for determining an alkali ion The present invention relates to ττ-conjugated fluoroionophores, methods for their preparation, and their use and a method for determining an alkali ion.

Background of the invention EP 0 881 487 A2 discloses a method for the determination of an alkali ion in biological samples like blood. The determination is performed by using a compound having a luminophoric and an ionophoric moiety. WO

2007/0044866 A2 describes chromoionophoric compounds comprising a tri- azaeryptand (TAC) K ⁺ ionophore conjugated to at least a first chromophoric moiety. These water-soluble fluorescent compounds are used for the detection of potassium. US 6,21 1 ,359 B1 discloses a triaza-cryptand useful as a luminescence indicator for alkali ions. US 2007/0259443 A1 and US

2007/0259444 A1 disclose chromoionophores and a method of determining potassium ions.

Recently 1 ,2,3-triazoM ,4-diyl fluoroionophores for Zn ²⁺ , ¹ Ni ²⁺ , ² Cu ²⁺ , ³ Hg ²⁺ , ⁴ Ag ⁺ , ⁴ and Al ³⁺ , ⁵ were generated by Cu(l) catalyzed reaction between an azide and an alkyne (CuAAC). In these fluoroionophores the 1 ,2,3-triazol-1 ,4- diyls serve in addition to the conventional function as covalent linkers as both, chelating ligand of the metal ions and as electronic transmitter of a coordination event to the fluorophore. ^{1 a} Only Zn ²⁺ and Al ³⁺ could be detected by fluorescent enhancement, whereas the other metal ions were analysed through fluorescence quenching. In these known 1 ,2,3-triazol-1 ,4-diyl fluoroionophores, no electronically conjugated signal transduction chain: ionophore - 1 ,2,3-triazole - fluorophore, is used. Either receptor and 1 ,2,3-triazole are connected through a deconjugated linker ^{1 ,2} or the triazole and the fluoro- phoric group are deconjugated. ^3,4,5

In a systematic investigation Diederich et al. showed the capacity of the 1 ,2,3-triazol-diyls as active ττ-linkers in Charge Transfer (CT) chromophores. ⁶ Such push-pull chromophores are only weak or even non fluorescent. The compounds and methods known show several disadvantages when used for the determination of potassium especially in biological samples like blood. A disadvantage of the known methods is that the cations can only be measured within a small concentration range. Further these methods also lack sensitivity and selectivity especially in the presence of sodium.

Therefore, it is an object of the present invention to provide a method for the determination of alkali metal cations in biological samples within a broad con- centration range, and with improved sensitivity and specificity.

Brief description of the invention

The inventors found that in simple CuAAC-generated systems the electronic conjugation of an alkali metal ion selective receptor which could be a N- phenylaza-crown ether or a /V-phenylaza-cryptand, and a fluorophore through a 1 ,2,3-triazol ττ-linker results in a perfect signal transduction chain for a simultaneous fluorescence quenching via both, an internal charge transfer (ICT) and a photoinduced electron transfer (PET). The fluorescence of the original fluorophore is basically nullified. Coordination of an alkali metal ion to the anilino-donor of the ionophoric unit interrupts this ττ-conjugation as well as the PET, resulting in a revival of the fluorescence, proportional to the metal ion concentration. This concentration dependant fluorescence enhancement allows the sensing of K ⁺ and Na ⁺ under simulated physiological conditions.

The problem of the invention is solved by the development of novel triazole- based fluoroionophores which show significant selectivity for Na ⁺ and K ⁺ in water though bearing a simple crown-ether-receptor or cryptand-receptor unit.

Scheme 1 Π-conjugated 1 ,2,3-triazole-based fluoroionophores To the best of the inventors knowledge no 1 ,2,3-triazole-based linker between receptor and fluorophore, giving a conjugated system exists to now.

Variation of the ionophoric unit offers a broad spectrum of detectable K ⁺ and Na ⁺ -concentrations, ranging from high concentration around 800 mM down to very low concentrations around 3 mM.

The new alkali metal ion 1 ,2,3-triazole-based fluoroionophores have great potential for application in fluorescent optode systems based blood analyzing equipment for methods and kits for the determination of K ⁺ and Na ⁺ concen- trations in biological systems, either in vitro or in vivo, using embodiments of the fluoroionophores according to the present invention.

The problem is solved according to the invention by providing a fluoroiono- phoric compound of the general formula I lonophore - ττ-Linker - Fluorophore (I) wherein

the lonophore is an anilino containing crown ether or cryptand with one or more anilino donor moieties as electron donors, forming a stable complex with an alkali metal ion

the TT-Linker is an aromatic or heteroaromatic conjugative linking moiety, and the Fluorophore is an electron acceptor moiety.

Preferred are compounds according to the invention, wherein the ionophore is selected from the group consisting of

wherein

n is a number selected from 0 and 1 ,

m is a number selected from 0, 1 , and 2, and

wherein the phenyl ring is optionally substituted with halogen, nitro, amino, hydroxyl, lower alkyi or lower alkoxy, wherein the lower alkyi or lower alkoxy are optionally substituted with halogen, nitro, amino, hydroxyl or lower alkyi or lower alkoxy, and

wherein the phenyl ring may optionally be a part of a condensed aromatic system, that is optionally substituted with halogen, nitro, amino, hydroxyl, or lower alkyl or lower alkoxy or phenyl, optionally substituted with halogen, ni- tro, amino, hydroxyl or lower alkyl or lower alkoxy.

Preferred are also compounds according to the invention, wherein the ττ- Linker is selected from the group consisting of an aromatic or heteroaromatic moiety, wherein the aromatic moiety refers to a 6- to 14-membered monocyclic, bicyclic or tricyclic aromatic hydrocarbon ring system that is unsubstituted or optionally substituted with one or more substituents, and wherein the heteroaromatic moiety refers to an aromatic heterocyclic ring of 5 to 14 mem- bers and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including monocyclic, bicyclic, and tricyclic ring systems, that is unsubstituted or optionally substituted with one or more substituents. Especially preferred are compounds, wherein the ττ-Linker is selected from the group consisting of phenyl and naphthyl, that are unsubstituted or optionally substituted with one or more substituents, and triazolyl, tetrazolyl, oxadi- azolyl, pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, naphthothiazolyl, quinoxalinyl, that are unsubstituted or optionally substituted with one or more substituents. Especially preferred are compounds according to the invention, wherein the TT-Linker is selected from the group consisting of isomeric disubstituted 1 ,2,3- triazoles, preferably:

wherein

10 is the ionophore according to claim 1 ;

Fl is the fluorophore according to claim 1 ;

R ₉ is selected from hydrogen, halogen, nitro, amino, hydroxyl, lower alkyl and lower alkoxy, optionally substituted with halogen, nitro, amino, hydroxyl or lower alkyl or lower alkoxy.

Very specially preferred are compounds according to the invention, wherein the TT-Linker is selected from the group consisting of substituted 1 ,4-triazoles, namely

Fl

wherein IO is the ionophore and Fl is the fluorophore.

Preferred are compounds according to the invention, wherein the fluorophore moiety is represented by the formula wherein

R ¹ , R ⁴ , R ⁵ = H, lower alkyl, CF ₃ , MeO, halogen, N0 ₂ , CN,

R ₂ , R3 = H, NH ₂ , N(lower alkyl) ₂ , lower alkyl, optionally substituted with car- boxyl or carbonyl, diethyl amino

R ⁶ = alkinyl, azide.

Preferred are also compounds according to the invention, wherein the fluoro- phore moiety is selected from the group consisting of

wherein

n is an integer ranging from 0 to 15; R ₈ is selected from hydrogen, lower alkyl or lower alkoxy;

and which are optionally substituted with halogen, nitro, amino, hydroxyl, lower alkyl or lower alkoxy.

Especially preferred are the following compounds of the invention according to general Formula (I), namely

Coumarin fluoroionophores

Potassium selective

3-{4-[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- thylamino)-2H-chromen-2-one

3-{1 -[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- triazol-4-yl}-7-(diethylamino)-2H-chromen-2-one

o.

O ^'

7-(diethylamino)-3-{1 -[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}-2H-chromen-2-one

7-(diethylamino)-3-{4-[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-1 -yl}-2H-chromen-2-one

7-(diethylamino)-3-{4-3-[2-methoxyethoxy)-4{bis(4-methylb enzo[5, 6, 17, 18](0, N, N', 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 1 ]-phen-4-yl]-1 H-1 .2.3- triazol-1 yl}-2H-chromen-2-one

7-(diethylamino)-3-{4-[3-(2-methoxyethoxy]-4{bis(4-methyl benzo[5, 6, 17, 18](0, N, N', 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 1 ]-phen-4-yl]-1 H-1 .2.3- triazol-1 yl}-2H-chromen-2-one

Sodium selective

3-{4-[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- triazol-1 -yl}-7-(diethylamino)-2H-chromen-2-one o o

3-{1 -[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- thylamino)-2H-chromen-2-one

7-(diethylamino)-3-{4-[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13- n-13-yl)phenyl]-1 H-1 ,2,3-triazol-1 -yl}-2H-chromen-2-one

7-(diethylamino)-3-{1 -[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13- azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}-2H-chromen-2-one

7-(diethylamino)-3-{4-[3-methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](O, N, NT, O'H-1, 7, 16, -triaza-cryptand-[3, 2, 1]-phen-4-yl]-1 H-1.2.3-triazol-1yl}-2H- chromen-2-one

7-(diethylamino)-3-{1-[3methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](O, N, N', O'H-1, 7, 16, -triaza-cryptand-[3, 2, 1]-phen-1-yl]-1H-1.2.3-triazol-1 l}-2H- chromen-2-one

Naphthalimid fluoroionophores

Potassium selective

6-{4-[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- H-benzo[de]isoquinoline-1 ,3(2H)-dione

6-{1 -[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- triazol-4-yl}-2-ethyl-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{4-[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-1 -yl}-1 H- benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{1 -[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}-1 H- benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{4-[3-(2-methoxyethoxy)-4{bis(4-methylbenzo[5, 6, 17, 18](O, N, N', O'H-1 , 7, 16, -triaza-cryptand-[3, 2, 2]-phen-1 -yl]-1 H-1 .2.3-triazol 1 -yl}-1 H- benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{1 -[3-(2-methoxyethoxy)-4{bis(4-methylbenzo[5, 6, 17, 18](O, N, N', O')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 2]-phen-4-yl]-1 H-1 .2.3-triazol 1 -yl}-1 H- benzo[de]isoquinoline-1 ,3(2H)-dione

Sodium selective

6-{4-[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- triazol-1 -yl}-2-ethyl-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione

6-{1 -[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- -1 H-benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{4-[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13- -yl}-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{1 -[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13- -triazol-4-yl}-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione

2-ethyl-6-{4-[3-methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]-1, 7, 16, -triaza-cryptand-[3, 2, 1]-phen-1-yl]-1 H-1.2.3-triazol 1-yl}-1H- benzo[de]isoquinoline-1,3(2H)-dione

2-ethyl-6-{1-[3-methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]-1, 7, 16, -triaza-cryptand-[3, 2, 1]-phen-4-yl]-1 H-1.2.3-triazol 1-yl}-1H- benzo[de]isoquinoline-1,3(2H)-dione

7-BODIPY fluoroionophores

Potassium selective

7-{4-[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- triazol-1 -yl}-5,5-difluoro-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'- borinin-4-ium-5-uide

7-{1 -[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3- triazol-4-yl}-5,5-difluoro-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'- f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7-{4-[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-l -yl}-1 ,3,9, 10-tetramethyl- -c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7-{1 -[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}-1 ,3,9, 10-tetramethyl- 5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7 {4-[3-(2-methoxyethoxy]-4(bis(4-methylbenzo[5, 6, 17, 18](0, N, N\ 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 2]-phen-4-yl]-1 H-1 .2.3-triazol -1 - yl}-4-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium- 5-uide

5,5-difluoro-7 {1 -[3-(2-methoxyethoxy]-4(bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 2]-phen-1 -yl]-1 H-1 .2.3-triazol -1 - yl}-4-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium- 5-uide

Sodium selective

7-{4-[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- triazol-1 -yl}-5,5-difluoro-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'- f][1 ,3,2]diazaborinin-4-ium-5-uide

7-{1 -[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3- triazol-4-yl}-5,5-difluoro-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'- f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7-{4-[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-1 yl)phenyl]-1 H-1 ,2,3-triazol-l -yl}-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2' f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7-{1 -[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-1 yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}-1 ,3,9, 10-tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2' f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7 {4-[3-methoxy-4(bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]- 1 , 7, 16, -triaza-cryptand-[3, 2, 1 ]-phen-4-yl]-1 H-1 .2.3-triazol -1 -yl}-4-1 ,3,9, 10- tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-7 {1 -[3-methoxy-4(bis(4-methylbenzo[5, 6, 17, 18](O, N, N', O')-]- 1 , 7, 16, -triaza-cryptand-[3, 2, 2]-phen-1 -yl]-1 H-1 .2.3-triazol -1 -yl}-4-1 ,3,9, 10- tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

