7.58-7.6 (2 H, m) ; 7.95 (1 H, d, J = 8 Hz); 8.04-8.08 (2 H, m) ; 8,2 (1 H, t, J - 8 Hz); 8.60 (1H, d, J - 1 Hz); 8.67 (1 H, d, = 8 Hz); 8.70 (1 H, s); 8.83 (1 H, d, J = 1 Hz)

Example 28. The synthesis of 2- (6\' -aminomethyl- \' -phenyl-2\' pyridyl) -4-(6\' \' -aminomethy1-2\' \' -pyridyl) thiazole penta hydrochloride (28) .

This compound (28) was synthesized using a method analogou to the synthesis described in Example 21. The yield was 71 % UV (λ_ _aX in ethanol) : 318 & 251 nm

Example 29. The synthesis of 2—{6\'-[N,N-bis (tert-butoxycarbo nylmethyl) aminomethy1]— \'-phenyl-2\'-pyridyl}-4-{6\' \'—[N,N bis ( ert-butoxycarbonylmethyl) aminomethyl]—2\' \' -pyridyl} thiazole (29) .

This compound (29) was synthesized using a method analogou to the synthesis described in Example 22. The yield was 89 % -E NMR (60 MHz, CDC1 ₃ ) : 1.47 (36 H, s) ; 3.54 (4 H, s) ; 3.58 ( H, s); 3.95 (2 H, s); 4.18 (2 H, s) ; 7.42-7.94 (8 H, m) ; 8.1 (1 H, d, J = 2Hz); 8.20 (1 H, s) ; 8.43 (1 H, d, J = 2 Hz)

Example 30. The synthesis of 2-{6\'-[N,N-bis (carboxymethyl) aminomethyl]-4\'-phenyl-2\'-pyridyl}— -{6\' \'-[N,N-bis (carboxy methyl)aminomethyl] -2 \'\'-pyridyl}thiazole (30) .

This compound (30) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 91 % ^X H NMR (60 MHz, DMSO-d ₆ ) : 3.67 (4 H, s) ; 3.73 (4 H, s) ; 4.2

(4 H, s); 7.47-7.72 (4 H, ) ; 7.77-8.25 (5 H, m); 8.40-8.4

(2 H, m)

UV (λ _nax in water as free ligand) : 331 (sh) & 252 nm

UV (λ _j . _ax in water as europium(III) chelate) : 334, 288 & 251 n

Example 31. The synthesis of 4-(p-nitrobenzyl)pyridine N-oxid

(31) .

m-Chloroperbenzoic acid (50-55 %, 74.7 g, about 190 mmol) wa added to a cold mixture of 4—(p-nitrobenzyl)pyridine (21.4 g, 100 mmol) and dichloromethane (250 ml) . After stirring for tw hours, water (200 ml) was added and the reaction mixture wa alkalized with solid sodium carbonate. Fractions were sepa rated and the water fraction was extracted with a mixture o ethanol and chloroform (4 :: 150, 1/2) . The combined organi

fractions were dried with sodium sulfate. The yield was 19. g (83 %) .

^** H NMR (60 MHz, CDC1 ₃ ) : 4.07 (2 H,s) ; 7.08 (2 H, d, J = 7 Hz); 7.35 (2 H, d, J = 9 Hz); 8.17 (2 H, d, J = 7 Hz); 8.22 (2 H, d, J = 9 Hz)

UV (^ _i8 in ethanol) : 273 nm

Example 32. The synthesis of 2-cyano-4-(p-nitrobenzyl) pyridin (32) .

Trimethylsilylcyanide (50 ml, 400 mmol) was added to a mixtur of compound 31 (18.2 g, 79.0 mmol) and dichloromethane (16 ml) . After stirring for five minutes, benzoyl chloride (20 ml, 160 mmol) were added and the reaction mixture was stirred fo half an hour. Water (160 ml) and solid potassium carbonate (5 g) was added and the reaction mixture was stirred for half a hour. Fractions were separated and the water fraction wa extracted with dichloromethane (2 x 100 ml) . The organi fractions were dried with sodium sulfate and the product wa crystallized from toluene. The yield was 10.0 g (53 %) . ^l E NMR (60 MHz, CDC1 ₃ ) : 4.14 (2 H, s); 7.32 (1 H, d, J = Hz); 7.33 (2 H, d, J = 9 Hz); 7.48 (1 H, s); 8.23 (2 H, d, = 9 Hz); 8.64 (1 H, d, J = 5 Hz) UV (λ^ _ax i ethanol) : 266 nm

Example 33. The synthesis of 4- (p-nitrobenzyl)pyridine-2 tiocarboxamide (33) .

Absolute ethanol saturated with ammonia (20 ml) was added t a cold mixture of compound 32 (4.8 g, 20 mmol), absolut ethanol (100 ml) and dichloromethane (80 ml) . The mixture wa saturated with hydrogen sulfide. After stirring for overnight, the solution was concentrated. The cold mixture was filtere and washed with cold ethanol. A suspension of the soli material in chloroform (100 ml) was filtered, washed wit

chloroform and the filtrate was evaporated to dryness. Th yield was 3.3 g (60 %) .

^X H NMR (60 MHz, CDC1 ₃ ) : 4.17 (2 H, s) ; 7.19 (1 H, dd, J « 1 5 Hz) ; 7.35 ( 2 H, d, J = 8 Hz) ; 8.19 (2 H, d, J = 8 Hz) ; 8.4 (2 H, d, J = 5 Hz); 8.60 (1 H, d, J = 1 Hz) mol wt (MS) : 273 (JJT) UV (λ^ in ethanol) : 317 & 271 n

Example 34. The synthesis of 2-[4\'-(p-nitrobenzyl)-2\'-pyrid yl]-4-(2"-pyridyl)thiazole (34).

This compound (34) was synthesized using a method analogou to the synthesis described in Example 25. The yield was 61 - after a crystallization from methanol. -K NMR (60 MHz, CDC1 ₃ ) : 4.20 (2 H, s) ; 7.11-7.50 (4 H, m) 7.69-8.31 (6 H, m); 8.55-8.71 (2 H, m) UV λ^ in ethanol): 315(sh) , 285 & 246 n

Example 35. The synthesis of 2-[4\'-(p-nitrobenzyl) —2\'-pyrid yl]-4-(2"-pyridyl)thiazole N\',N"-dioxide (35).

