INTRODUCTION TO
EXPERIMENTAL
SECTION
45 The studies about the 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one derivatives we mentioned in the introduction showed that the presence of a pendant 3-phenyl ring seems to be a crucial requirement to gain A2B AR/A2A
AR selectivity, as this phenyl hampers the interaction with A2A AR; all the
compounds featuring such a substitution were completely inactive at this latter subtype. 79
Stating this consideration, an in-depth analysis of the data reported in Table
2 (in the Introduction) shows that the affinity and selectivity profile for
ARs within the 3-phenyl and 3-(2-furyl) subseries strictly depends on the substitution at N10.
46 46e
The 3-(2-furyl)-N10-unsubstituted compound shows comparable affinity at
A1, A2A and A2B ARs, probably due to the absence of groups able to
discriminate between the different binding sites. Insertion at N10 of the
small lipophilic methyl, as well as of the phenyl ring or benzyl group, lowers affinity at both A1 and A2A ARs; an effect that becomes even more evident
when a chlorine atom is placed at the 7-position (X) of the 3-(2-furyl)-N10
-phenyl compound, making it completely inactive at A1 and A2A ARs.79
Insertion at N10 of a methyl, phenyl, or benzyl group generally increases A 1
AR affinity and maintains A2B AR affinity in the nanomolar range, thus
reducing A2B/A1 selectivity. Insertion of a chlorine at 4-position of the N10
-phenyl ring of 46e causes a loss in A1 AR affinity while maintaining high A2B
46 Taken together, these data suggest that the lipophilic area hosting the N10
-substituent in the A2B AR is more tolerant with respect to the corresponding
pocket in A1 AR binding site.
Finally, insertion of a chlorine at the 7-position of the triazinobenzimidazole nucleus of 46e produces a derivative, which is the most potent and selective of all the compounds investigated., which a IC50 of 3.10 nM at A2B AR and no
activity at A1, A2A and A3 ARs.79
Bearing in mind the conclusion that have been drawn from these studies we decided to design and synthesize new A2B AR probes featuring fluorescent
moiety to specifically target this receptor subtype.
The fluorescently labeled ligands represent a safe, fast, and not too much expensive way in probing the ligand-receptor complex. As a further advantage, fluorescent probes can be displaced from their binding sites of a target receptor by no-fluorescent ligands, allowing the identification of the sites recognized by the ligands. Fluorescent agents with high specificity and attractive spectroscopic properties are therefore needed in the field of biomedical research.
We designed the final compounds (71, 72, 77 and 78) bearing the fluorescent moiety linked to N 1- or N 10-alkyl chain.
We selected the well-known 4-chloro-7-nitrobenzofurazan (NBD) group as the fluorophore, because its small size does not generally affect affinity of the parent ligand. Moreover, NBD-containing compounds typically exhibit a low quantum yield in an aqueous solution, but they become highly fluorescent in nonpolar solvents or when bound to membranes or to hydrophobic clefts in proteins. 99
In addition, the length of the spacer alkyl chain was modified to introduce some flexibility that would favor self-adaptation of the ligands into the receptor binding site.
47 The key intermediates for the preparation of the target compounds were represented by derivatives 65 and 66 that were synthesized as outlined in
Scheme 1. The N10
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (65) and N1
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one (66) were prepared in
four steps starting from the commercially available 2-chloro-1H -benzimidazole (60) (Scheme 1), that was reacted with hydrazine hydrate at 180°C for 6h in a Pyrex capped tube yielding the 2-hydrazinyl-1H -benzimidazole (62). This latter compound was then refluxed for 2h with benzoyl formic acid to yield the 2-(2-(1H-benzimidazol-2-yl)hydrazono)-2-phenylacetic acid (63). The tricyclic system 3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (64) was formed by the cyclization of (63) in refluxing glacial acetic acid for 12h. Treatment of (64) with sodium hydride and ethyl bromo acetate induce the alkylation at the N10 or N1, affording the
regioisomer derivatives (65, yield 70%) and (66, yield 30%), respectively, which were separated and purified by flash cromatography (toluene/acetonitrile=9/1 as eluting system). Yield, melting point and spectral data of compounds 62, 63 and 64 were in agreement with those reported in literature.100,101 The reaction yields and spectroscopy characteristics of
48
Scheme 1
N H N Cl 180° + H2N.NH2 . H2O N H N NHNH2 COCOOH abs EtOH N H N N H N C HOOCGlacial acetic acid
N H N N N O NaH anhydrous DMF N N N N O CH2COOEt N N N N O + CH2COOEt N H NH N N C HOOC 60 61 62 64 63 65 66 N N N H N O BrCH2COOEt
49 As schematized in Scheme 2, the N10
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (65) was condensed
with N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine in a Pyrex capped tube al 80°C for 3h. The derivatives thus formed, N10
-[Boc- (aminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (67) and N10
-[Bpc-(aminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (68), were purified by
flash-chromatography (AcOEt/Hexane=5/5 as eluting system). Compounds (67) and (68) were then treated with trifluoroacetic acid to remove the protecting group (Boc), freeing in this way the amino moiety. The amino compounds formed, N10
-[(aminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (69) and N10
- [(aminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (70), were then reacted with the fluorescent
4-chloro-7-nitrobenzofurazan in anhydrous DMF, at 0°C and in the presence of triethylamine which is needed to neutralize the hydrochloric acid that gets free in the reaction, yielding the target N10
-((4-(4-
nitrobenzo[1,2,5]oxadiazol-7-ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (71) and N10
-((6-(4-
nitrobenzo[1,2,5]oxadiazol-7-ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (72), that were purified by flash-chromatography (AcOEt as eluent). The reaction yields and spectroscopy characteristics of compounds (67-72) are listed in the Experimental Section.
50
Scheme 2
N N N N O CH2COOEt BOC-NH-(CH2)n-NH2 65 N N N N O CH2CNH(CH2)nNHCOC O O CH3 CH3 CH3 67: n=4; 68: n=6 CF3COOH N N N N O CH2CNH(CH)nNH2 O 69: n=4; 70: n=6 NBD-Cl NEt3/DMF N N N N O CH2CNH(CH)nNH O NO2 N O N 71: n=4; 72: n=6 n=4; n=651 In order to synthesize the other two final compounds, we started from the N1-substituted regioisomer, N1
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one (66), using the same
experimental conditions depicted in Scheme 2. Briefly, compound (66) was condensed with N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine
(Scheme 3), yielding N1
-[Boc-(aminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (73) and N1
-[Boc- (aminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (74) that were purified by flash-chromatography
(AcOEt/Hexane=5/5 as eluting system). The protector group (Boc) was removed afterwards, obtaining N1
-[(aminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (75) and N1
- [(aminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (76). When the fluorescent moiety
4-chloro-7-nitrobenzofurazan was finally added, we achieved our final compounds, N1
- ((4-(4-nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (77) and N1
-((6-(4-
nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-[4(10H)-one] (78) that were
purified by flash-chromatography (AcOEt as eluent). The reaction yields and spectroscopy characteristics of compounds (73-78) are listed in the Experimental Section.
52