10-phenyl-BODIPY-fluoroionophores

Potassium selective

10-(4-{4-[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H- 1 ,2,3-triazol-1 -yl}phenyl)-5,5-difluoro-1 ,3,7,9-tetramethyl-5H-dipyrrolo[1 ,2- c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

10-(4-{1 -[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl]-1 H- 1 ,2,3-triazol-4-yl}phenyl)-5,5-difluoro-1 ,3,7,9-tetramethyl-5H-dipyrrolo[1 ,2- azaborinin-4-ium-5-uide

5,5-difluoro-10-(4-{4-[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16 azacyclooctadecan-16-ylphenyl]-1 H-1 ,2,3-triazol-l -yl)phenyl\-1 ,3,7,9- tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-l 0-(4-{1 -[3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13-pentaoxa-16- azacyclooctadecan-16-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}phenyl)-1 ,3,7,9- tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro 10-(4-{4-[3-(2-methoxyethoxy)-4(bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 2]- phen-4-yl]-1 H-1 .2.3- triazol 1 -yl}1 ,3,7,9-tetramethyl-5H-dipyrrolo)(1 ,2-c: 1 ',2'-f)(1 ,3,2)diazaborinin- 4-ium-5-uide

5,5-difluoro 10-(4-{1 -[3-(2-methoxyethoxy)-4(bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')-]-1 , 7, 16, -triaza-cryptand-[3, 2, 2]- phen-4-yl]-1 H-1 .2.3- triazol 4-yl}1 ,3,7,9-tetramethyl-5H-dipyrrolo)(1 ,2-c: 1 ',2'-f)(1 ,3,2)diazaborinin- 4-ium-5-uide

Sodium selective

10-(4-{4-[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H- 1 ,2,3-triazol-1 -yl}phenyl)-5,5-difluoro-1 ,3,7,9-tetramethyl-5H-dipyrrolo[1 ,2- aborinin-4-ium-5-uide

10-(4-{1 -[4-(1 ,4,7, 10-tetraoxa-13-azacyclopentadecan-13-yl)phenyl]-1 H- 1 ,2,3-triazol-4-yl}phenyl)-5,5-difluoro-1 ,3,7,9-tetramethyl-5H-dipyrrolo[1 ,2- c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro-10-(4-{4-[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13- azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3-triazol-l -yl}phenyl)-1 ,3,7,9-

5,5-difluoro-10-(4-{1 -[3-methoxy-4-(1 ,4,7, 10-tetraoxa-13- azacyclopentadecan-13-yl)phenyl]-1 H-1 ,2,3-triazol-4-yl}phenyl)-1 ,3,7,9- tetramethyl-5H-dipyrrolo[1 ,2-c: 1 ',2'-f][1 ,3,2]diazaborinin-4-ium-5-uide

5,5-difluoro 10-(4-[3-methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](0, N, N\ 0')- ]-1 , 7, 16, -triaza-cryptand-[3, 2, 1 ]-phen-4-yl]-1 H-1 .2.3-triazol -1 -yl}-(1 ,3,7,9- tetramethyl-5H-dipyrrolo)(1 ,2-c: 1 ',2'-f)(1 ,3,2)diazaborinin-4-ium-5-uide

5,5-difluoro 10-(1 -[3-methoxy-4{bis(4-methylbenzo[5, 6, 17, 18](0, N, N', 0')- ]-1 , 7, 16, -triaza-cryptand-[3, 2, 1 ]-phen-1 -yl]-1 H-1 .2.3-triazol -1 -yl}-(1 ,3,7,9- tetramethyl-5H-dipyrrolo)(1 ,2-c: 1 ',2'-f)(1 ,3,2)diazaborinin-4-ium-5-uide

A further object of the present invention is a complex, consisting of a com- pound according to present invention as given in general Formula (I) and an alkali metal cation. The compounds of the present invention comprise an ionophore that has the capability to bind metal ions. This capability is given by the crown ether or the cryptand moiety of the compounds according to the invention.

According to the invention a complex is preferred, wherein the alkali metal cation is selected from the group consisting of Na ⁺ and K ⁺ .

An object of the present invention is also a method for the determination of metal cations in a sample, comprising the steps of

a) contacting the metal cations with at least one compound according to the present invention,

b) forming a complex according to another object of the invention, whereupon fluorescence and/or luminescence appears or changes,

c) measuring the resulting fluorescence and/or luminescence.

According to the invention a method is preferred, wherein the metal cations are Na ⁺ or K ⁺ , or a combination thereof. According to the invention a method is especially preferred, wherein the at least one compound according to the invention selectively complexes the metal cations to be determined. Lastly, another object of the invention is the use of a compound according to the invention in a method according to the invention for the quantitative determination of metal cations in a sample.

Brief description of the figures

The figures show fluorescence spectra of preferred embodiments of the present invention.

Fig. 1 Fluorescence spectra of 1 in MeCN (bottom line) upon addition of 0- 2.5 mM NaPF ₆ (middle line) and 0-1 .16 mM KPF ₆ (top line), respectively.

Fig. 2 Fluorescence measurements of 1 (λ _βχ = 424 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)]. a) fluorescence spectra in the presence of 0-160 mM Na ⁺ (bottom lines) and K ⁺ (top lines), respectively and b) corresponding FEF with error bars in the presence of 0-160 mM K ⁺ or Na ⁺ after repeating the experiment with n = 4.

Fig. 3 K ⁺ Sensitivity of 1 (λ _βχ = 424 nm) under simulated physiological conditions, 0-2000mM KCI, saturation at 1000 mM.

Fig. 4 K ⁺ Sensitivity of 5 (λ _βχ = 424 nm) under simulated physiological condi- tions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM

Tris)] in the presence of 0-160 mM K ⁺ .

Fig. 5 Fluorescence measurements of 5 (λ _βχ = 421 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)]. a) fluorescence spectra in the presence of 0-160 mM

KCI and 180-0 mM NaCI, respectively and b) corresponding FEF in the presence of 0-160 mM K ⁺ or Na ⁺ .

Fig. 6 K ⁺ Sensitivity of 6 (A _ex = 367 nm) under simulated physiological condi- tions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)] in the presence of 0-160 mM KCI. Fig. 7 Fluorescence measurements of 6 (A _e = 367 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)]. a) fluorescence spectra in the presence of 0-160 mM KCI and 180-0 mM NaCI, respectively and b) corresponding FEF in the presence of 0-160 mM K ⁺ or Na ⁺ .

Fig. 8 K ⁺ Sensitivity of 14 (A _ex = 493 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)] in the presence of 0-180 mM Na ⁺ .

Fig.9 Fluorescence measurements of 14 (A _ex = 430 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)], fluorescence spectra in the presence of 0-180 mM K ⁺ plus Na ⁺ .

Fig. 10 K ⁺ Sensitivity of 15 (A _ex = 493 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)] in the presence of 0-180 mM Na ⁺ .

Fig. 11 Fluorescence measurements of 15 (A _ex = 422 nm) under simulated physiological conditions [180 -0 mM choline chloride, 2mM Ca ²⁺ , 2mM Mg ²⁺ , pH=7.2 (10mM Tris)], fluorescence spectra in the presence of 0-180 mM K ⁺ plus Na ⁺ .

Detailed description of the invention

As used herein, the terms have the following meaning: The term "alkyi" or "lower alkyi" as used herein refers to a straight or branched chain, saturated hydrocarbon having the indicated number of carbon atoms. For example, (C1 -C6) alkyi is meant to include, but is not limited to methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyi or lower alkyi group can be unsubstituted or optionally substituted with one or more substituents. The term "alkoxy" or "lower alkoxy" as used herein refers to an -O-alkyI group having the indicated number of carbon atoms. For example, a (C1 -C6) alkoxy group includes -O-methyl, -O-ethyl, -O-propyl, -O-iospropyl, -O-butyl, -O-sec- butyl, -O-tert-butyl, -O-pentyl, -O-isopentyl, -O-neopentyl, -O-hexyl, -O- isohexyl, and -O-neohexyl.

The term "aromatic" as used herein refers to an aromatic or heteroaromatic moiety. An "aromatic" moiety refers to a 6- to 14-membered monocyclic, bi- cyclic or tricyclic aromatic hydrocarbon ring system. Examples of an aromatic group include phenyl and naphthyl. An aromatic group can be unsubstituted or optionally substituted with one or more substituents. The term "heteroaromatic" as used herein refers to an aromatic heterocyclic ring of 5 to 14 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including monocyclic, bicyclic, and tricyclic ring systems. Representative heteroaromatics are triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl, benzothio- phenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, ben- zimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyri- dazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, naphthothiazolyl, quinoxalinyl. A heteroaromatic group can be unsubstituted or optionally substituted with one or more substituents. The term "halogen" as used herein refers to -F, -CI, -Br or -I.

As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), and sulfur (S). As used herein, the term "BODIPY" is an acronym for the compound class of boron-dipyrromethanes. The new ττ-conjugated 1 ,2,3-triazol-l ,4-diyl fluoroionophores according to the present invention generated via Cu(l) catalyzed [3+2] cycloaddition show high fluorescence enhancement factors in the presence of Na ⁺ and K ⁺ in MeCN and high selectivity towards Na ⁺ and K ⁺ under simulated physiological conditions.

Preferred embodiments of the present invention are compounds 1 and 2 as given in Scheme 1. The inventors found that in the simple CuAAC-generated fluoroionophore 1 , the electronic conjugation of the /V-phenylaza-18-crown-6 ether and the 7-diethylaminocoumarin fluorophore through a 1 ,2,3-triazol-l ,4- diyl π-linker results in a perfect signal transduction chain for the sensing of Na ⁺ and K ⁺ . In MeCN high cation-induced FEFs were obtained for 1 (FEF _Na +: 58; FEF _K +: 27). The signal transduction in 1 also works nicely under simulated physiological conditions. In the presence of 160 mM K ⁺ a FEF of 2.5 was observed, whereas the same concentration range of Na ⁺ resulted in an almost negligible fluorescence enhancement. The inventors assume, that compound 1 is a PET-fluoroionophore with a virtual spacer between the ani- linotriazole electron donor unit and the coumarin electron acceptor moiety.

Scheme 1 Conjugated fluoroionophores 1 and 2 with 1 ,2, 3-triazol-1 ,4-diyl as the TT-linker, and reference compounds 3 and 4. ( < - indicates the binding position). The CuAAC of the ethinyl-functionalized /V-phenylaza-18-crown-6 ether ⁷ with 3-azido-7-diethylaminocoumarin ⁸ afforded 1 ,2,3-triazole-fluoroionophore 1. The triazole-isomer 2 was obtained by the reaction of the azido- functionalized /V-phenylaza-18-crown-6 ether ⁹ with 3-ethinyl-7-diethylamino- coumarin ¹⁰ . Dye 3, in which the crown has been replaced by a diethylamino group was synthesised in order to represent a reference compound for 1. A further reference compound for 1 is compound 4, in which the p-phenylen linker between aza-18-crown-6 and triazole ring is replaced by a deconju- gated methylene group. Compound 4 was obtained by the reaction of N- propargylaza-18-crown-6 ether ¹¹ with 3-azido-7-diethyl-aminocoumarin. The new 1 ,4-disubstituted 1 ,2,3-triazoles 1 -4 are stable in solutions of MeCN and DMSO at room temperature for many weeks. UV-irradiation over a period of several hours did not show any decomposition of the fluoroionophores 1 and 2

The fluorescence quantum yield of 1 in MeCN is extremely low (Φί(1 )= 0.008, Table 1 ). The fluorescence spectra of 1 in the presence of Na ⁺ and K ⁺ , respectively (Fig. 1 ) show the impressive fluorescence enhancement upon coordination of the alkali metal ions. High FEFs could be determined for Na ⁺ (FEF=58) and for K ⁺ (FEF=27). ¹²

Photophysical properties of 1-4 in MeCN. ligand 1 2 3 4

<£r(ligand) ^c 0.008 0.03 0.017 0.56

<£r(ligand + Na ⁺ ) ^d complex) 0.62 0.79 0.018 0.55

<£r(ligand + K ⁺ ) ^d 0.23 0.40 0.016 0.58 ^a Emission maxima, excitation maxima, ^c fluorescence quantum yields (Φτ), ^d Of were determined in the presence of 40 mM of NaPF ₆ or 40 mM KPF ₆ respectively.