This compound (35) was synthesized using a method analogou to the synthesis described in Example 19. The yield was 82 ^~ . . ^X H NMR (60 MHz, DMSO-d ₆ ) : 4.32 (2 H, s) ; 7.33-7.55 (4 H, m); 1.12-1 .89 (4 H, m) ; 8.13-8.69 (4 H, m) UV (λ, _^ in ethanol) : 326 & 253 nm

Example 36. The synthesis of 2-[6\'-cyano-4\'-(p-nitrobenzyl) 2\'-p ridy1]-4-(6"-cyano-2"-pyridyl)thiazole (36) .

This compound (36) was synthesized using a method analogous to the synthesis described in Example 20. After the addition of 10 % potassium carbonate, the mixture was extracted several times with chloroform. The combined organic fractions were dried with sodium sulfate. The vield was 69 .

^: H NMR (60 MHz, CDC1,) : 4.21 (2 H, s); 7.27-7.60 (5 H, m; 7.81-8.20 (4 H, m) ; 8.33 (1 H, s)

Example 37. The synthesis of 2-[6\' -aminomethyl-4\' - (p-nitro benzyl) -2\'-pyridyl] -4- (6\' \' -aminomethy1-2\' \' -pyridyl) thiazol pentahydrochloride (37) .

This compound (37) was synthesized using a method analogou to the synthesis described in Example 21. After the additio of methanol saturated with hydrogen chloride and stirring fo one hour, the solution was evaporated to dryness. The residu was triturated with cold tetrahydrofuran and filtered. Th yield was 63 %. UV (λm _ax in water) : 315 (sh) , 284 & 242 nm

Example 38. The synthesis of 2-{ 6\'-[N,N-bis (tert-butoxy carbonylmethyl) aminomethyl]-4\' -(p-nitrobenzyl) -2\'-pyridyl}-4 { 6\' \' - [N,N-bis (tert-butoxycarbonylmethyl) aminomethyl] -2\' \' pyridyl}thiazole (38) .

This compound (38) was synthesized using a method analogou to the synthesis described in Example 22. The product wa purified with flash chromatography (silica, petroleum ether ethyl acetate, 5/2) . The yield was 24 %.

\'H NMR (60 MHz, CDC1 ₃ ) : 1.46 (36 H, s); 3.52 (8 H, s); 3.80 ( H, s); 3.95 (2 H, s) ; 4.08 (2 H, s); 7.17-8.17 (10 H, m)

Example 39. The synthesis of 2-{ 6\' -[N, -bis (carboxymethyl) aminomethyl] -4 \' - (p-nitrobenzyl) -2\' -pyridyl }-4-{6\'\'-[N,N bis (carboxymethyl) aminomethyl] -2\' \' -pyridyl }thiazole (39) .

This compound (39) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 100 h ^: H NMR (400 MHz, DMSO-d.-) : 3.55 (4 K, s); 3.60 (4 K, s); 3.9

(2 H, s); 4.06 (2 H, s) ; 4.10 (2 H, s); 7.52-7.65 (7 E, ) ;

8.09-8.22 (3 H, m)

UV (λ^ in water as free ligand) : 285, 265 & 245 nm

UV (λ^ _ax in water as europium(III) chelate) : 320 (sh) & 285 nm

CF _j COOH

41

EuCl,

42

Scheme 7. The synthesis of compound 42

Example 40. The synthesis of 2-{4\'-(p-aminobenzyl) -6\'-[N,N bis (tert-butoxycarbonylmethyl) aminomethyl] -2\'-pyridyl}-4-{6\' \' [N,N-bis (tert-butoxycarbonylmethyl) aminomethyl] -2\' \'-pyridyl} thiazole (40) .

A mixture of compound 38 (150 mg, 0.17 mmol) , 10 % palladiu on carbon (10 mg) and methanol (30 ml) was stirred for fiv hours under hydrogen atmosphere (690 kPa) . After filtratio and evaporation of the filtrate, the product was purified wit flash chromatography (silica, petroleum ether/ethyl acetate 5/3.) . The yield was 40 mg (27 %) .

_{3 8}

^; H NMR (60 MHz , CDC1 ₃ ) : 1 . 45 (36 H, s) ; 3 .51 (8 H, s ) ; 3 - 77 H, s ) ; 3. 92 (2 H, s ) ; 4 . 06 (2 H, s) ; 6. 64-8 . 03 ( 9 H, m) ; 8 . (1 H, s )

Example 41. The synthesis of 2-{4\'—(p-aminobenzyl)-6\'-[N, bis(carboxymethyl)aminomethyl]-2\'-pyridyl}-4-{6\' \'-[N,N-bis (carboxymethyl)aminomethyl]-2\'\'-pyridyl}thiazole (41) .

This compound (41) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 100 % UV (λ _aax in water as free ligand) : 315 & 290 nm UV (^ _ax in water as europium(III) chelate) : 325 & 290 nm

Example 42. The synthesis of europium(III) chelate of 2-{4\' [p-(4,6-dichlorotriazon-2-ylamino)benzyl)-6\'-[N,N-bis(carbo xy methyl)aminomethyl]-2\'-pyridyl}-4—{6\' \'-[ _f N-bis (carboxy methyl)aminomethyl] -2\' \'-pyridyl}thiazole (42) .

The compound 41 (25 mg, 40 μmol) was dissolved in water (70 μl) and the pH was adjusted to 6.5 with solid sodium bicarbo nate. Europium (III) chloride (22 g, 60 μmol) in water (20 μl) was added during 15 minutes and the pH was maintained a 5—7. After stirring for 1.5 hours, the pH was raised to 8. with 1 M sodium hydroxide and the precititate was filtere off. The filtrate was triturated with acetone, the precipitat was filtered and washed with acetone. A mixture of 2,4,6 trichlorotriazine (2 mg, 10 μmol) , acetone (100 μl) and wate (100 μl) was added to a solution of the europium(III) chelat (8 mg, 10 μl) and 0.1 M sodium acetate (150μl, pH 4.9) . Afte stirring for 15 minutes, the reaction mixture was triturate with acetone. The precipitate was filtered off, washed wit acetone and dried in exsiccator. UV (?^ _ax in water) : 331, 287 & 250 nm

m-ClPBA

43 R = H 50 R = NO,

(CH,) ₃ SiCN, PhCOCl

I I

O O ^"

44, 51

45, 52

BrCHXOO- tert-Bu

46, 53

47 , 54

Ammonium formate , Pd/C

H,, Pd/C

48

55

CF,COOH

49

56

Scheme z . The svnthesis of compounds 49 and 56

Example 43. The synthesis of 1-benzyl—3,5-di (2\' -pyridyl) 1,2,4-triazole (43) .

A mixture of 3,5-di (2\'-pyridyl)-1,2, 4-triazole (1.12 g, 5.00 mmol), potassium carbonate (1.38 g, 10.0 mmol), benzylchlorid

(0.63 g, 5.0 mmol) and acetonitrile (65 ml) was refluxed fo

2.5 hours. The reaction mixture ^\' was filtered and the filtrate was evaporated to dryness. The product was purified with flash chromatography (silica, 1 % methanol in dichloromethane) . The yield was 1.05 g (67 %) .

¹ H NMR (60 MHz, CDC1 ₃ ) : 6.20 (2 H, s) ; 6.93-7.38 ( 6 H, m) ; 7.58-7.93 (3 H, m) ; 8.15-8.79 (4 H, m)

Example 44. The synthesis of 1-benzy1-3,5-di (2\'-pyridyl)- 1,2,4-triazole N\' ,N\'-dioxide (44) .

This compound (44) was synthesized using a method analogous to the synthesis described in Example 19. The reaction time was 11 days at room temperature. The product was purified with flash chromatography (silica, 2, 5 and 10 % methanol in chloroform) . The yield was 62 %.

^X H NMR (60 MHz, CDC1,) : 5.76 (2 H, s) ; 7.14-7.41 (9 H, m) ; 7.97-8.45 (4 H, )

Example 45. The synthesis of l-benzyl-3,5-bis (6\'-cyano-2\'- pyridyl)-!,2,4-triazole (45) .

This compound (45) was synthesized using a method analogous to the synthesis described in Example 36. The yield was 71 ^\' ■:. ^l R NMR (400 MHz, CDC1,) : 6.15 (2 H, s); 7.26 (1 H, t, J = " Hz) : 7.32 (2 H, t, J = 7 Hz); 7.42 (2 H, d, J = 7 Hz); 7.75- 7.78 (2 K, m) ; 7.79 (1 H, t, J ^* = 8 Hz); 8.01 (1 H, t, J = 8 Hz); 8.47 (1 H, dd, J = 1 & 8 Hz) ; 8.64 (1 H, dd, J = 1 & 8 Hz)

43

Example 46. The synthesis cf 3, 5-bis (6\' -aminomethy1-2\' -pyri dyl)-1-benzyl-l, 2, 4-triazole pentahydrochloride (46) .

This compound (46) was synthesized using a method analogou to the synthesis described in Example 37. The yield was 10

^: H NMR (400 MHz, CDC1 ₃ ) : 4.26 (2 ^\' H, s) ; 4.28 (2 H, s); 5.87 ( H, s); 7.03-7.50 (m, 7 H) ; 7.88-8.03 (4 H, m)

Example 47. The synthesis of l-benzyl-3, 5-bis{6\'-bis [N,N bis (tert-butoxycarbonylmethyl) aminomethyl] -2\'-pyridyl}-l, 2,4 triazole (47) .

This compound (47) was synthesized using a method analogou to the synthesis described in Example 22. The product wa purified with flash chromatography (silica, petroleum ether/ ethyl acetate, 1/1) . The yield was 39 %.

^■* H NMR (60 MHz, CDC1 ₃ ) : 1.45 (36 H, s); 3.47 (4 H, s); 3.52 (

H, s); 4.08 (2 H, s) ; 4.21 (2 H, s); 6.20 (2 H, s); 7.13-7.3 (5 H, m) ; 7.54-7.83 (4 H, m) ; 7.95-8.33 (2 H, m)

Example 48. The synthesis of 3, 5-bis{ 6\'-bis [N, -bis (tert butoxycarbonylmethyl) aminomethyl] -2\' -pyridyl}-l, 2, 4-triazol (48) .

A mixture of compound 47 (0.44 g, 0.53 mmol), 10 % palladiu on carbon (0.25 g) , ammonium formate (about 1.0 g, 15 mmol), methanol (10 ml) under nitrogen atmosphere was stirred severa days at room temperature. The reaction mixture was filtere and the filtrate was evaporated to dryness. The residue wa dissolved in chloroform ( 50 ml) , washed with water (20 mi) and dried with sodium sulfate. The yield was 0.34 g (87 ) . ^: H NMR ^{^} (60 MHz, CDC1 ₃ ) : 1.46 (36 K, s); 3.35 (4 H, s); 3.52 ( H, s); 4.12 (2 H, s); 4.17 (2 H, s); 7.57-8.25 (6 H, m)

Example 9. The synthesis of 3,5-bis(6\'-bis[N,N-bis(carboxy¬ methyl)aminomethyl]-2\'-pyridyl}-1,2,4-triazole (49) .

This compound (49) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 100 %. UV (λ _aax in water as free ligand) : 284 & 232 nm UV (^ _x in water as europium chelate) : 288 & 233 nm

Example 50. The synthesis of l-(m-nitrobenzyl)-3,5-di(2\'- pyridyl)-1,2,4-triazole (50).

This compound (50) was synthesized using a method analogous to the synthesis described in Example 43. The yield was 78 %. -E NMR (60 MHz, CDC1 ₃ ) : 6.27 (2.H, s) ; 7.20-7.86 (6 H, m) ; 7.91-8.52 (4 H, m) ; 8.62-8.80 (2 H, m)

Example 51. The synthesis of l-(m-nitrobenzyl)-3,5-di .2\' - pyridyl)-1,2, -triazole N\',N\'-dioxide (51).