The fluorescence quantum yield of the constitutional isomer 2 in MeCN is higher than that of 1 (Φτ(2)= 0.03, Table 1 ). The fluorescence of 2 is also enhanced in the presence of increasing concentrations of Na ⁺ and K ⁺ (Fig. 7 and 8, respectively). However the observed FEFs for Na ⁺ (FEF=17) and K ⁺ (FEF=13) are significantly lower than for the isomer 1. The reference compound 4 consists of a poorer PET donor due to the aliphatic amine which is electronically separated from the triazole. Hence 4 has a high quantum yield (O _f (4) = 0.56, Table 1 ) and is therefore less affected by the presence of Na ⁺ and K ⁺ . The inventors further investigated the influence of Na ⁺ and K ⁺ on the fluorescence of 1 under simulated physiological conditions. The ligand was exposed to aqueous solutions in the physiological interesting concentration range of 0- 160 mM Na ⁺ or K ⁺ , respectively. Additionally, the solutions contained 2 mM Ca ²⁺ and 2 mM Mg ²⁺ . The pH was adjusted to 7.2 with 10 mM Tris and a constant ionic strength of 180 mM was maintained with choline chloride. Under these conditions, the fluorescence of 1 (Aex = 424 nm, Aem = 500 nm, O _f = 0.07) is hardly increased in the presence of Na ⁺ whereas increasing concentrations of K ⁺ resulted in a modest fluorescence enhancement (Fig. 2a). In the presence of 160 mM K ⁺ a FEF of 2.5 is observed. The fluorescence measurements were repeated four times showing only very little variations in the FEF values (Fig. 2b). It is noteworthy, that the presence of physiological important extracellular cations, such as Mg ²⁺ and Ca ²⁺ does not affect the signal response. In comparison, the FEF of 1 for K ⁺ in MeCN is clearly smaller than the FEF in water under simulated physiological conditions. This can be explained by the significantly smaller stability constants of the K ⁺ complex with the N- phenylaza-18-crown-6 in water [IgK (H ₂ 0) > 0.5; IgK (MeCN) = 3.95+0.08]. The fact that the fluorescence of 1 in water is exclusively enhanced in the presence of K ⁺ and not by Na ⁺ can be rationalized by the stronger hydration enthalpy of Na ⁺ . However, the fluorescence enhancement of 1 in the presence of K ⁺ under simulated physiological conditions shows that the signaling transduction chain in this fluoroionophore works well in water. The dissociation constant (Kd) of 1 + K ⁺ amounts to ~ 260 mM in solutions which approximates physiological ionic strength. To measure intracellular or extracellular concentrations of K ⁺ (Kd around 140 or 4 mM) tuning of probe 1 towards a higher complex stability while maintaining the selectivity will be necessary. To investigate the pH-sensitivity of 1 , the fluorescence intensity was measured in water at different pH values. The resulting pKa of 1 is near 4.5 meaning that the triazole-substituted N-phenylaza-18-crown-6 is less pH-sensitive than the N(o-methoxyphenyl)aza-15-crown-5- naphthalimide Na ⁺ - fluoroionophore (pKa ~ 5.5) of the authors He et al. ¹³

In summary, the inventors have shown for the first time that an electronically conjugated 1 ,2,3-triazole-fluoroionophore consisting of the signaling transduction chain: anilino-ionophore - 1 ,2,3-triazol-1 ,4-diyl - fluorophore, works as an effective sensor for Na ⁺ and K ⁺ in acetonitrile with high cation-induced FEFs. Under simulated physiological conditions 1 selectively detects K ⁺ with the modest FEF being limited by the rather simple receptor unit. The inventors found that the substitution of ionophore and fluorophore on the 1 ,2,3- triazole ring has a basic influence on the quality of the fluoroionophore. Recently He et al. ^13a and the Verkman group ^13b designed fluorescence switch-on PET sensors with high K ⁺ /Na ⁺ selectivity and sensitivity for physiological K ⁺ in the concentration range of 0-40 mM. In these K ⁺ fluoroionopho- res a [3.2.2]-cryptand represents the ionophore. A drawback of the [3.2.2]- cryptand fluoroionophores though, is the very expensive synthetic proce- dure. ¹³⁰

The inventors developed simple lariat aza-18-crown-6 ionophores, which show a high K ⁺ /Na ⁺ selectivity and sensitivity under simulated physiological conditions. These novel lariat aza-18-crown-6 ionophores are also an object of the present invention.

The further preferred embodiments of the compounds of the present invention containing a 3(2-methoxyethoxy)phenyl-aza-18-crown-6 as K ⁺ /Na ⁺ selective and sensitive ionophore are given in Scheme 3.

Scheme 2 Conjugated K ⁺ -fluoroionophores 5 to 13 with 1 ,2,3-triazol-l ,4-diyl as the TT-linker and either a phenyl-aza-18-crown-6 or a 3(2 methoxyeth- oxy)phenyl-aza-18-crown-6 as ionophoric unit

Compounds 6 to 9 have a 2-methoxyethoxy - lariate chain in close proximity to the aza-18-crown-6. This lariate group provides a higher binding selectivity for K ⁺ . The fluorescence spectra are shown in Figures 4 to 7. According to the invention Na ⁺ -fluoroionophores 14 and 15 with a 1 , 2,3- triazol-1 ,4-diyl as the ττ-linker (Scheme 4) are provided. These fluoroionophores show high Na ⁺ /K ⁺ -selectivity under simuated physiological conditions. The fluorescence spectra are shown in Figures 8 to 11.

The following examples explain the present invention in more detail. The embodiments described in the examples are not limiting the scope of the invention. The examples should only serve as preferred embodiments and a skilled artesian will derive other embodiments from those examples without any undue burden.

Examples Fluorescence measurements

Fluorescence titration spectra of the compounds (c = 5 10 ^"6 mol l ^"1 ) in acetoni- trile were recorded 5 min after the addition of 0.02 ml of volumetric standard solution of NaPF ₆ or KPF ₆ (c = 5 10 ^"5 -5 10 ^"2 mol l ^"1 ), respectively. Titration was continued until no change in fluorescence enhancement was observed. Fluorescence Quantum yields were determined using a PL Quantum Yield measurement System C9920-2 of Hamamatsu, Japan. Fluorescence spectra in aqueous solutions were carried out in buffered saline solutions (10 mM Tris-buffer, pH = 7.2) using a 1 mM ligand solution in DMSO. The water used was purified by a Milli-Q-Deioniser from Millipore ^® . To obtain physiological conditions, the salt solutions contained constant con- centrations of CaC (2 mM) and MgCI ₂ (2 mM). The physiological solutions were varied with regard to the concentrations of respectively KCI or NaCI and constant ionic strength was adjusted to a typical ionic strength in physiological systems (180 mM) by choline chloride. The dye was exposed to different saline solutions containing 0, 5, 10, 20, 40, 80, 160 mM KCI or NaCI, respectively (Table 2 and 3). Each salt solution was mixed with 1 mM DMSO solution of the compound (990/10 v/v) to give a final ligand concentration of 10 μΜ. For each measurement, a freshly prepared dye mixture was used and every experiment was repeated with n = 4. Fluo- rescence response of the compounds was recorded exciting at the given wavelengths.

Table 2. Composition of the different aqueous solutions under simulated

ological conditions with regard to K ⁺ - selectivity.

KCI/ mM Choline CI/ mM MgCI ₂ / mM CaCI ₂ / mM

0 180

5 175

10 170

20 160

40 140

80 100

160 20 Table 3. Composition of the different aqueous solutions under simulated physiological conditions with regard to Na ⁺ - selectivity.

NaCI/ mM Choline CI/ mM MgCI ₂ / mM CaCI ₂ / mM

0 180 2 2

5 175 2 2

10 170 2 2

20 160 2 2

40 140 2 2

80 100 2 2

160 20 2 2

General Methods and Procedures of Preparation of Compounds

All commercially available chemicals were used without further purification. Solvents were distilled prior use. ¹ H and ¹³ C NMR spectra were recorded on 300 MHz, 500MHz or 600MHz instruments. Data are reported as follows: chemical shifts in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets), integration, coupling constant (Hz). ESI spectra were recorded using a Micromass Q-TOF micro mass spectrometer in a positive electrospray mode. IR spectra were recorded using a Thermo Nicolet NEXUS FTIR instrument.

Air/water-sensitive reactions were performed in oven-dried glassware under an argon atmosphere. Column chromatography was performed with S 1O2 (Merck Silica Gel 60 (0.04-0.063 mesh)).

Preparation of precursors The preparation of 3-azido-7-diethylaminocoumarine (60), ¹ 3-ethinyl-7- diethylaminocoumarine (61 ), ¹⁵ /V-phenylaza-18-crown-6 ether ¹⁶ and N-(4- formyl)phenylaza-18-crown-6 ether ¹⁷ has been described previously. 16-(4-ethynylphenyl)-1 ,4, 7, 10, 13-pentaoxa-16-azacvclooctadecane (27)

25 26 27

Λ/-4-(2,2-Ρί ΓοηιονίηνΙ)ρΙΐΘηνΐ3Ζ3-18-θΓθ\Λ/η-6 ether (26)

The preparation of 27 followed the literature and references therein according to a modified procedure. ¹⁸

A suspension of zinc (1 .69 g, 25.9 mmol) and carbon tetrabromide (8.59 g, 25.9 mmol) in dry CH2CI2 (40 ml) was cooled to -15°C with an ice-salt bath before a solution of triphenylphosphine (6.79 g, 25.9 mmol) in dry CH2CI2 was added dropwise. The resulting yellow green mixture was kept at -15°C for 30 min and was then stirred at RT for 3 h whereupon a solution of 25 (4.31 g, 1 1 .7 mmol) in dry CH ₂ Cl2 was added dropwise. The red brown mixture was stirred at RT for 2 h and then water (100 ml) was added. The organic phase was separated and washed with water (3 χ 75 ml). The combined organic layer were dried with MgS0 ₄ and concentrated to give 26 as a light brown oil that was directly used for the next step.

Note that the product decomposes when concentrated to dryness resulting in a green solid. Storage at -20°C is recommended.

HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 522.04, 524.05, 526.04; found, 522.1 1 , 524.1 1 , 526.1 1 .

26 (2.91 g, 5.56 mmol) was dissolved in 25 ml dry THF and set under argon atmosphere immediately. It was cooled to -78°C before n-butyllithium (7.5 ml, 1 .6 M, solution in hexane) was added via a syringe. After stirring for 1 h at - 78°C the solution was allowed to warm up to room temperature and stirring was continued at this temperature for 1 h before H ₂ 0 (15 ml) was added cautiously. The reaction mixture was extracted with 3xCH ₂ Cl2 and the combined organic layers were dried with MgS0 ₄ and concentrated to give 27 as a red brown oil, which was purified via chromatography (silica gel, CHCIs/MeOH, 95/5 v/v). Yield: 20%, overall.

¹ H NMR (CDCI _3, 300 MHz): δ = 2.93 (s, 1 H), 3.6 (m, 24H), 6.56 (d, 2H, J = 9.23 Hz), 7.28 (d, 2H, J = 8.85 Hz); ¹³ C NMR (CDCI ₃ , 75 MHz): δ = 51 .66, 68.94, 71 .15, 75.12, 85.17, 108.82, 1 1 1 .62, 133.73, 148.45; HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 364.21 ; found, 364.26; IR(ATR, cm ^"1 ): 718, 692, 1 1 15, 2097, 2868, 3054.

16-(4-azidophenyl)-1 ,4, 7, 10, 13-pentaoxa-16-azacyclooctadecane (28)

The preparation of the /V-(4-nitro)phenylaza-18-crown ether and subsequent hydrogenation to the corresponding amine followed the literature. ¹⁹

28 The synthesis of 28 followed the literature according to a modified procedure. ²⁰ /V-Anilino-4-aza-18-crown-6 ether (4.03 g, 1 1 .4 mmol) was dissolved in 90 ml HCI (4M) and cooled to 0°C. A solution of NaN0 ₂ (0.784 g,

1 1 .4 mmol) in 45 ml H ₂ 0 was added dropwise. The mixture was stirred for 10 min before a solution of NaN ₃ (1 .1 g, 17 mmol) in 45 ml H ₂ 0 was added drop wise. Stirring was continued for another 10 min at 0°C before the orange solution was allowed to warm up to room temperature and it was stirred for 14 h at ambient temperature. The reaction mixture was brought to pH = 7 with Na2C03, was extracted with 3x180ml CHC and the combined organic layers were dried with MgS0 ₄ . The solvent was removed under reduced pressure to yield 28 as brown oil (1 .31 g, 30.3 %). The product was used for the next reaction step without further purification. ¹ H NMR (CDCI3, 300 MHz): δ = 3.57-3.69 (m, 24H), 6.68 (d, 2H, J = 8.854 Hz), 6.88 (d, 2H, J = 9.042 Hz); ¹³ C NMR (CDCI ₃ , 75 MHz): δ = 51 .48, 68.74, 70.70, 1 12.97, 1 19.94, 127.55, 145.98;

HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 381. 21 ; found, 381.34, calcd for (M-N ₂ ) ⁺ 353.21 ; found, 353.30; IR (ATR, cm ^"1 ): 1 100 , 1508, 2120, 2097, 2867.

16-(prop-2-ynyl)-1,4, 7, 10, 13-pentaoxa-16-azacyclooctadecane (29)

29

The synthesis followed the literature according to a modified procedure. Monoaza-18-crown-6 ether ²² (0.6 g, 2.28 mmol) and propargylbromide (0.206 ml, 2.74 mmol) were dissolved in dry acetonitrile (70 ml) before CS2CO3 (1 .48 g, 4.56 mmol) was added. The suspension was stirred overnight at 85°C. After cooling to room temperature, the suspension was filtered and the filtrate was concentrated. The residue was purified by column chromatography on silica gel eluting with CHC /MeOH (9/1 v/v) yielding 29 as a red oil (64 %)

¹ H NMR (CDCI3, 300 MHz): δ = 2.18 (t, 1 H, J = 2,45Hz), 2.82 (t, 4H, J = 5.28Hz), 3.61 -3.74 (m, 22H); ¹³ C (CDCI ₃ , 75 MHz): δ = 43.42, 53.29, 68.94, 70.52, 73.61 , 78.85; IR (ATR, cm ^"1 ): 1 1 15, 2867, 3189; HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ ,302.20; found, 302.21 .

N, N-dieth l-4-eth vn ylaniline (32)

30 31 32 The synthesis followed the literature according to a modified procedure.

1 , 1 -Dibromo-2-(4-/V,/V-diethylaminophenyl)ethane (31):

A mixture of zinc (4.9 g, 75 mmol), triphenylphosphine (19.67 g, 75 mmol), and carbon tetrabromide (24.87 g, 75 mmol) in dry dichloromethane (250 ml) was sonicated for 2 h while cooling with an ice bath. N,N- diethylaminobenzaldehyde (30) (5.30 g, 29.9 mmol) was added to the grayish suspension and it was stirred overnight resulting in a brownish suspension. The mixture was concentrated and petroleum ether (500 ml) was added, whereupon a tarry precipitate formed. It was washed with 1/1 CH ₂ Cl2/light petroleum ether (2 χ 100 ml). The combined organic phases were concentrated, and the residue was chromatographed on silica gel (150 g), eluting with 1/1 CH ₂ CI ₂ /hexane to give 31 as a yellow oil (7.3 g, 21 .0 mmol, 70 %). The intermediate product was kept at -20°C until the next step.

1H NMR (CDCIs, 300 MHz): δ = 1 .17 (t, 6H, J = 6.94 Hz, CH ₃ ), 3.37 (q, 4H, J = 6.94 Hz, CH ₂ ), 6.62 (d, 2H, J = 7.88 Hz, Ar-H), 7.32 (s, 1 H, CH), 7.49 (d, 2H, J = 8.21 Hz); HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ , 331 .96, 333.96, 335.96; found, 331.93, 333.93, 335.94. N, N-dieth yl-4-eth yn yl aniline (32):

A solution of 31 (4.3 g, 12.4 mmol) in dry THF (93 ml) was cooled to -78°C before n-butyllithium ( 19 ml, 15% in hexane) was added dropwise. It was stirred for 45 min at this temperature before the reaction mixture was allowed to warm up to RT. After stirring for 1 h at RT, water (7ml) was added slowly. The solvent was removed and the residue was taken up in diethyl ether. The organic layer was washed with water (30 ml) and brine (30 ml) and dried over MgS0 ₄ . Concentration of the ether phase gave 32 as orange oil that was used in the next step without further purification (1 .29 g, 6.96 mmol, 56.2 %). ¹ H NMR (CDCI3, 300 MHz): δ = 1 .16 (t, 6H, J = 7.25Hz), 2.97 (s, 1 H), 3.37 (q, 4H, J = 7.25Hz,), 6.57 (d, 2H, J = 9.14Hz), 7.34 (d, 2H, J = 9.14Hz), ¹³ C (CDCI3, 75 MHz): δ = 12.46, 44.26, 74.41 , 85.01 , 107.47, 1 10.94, 133.39, 147.73, HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ , 174.13; found, 174.16. 13-(4-Ethynyl-2-methoxyphenyl)-1 ,4,7, 10-tetraoxa-13-azacvclopentadecane {40}

The synthesis followed the literature according to a modified procedure. Into a stirred mixture of 860 mg (2,43 mmol) Aldehyd 37 and 673 mg (4,86 mmol) K2CO3 in 35 ml dry Methanol was added 564 mg (2,93 mmol) di- methyl-1 -diazo-2-oxopropylphosphonate (Bestmann-Ohira Reagent). This mixture was stirred for 20 hours at ambient temperature. After addition of 60 ml CHCI3 and extraction 3 times with water, the organic layer was separated, dried with MgS0 ₄ and concentrated in vacuum. The residue was purified by column chromatography on silica with CHCI3/CH3OH (95/5) as eluent. The product was obtained as a yellow oil (435 mg, 51 %).

1H NMR (300 MHz, CDCI3): δ = 2.99 (s, 1 H), 3.44-3.48 (m, 4H), 3.58-3.66 (m, 16H), 3.77 (s, 3H),

¹³ C NMR (75 MHz, CDCI3): δ = 52.85, 55.37, 69.94, 70.25, 70.34, 70.85, 75.57, 84.15, 1 13.94, 1 15.13, 1 19.06, 125.21 , 140.93, 151 .32

MS (El) m/z (%): 349(100) [M] ⁺ .

IR (ATR, cm ^"1 ): 2923 (s), 2855 (s), 2102 (m), 1507 (s), 1253 (s), 1 1 10 (s). Elemental analysis (%) calcd. for C19H27NO5 (349.42): C 65.31 , H 7.79, N 4.01 ; found C 65.12, H 7.38, N 3.98

13-(4-Azido-2-methoxyphenyl)-1 ,4,7, 10-tetraoxa-13-azacvclopentadecane (43)

36 41 42 43

The synthesis of 43 followed the literature according to a modified proce- dure. ²⁵

The appropriate aminocrownether (42) (807 mg, 2.37 mmol) was dissolved in 18 ml 4 M HCI and cooled. At 0°C was slowly added a solution of 163,6 mg (2.37 mmol) sodium nitrite in 10 ml water. The solution was stirred for 10 minutes at 0°C. Then was added a solution of 230 mg (3,53 mmol) sodium azide in 10 ml water. The resulting solution was stirred for further 10 min at 0°C and led warm up to room temperature. It was stirred overnight. The mixture was brought to pH 7 with potassium carbonate and extracted 3 times with CHCI3. The combined organic layers were dried with MgS0 ₄ and concentrated in vacuum. The residue was purified by column chromatography on silica with CHCI3/CH3OH (95/5) as eluent. The product was obtained as a darkbrown oil (265 mg, 31 %).

¹ H NMR (300 MHz, CDCI3): δ = 3.39-3.4 (m, 4H), 3.60-3.68 (m, 16H), 3.79 (s, 3H)

¹³ C NMR (75 MHz, CDCI3): δ = 53.13, 55.47, 70.08, 70.37, 70.48, 70.93,

103.25, 1 10.61 , 122.16, 133.87, 137.25, 153.89

MS (El) m/z (%): 366 (12) [M] ⁺ , 338 (100)[M-N ₂ ] ⁺ .

IR (ATR, cm ^"1 ): 2858 (s), 2102 (s), 1504 (s), 1234 (s), 1 105 (s).

N-n-butyl-4-ethinyl-1 ,8-naphthalimide (50)

N-n-butyl-4-bromo- 1, 8-naphthalimide

9

The reaction followed the literature according to a modified procedure. ²⁶ In a dry 250-ml round bottom flask, under a stream of argon gas, were placed the 4-bromo-1 ,8-naphthalic anhydride (95 w%, 1 .35 g, 4.63 mmol, 1 equiv..) and 1 -butylamine (99 w%, 410.35 mg, 5.5 mmol, 1 .2 equiv..) in dry ethanol (96 ml) and the reaction mixture was heated at 60 °C for 16 h. After cooling to room temperature, the solid was filtered and washed with 200 ml H ₂ 0 to give the product as a light yellow solid. Yield: 0.800 mg, 52 %, mp: 100 - 102 °C (Lit.: 103-105 °C, R _f = 0.8

(n-hexane/ethyl acetate = 4: 1 v/v); H NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.63 (d, 1 H, J = 7.29, H ¹ ), 8.53 (d, 1 H, J = 8.52, H ³ ), 8.39 (d, 1 H, J = 7.86, H ⁴ ), 8.01 (d, 1 H, J = 7.9, H ⁵ ), 7.82 (tr, 1 H, J = 8.46, H ² ), 4.16 (t, 2H, J = 7.55, H ⁶ ), 1.71 (qu, 2H, J = 7.5, H ⁷ ), 1 .45 (sx, 2H, J = 7.5, H ⁸ ), 0.97 (t, 3H, J = 7.32, H ⁹ ); ³ C NMR, (CDCI ₃ , 75 MHz) δ (ppm): 163.74, 163.71 , 133.30, 132.1 1 , 131 .30, 131 .21 , 130.74, 130.28, 129.12, 128.19, 123.30, 122.45, 40.53, 30.31 , 20.52, 13.98; -n-Butyl-4-(trimethylsilylethinyl)- 1, 8-naphthalimide

1

9

A dry 100-ml two-necked round-bottom flask was set under argon atmosphere before /V-n-Butyl-4-Bromo-1 ,8-naphthalimide(0.8 g, 2.4 mmol, l equiv.), THF (8 ml_), Pd(PPh ₃ ) ₂ CI ₂ (33.8 mg, 0.048 mmol, 0.02 equiv.), PPh ₃

(25.3 mg, 0.096 mmol, 0.04 equiv..), Cul (18.3 mg, 0.096 mmol, 0.04 equiv..) and dry TEA (8 ml) were added followed by the trimethylsilylacetylene

(331 .1 mg, 3.37 mmol, 1 .4 equiv..). It was refluxed for 6 h. Then the reaction mixture was allowed to cool down to room temperature and the solvent was evaporated under reduced pressure. The crude product was purified by col- umn chromatorgraphy (50 x 3 cm, silica gel, CH2CI2 = 10: 1 v/v) to give the productas a yellow solid.

Yield: 0.780 g, 92 %, mp: 136.2-138.8 °C, R _f = 0.87; H NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.61 (dd, 2H, H ⁴ , H ³ ), 8.50 (d, 1 H, J = 7.62, H ¹ ), 7.88 (d, 1 H, J = 7.62, H ⁵ ), 7.8 (t, 1 H, J = 7.62, H ² ), 4.17 (t, 2H, J = 7.5, H ⁶ ), 1 .71 (qu, J = 7.5, 2H, H ⁷ ), 1 .37-1 .51 (m, 2H, J = 7.53H ⁸ ), 0.97 (t, 3H, J = 7.3, H ⁹ ), 0.36 (s, 9H, H ¹⁰ ); ³ C NMR, (CDCI ₃ , 75 MHz) δ (ppm): 164.14, 163.86, 132.49, 131 .93, 131 .68, 131 .32, 130.35, 128.07, 127.63, 127.37, 123.1 1 , 122.53, 105.37, 101 .44, 40.48, 30.36, 20.54, 13.99, 0.002;

N-n-Butyl-4-ethinyl-l, 8-naphthalimide (50)

/V-n-Butyl-4-(trimethylsilylethinyl)-1 ,8-naphthalimide (710 mg, 2.03 mmol) was placed into a 100-ml round-bottom flask and in dry methanol (50 ml) and dry K2CO3 (1 .09 g, 7.92 mmol, 3.9 equiv.) were added. It was heated for 16 h at room temperature. CH2CI2 (40 ml) was added to the reaction mixture and the organic phase was washed with water (2x 20 ml). The combined organic phases were dried over Na ₂ S0 ₄ and concentrated in vacuo. The crude prod- uct was purified by column chromatography (50 x 3 cm, silica gel,

CH ₂ Cl2/ethyl acetate = 10: 1 V/V) to give the product as a yellow solid.

Yield: 0.200 g, 35 %, mp.: 127.5-130.8 °C, R _f = 0.81 ; H NMR, (CDCI ₃ , 600 MHz) δ (ppm), J (Hz): 8.65 (d, 1 H, J = 8.43, H ³ ), 8.63 (d, 1 H, J = 7.32, H ⁴ ,), 8.53 (d, 1 H, J = 7.56, H ¹ ), 7.93 (d, 1 H, J = 7.56, H ⁵ ), 7.82 (t, 1 H, J = 8.25, H ² ), 4.17 (t, 2H, J = 7.62, H ⁶ ), 3.73 (s, 1 H, H ¹⁰ ), 1.71 (qu, 2H, J = 7.56, H ⁷ ), 1 .44 (sx, 2H, J = 7.56, H ⁸ ), 0.97 (t, 3H, J = 7.38, H ⁹ ); ³ C NMR, (CDCI3, 75 MHz) δ (ppm): 164.05, 163.78, 132.24, 132.12, 131.77, 130.245, 128.09, 127.81 , 126.30, 123.24, 123.07, 122.01 , 86.52, 80.51 , 40.50, 30.36, 20.52, 13.95; 6-azido-N-ethyl-1 , 8-naphthalimide (51 ) 27

N-n-ethyl-4-bromo- 1, 8-naphthalimide

The synthesis followed the literature according to a modified procedure.' A suspension of 4-bromo-1 ,8-naphthalic anhydride (2g, 7.2 mmol, l equiv.) and ethylamine (70 w% in water ,0.69 ml, 8.66 mmol, 1 .2 equiv.) in 100 ml 1 .4-dioxane was refluxed for 6 h giving a brownish solution. The solution was cooled to room temperature before it was poured onto 300 ml ice water re- suiting in an off white precipitate. After complete melting of the ice, the precipitate was collected by filtration and was washed with water. The resulting solid was dried in vacuo in a desiccator equipped with CaC to give the product as an off white solid which was directly used in the next step.