This compound (51) was synthesized using a method analogous to the synthesis described in Example 44. The product was purified with flash chromatography (silica, 3, 7 and 10 « methanol in chloroform) . The yield was 51 %. ^** H NMR (60 MHz, CDC1 ₃ ) : 5.80 (2 H, s) ; 7.05-8.05 (10 H, m) ; 8.32-8.53 (1 H, ) ; 9.29 (1 H, m) mol wt (MS) : 390 (M ^÷ )

Example 52. The synthesis of 3,5-bis (6\'-cyano-2\'-pyridyl)-1- (m-nitrobenzyl)-1,2,4-triazole (52) .

This compound (52) was synthesized using a method analogous to the synthesis described in Example 36. The product was purified with flash chromatography (silica, ethyl acetate) . The yield was 55 - ^" -. -E NMR (60 MHz, CI.CI- : 6.24 (2 H, ε; ; 7.52-3.24 (8 H, ) ;

8.48 (1 H, dd) ; 8.70 (1 H, dd)

UV (λ _^ in ethanol) : 287 (sh) & 254 nm mol wt (MS) : 408 (M ⁺ )

Example 53. The synthesis of 3, 5-bis (6\' -aminomethyl-2\' -pyri dyl)-1-(m-nitrobenzyl) -1, 2, 4-triazole pentahydrochloride (53) .

This compound (53) was synthesized using a method analogou to the synthesis described in Example 37. The yield was 82 % . ^: H NMR (60 MHz, CDC1 ₃ ) : 4.25 (4 H, s); 5.97 (2 H, s); 7.20 8.05 (10 H, m)

Example 54. The synthesis of 3, 5-bis{ 6\'-bis [N,N-bis (tert butoxycarbonylmethyl) aminomethyl] -2\' -pyridyl }-1- (m-nitro benzyl) -1, 2, 4-triazole (54) .

This compound (54) was synthesized using a method analogou to the synthesis described in Example 22. The product wa purified with flash chromatography (silica, petroleum ether ethyl acetate, 1/1) . The yield was 42 %. ^l E NMR (60 MHz, CDC1,) : 1.44 (18 H, s); 1.45 (18 H, s); 3.4 (4 H, s); 3.52 (4 H, s); 4.10 (2 H, s); 4.22 (2 H, s); 6.3 (2 H, s) ; 7.30-8.39 (10, m) UV (λ.-. _ax in ethanol) : 283 & 237 nm

Example 55. The synthesis of 1- (m-aminobenzyl) -3, 5-bis{ 6\' bis [N,N-bis (tert-butoxycarbonylmethyl) aminomethyl] -2\' -pyrid yl}-I,2,4-triazole (55) .

This compound (55) was synthesized using a method analogou to the synthesis described in Example 40. The product wa purified with flash chromatography (silica, 5 \ methanol i chloroform) . The yield was 85 % .

^: H NMR (60 MHz, CDC1,) : 1.45 (36 H, s); 3.48 (4 H, s); 3.52 ( H, S); 4.10 (2 H, 3); 4.22 (2 H, s); 6.1C (2 H, s); 5.41-~.C

46

(4 H, m) ; 7 . 70-8 . 33 (6 H, )

Example 56. The synthesis of 1—(m-aminobenzyl)-3,5-bis{6\' bis[N, —bis (carboxymethyl) aminomethyl] -2\'-pyridyl}-1,2,4 triazole (56) .

This compound (56) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 100 % ^l H NMR (400 MHz, DMSO-d ₆ ) : 3.54 (4 H, s) ; 3.62 (4 H, s) ; 4.0 (2 H, s); 4.14 (2 H, s); 6.10 (2 H, s); 6.80-6.88 (2 H, m)

6.96-7.02 (1 H, m) ; 7.19-7.23 (1 H, m) ; 7.69 (1 H, d) ; 7.7

(1 H, d); 7.97 (1 H, t) ; 8.04 (1 H, t) ; 8.05 (1 H, d) ; 8.1

(1 H, d)

UV (λ„ _aχ in water as free ligand) : 282 & 234 nm UV (^ _x in water as europium(III) chelate) : 293, 282. & 236 n

MedlDCl,

56

57a M ^* = Eu ³ \'

M ^5* = Tb ^J* 57b

Scheme 5 . The svnthesis of compounds 57* and 57b

Example 57a. The synthesis of europium(III) chelate of l-[-m- (4, 6-dichlorotriazon-2-ylamino)benzyl] -3, 5-bis{ 6\'-bis [N,N-bis- (carboxymethyl) aminomethyl] -2\'-pyridyl}-l, 2, 4-triazole (57a) .

This compound (57a) was synthesized using a method analogous to the synthesis described in Example 42. UV (^ in water) : 295 (sh), 282 & 236 nm.

Example 57b. The synthesis of terbium(III) chelate of l-[m- (4, 6-dichlorotriazon-2-ylamino)benzyl] -3, 5-bis{6\'-bis [N,N-bis- (carboxymethyl) aminomethyl] -2\'-pyridyl}-l, 2, 4-triazole (58) .

This compound (57b) was synthesized using a method analogous to the synthesis described in Example 42. UV (λ^ _ax in water) : 295 (sh) , 282 & 237 nm.

58

m-ClPBA

one isomer shown

BrCH,COO- tert-Bu H ^, , Pd/C

64

Ammonium formate CF,COOH „ 66 •• 67

Pd/C

68

Scheme 10. The synthesis of compound 68

Example 58. The synthesis of 2-pyridylhydrazide (58) .

Hydrazine hydrate (17.5 ml, 500 mmol) in ethanol (50 ml) was added to a solution of ethyl 2-pyridinecarboxylate (7.53 g,

50.0 mmol) and ethanol (25 ml) . After evaporation and coevapo- ration with toluene, the product was crystallized from toluene. The yield was 6.1 g (86 %) .

^X H NMR (60 MHz, DMSO-d _s ) : 4.49 (3 H, bs) ; 7.44-7.74 (1 H, m); 7.94-8.04 (2 H, m) ; 8.56-8.68 (1 H, m)

UV (λ _nax in ethanol) : 265 δ 216 nm

Example 59. The synthesis of 3—[4\'-(p-nitrobenzyl)—2\'-pyrid¬ yl]-5-(2"-pyridyl)-1,2,4-triazole (59) .