Yield: 1 .72g, 78 %, mp: 145-146 °C, H NMR, (CDCI ₃ , 500 MHz) δ (ppm), J (Hz): 8.62 (dd, 1 H, H ¹ ), 8.34 (dd, 1 H, H ³ ), 8.37 (d, 1 H, J = 7.85, H ⁴ ), 8.0 (d, 1 H, J = 7.80, H ⁵ ), 7.81 (tr, 1 H, J = 7.35, H ² ), 4.22 (q, 2H, J = 7.15, H ⁶ ), 1 .32 (tr, 3H, J = 7.10, H ⁷ ); MS (El): m/z calcd.: 303; found, 303+305: [m/z- ^• CH ₂ CH ₃ ] ⁺ calcd. :275, found 275+277; 6-azido-N-eth l-1 , 8-naphthalimide (51)

7 The synthesis followed the literature and references therein. In a 25-ml round-bottom flask was placed the /V-n-ethyl-4-bromo-1 ,8-naphthalimide (0.5 g, 1.64 mmol, 1 equiv.) and sodium azide (0.53 g, 8.2 mmol, 5 equiv.). Then /V-methylpyrrolidinone (7ml) was added and the mixture was heated at 1 10 °C for 1 .5 h. The solution was allowed to cool to room temperature and diluted with 25 ml water. It was extracted with ethyl acetate (3x20 ml) and the combined organic phases were washed with brine. The organic phase was dried over MgS0 ₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, hexane/ethyl ace- tate = 4: 1 v/v) to give the product as a yellow solid.

Yield: 0.254 g, 58 %, H NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.62 (dd, 1 H, H ¹ ), 8.56 (dd, 1 H, H ⁴ ), 8.41 (dd, 1 H, H ³ ), 7.72 (m, 1 H, H ² ), 7.45 (dd, 1 H, H ⁵ ), 4.23 (q, 2H, J = 7.14, H ⁶ ), 1.32 (tr, 3H, J = 7.08, H ⁷ ); ³ C NMR, (CDCI ₃ , 75 MHz) δ (ppm): 163.92, 163.51 , 143.50, 132.26, 131 ,74, 129.27, 128.82, 126.96, 124.49, 122.85, 1 19.15, 1 14.78, 35.65, 13.48; MS (El): m/z calcd.:266; found, 266; [m/z-'N ₂ ] ⁺ calcd.:238, found 238; IR (KBr, cm ^"1 ): 2131 (s, N ₃ ).

10-ethynylanthracene (52)

The synthesis followed the literature.

Yield: 78 %, H NMR, (CDCI ₃ , 300 MHz) δ (ppm)): 8.58 (dd, 2H, H ¹ ), 8.46 (s, 1 H, H ⁵ ), 8.02 (dd, 2H, H ⁴ ), 8.62-8.45 (m, 4H, H ² ' ³ ), 3.99 (s, 1 H, H ⁶ ) General procedure for the synthesis of azidoanthracene derivatives -(azidomethyl)-anthracene (53)

5

The synthesis followed the literature according to a modified procedure. A suspension of chloromethylanthracene (1 g, 4.41 mmol, 1 equiv.) and sodium azide (0.43g, 6.62 mmol, 1.5 equiv.) in 30 ml acetonitrile was refluxed for 5 h. After the reaction was complete (monitoring via DC) the reaction mixture was cooled to room temperature and the resulting solid was filtered off. The solution was concentrated in vacuo and the resulting yellow solid was purified by column chromatography on silica gel (n-hexane/ethyl acetate = 3: 1 v/v).

Yield: 0.92 g (90%), mp.: 78-80°C, ¹ H-NMR, (CDCI ₃ , 500 MHz) δ (ppm), J (Hz): 8.5 (s, 1 H, H ⁵ ), 8.29 (d, 2H, J = 8.85, H ¹ ), 8.05 (d, 2H, J = 8.40, H ⁴ ), 7.60 (m, 2H, H ² ), 7.52 (m, 2H, H ³ ), 5.31 (s, 2H, H ⁶ ); ¹³ C-NMR, (CDCI ₃ , 125 MHz) δ (ppm): 131 .45, 130.79, 129.39, 129.09, 126.94, 125.87, 125.30, 123.31 , 46.42; MS (El): m/z calcd.:233; found, 233; FT-IR (KBr), cm ^"1 : 2095 (s, -N ₃ ), 1444 (m, -CH ₂ -).

10-(azidomethyl)-9-carbonitrilanthracene (54)

The Synthesis of the 10-Bromomethyl-9-cyanoanthracene via 10-Methyl-9- cyanoanthracene followed the literature. ³⁰

10-meth l-9-cyanoanthracene

The product was collected after crystallization from acetic anhydride in form of yellow needles.

Yield: 81 %, mp: 207-208 °C; H-NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.38 (d, 2H, J = 8.61 , H ¹ ), 8.29 (d, 2H, J = 8.79, H ⁴ ), 7.69-7.5 (m, 4H, H ² ' ³ ), 3.09 (s, 3H, H ⁵ ); ³ C-NMR, (CDCI3, 75 MHz) δ (ppm): 138.24, 132.98, 129.51 , 128.45, 126.29, 126.17, 125.48, 1 17.83, 104.41 , 14.91 ; HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 218.10; found, 218.15.

10-bromomethyl-9-cyanoanthracene

Recrystallization from n-hexanes/CHC at -20°C gave the product as a yellow solid (needles).

Yield: 90, 1 % mp.: 248-254 °C, Rf = 0.33 (n-hexanes/ethyl acetate = 8:2 v/v); H-NMR, (CDCI3, 300 MHz) δ (ppm), J (Hz):8.49-8.43 (m, 2H, H ¹ ), 8.39-8.31 (m, 2H, H ⁴ ), 7.77-7.69 (m, 4H ² ' ³ ), 5.46 (s, 2H, H ⁵ ); ³ C-NMR, (CDCI ₃ , 75 MHz) δ (ppm): 135.07, 133.18, 128.95, 128.85, 127.68, 126.45, 124.42, 1 17.20, 107.84, 24.97; FT-IR (KBr,cm ^"1 ): 3051 (w, Br-CH ₂ -), 2212 (s, -C≡N), 1444 (m, -CH ₂ -).

10-(azidomethyl)-9-carbonitrilanthracene (54)

The synthesis followed the procedure of the preparation of 9-(azidomethyl)- anthracene as described above. The crude product was recrystallized from CH ₂ CI ₂ /MeOH to give the product as yellow fluffy needles. Yield: 39 %, R _f = 0AV, ¹ H-NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.48 (d, 2H, J = 8.84, H ¹ ), 8.35 (d, 2H, J = 8.13, H ), 7.79-7.65 (m, 4H, H ² ' ³ ), 5.33 (s, 2H, H ⁵ ); ¹³ C-NMR, (CDCI ₃ , 75 MHz) δ (ppm): 133.04, 132.90, 129.94, 128.85, 127.84, 126.52, 124.48, 1 17.07, 108.08, 46.29; HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 259.10; found, 259.15; FT-IR (KBr) u ( cm ^"1 ): 2213 (s, - C≡N), 2100 (s, -N ₃ ), 1444 (m, -CH ₂ -).

9-(azidomethyl)-1 O-methylanthracene (55)

9- 10-methyl)-bromomethyl anthracene

b

A 500-ml round-bottom flask, equipped with condenser, thermometer and stirbar was set under argon atmosphere and the anthraquinone (10.5 g, 0.050 mol) and dry THF (375 ml) were added. The mixture was cooled to -78 °C before a solution of methyl lithium (1 10 ml, 1 .4 M in diethylether,

0.151 mol, 3.05 equiv..) was added cautiously via a syringe. The reaction mixture was slowly allowed to warm up to room temperature and was stirred at this temperature for 2h before water (100 ml) was added. The mixture was extracted with CH2CI2 (2 x 100 ml) and the combined organic phases were concentrated to give the 9, 10-dihydroxy-9, 10-dimethylanthracene as a white solid which was used in the next step without further purification. It was dissolved in THF (250 ml) and a solution of THF (130 ml) and HBr

(150 ml, 48 w%) was added drop wise. The reaction mixture was stirred at ambient temperature for 30 min where upon yellow crystals formed. The crystals were collected by filtration, washed with water and dried under vac- uum to give the final product.

Yield: 10.16 g, 72 %, mp: 177.5-179.0 °C, R _f = 0.25 (CHCI ₃ /MeOH = 97:3 v/v); H-NMR, (CDCI3, 300 MHz) δ (ppm), J (Hz): 8.35 (t, 4H, J = 7.56, H ^{1 , 5} ), 7.65 (tr, 2H, J = 6.9, J = 8.2, H ³ ), 7.54 (tr, 2H, J = 6.9, J = 8.2, H ⁴ ), 5.55 (s, 2H, H ¹ ), 3.09 (s, 3H, H ⁶ ); ³ C-NMR, (CDCI ₃ , 75 MHz) δ (ppm): 133.30, 130.28, 129.58, 126.39, 126.32, 125.67, 125.39, 124.26, 27.97, 14.74;

HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 286.19; found, 286.32;

9- azidomethyl)- 10-methylanthracene (55)

6

The synthesis followed the procedure of the preparation of 9-(azidomethyl)- anthracene as described above.

Yield: 90 %, R _f = 0.32; H-NMR, (CDCI ₃ , 300 MHz) δ (ppm), J (Hz): 8.39-8.31 (m, 4H, H ² ' ⁵ ), 7.63-7.53 (m, 4H, H ^3, ), 5.34 (s, 2H, H ¹ ), 3.14 (s, 3H, H ⁶ ); ³ C- NMR, (CDCIs, 75 MHz) δ (ppm): 132.91 , 130.62, 130.03, 126.44, 125.74, 125.22, 124.29, 46.69, 14.64; MS (El): m/z:247; [m/z-'N ₂ ]= 218; [m/z- ^• CH ₂ N ₃ ]= 205; FT-IR (KBr), cm ^"1 : 2214 (s, C≡N), 21 14 (s, N ₃ ), 1444 (m, CH ₂ ).

N.N-bis(2-hvdroxyethyl)-2-methoxyethyloxyaniline

2

The Synthesis followed the literature according to a modified procedure. ³² A mixture of /V-(2-methoxyethoxy)-2-nitrobenzole ³³ (21 .9 g; 0.131 mol; 1 equiv.). 2-chlorethanol (52.77g; 0.656 mol; 5 equiv.) and CaC0 ₃ (18.37; 0.184 mol; 1 .4 equiv.) in dist. water (300 ml) was stirred for 6 days at 60 °C. After cooling to room temperature, Na ₂ C0 ₃ (75. Og; 0.7 mol; 5.34 equiv.) was added and it was stirred for 40 Min at 60 °C after which the solid is filtered off. The aqueous phase is extracted tertbutyl-methyl-ether (3x500ml). The combined organic phases are dried over MgS0 ₄ and concentration in vacuo gave the crude product as a brownish oil, which was purified by column chromatography (silica gel. ethyl acetate). Yield: (30.1 g. 90 %).

H NMR (CDCIs. 300 MHz): δ = 3.15 (tr. 4H. H ⁶ ). 3.43 (s. 3H. H ⁹ ). 3.47 (tr. 4H. H ⁵ ). 3.74 (tr. 2H. H ⁸ ). 4.1 1 (tr. 2H. H ⁷ ). 6.91 (dd. 1 H. H ⁴ ). 6.98 (tr. 1 H. H ² ). 7.1 1 (tr. 1 H. H ³ ). 7.22 (d. 1 H. H ¹ ); ¹³ C NMR (CDCI ₃ . 75 MHz): δ = 57.93; 58.93; 59.66; 67.80; 70.70; 1 13.33; 122.23; 125.51 ; 126.01 ; 139.28; 155.25; HRMS ( ⁺ ESI): m/z calc. for (M+H) ⁺ . 256.15; found. 256.13. 2-methoxyethoxyphenylaza-18-crown-6-ether

The synthesis followed the literature according to a modified procedure.