A mixture of compounds 32 (4.80 g, 20.0 mmol) and 58 (2.74 g, 20.0 mmol) was stirred for 24 hours at 160 ^e C. The reaction mixture was treated several times with hot toluene and the combined toluene fractions were evaporated to dryness. The residue was dissolved in 10 methanol in chloroform, filtered

-brought a short silica column and the filtrate was evaporated to dryness. The yield was 5.6 g (78 %) .

^X H NMR (400 MHz, CDC1 ₃ ) : 4.17 (2 H, s); 7.20 (1 H, d, J = 4.7 Hz); 7.33-7.39 (1 H, m) ; 7.39 (2 H, d, J = 8.4 Hz); 7.85-7.90 (1 H, m) ; 8.18 (2 H, d, J = 8.4 Hz); 8.25 (1 H, s); 8.35 (1 H, d, J = 8.1 Hz); 8.72 (1 H, d, J = 5.0 Hz); 8.78 (1 H, d, ^\' J = 4.7 Hz) UV (λ^ _x in ethanol) : 275 nm

Example 60. The synthesis of 1 (and 2) -benzyl-3-[4\'- (p-nitro¬ benzyl) -2\'-pyridyl] -5-(2\' \'-pyridyl) -1, 2, 4-triazole (60) .

This compound (60) was synthesized using a method analogous to the synthesis described in Example 43. The yield was 76 %. ^X H NMR (60 MHz, CDC1 ₃ ) : 4.12 (2 H, s) ; 6.19 (2 H, s) ; 6.86- 8.73 (16 H, m) UV (λ _nax in ethanol) : 279 nm

Example 61. The synthesis of 1 (and 2) -benzyl-3-[4\'-(p-nitro- benzyl)-2\'-pyridyl]-5-(2"-pyridyl)-l,2,4-triazole N\',N"- dioxide (61) .

This compound (61) was synthesized using a method analogous to the synthesis described in Example 44. The product was purified with flash chromatography (silica, 2, 5 and 10 methanol in chloroform) . The yield was 50 %.

^: H NMR (400 MHz, CDC1 ₃ , aliphatic area, isomer 1) : 3.96 (2 H, s) ; 5.79 (2 H, s)

^: H NMR (400 MHz, CDC1 _. ,, aliphatic area, isomer 2) : 4.10 (2 H, s) ; 5.75 (2 H, s)

Example 62. The synthesis of 1 (and 2)-benzyl-3-[6\'-cyano-4\' (p-nitrobenzyl)-2\'-pyridyl]—5-(6\' \'-cyano-2\' \'-pyridyl)—1,2,4 triazole (62) .

This compound (62) was synthesized using a method analogou to the synthesis described in Example 36. The product wa purified with flash chromatography (silica, 5/3, 1/1 and ^* 0/ petroleum ether/ethyl acetate) . The yield was 51 %. ^l E NMR (400 MHz, CDC1 ₃ , isomer 1): 4.24 (2 H, s); 6.13 (2 H, s); 7.27 (1 H, t, J - 7 Hz); 7.31 (2 H, t, J = 7 Hz); 7.39 ( H, d, J = 7 Hz); 7.41 (2 H, d, J = 9 Hz); 7.54 (1 H, s) ; 7.7 (1 H, d, J = 8 Hz); 8.00 (1 H, d, J = 8 Hz); 8.24 (2 H, d, = 9 Hz); 8.29 (1 K, s) ; 8.60 (1 H, d, J = 8 Hz) -E NMR (400 MHz, CDC1 ₃ , isomer 2): 4.20 (2 H, s) ; 6.13 (2 H, s); 7.26 (1 H, t, J = 7 Hz); 7.31 (2 H, t, J = 7 Hz); 7.37 (2 H, d, J = 9 Hz); 7.40 (2 H, d, J « 7 Hz); 7.50 (1 H, s) ; 7.77 (1 H _r d, J = 8 Hz); 7.99 (1 H, t, J = 8 Hz); 8.22 (2 H, d, = 9 Hz); 8.45 (1 H, d, J = 8 Hz); 8.54 (1 H, s) UV (λ^ _χ in ethanol) : 263 nm

Example 63. The synthesis of 3-[6\'-aminomethyl-4\'-(p-nitro¬ benzyl)-2\'-pyridyl]-5-(6\' \'-aminomethy1-2\' \'-pyridyl)—1 (and 2)- benzyl—1,2,4-triazole pentahydrochloride (63).

This compound (63) was synthesized using a method analogous to the synthesis described in Example 37. The yield was 88 %. UV (λ _^x n water) : 280 & 235(sh)

Example 64. The synthesis of I(and 2)-benzyl-3-{6\'-[N,N- bis ( ert-butoxycarbonylmethyl)aminomethyl]—4\'-(p-nitrobenzyl)- 2\'-pyridyl}-5-{6\' \'-[ ,N-bis( ert-butoxycarbonylmethyl)amino¬ methyl]-2"-pyridyl}-l,2,4-triazole (64) .

This compound (64) was synthesized using a method analogous to the svnthesis described in Example 22. The product was

purified with flash chromatography (silica, petroleum ether/ ethyl acetate, 1/1) . The yield was 44 %.

^■ -H NMR (400 MHz, CDC1 ₃ , isomer 1, aliphatic area) : 1.45 (36 H, s); 3.46 (4 H, s); 3.48 (4 H, s); 4.06 (2 H, s); 4.15 (2 H, s) ; 4.19 (2 H, s) ; 6.19 (2 H, s)

^** H NMR (400 MHz, CDC1 ₃ , isomer 2, aliphatic area) : 1.45 (36 H,- s); 3.44 (4 H, s); 3.51 (4 H, s); 4.05 (2 H, s); 4.12 (2 H, s) ; 4.20 (2 H, s); 6.19 (2 H, s) UV (λ^ _* in ethanol) : 283 & 250 nm

Example 65. The synthesis of 1 (and 2) -benzyl-3-{ 6\' -[N,N-bis- (tert-butoxycarbonylmethyl) aminomethyl]-4\'-(p-aminobenzy1) -2\'- pyridyl }-5—{ 6\' \' - [N, N-bis (tert-butoxycarbonylmethyl) amino¬ methyl] -2\' \'-pyridyl}-l, 2, 4-triazole (65) .