The /V./V-bis(2-hydroxyethyl)-2-methoxyethyloxyaniline (15.46 g; 60.55 mmol; l equiv.) was dissolved in 440 ml dry acetonitrile and set under argon atmosphere. Under a stream of argon sodiumhydride (80% ig; 4.5 g; 2.86 equiv.) was added to the reaction mixture over a period of 1 h. The 1 , 17- ditosyl-3,6,9, 12, 15-pentaoxaheptadecane (30.4 g; 60.55 mmol; l equiv.) was dissolved in 216 ml dry acetonitrile and added dropwise to the refluxing reaction mixture over 4 h. It was heated to reflux for 1 1 h before the yellow suspension was allowed to cool to room temperature. It was filtered and the solvent was removed to give a brown oil. which was purified by column chromatography (silica gel. CHCI ₃ . MeOH, 95/5, v/v )to give the product as a light brown oil.

¹ H NMR (CDCI3. 300 MHz): δ = 3.41 (s. 3H. H ¹³ ). 3.45-3.66 (m. 24H. H ^5"10 ). 3.73 (tr. 2H. J = 4.9 Hz. H ¹² ). 4.10 (tr. 2H. . J = 4.9 Hz. H ¹³ ). 6.82-7.12 (m. 4H. H ^1"4 ); ¹³ C NMR (CDCI3. 75 MHz): δ = 56.70; 59.91 ; 59.83; 67.83; 69.57- 71 .18; 1 14.39; 121 .24; 122.21 ; 124.21 ; 151 .71 ; 153.9; HRMS ( ⁺ ESI): m/z ber. fur (M+H) ⁺ . 255.16; gefunden. 255.13.

N-2-methoxyethoxy-4-nitrophenylaza-18-crown-6-ether

The synthesis followed the literature according to a modified procedure. ³⁵ The 2-methoxyethoxyphenylaza-18-crown-6-ether (4.4g; 10.65 mmol;

l equiv.) was dissolved in a mixture of dist. water (340 ml) und glacial acetic acid (34 ml) before a solution of NaN0 ₂ (0.81 ; 1 1.7 mmol; 1 .1 equiv.) in de- ionized water was added drop wise over a period of 10 min. It was stirred for 16 h at room temperature. The deep orange reaction mixture was neutralized with LiOH followed by extraction with CH ₂ CI ₂ (3x100ml) The combined organic phases were dried over MgS0 ₄ and the solvent was removed in vacuo to give the crude product as an orange oil. Purification by column chromatography yielded the product as an orange oil (silica gel. CH ₃ CI/MeOH, 95/5, v/v) (1 .1 g. 22.4 %).

H NMR (CDCI ₃ . 300 MHz): δ = 3.40 (s. 3H. H ¹² ). 3.60-3.70 (m. 24 H. H ^4"9 ). 3.73 (m, 2H, H ¹¹ ). 4.14 (m, 2H, H ¹⁰ ). 6.88 (d. 1 H. J = 9.04 Hz. H ³ ). 7.65 (d, 1 H. J = 2.5 Hz, H ¹ ). 7.80 (dd, 1 H, H ² ); HRMS ( ⁺ ESI): m/z calc. (M+H) ⁺ .

459.23; found, 459.26. ³ C NMR (CDCI ₃ . 75 MHz): δ = 52.83, 58.82, 67.91 , 69.93, 70.52, 70.55, 72.57, 70.64, 70.69, 70.77, 108.20. 1 15.65, 1 18.57, 139.07 , 146.32, 148.38; HRMS ( ⁺ ESI): m/z calc. for (M+H) ⁺ . 459.23; found. 459.25. /V-3-(-2-methoxyethoxyphenylaza-18-crown-6-)aniline

The /V-2-methoxyethoxy-4-nitrophenylaza-18-crown-6-ether (0.22g. 0.48 mmol) was dissolved in dry methanol (30 ml) After addition of Pd/C (30 mg) it was hydrated in an autoclave for 16 h at 75 bar. The catalyst was filtered of through a bed of Celite® and the solvent was removed in vacuo to yield a colorless oil (0.2g; 97%). Note that the compound decomposes quickly when exposed to air, resulting in a purple colour. That's why it was used in the next step without further purification.

HRMS ( ⁺ ESI): m/z calc. for (M+H) ⁺ . 429.26; found. 429.20. /V-4-(azido)-2-methoxyethoxyphenylaza-18-crown-6-ether (56)

Due to the instability of the /V-3-(-2-methoxyethoxyphenylaza-18-crown- 6-)aniline. the reaction was carried out under argon atmosphere and in oven dried glassware. The aqueous solutions were degassed and flushed with argon prior use.

The /V-3-(-2-methoxyethoxyphenylaza-18-crown-6-)aniline (0.2 g;0.467 mmol; 1 equiv.) was dissolved in HCI (3.6 ml; 4M) gelost and cooled to 0°C before a solution of NaN02 (32 mg; 0.467 mmol; 1 equiv.) in 1 .8 ml H ₂ 0 was slowly added via a syringe. The reaction mixture was stirred at this temperature for 10 min. before a solution of NaN ₃ (0.45 mg. 0.7 mmol; 1 .5 equiv.) in 1 .8 ml H ₂ 0 was added dropwise followed by 10 in of stirring at 0°C. The solution was allowed to warm up to room temperatur and stirred at ambient temperatuer for 14 h. The reaction mixture was neutralized with

U2CO3 before it was extracted with 3x50ml CHCI3. The organic layers were combined, dried with MgS0 ₄ , filtered and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica gel. CH ₃ CI/MeOH (95/5) yielding the desired product as light yel- low oil (0.13 g. 61 .3 %).

H NMR (CDCI ₃ . 300 MHz): δ = 3.4 (s. 3H. H ¹² ). 3.52-3.68 (m. 24H. H ^4"9 ). 3.75 (tr. 2H. J = 4.9 Hz. H ¹¹ ). 4.10 (tr. 2H. J = 4.9 Hz. H ¹⁰ ). 6.51 (s. 1 H. H ³ ). 6.60 (d. 1 H. J = 8.48. H ² ). 7.09 (d. 1 H. J = 8.48 Hz. H ² ); ³ C NMR (CDCI ₃ . 75 MHz): δ = 53.18. 59.06. 67.82. 69.76. 70.31 . 70.55. 70.72. 70.61 . 71 .00. 105.02. 1 1 1 .48. 123.52. 134.51 . 136.95. 153.87; HRMS ( ⁺ ESI): m/z calc. for (M+H) ⁺ found. 455.17.

IR (ATR. cm ^"1 ): 2106 (strong. -N ₃ )

6-ethynyl-2-hexadecyl-1.8-naphthalimid (57)

The synthesis of the 6-ethynyl-2-hexyl-1 .8-naphthalimid_followed the literature according to a modified procedure. ³⁶

6-bromo-2-hexadecyl- 1.8-naphthalimid

The 4-bromonaphthalene anhydride (1 .0 g. 3.61 mmol. 1 Eq.) and n- butylamine (1 .04 g. 4.32 mmol. 1 .2 Aq.) were stirred in dry ethanol (96 ml) at 60 C for 16 h. The reaction mixture was allowed to warm up to room temperature and before the solid was filtered off. It was washed with 200 ml H ₂ 0 and dried in high vacuo to give the product as a light yellow solid (1 .74 g; 93 %)

H NMR (CDCI ₃ . 300 MHz): δ = 0.89 (tr. 3H. H ¹ ). 1 .25-1 .48 (m. 24H. H ^2"4 ). 1 .73 (qu. 2H. H ⁵ ). 4.17 (tr. 2H. H ⁶ ). 7.85 (tr. 1 H. J = 7.35 Hz. H ⁸ ). 8.06 (d. 1 H. J = 7.72 Hz. H ¹¹ ). 8.57 (d. 1 H. J = 7.91 Hz. H ¹⁰ ). 8.57 (dd. 1 H. H ⁷ ). 8.66 (d. 1 H. J = 7.35 Hz. H ⁹ ); ³ C NMR (CDCI ₃ . 75 MHz): δ = 14.50. 23.08. 27.52. 28.47. 29.75. 29.95. 30.00. 30.03. 30.05. 30.06. 30.09. 32.32. 41 .02. 122.69. 123.55. 128.42. 129.34. 130.50. 130.96. 131 .43. 131 .52. 132.33. 133.50. 163.90. 163.92; MS (El): m/z calcd.: 505; found. 499+501 ;

2-hexadecyl-6-((trimethylsilyl)ethynyl)-1.8-naphthalimid

Under argon atmosphere, to a dry flask provided with the 6-bromo-2- hexadecyl-1.8-naphthalimid (0.916 g; 1.83 mmol; 1 equiv.). Pd(PPh ₃ )2CI ₂ (26.0 mg. 0.037 mmol. 0.02 equiv.). PPh ₃ (19.0 mg. 0.037 mmol.

0.04 equiv.). Cul (14.0 mg. 0.037 mmol. 0.04 equiv.) and dry triethylamine were added. Dry THF (10 ml_) was transfered into the flask via a canula followed by the dropwise addition of trimethylsilylacetylene (0.252 mg.

2.56 mmol. 1 .4 equiv.). The reaction mixture was heated to reflux for 6 h and then allowed to cool down to room temperature. The solvent was removed and the residue was taken up CHCI3. Water was added and it was extracted with 3x30ml CHCI3. The organic layers were combined, dried over MgS0 ₄ and filtered. The solvent was removed in vacuo to yield the desired product quantitatively as a grey solid (0.94 g. 100 %).

H NMR (CDCI3. 300 MHz): δ = 0.35 (s. 9H. J = 6.97 Hz. H ¹² ). 0.86 (tr. 3H.

H ¹ ). 1 .23-1 .34 (m. 24H. H ^2"4 ). 1.71 (qu. 2H. J = 7.54 Hz. H ⁵ ). 4.14 (tr. 2H. J = 7.72 Hz. H ⁶ ). 7.80 (tr. 1 H. J = 7.35. H ⁸ ). 7.89 (d. 1 H. J = 7.35 Hz. H ⁷ ). 8.51 (d.

1 H. J = 7.72Hz H ¹¹ ). 8.62 (dd. 2H. H ^{9 10} ); ³ C NMR (CDCI ₃ . 75 MHz): δ =

9.08. 14.03. 22.64. 27.13. 28.12. 29.31 . 29.34. 29.51 . 29.56. 29.60. 29.62.

29.63. 29.65. 31.89. 40.57. 46.15. 101 .32. 105.20. 122.45. 123.36. 127.46.

130.16. 131 .15. 131 .48. 132.29. 163.163.94; MS (El): m/z calcd.: 517; found. 517;

6-ethynyl-2-hexadecyl-1.8-naphthalimid (57)

12

The 2-hexadecyl-6-((trimethylsilyl)ethynyl)-1 .8-naphthalimid (198 mg.

0.383 mmol; 1 equiv.) was dissolved in 10 ml dry methanol, dry

K ₂ C0 ₃ (212 mg. 1 .53 mmol. 4 equiv.) was added and it was stirred for 50 h at roomtemperature The red brown suspension was filtered and the collected solid was purified by column chromatography (80 x 3 cm. Kieselgel.

Dichlormethan/ Essigsaureethylester = 95:5 v/v) to give the pure product as a light yellow solid (0.91 g. 53 %).

H NMR (CDCI ₃ . 300 MHz): δ = 0.87 (tr. 3H. J = 6.78Hz. H ¹ ). 1 .24-1 .44 (m. 26H. H ^2"4 ). 1 .72 (qu. 2H. J = 7.53Hz. H ⁵ ). 3.73 (s. 1 H. H ¹² ). 4.19 (tr. 2H. J = 7.53Hz. H ⁶ ). 7.82 (tr. 1 H. J = 7.35Hz. H ⁸ ). 7.96 (d. 1 H. J = 7.72Hz. H ¹¹ ). 8.26 (d. 1 H. J = 7.53Hz. H ¹⁰ ). 8.64 (dd. 2H. H ^{7 9} ); ³ C NMR (CDCI ₃ . 75 MHz): δ = 14.22. 22.83. 27.32. 28.30. 29.50. 29.52. 29.70. 29.56. 28.80. 29.82. 29.85. 32.08. 40.79. 80.56. 86.50. 123.16. 123.30. 126.31 . 127.80. 128.14. 130.24. 131.76. 131 .79. 132.16. 132.23. 163.76. 164.03; MS (El): m/z calcd.: 445; found. 445;

General Procedure of the CuAAC reaction

The Cu(l) catalyzed reaction between an azide and an alkyne (CuAAC) has been performed as follows: All reactions were performed on a mmol-scale. To a solution of azide (1 eq) and alkyne (1 eq) in THF/H ₂ 0 (3/1 ) was added Cul (5 mol%) and sodium ascorbate (2.5 mol%). It was stirred overnight at 50°C.

The THF was removed under reduced pressure and the residue was taken up in CHCI3. It was washed with water and the organic phase was dried over MgS0 ₄ . The organic phase was concentrated and the residue was chroma- tographed on silica gel eluting with CH ₂ Cl2/MeOH (95/5).

Example 1

Synthesis of 3-{4-[4-(1 ,4,7, 10, 13-pentaoxa-16-azacvclooctadecan-16- vOphenylH H-1 ,2,3-triazol-1 -yl)-7-(diethylamino)-2H-chromen-2-one (1 ):

27 33

The synthesis followed the general procedure of the CuAAC reaction.

Yield: 49 %, ¹ H NMR (CDCI _3, 500 MHz): δ = 1 .23 (t, 6H, J = 7.25Hz), 3.44 (q, 4H, J = 7.25Hz,), 3.64-3.67 (m, 20H), 3.72 (t, 4H, J = 5.68Hz ), 6.55 (s, 1 H), 6.66 (dd, 1 H), 6.75 (d, 2H, J = 8.52Hz), 7.41 (d, 1 H, J = 8.83Hz), 7.74 (d, 2H, J = 8.83Hz), 8.15 (s, 1 H), 8.38 (s, 1 H); ¹³ C (CDCI ₃ , 75 MHz): δ = 13.53, 46.05, 52.38, 69.78, 71.89, 98.08, 108.27, 1 10.91 , 1 12.67, 1 18.12, 1 19.22, 1 19.70, 127.96, 130.95, 135.34, 148.82, 149.06, 152.40, 156.79, 158.02; HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ , 622.32; found, 623.33; UV/Vis

(Acetonitrile), A _max (ε) = 410 nm (21228 M ^"1 cm ^"1 ), A _max (ε) = 288 nm (2021 1 M ^"1 cm ^"1 ).

Example 2

Synthesis of 3-{1 -[4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16- yl)phenyl1-1 H-1 ,2,3-triazol-4-yl)-7-(diethylamino)-2H-chromen-2-one (2):

2

28 34

The synthesis followed the general procedure of the CuAAC reaction.

Yield: 55 %, ¹ H NMR (CDCI _3, 600 MHz): δ = 1 .23 (t, 6H, J = 7.25Hz), 3.44 (q, 4H, J = 7.25Hz,), 3.63-3.67 (m, 20H), 3.72 (t, 4H, J = 5.68Hz), 6.53 (s, 1 H), 6.62 (dd, 1 H), 6.76 (d, 2H, J = 8.48Hz), 7.41 (d, 1 H, J = 8.85Hz), 7.76 (d, 2H, J = 8.48Hz), 8.60 (s, 1 H), 8.64 (s, 1 H); ¹³ C (CDCI ₃ , 150 MHz): δ = 12.54, 44.94, 51 .51 , 68.58, 70.84, 97.13, 108.83, 109.44, 1 10.87, 1 1 1.93, 120.54, 122.12, 126.56, 129.60, 138.51 , 142.06, 148.26, 150.82, 156.1 1 , 160.79; HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ , 622.32; found, 623.35; UV/Vis

(Acetonitrile), A _max (ε) = 413 nm (40057 M ^"1 cm ^"1 ), A _max (ε) = 293 nm (18136 M ^"1 cm ^"1 ).

Example 3 (reference compound) Synthesis of A/,/V-Diethyl-4-ri -(7-diethylaminocoumann-3-yl)-1 /-/-1 , 2, 3- triazo -4-yl)1aniline (3):

32 33

The synthesis followed the general procedure of the CuAAC reaction.

Yield: 60 %, ¹ H NMR (CDCI ₃ , 300 MHz): δ = 1 .17-1 .27 (m, 12H), 3.37-3.50 (m, 8H), 6.57 (s, 1 H), 6.68 (dd, 1 H), 6.75 (d, 2H, J = 8.48Hz), 7.42 (d, 1 H, J = 8.85Hz), 7.76 (d, 2H, J = 8.1 Hz), 8.44 (s, 1 H), 8.65 (s, 1 H), ¹³ C (CDCI ₃ , 75 MHz): δ = 12.43, 12.62, 29.69, 44.39, 44.98, 97.08, 107.28, 109.99, 1 1 1.80, 1 17.31 , 1 17.57, 1 18.61 , 127.06, 129.91 , 134.15, 147.75, 148.25, 151.41 , 155.72, 157.01 ; HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ , 431 .23; found, 432.32; UV/Vis (acetonitrile), A _max (ε) = 410 nm (29501 M ^"1 cm ^"1 ), 248 nm (22823 M ^"1 cm ^"1 ).

Example 4 (reference compound)

Synthesis of 3-(4-((1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16- yl)methyl)-1 H-1 ,2,3-triazol-1 -yl)-7-(diethylamino)-2H-chromen-2-one (4):

29 33

The synthesis followed the general procedure of the CuAAC reaction. Yield: 23 %, ¹ H NMR (CDCI ₃ , 300 MHz): δ =1 .14 (t, 6H, J = 7.16Hz), 2.70 (m, 4H), 3.36 (q, 4H, J = 7.16Hz,), 3.54-3.68 (m, 20 H), 3.79 (s, 2H), 6.42 (s, 1 H), 6.60 (dd, 1 H), 7.40 (d, 1 H, J = 9.04Hz), 8.24 (s, 1 H), 8.33 (s, 1 H); ¹³ C

(CDCI3, 75 MHz): δ = 12.76, 45.34, 49.51 , 53.78, 67.91 , 69.72, 97.16, 107.21 , 1 10.60, 124.15, 129.08, 130.67, 131 .26, 136.14, 152.12, 156.27, 127.46 ; HRMS ( ⁺ ESI): m/z calcd. for (M+H) ⁺ ,560.31 ; found,560.43; UV/Vis (acetonitrile), A _max (ε) = 408 nm (10534 M ^"1 cm ^"1 ), 246 nm (1 1 149 M ^"1 cm ^"1 ).

Example 5

Synthesis of 7-(diethylamino)-3-(4-(3-methoxy-4-(1 ,4,7, 10-tetraoxa-13- azacyclopentadecan-13-yl)phenyl)-1 H-1 ,2,3-triazol-l -yl)-2H-chromen-2-one

14

A mixture of compound 40 (130 mg, 0.37 mmol), 3-azido-7- diethylaminocoumarin 60 (96.1 mg, 0.37 mmol), copper sulfate pentahydrate (4.6 mg, 5 mol%) and sodium ascorbate (7.3 mg, 10 mol%) in 6 ml

THF/water (2/1 ) was stirred at 60 °C for 48 hours. To the reaction mixture was added water (5 mL) and it was extracted 3 times with CHCl3 (3*10 mL). The combined organic layers were dried with MgS0 ₄ and concentrated in vacuum. The residue was purified by column chromatography on silica with CHCI3/CH3OH (95/5) as eluent. The product was obtained as a dark yellow oil, which crystallised upon standing in a freezer. (90 mg, 40%).

1H NMR (500 MHz, CDCI3): δ = 1 .24 (t, 6H, ³ J = 7.09 Hz, 1-H), 3.45 (q, 4H, ^Z J = 7.09 Hz, 2-H), 3.51 -3.55 (m, 4H, 21 -H), 3.64-3.70 (m, 16H, 22-H, 23-H, 24-H, 25-H), 3.93 (s, 3H, 20-H), 6.56 (d, 1 H, ⁴ J = 2.21 Hz, 3-H), 6.68 (dd, 1 H, ³ J = 8.83 Hz, ⁴ J = 2.36 Hz, 5-H), 7.17 (d, 1 H, ³ J = 7.09 Hz, 16-H), 7.38 (d, 1 H, ³ J = 7.88 Hz, 15-H), 7.43 (d, 1 H, ³ J = 8.99 Hz, 6-H), 7.46 (s, 1 H, 19- H), 8.44 (s, 1 H, 9-H), 8.74 ppm (s, 1 H, 12-H); ¹³ C NMR (125 MHz, CDCI3); δ = 12.39 (C1 ), 44.96 (C2), 53.05 (C21 ), 55.63 (C20), 70.08, 70.37, 70.49,

70.90 (C22, C23, C24, C25), 97.00 (C3), 107.13 (C7), 109.26 (C19), 1 10.05 (C5), 1 16.98 (C10), 1 18.38 (C15), 1 19.63 (C12), 120.52 (C16), 124.07 (C18), 129.96 (C6), 134.34 (C9), 139.92 (C13), 147.68 (C14), 151 .50 (C4), 152.66 (C17), 155.74 (C8), 156.95 ppm (C1 1 )

ESI-MS: m/z calcd. for [M+H] ⁺ 608.31 ; found 608.38;

IR (KBr, cm ^"1 ): 1 130 (s), 1239 (s), 1602 (s), 1728 (s), 2855 (s), 2923 (s) UV/Vis (CH3CN): Amax (ε) = 267 (4352), 413 nm (8649).

Example 6

Synthesis of 7-(diethylamino)-3-( 1 -(3-methoxy-4-( 1 ,4,7, 10-tetraoxa-13- azacyclopentadecan-13-yl)phenyl)-1 H-1 ,2,3-ΐπ3ζοΙ-4-νΙ)-2Η-οΙΐΓοηΐθη-2-οηθ

15 A mixture of compound 43 (1 17.5 mg, 0.32 mmol), 3-acetylen-7- diethylaminocoumarin 61 (77.4 mg, 0.32 mmol), Cu/C (16 mg, 20 mol%) and Triethylamin (32.5 mg, 0.32 mmol) was stirred in THF (4 ml_) for 48 hours. Cu/C was filtered off through Celite, washed several times with CHCI3 and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica with CHCI3/CH3OH (95/5) as eluent. The product was obtained as a dark yellow oil, which crystallised upon standing in a freezer. (91 mg, 47%).

1H NMR (300 MHz, CDCI3): δ = 1 .22.(t, 6H, ³ J = 7.06 Hz, 1-H), 3.43 (q, 4H, ³ J = 7.06 Hz, 2-H), 3.51 -3.55 (m, 4H, 21 -H), 3.65-3.69 (m, 16H, 22-H, 23-H, 24-H,25-H), 3.91 (s, 3H, 20-H), 6.54 (d, 1 H, ⁴ J = 1 .98 Hz, 3-H), 6.63 (dd, 1 H, ³ J = 8.76 Hz, J = 2.31 Hz, 5-H), 7.13-7.23 (m, 2H, 15-H, 16-H), 7.32 (s, 1 H, 19-H), 7.41 (d, 1 H, ³ J = 8.85 Hz, 6-H), 8.65 (s, 1 H, 9-H), 8.66 ppm (s, 1 H, 13- H); ¹³ C NMR (75 MHz, CDCI3): δ = 12.42 (C1 ), 44.82 (C2), 53.03 (C21 ),

55.78 (C20), 70.03, 70.42, 70.94, (C22, C23, C24, C25), 97.03 (C3), 104.73 (C19), 108.67 (C7), 109.32 (C5), 1 10.59 (C14), 1 12.45 (C16), 120.40 (C15), 122.05 (C13), 129.49 (C6), 131 .15 (C10), 138.49 (C9), 140.25 (C12), 142.14 (C18), 150.78 (C4), 152.83 (C17), 156.00 (C8), 160.62 ppm (C1 1 )

ESI-MS: m/z calcd. for [M+H] ⁺ 608.31 ; found 608.35;

IR (KBr, cm ^"1 ): 1 130 (s), 1230 (s), 1600 (s), 1694 (s), 1720 (s), 2861 (s), 2969 (s)

UV/Vis (CH3CN): Amax (ε) = 259 (14131 ), 410 nm (34604)

Elemental analysis (%) calcd. for C32H41 N5O7 (607.70): C 63.25, H 6.80, N 1 1 .52; found C 62.91 , H 6.69, N 1 1 .12.

Example 7

Synthesis of 6-(1 -(4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16- yl)phenyl)-1 H-1 ,2,3-triazol-4-yl)-2-butyl-1 H-benzordelisoquinoline-1 ,3(2H)- dione (11 ):

The synthesis followed the general procedure of the CuAAC reaction using precursors 28 and 50.

The crude product was purified by column chromatography (65 x 2 cm, silica gel, CHCI ₃ /MeOH = 98:2 v/v) to yield as a yellow solid.