This compound (65) was synthesized using a method analogous to the synthesis described in Example 40. The product was purified with flash chromatography (silica, 2 and 3 % methanol in chloroform) . The yield was 36 %. --H NMR (400 MHz, CDC1 ₃ , isomer 1) : 1.45 (36 H, s); 3.46 (4 H, s); 3.50 (4 H, s) ; 3.93 (2 H, s); 4.05 (2 H, s); 4.19 (2 H, s); 6.19 (2 H, s) ; 6.62 (2 H, d, J = 8 Hz); 7.01 (2 H, d, J = 8 Hz); 7.15-7.35 (5 H, m) ; 7.65 (1 H, s) ; 7.71 (1 H, d, J = 8 Hz); 7.79 (1 H, t, J = 8 Hz); 7.91 (1 H, s); 8.23 (1 H, d, J = 8 Hz) ^l E NMR (400 MHz, CDC1 ₃ , isomer 2) : 1.45 (36 H, s); 3.45 (4 H, s); 3.51 (2 H, s); 3.90 (2 H, s); 4.03 (2 H, s); 4.21 (2 H, s); 6.19 (2 H, s) ; 6.61 (2 H, d, J = 8 Hz); 6.98 (2 H, d, J = 8 Hz); 7.15-7.35 (5 H, m) ; 7.50 (1 H, s); 7.78 (1 H, t, J = 8 Hz); 7.83 (1 H, d, J = 8 Hz); 8.07 (1 H, d, J = 8 Hz); 8.12 (1 H, s)

Example 66. The synthesis of 3—{6\'—[N, -bis (tert-butoxycarbon ylmethyl)aminomethyl]-4\'-(p—aminobenzyl)-2\'-pyridyl}-5-{ 6\' \' [N,N-bis(tert-butoxycarbonylmethyl)aminomethyl] -2 ^r \'-pyridyl} 1,2,4-triazole (66).

This compound (66) was synthesized using a method analogou to the synthesis described in Example 48. The yield was 50 % ⁱ H NMR (60 MHz, CDC1 ₃ , aliphatic area): 1.45 (36 H, s) ; 3.5 (8 H, s); 3.91 (2 H, s); 4.09 (2 H, s) ; 4.16 (2 H, s)

Example 67. The synthesis of 3-{6\'—[N,N-bis (carboxymethyl) aminomethyl]-4\'-(p-aminobenzyl)-2\'-pyridyl}-5-{6\' \'-[N,N bis (carboxymethyl)aminomethyl]—2\' \'-pyridyl} ^\' —I,2,4-triazol (67) .

This compound (67) was synthesized using a method analogou to the synthesis described in Example 9. The yield was 100 %.

Example 68. The synthesis of terbium(III) chelate of 3-{6\' [N,N-bis (carboxymethyl)aminomethyl]-4\'—(p-isothiocyanato benzyl) -2\'—pyridyl}-5-{6\' \'-[N,N-bis (carboxymethyl) amino methyl]-2"-pyridyl}-l,2,4-triazole (68) .

The compound 68 (50 mg, 0.08 mmol) was dissolved in water (1.5 ml) and the pH was adjusted to 6.5 with solid sodium bicarbo¬ nate. Terbium(III) chloride (35 mg, 0.090 mmol) in water (0.5 ml) was added during 15 minutes and the pH was maintained at 5—7. After stirring for 1.5 hours, the pH was raised to 8.5 with 1 M sodium hydroxide and the precipitate was filtered off. The filtrate was triturated with acetone, the precipitate was filtered and washed with acetone. An aqueous solution of the precipitate (2.5 ml) was added during 15 minutes to a mixture of thiophosgene (25 ui, 0.32 mmol) , sodium bicarbonate (34 g, 0.40 mmol) and chloroform (2.5 ml) . After stirring for

one hour, the fractions were separated and the water fractio was washed with chloroform (3 x 1.0 ml) . The aqueous solutio was triturated with acetone, the precipitate was filtered an washed with acetone. The vield was 38 m (55 %) .

CH,COOCH,Br

(CH ₃ ) ₃ SiCN, PhCOCl

72

scheme m < Tmhi-.e synthesis cf compound 72

Example 69. The synthesis of 2,4-di(2\'-pyridyl)imidazole (69) .

A mixture of 2-pyridinecarboxamidine hydrochloride (4.85 g, 31.0 mmol), 2-bromoacetylpyridine (5.00 g, 25.0 mmol), N,N- diisopropylethylamine (8.8 ml, 50 mmol) and chloroform (50 mi) was refluxed for two hours. The reaction mixture was washed with 5 % sodium bicarbonate (20 ml) , water (2 x 20 ml) and dried with sodium sulfate. The product was purified with flash chromatography (silica, ammonia/5 % methanol in chloroform, first 0/1 then 1/49) . The yield was 2.28 g (41 %) . M.p. 145°C. ^l E NMR (400 MHz, CDC1 ₃ ) : 7.15-7.18 (1 H, m) ; 7.27-7.30 (1 H, m); 7.72 (2 H, t) ; 7.80 (2 H, t) ; 8.22 (1 H, d) ; 8.58-8.59 (2 H, d) UV (λ _aax ethanol) : 307, 260 & 227 nm

Example 70. The synthesis of l-acetoxymethyl-2,4-di (2\'-pyri¬ dyl)imidazole (70).

A mixture- of compound 69 (0.50 g, 2.3 mmol), potassium car- bonate (0.48, 3.4 mmol), bromomethyl acetate (1.03 g, 6.80 mmol) in acetonitrile (20 ml) was refluxed for five hours. After filtration the filtrate was evaporated to dryness. The product was purified with flash chromatography (silica, 2 and 5 % methanol in chloroform) . The yield was 0.40 g (59 %) . ⁱ H NMR (400 MHz, CDC1 ₃ ) : 2.07 (3 H, s) ; 6.69 (2 H, s) ; 7.16- 7.19 (1 H, m); 7.27-7.30 (1 H, m) ; 7.75 (1 H, dt, J = 1 & 8 Hz); 7.81 (1 H, dt, J = 1 & 8 Hz); 7.92 (1 H, s); 8.07 (1 H, d, J = 8 Hz); 8.35 (1 H, d, J = 8 Hz); 8.59-8.61 (2 H, m) UV (λ-, _ax ethanol) : 294, 257 & 223 nm

Example 71. The synthesis of 1—acetoxymethyl—2, -di(2\'-pyri¬ dyl)imidazole N\' ,N\'-dioxide (71).

This compound (71) was synthesized using a method analogous to the svnthesis described in Example 44. The product was

purified with flash chromatography (silica, 2, 5 and 10 -ϊ methanol in chloroform) . The yield was 20 %.

-■H NMR (400 MHz, CDC1 ₃ ) : 1.98 (3 H, s); 6.14 (2 H, s); 7.15 ⁽ 1

H, dt); 7.32 (1 H, t) ; 7.40-7.43 (2 H, m) ; 7.72-7.75 ⁽ 1 H, m ^{) ;}

8.31-8.35 (3 H, m) ; 8.92 (1 H, s)

UV (λ _ax in ethanol ) : 289 ( sh) , 254 & 220 nm

Example 72. The synthesis of l-acetoxymethyl-2, 4-bis (6\' -cyano 2\'-pyridyl) imidazole (72) .

This compound (72) was synthesized using a method analogou to the synthesis described in Example 36. The product wa purified with flash chromatography (silica, petroleum ether ethyl acetate, 5/3) . The yield was 52 %.

--H NMR (400 MHz, CDC1 ₃ ) : 2.11 (3 H, s) : 6.61 (2 H, s) ; 7.57 ⁽ H, d, J = 8 Hz); 7.71 (1 H, d, J = 8 Hz); 7.86 (1 H, t, J 8 Hz); 7.97 (1 H, t, J = 8 Hz); 8.04 (1 H, s); 8.25 ⁽ 1 H, d J - 8 Hz); 8.57 (1 H, d, J = 8 Hz) UV (λ^ _x in ethanol) : 301 & 272 nm

73

74

Scheme 12. The synthesis of compound 76

Example 73. The synthesis of 2, 6-bis(4\'-methylthiazol-2\'-yl)- pyridine (73) .

A mixture of 2,6-pyridinedithiodicarboxamide (0.74 g, 3.8 mmol), chloroacetone (0.70 ml, 8.7 mmol) and ethanol (15 ml) was refluxed overnight. Solid material was filtered and washed with ethanol. The suspension of the hydrochloric salt of the product in hot water (50 ml) was alkalized with solid sodium carbonate. The product was filtered and washed with water. The yield was 0.63 g (61 %) . -& NMR (400 MHz, CDC1 ₃ ) : 2.54 (6 H, s) ; 7.03 (2 H, s) ; 7.86 (1 H, t, J = 7.8 Hz); 8.15 (2 H, d, J = 7.8 Hz) UV (λ _^ in ethanol) : 330, 306 & 232 nm

Example 74. The synthesis of 2,6-bis (4\'-bromomethylthiazol-2\'- yl)pyridine (74) .

A mixture of compound 73 (0.63 g, 2.3 mmol), N-bromosuc¬ cinimide (0.90 g, 5.1 mmol), dibenzoylperoxide (56 mg, 0.2 mmol) and carbon tetrachloride (15 ml) was refluxed overnight. The reaction mixture was filtered and the filtrate was

evaporated to dryness. The product was purified with flash chromatography (silica, 2 % methanol in chloroform) . The yield was 82 %.

^: H NMR (400 MHz, CDC1 ₃ ) : 4.65 (4 H, s); 7.44 (2 H, s); 7.93 (1 H, t, J = 9 Hz); 8.24 (2 H, d, J = 9 Hz) UV (-- _ax in ethanol) : 328 & 302 nm

Example 75. The synthesis of 2, 6-bis {4\' -[N,N-bis (tert-butoxy- carbonylmethyl) aminomethyl] thiazol-2\' -yl}pyridine (75) .

This compound (75) was synthesized using a method analogous to the synthesis described in Example 8. The product was purified with flash chromatography (silica, petroleum ether/ ethyl acetate, first 10/1, then 5/1) . The yield was 31 %. ^l E NMR (400 MHz, CDC1 ₃ ) : 1.48 (36 H, s); 3.53 (8 H, s) ; 4.16 (4 H, s); 7.42 (2 H, s) ; 7.86 (1 H, t, J = 8.0 Hz); 8.18 (2 H, d, J = 8.0 Hz) UV (λ^ _ax in ethanol) : 329 & 299 nm

Example 76. The synthesis of 2, 6-bis {4\'-[N,N-bis (carboxy¬ methyl) aminomethyl]thiazol-2\'-yl}pyridine (76) .

This compound (76) was synthesized using a method analogous to the synthesis described in Example 9. The yield was 75 %.

^: H NMR (400 MHz, DMSO-d ₆ ) : 3.85 (8 H, s); 4.33 (4 H, s); 7.90

(2 H, s) ; 8.18-8.22 (3 H, m)

UV (λ _j . _ax in water as free ligand) : 323 & 287 nm UV (λ^ _ax in water as europium(III) chelate) : 341 & 278 nm

77

78

Scheme 13. The synthesis of compound 78

Example 77. The synthesis of 2,6-bis(N-hydroxycarboximid- amido)pyridine (77) .

A mixture of 2, 6-dicyanopyridine (8.03 g, 62.2 mmol), hydrox- ylamine hydrochloride (10.4 g, 150 mmol), sodium acetate (13.8 g, 168 mmol) and water/ethanol (200 ml, 1/5) was refluxed for

45 minutes. After evaporation to dryness the residue was triturated with water, filtered and washed with water. The yield was 11.43 g (94 %) .

^X H NMR (60 MHz, DMSO-d : 6. 26 (4 H, bs ) ; 7 . 70-7 . 87 ( 3 H, m) ; 9 . 83 (2 H, S )

UV (>^ _ax i ethanol) : 301 nm

Example 78. The synthesis of 2, 6-bis (5\'-methyl-1\' , 2\' , 4\'-oxadi azol-3\'-yl)pyridine (78).