Yield: 0.086 g, 49 %, mp: 136.1 -139.0 °C, R _f = 0.52; H NMR, (CDCI ₃ , 600 MHz) δ (ppm), J (Hz): 9.1 (dd, 1 H, H ⁹ ), 8.63 (dd, 1 H, H ⁷ ), 8.61 (d, 1 H, J = 8.55, H ⁵ ), 8.26 (s, 1 H, H ¹⁰ ), 7.99 (d, 1 H, J = 7.56, H ⁶ ), 7.79 (dd, 1 H, H ⁸ ), 7.61 , (d, 2H, J = 9.12, H ¹¹ ), 6.81 (d, 2H, J = 9.12, H ¹² ), 4.18 (t, 2H, J = 7.62, H ⁴ ), 3.74-3.64 (m, 24H, H ^13"18 ), 1 .76-1 .69 (m, 2H, H ³ ), 1 .45 (sx, 2H, J = 7,62, H ² ), 0.98 (t, 3H, J = 7.38, H ¹ ); ³ C NMR, (CDCI ₃ , 150 MHz) δ (ppm): 164.34, 164.06, 148.68, 146.15, 145.66, 134.32, 132.92, 131 .52, 130.81 , 129.40, 128.95, 127.48, 127.35, 125.96, 122.94, 122.56, 121 .52, 120.06, 1 12.96, 1 1 1 .99, 70.98-70.82,68.8, 68.60, 51.59, 40.41 , 30.32, 20.52, 13.99; HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 658.32; found, 658.35;

Example 8

Synthesis of 6-(4-(4-(1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecan-16- yl)phenyl)-1 H-1 ,2,3-triazol-1 -yl)-2-ethyl-1 H-benzordelisoquinoline-1 ,3(2H)- dione (10):

The synthesis followed the general procedure of the CuAAC reaction using precursors 27 and 51.

The crude product was purified by column chromatography (65 x 2 cm, silica gel, CHCI ₃ /MeOH = 95:5 v/v) to yield as an orange solid.

Yield: 0.024 g, 19 %, mp: 213.6-216.4 °C, R _f = 0.14; H NMR, (CDCI ₃ , 600 MHz) δ (ppm), J (Hz): 8.71 (t, 2H, J = 7.47 H ³ , H ⁵ ), 8.36 (d, 1 H, J = 8.1 , H ⁷ ), 8.09 (s, 1 H, H ⁸ ), 7.89 (d, 1 H, J = 7.47, H ⁴ ), 7.83 (tr, 1 H, J = 5.26, H ⁶ ), 7.79, (d, 2H, J = 8.7, H ⁹ ), 6.79 (d, 2H, J = 8.82, H ¹⁰ ), 4.28 (q, 2H, J = 7.12, H ² ), 3.74-3.68 (m, 24H, H ^11"16 ), 1 .36 (t, 3H, J = 7.12, H ¹ ); ³ C NMR, (CDCI ₃ , 150 MHz) δ (ppm): 163.68, 163.16, 149.13, 148.48, 138.59, 132.29, 130.82, 129.87, 129.29, 128.63, 127.31 , 126.71 , 123.93, 123.46, 123.19, 120.20, 1 17.23, 1 12.01 , 71 .04-70.93, 68.82, 51 .50, 35.93, 13.47; HRMS ( ⁺ ESI): m/z calcd for (M+H) ⁺ , 630.29; found,630.14 Example 9

Synthesis of 16-(4-(4-(anthracen-9-yl)-1 H-1 ,2,3-triazol-1 -vQphenyQ- 1 4,7, 10, 13-pentaoxa-16-azacyclooctadecane (5):

The synthesis followed the general procedure of the CuAAC reaction using precursors 28 and 52.

The crude product was purified by column chromatography (65 x 2 cm, silica gel, CHCI ₃ /MeOH = 95:5 v/v) to yield as a light brown oil.

Yield: 23%, ¹ H NMR (CDCI ₃ , 300 MHz): δ = 3.53-3.758 (m, 24H, H ^9"14 ), 6.31 (d, 2H, J = 9.231 , H ⁸ ), 7.40-7.55 (m, 4H, H ⁴ ' ² ), 7.7 (d, 2H, J = 9.231 , H ⁷ ), 8.03 (m, 4H, H ⁵ ' ² ), 8.1 (s, 1 H,H ⁶ ), 8,57 (s, 1 H, H ¹ ); ¹³ C (CDCI ₃ , 75 ΜΗζ):δ = 51 .16, 68.37, 70.85, 1 1 1 .24, 1 19.95, 122.07, 124.54, 125.21 , 125.48, 126.07, 126.89, 128.64, 131 .02, 131.34, 145.62, 148,02; HRMS ( ⁺ ESI): m/z calcd. (M+H) ⁺ , 583.34; found, 583.29; UV/Vis (MeCN,) A _max (ε) = 388 nm (5596 M ^"1 cm ^"1 ), 420 nm (5782 M ^"1 cm ^"1 ).

Example 10

Synthesis of 7-(diethylamino)-3-(1 -(3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13- pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-1 H-1 ,2,3-triazol-4-yl)-2H- chromen-2-one 7)

The synthesis followed the general procedure of the CuAAC reaction using precursors 56 and 61.

The crude product was purified by column chromatography (65 x 2 cm, silica gel. CHCI ₃ /MeOH = 95:5 v/v) to yield as a yellow oil.

Yield: 13%, HRMS ( ⁺ ESI): m/z calc. (M+H) ⁺ . 696.36; found. 696.47.

Example 11

Synthesis of 16-(4-(4-(anthracen-9-yl)-1 H-1 ,2,3-triazol-1 -yl)-2-(2- methoxyethoxy)phenyl)-1 ,4,7, 10, 13-pentaoxa-16-azacyclooctadecane (6)

The synthesis followed the general procedure of the CuAAC reaction using precursors 56 and 52. The crude product was purified by column chromatography (65 x 2 cm, silica gel. CHCI ₃ /MeOH = 98:2 v/v) to yield 5 as a light brown oil.

Yield: 19.3 %; H NMR (CDCI ₃ . 600 MHz): δ = 3.35-3.75 (m. 27H). 3.81 (tr. 2H). 4.28 (tr. 2H). 7.31 (d. 1 H). 7.45 (m. 5H). 7.58 (s. 1 H). 7.91 (d. 2H). 8.05 (d. 2H). 8.21 (s. 1 H). 8.55 (s. 1 H);

Example12

Synthesis of 2-hexadecyl-6-(1 -(3-(2-methoxyethoxy)-4-(1 ,4,7, 10, 13- pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-1 H-1 ,2,3-triazol-4-yl)-1 H- benzo[de]isoquinoline-1 ,3(2H)-dione (9)

The synthesis followed the general procedure of the CuAAC reaction using precursors 56 and 57.

The crude product was purified by column chromatography (65 x 2 cm, silica gel. CHCI ₃ /MeOH = 95:5 v/v) to yield 9 as a light brown oil.

Yield: 24 %; ¹ H NMR (CDCI _3. 600 MHz): δ = 0.9 (tr. 3H). 1 .27 (m. 24 H). 1 .39 (qu. 2H). 1.47 (qu. 2H). 1 .78 (qu. 2H). 3.40-3.78 (m. 27 H). 3.83 (tr. 2H). 4.28 (tr. 2H). 7.21 (d. 1 H). 7.35 (d. 1 H). 7.50 (s. 1 H). 7.86 (tr. 1 H). 8.09 (d. 1 H). 8.49 (s. 1 H). 8.7 (tr. 2H). 9.17 (d. 1 H).

Example 13

Synthesis of 7-(diethylamino)-3-{4-3-[2-methoxyethoxy)-4{bis(4- methylbenzo[5, 6, 17, 181(0, N, N', O'H-1 , 7, 16, -triaza-crvptand-[3, 2, 1 ]- phen-4-yl]-1 H-1 .2.3-triazol-1 yl)-2H-chromen-2-one (13)

The synthesis followed the general procedure of the CuAAC.

Yield: 62%, HRMS ( ⁺ ESI): m/z calc. (M+H) ⁺ . 976.52; found. 976.59.

References

1 a) E. Tamanini, A. Katewa, L. M. Sedger, M. H. Todd and M. Watkin- son, Inorg. Chem,. 2009, 48, 319; b) E. Tamanini, K. Flavin, M. Mote- valli, M. H. Todd and M. Watkinson, Inorg. Chem., 2010, 49, 3798; c) S.Huang, R. J. Clark, L. Zhu, Org. Lett. 2007, 9, 4999;

2 S. Maisonneuve, Q. Fang, J. Xie, Tetrahedron, 2008, 64, 8716.

3 K.Varazo, F. Xie, D. Gulledge, Q. Wang . Tetrahedron Lett., 2008, 49,

5293.

4 H.-C. Hung, C.-W. Cheng, Y.-Y. Wang, Y.-J. Chen, W.-S. Chung, Eur.

J. Org. Chem., 2009, 15, 6360.

5 D. Maity, T. Govindaraju, Inorg. Chem., 2010, 49, 7229.

6 P. D. Jarowski, Y.-L. Wu, B. Schweizer, F. Diederich, Org. Lett., 2008,

10, 3347.

7 J. D. Lewis and J. M. Moore, Dalton Trans., 2004, 1376.

8 K. Sivakumar, F. Xie and Q. Wang, Org. Lett, 2004, 6, 4603.

9 J. W. Sibert and V. Lynch, Inorg. Chem., 2007, 46, 10913; J. W. Si- bert, P. B. Forshee, Inorg. Chem., 2002, 41(23), 5928.

10 D.-N. Lee, G.-J. Kim, H.-J. Kim, Tetrahedron Lett, 2009, 50, 4766.

1 1 E. Tamanini, S. E. J. Rigby, M. Motevalli, M. H. Todd, M. Watkinson, Chem. Eur, J. 2009, 15, 3720.

12 The FEFs were determined by division of the relative fluorescence intensities at the A _max of the complexed and free fluoroionophores. 13 a) H. He, M. A. Mortellaro, M. J. P. Leiner, R. J. Fraatz, J. K. Tusa, J.

Am. Chem. Soc, 2003, 125, 1468; b) P. Padmawar, X. Yao, O. Bloch, G. T. Manley, A. S. Verkman, Nat. Methods., 2005, 2, 825; R. D.

Carpenter, A. S. Verkman, Org. Lett, 2010, 12, 1 160; c) H. He, M. A. Mortellaro, M. J. P. Leiner, S. T. Young, R. J. Fraatz, J. K. Tusa, Anal. Chem., 2003, 75, 549.

14 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q.

Wang, Org. Lett, 2004, 6, 4603-4606. 15 a) J.-S. Wu, W.-M. Liu, X.-Q. Zhuang, F. Wang and S.-T. Lee, Org. Lett, 2007, 9, 33-36.

b) D.-N. Lee, G.-J. Kim, H.-J. Kim, Tetrahedron Lett, 2000, 50, 4766- 4768.

16 D. Jimenez, R. Martinez-Manez, F. Sancenon, and E.Garcia-Breijo, Eur. J. Inorg. Chem. 2005, 2393-2403

17 J. P. Dix and F. Vogtle, Chem. Ber. 1980, 113, 457.

18 J. D.Lewis, J. M. Moore, Dalton Trans., 2004, 1376-1385.

19 J. W. Sibert, V. Lynch, Inorg. Chem., 2007, 46, 10913-10925.

20 P. A. S. Smith and J. H. Boyer, Org. Synth., 1963, 4, 74.

21 E. Tamanini, S. E. J. Rigby, M. Motevalli, M. H. Todd, M. Watkinson, Chem. Eur. J. 2009, 15, 3720-3728.

22 M. Hirokazu; S. Furuyoshi, Y. Nakatsuji, M. Okahara. Bull. Chem. Soc.

Jpn.1983, 56, 212-218.

23 R. Wortmann, C. Glania, P. Kramer, R. Matschiner, J. J. Wolff, S.

Kraft, B. Treptow, E. Barbu, D. Langle and G. Gorlitz, Chem. Eur. J., 1997, 3, 1765-1773.

24 Synlett 1996, 521 -522

25 Chem. Commun. 201 1 , 47, 4685-4687

26 Huimin Guo, Maria L. Muro-Small, Shaomin Ji, Jianzhang Zhao, Felix N. Castellano, Inorg. Chem. 2010, 49, 6802-6804

27 T. Gunnlaugsson, Paul. E. Kruger and G. M. Hussey, J. of. Org.

Chem., 2005, 70(25), 10875-10878.

28 Q. Xiao, T. Brown, Tetrahedron, 2007, 63, 3483.

29 K.-C. Chang, l.-H. Su, A. Senthilvelan, W.-S. Chung, Org. Lett. 2007, 9(17), 3363-3366

30 A. Torrado, B, Imperial!, J. Org. Chem. 1996, 61 , 8940-8948

31 Liu et al., Journal of Polymer Science: Part A: Polymer Chemistry

2001 , 39, 1495-1504 Kreicberga. E. Jecs. V. Kampars. Rigas Tehniskas Universitates Zi- natniskie Raksti. Serija 1 : Matehalzinatne un Lietiska Kimija. 2005. 10. 46-53.

H. He und J. K. Tusa. J. Am. Chem. Soc. 2003. 125. 1468-1469 D. Jimenez und E.Garcia-Breijo. Eur. J. Inorg. Chem. 2005. 2393- 2403

T. Gunnlaugsson und M. Nieuwenhuyzen. J. Chem. Soc. Trans. 1 . 2002. 1954-1962

H. Guo und F. N. Castellano. Inorg. Chem. 2010. 49. 6802-6804.