A mixture of compound 77 (11.1 g, 57.0 mmol), acetic anhydrid (34.9 g, 342 mmol) and toluene (150 ml) was refluxed over night. After evaporation to the half of the original volume the solid material was filtered and washed with toluene. Th yield was 11.6 g (84 %) . ^l E NMR (400 MHz, CDC1 ₃ ) : 2.71 (6 H, s) ; 8.03 (1 H, t, J = 7. Hz); 8.21 (2 H, d, J = 7.7 Hz) UV (λ _^ax in ethanol) : 282 & 233 nm

Eu(OAc.

79

Scheme 14. The synthesis of compound 79

Example 79. The synthesis of europium(III) cryptate (79).

A mixture of europium(III) acetate (78 mg, 0.24 mmol), tri methvl orthoformate (1.2 ml) and dry acetonitrile (6 ml) wa

3

62 refluxed for two hours. After addition of 1,7,10,16-tetraox 4,13-diazacyclooctadecane (62 mg, 0.24 mmol) the reacti mixture was refluxed for 15 minutes. A suspension of compou 12 (130 mg, 0.24 mmol) and dry acetonitrile (4 ml) was add and the reaction mixture was refluxed for 24 hours. T product was filtered and washed with acetonitrile. The yie was 70 mg (37 %) .

Example 80. The synthesis of terbium(III) chelate of 3-{6\' [N, -bis(carboxymethyl)aminomethyl]-4\'-[p-( ,6-dichlorotri zon-2- lamino)benzyl]-2\'-pyridyl}-5-{6\' \'-[N,N-bis (carbox meth l)aminomethyl]-2"-pyridyl}-1,2,4-triazole (80) .

This compound (80) was synthesized from the compound 67 usi a method analogous to the synthesis described in Example 57b. Example 81. The luminescence properties of europiu (III) a terbium(III) chelates of the compound 49.

The relative luminescence yield φ _rβl of the europium(III) an terbium(III) chelates of the compound 49 were measured i equimolar 10" ⁵ M solutions of the compound 49 and the corre sponding lanthanide ion. Luminescence measurements were don on a Perkin-Elmer LS-5* spectrofluorometer using the phospho rescence mode which allowed the decay curves of the lanthanid luminescence to be measured. The luminescence yield i reported relative to the luminescence of the uncomplexe lanthanide cation (Ln) using the equation:

6 \' _r r _e e _l l =

^J - r-Cche-\'"-c---- where I _che and 1^ are the preexponenti≤.1 terms of the emissio decay curves for the chelated and uncomplexed lanthanid cation, respectively (614 nm for europium and 544 nm fo terbium) . The excitation wavelength for the uncomplexe europium(III) was 395 nm and for terbium(III) 370 nm. C-__ an Cw--. are the concentrations-of free and complexed lanthanid

cation, respectively, and k _Ln and k _che the corresponding decay constants. For the europium(III) complex of the compound 49 the relative luminescence yield was 1.3xl0 ⁵ and for the ter¬ bium(III) complex it was 5.8xl0 ⁵ . The excitation wavelength for europium(III) was 280 nm and for terbium (III) 310 nm.

Example 82. The luminescence properties of europium (III) an terbium(III) chelates of the compound 30.

For the europium(III) complex of the compound 30 the relative luminescence yield was 8.9xl0 ⁵ . The excitation wavelength was 336 nm. For terbium(III) the corresponding values were 2.8xl0 ³ and 260 nm.

Example 83. The luminescence properties of the europium (III) chelate of the compound 76.

For the europium(III) complex of the compound 76 the relative luminescence yield was 5.7xl0 ⁵ . The excitation wavelength was 340 nm.

Example 84. The labelling of Rabbit-anti-mouse IgG with the compound 68.

IgG fraction of rabbit-anti-mouse-IgG (RaM) (Dako, Denmark) was purified into a labelling buffer consisting of 50 mM carbonate (pH 9.3) . RaM fractions (1 mg each) were labelled with the chelate 68 using a 100, 300 and 1200 fold molar excess of the chelate in the labelling buffer, incubating at room temperature for 16 hours. Thereafter the IgG-conjugates were purified with a combined column of Trisacryl* GF5

(Reactifs IBF, France) and Sephacryl* S-300 (Pharmacia

Biosystems, Sweden) , eluting with tris-buffered salt solution

(50 mM, pH 7.75) . Both the partly aggregated and monomeric IgG fractions were collected and analyzed for protein and terbium(III) concentration. Terbium(III) concentrations wer measured with a modified DELFI * system (Wallac, Finland) using 2, , 6-trimethoxyphenyIdipicoiinic acid as the fluorege nic liσand. The incorporation yields varied between 12.2 an

69 terbiu (III)/IgG.

Example 85. The relative luminescence of terbium(III)-labelle antibobies.

The relative luminescence of labelled antibodies were measure in different buffers in the pH range between 3.2 and 11.7. Th highest luminescences were achieved at neutral pH, at pH belo 5 luminescence decreased rapidly, at pH over 9 the decreas was slow. The luminescence was not quenched by innerfilte effect. The excitation maximum was at 310 nm, producing the typical emission lines at 490 and 544 nm (in addition to mino lines at longer wavelenght) . The quantum yield compared to the system in Example 83 approached 25 % and the decay time was 1.46 ms (in buffer) .

Example 86. The immunoassay with terbium(III)-labelled anti¬ bodies.

The immunoreactivity of the present terbium(III)-labelled antibodies were.tested in a model assay system consisting of polystyrene microtitration strips physically coated with monoclonal antibodies (MIgG) using albumin (BSA) coated strips as a control. The strips were incubated in assay buffer (Wallac) containing varying amounts of terbium(III)-labelled RaM. The specific signal ranged from 3000 cps with 1 ng/ml of the terbium(III)-RaM to 1,000,000 cps with 10 μg/ml of terbiu (III)-RaM.

Terbium(III)—labelled RaM was also tested in an allergy- specific IgE binding test using a matrix (CAP* matrix, Phar¬ macia Diagnostics) immobilized with allergenic material. The binding of specific IgE from patient serum was visualized by staining with mouse-anti-human IgE and subsequently with terbium(III)-labelled RaM. The stained matrix was examined with a time—resolved fluorescence microscope and the signal recorded with a CCD camera.